key: cord-0037911-4pqabi1d
authors: nan
title: Poster Presentations
date: 2016-09-14
journal: Cytokine
DOI: 10.1016/j.cyto.2016.07.021
sha: f2f841ce20aa58b0dfdbee95941c7b20970752aa
doc_id: 37911
cord_uid: 4pqabi1d

nan

Results: RNAseq and bioinformatic analyses revealed that IL-1b production associates with liver disease linked with expression of a range of IL-1b-responsive inflammatory genes known to support tissue remodeling, leukocyte homing and activation, and proinflammatory cytokine production and amplification. Further, we found that hepatic macrophages, known as Kupffer cells, are a major source of liver IL-1b. Typically, IL-1b production is a tightly regulated pathway requiring a priming step to trigger IL-1b transcription and an activating signal to promote mature IL-1b processing and release. We found that the HCV virion is efficiently phagocytosed by the Kupffer cell and human THP1 macrophage cell line to drive signaling through TLR7/MyD88 and NLRP3 inflammasome. This process triggers IL-1b expression and mature IL-1b protein release, respectively. We have now identified the specific HCV-virion component, HCV core protein, as a driver of NLRP3 inflammasome activation. HCV core protein modulates many cellular events such as reactive oxygen species production and calcium signaling, all of which have been shown to activate the NLRP3 inflammasome pathway. We found patient-derived core protein, isolated from patients with HCV infection, can stimulate NLRP3 inflammasome activation and trigger the release of IL-1b.

Conclusion: Our studies reveal HCV virion-associated core protein as the trigger of viral-induced NLRP3 inflammasome activation within hepatic macrophages. Our observations define the HCV-Kupffer cell interaction as a central node directing hepatic inflammation underlying HCV pathogenesis.

Introduction: Chemokine receptors and their ligands are expressed on several tumour and cancer cells. Their expression has been correlated with migration and metastasis of cancer/tumour cells. CCR2 is expressed under varied immune conditions in T cells, B cells, natural killer cells, basophils, and dendritic cells. Furthermore, CCR2 is expressed in cancer tissues and upregulation correlates with advanced cancer/tumour, metastasis and relapse. CCR2 is regulated at the genomic level by several transcription factors which upon binding to the promoter of the gene, leads to either an upregulation or downregulation of the gene. One of the several proteins and pathways is the JAK-STAT3/5 pathway. The JAK-STAT3/5 pathways play a crucial role in cell differentiation, proliferation and thus has a critical role in cancers/tumours. The JAK-STAT3/5 pathway is regulated by cytokines including IL-2, IL-15 and IL-6 amongst several others. IL-2 has been well studied in cancer immunotherapy trials and current immunotherapy trials are also taking place with IL-15 in man. However, the precise mechanism of action and the cascade of proteins and events involved are not completely understood. This study, aims to investigate the regulation of the JAK-STAT pathway by IL-2 and IL-15 and its role in CCR2 mediated cancer metastasis in prostate cancer.

Methods: Preliminary studies on the JAK-STAT5 regulated genes using quantitative real time PCR and quantitative PCR in concert with bioinformatic analysis on publicly available databases have identified CCR2 as an IL-2 regulated STAT5 target gene. Interestingly, preliminary studies on cancer databases have shown activation of CCR2 through several other hormones and growth factors.

Results: The STAT5 binding site identified within the CCR2 gene, mapped to the promoter of the gene and its co-ordinates on the UCSC genome browser (hg38) are chr3:46,351,020-46,351,025 and chr3:463,520,53-463,520,58. The two sites have differential binding of the STAT5 complex. The CCR2 gene is regulated by IL-2 in T cells and public databases have shown an upregulation in solid cancers. Its role in prostate cancer is under investigation.

Conclusion: CCR2 plays an important role in cancer metastasis. Prostate cancer is the most common cancer in men and unfortunately, tumour cells may develop resistance to therapy and become metastatic. This then becomes a major obstacle, limiting the success in prostate cancer treatment. The complicated crosstalk, both genomic and nongenomic, between transcription factors, immune cells and cytokines/growth factors is considered to be a crucial factor contributing to metastasis. Although several mechanisms have been proposed, so far none of them can be defined as the rationale behind the phenomena of metastasis. This study, is in progress to delineate the role of the IL-2/IL-15 regulated JAK-STAT5 pathway in regulating the CCR2 gene and thus the CCL2-CCR2 axis.

References [1] Lim SY, Yuzhalin AE, Gordon-Weeks AN, Muschel RJ. [2] Bishop JL, Thaper D, Zoubeidi ACancers (Basel). 2014 Apr 9;6 (2):829-859. [3] Introduction: Despite significant research efforts, breast cancer is still the most common malignancy in women and the most common cause of cancer death worldwide. A major challenge in breast cancer treatment is disease heterogeneity that is contributed by many factors including intrinsic cell factors, microenvironment, angiogenesis and tumor-specific immune responses. Interferon regulatory factor 5 (IRF5) is a transcription factor that controls inflammatory and immune responses. Analysis of over 3000 human breast cancer tissues revealed that high expression of IRF5 correlates with increased survival and lower incidence of metastasis, whereas the lower quartile of IRF5 expression is a significant marker of poor prognosis for recurrence-free survival. These data support a tumor suppressor role for IRF5. The main purpose of this study was to generate a murine model of spontaneous mammary tumorigenesis in order to determine whether loss of IRF5, as found in human breast tumor tissue, contributes to disease onset and/or progression.

Methods: Irf5 knockout (ko) mice were backcrossed to BALB/C mice to obtain an F10 generation of Irf5ko Balb/c mice that retain wildtype Dock2 expression. Wild-type BALB/c mice are susceptible to spontaneous mammary tumorigenesis at a low incidence rate. The effect(s) of Irf5 deficiency on normal mammary gland development and mammary tumorigenesis was examined.

Results: In mammary glands harvested from one-year-old female virgin ko mice (n = 20), we found an increased incidence (15%) of mammary carcinoma in situ as compared to their wild-type littermates (5%; n = 20). Of interest, the number of tumor bearing mice was significantly higher (38%) in retired ko female breeders (n = 8); however, there were no abnormalities detected in one-yearold male ko virgins or breeders (n = 6). Cellular and molecular analysis of female ko and wild-type littermates revealed that Irf5 deficiency enhanced mammary epithelial cell proliferation as indicated by an increased number of Ki67 expressing cells, and resulted in abnormal hyperbranching, which may contribute to mammary tumorigenesis. Irf5 ko breeders also showed impaired mammary gland involution after weaning, which has been shown to facilitate mammary tumor formation in humans.

Conclusion: Taken together, these findings demonstrate that IRF5 is a gender-specific tumor suppressor in breast cancer. In this ongoing study, we are further investigating the intrinsic and extrinsic Results: We could show that the cleavage site that is used by the metalloprotease ADAM17 in vitro is the same that we found in vivo. Mutation of a single amino-acid residue within the stalk region of the IL-6R was sufficient to completely abrogate IL-6R proteolysis. We further found that N-and O-glycosylation was dispensable for signaling of the IL-6R, as an IL-6R variant completely devoid of glycans was still transported to the cell surface and was biologically active. In contrast, proteolysis of the IL-6R was orchestrated by an N-and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1, and proteolysis of the glycan-deficient IL-6R mutant was significantly impaired.

Conclusion: We identified for the first time a soluble form of the IL-6R in human serum that is generated by a protease. Mapping of the occupancy of all N-and O-glycosylation sites of the sIL-6R allowed us to describe glycosylation as dispensable for trafficking, stabilization and signaling of the IL-6R, but an important regulatory mechanism in terms of proteolysis. Introduction: Breast cancer is a heterogenous disease whose progression from atypical ductal hyperplasia to ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) is regulated by the aberrant expression of multiple mediators produced by the mammary tumor itself and the adjacent reactive stroma. We previously found that expression of the transcription factor interferon regulatory factor 5 (IRF5) was down-regulated in different stages of human DCIS and IDC, with loss of IRF5 expression correlating with metastasis. IRF5 is a critical mediator of the host immune response to pathogens and cellular response to DNA damage. It has recently been shown to play a role in macrophage polarization, with high levels expressed in M1 (proinflammatory) macrophages and low levels in M2 (anti-inflammatory). We also found that restoring IRF5 expression in the metastatic human mammary epithelial cell line MDA-MB-231 led to the recruitment of CXCR5(+) B and T cells to the tumor. These findings support a role for IRF5 in regulating tumor immunity. In order to directly address the in vivo function of IRF5 in the mammary tumor versus its function in the immune system, we generated syngeneic models of mammary tumorigenesis. Specifically, Irf5 +/+ and Irf5 -/-4T1 murine mammary tumor cells were generated and injected into mammary fat pads of Irf5 +/+ and Irf5 -/-BALB/c littermate mice. Kinetics of tumor formation, immune response and metastasis were monitored.

Methods: In vitro cell proliferation was measured by MTT assay and in vivo by flow cytometry analysis of Ki67 expression. In vitro cell migration and invasion were measured by transwell assays using non-coated and Matrigel-coated wells, respectively. Primary tumor growth and metastasis were measured by in vivo bioluminescence imaging and metastasis confirmed by counting metastatic nodules on the lung.

In vitro experiments showed that murine IRF5 re-expressed in 4T1 cells did not affect cell proliferation but reduced cell migration by around 30% and invasion by 50%. In vivo experiments examining IRF5-mediated tumor immunity showed a significant increase in 4T1 primary tumor growth and metastasis in Irf5 -/-BALB/c mice (10-fold bioluminescence intensity; 3-fold increase in metastatic nodules) as compared to wild-type littermates.

Conclusion: These data show that restoring murine IRF5 expression in murine mammary tumor cells inhibits cell migration and invasion, supporting similar tumor suppressor functions for murine IRF5 as seen with human IRF5. Similarly, IRF5 expression in the whole animal is important for regulating the establishment of primary tumors as well as metastasis to secondary sites. Together, these data indicate that IRF5 acts as a tumor suppressor in breast cancer through intrinsic control of tumor cell migration/invasion and extrinsic regulation of the tumor immune microenvironment. Thus, determining the pathways and mechanisms by which IRF5 influences tumor progression and tumor immunity will lead to the discovery of new biomarkers for prognosis and molecular targets for the treatment of breast cancer. nervous system neoplasms. Although advances in surgery, radiation and chemotherapy have improved overall survival, the lifelong sequelae of these treatments represents a major health care burden for patients, parents, and society. Recent progress in the molecular subtyping of medulloblastoma have revealed at least four distinct subsets of tumours (WNT, sonic hedgehog (SHH), Group 3 and Group 4) with dramatically different biology. Analysis of gene expression data reveal that elevated STAT3 expression correlates with significantly worse overall survival of medulloblastoma patients.

Methods: To define the role of STAT3 in development and progression of SHH medulloblastoma we crossed two independent mouse models of medulloblastoma to mice lacking STAT3 from the granule cell neurons which is the cell of origin of medulloblastoma.

To identify STAT3 dependent pathways that can be targeted therapeutically we screened phospho-protein antibody array and compound libraries.

Results: Conditional deletion of STAT3 specifically in cerebellar granule cells provides a significant survival advantage in mouse models of SHH medulloblastoma. Intriguingly, this protection is almost completely restricted to males.

Our phospho-protein arrays and compound libraries converge on those pathways required for proliferation and cell cycle which may prove to be viable therapeutic options for the treatment of medulloblastoma.

Conclusion: Elevated STAT3 expression correlates with poor overall survival of medulloblastoma patients. Conditional of STAT3 from the cell of origin in two independent mouse models of SHH medulloblastoma provides a significant survival advantage specifically in males. Moreover, we identify compounds that specifically kill medulloblastoma tissue in a STAT3-dependent manner. Introduction: PD-1/PD-L1 signaling is critical for the negative regulation of the immune response to colorectal cancer (CRC) progression, but little is known about the process by which these proteins are expressed aberrantly in the tumor microenvironment [1] . Moreover, conventional RNAi screening conducted in twodimensional (2D) monoculture methods are lacking in physiological relevance; a limitation that may be improved by recapitulating the tumor microenvironment in three-dimensional (3D) assay systems containing multiple cell types found in vivo [2] . Therefore, we hypothesized that RNAi screening for genes that are responsible for the negative regulation of the immune response would yield different results when the tumor microenvironment is recapitulated (3D) versus in monolayer culture (2D). These results could drastically impact the process of target identification and drug discovery by improving the reliability of hit selection.

Methods: The Ambion Silencer Ò Select Human Kinase siRNA Library V4 was screened in 3D in Corning 384 Well Black Clear Round Bottom Ultra-Low Attachment Spheroid Microplates against tumor spheroids comprised of a colorectal cancer cell line, DLD-1 (ATCC), at a density of 12.5 Â 10 3 cells/ml and a coculture of DLD-1 cells with murine fibroblasts, NIH3T3 (ATCC), at a 3:2 ratio (respectively) using a process of reverse transfection. Greiner Bio-One 384 Well Black Clear Flat Bottom Microplates were used for RNAi high-throughput screening in 2D using the same cell lines and similar conditions. Lipofectamine RNAiMAX Transfection Reagent was used according to manufacturer's protocols for efficient delivery of siRNA and plasmid expressing PD-L1 and a T cell receptor activator. Tumor spheroids were incubated for 16 h with recombinant Jurkat T cells 48 h posttransfection. These Jurkat cells express the firefly luciferase gene under the control of NFAT response elements with constitutive expression of human PD-1 (BPS Bioscience). Promega's ONE-Glo Luciferase Assay System was used for measurement of luciferase activity, while Promega's CellTiter-Glo Luminescent Cell Viability Assay was used for measurement of viability. Luminescence was quantified by the EnVision Multilabel Plate Reader by Perkin Elmer.

Results: Preliminary results from this robust, reproducible, assay suggest high transfection efficiencies, in both 2D and 3D, of siRNA and plasmid expressing PD-L1 and/or the TCR activator, introduced simultaneously.

Conclusion: We believe that this high throughput screening assay system is one of the first of its kind, and will lend itself to the discovery of novel targets involved in the negative regulation of the immune system by cancers that express the immune checkpoint blockade protein PD-L1.

Introduction: Tumor-targeted therapies that efficiently eliminate malignant cells and in the process engage the innate and adaptive immune system are desirable for preventing cancer recurrence. We have pioneered an oncolytic poliovirus therapy, PVSRIPO, which selectively targets and eliminates malignant cells. PVSRIPO is a recombinant polio:rhinovirus chimera, engineered to eliminate neuronal competence and is devoid of neuropathogenicity after intracerebral inoculation in non-human primates and in humans. PVSRIPO has cancer tropism due to widespread ectopic expression of the poliovirus receptor, CD155, in solid cancers. PVSRIPO is highly cytotoxic against malignant cells and is effective despite pre-existing neutralizing antibodies and viral cell killing proceeds in the presence of an active innate antiviral response. A first-in-human Phase-1 study with PVSRIPO at Duke University has shown remarkable promise in patients with recurrent glioblastoma (GBM), a uniformly lethal disease. PVSRIPO tumor cell killing is associated with the induction of danger-and pathogen-associated molecular patterns (DAMPs and PAMPs) via antiviral type I interferons (IFNs) and simultaneous non-lethal infection of antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs) via CD155. Type I IFNs are known to inhibit tumor proliferation, activate innate immune cells and bridge innate and adaptive immunity among other functions.

Methods: To understand immune events associated with poliovirus infection of APCs, we examined the effects of PVSRIPO treatment on the human macrophage cell line (THP1) and primary human monocyte-derived DCs. PVSRIPO-treated DCs were evaluated for expression of maturation/activation markers and compared to untreated immature and mature DCs. Moreover, we assessed the PVSRIPO vaccine effect in an in vitro human immunotherapy assay. Human DCs generated from HLA-A2+ donor cells were incubated with PVSRIPO-induced tumor cell lysate and then used to stimulate autologous T cells in vitro followed by a cytotoxic T lymphocyte (CTL) assay. In addition, we performed in vivo studies in nude mice and PVR transgenic mice, utilizing four different cancer cell lines, DU145, Sum149, CT2A and EO771.

Results: PVSRIPO infection of THP1 macrophages induces Mda5/ Stat1 and Stat1 phosphorylation and TNFa and IFNb release. Coculturing of DCs with PVSRIPO alone or PVSRIPO-induced tumor lysate stimulates DC activation and IL12 and TNFa production. Human DCs co-incubated with PVSRIPO-induced tumor cell lysate, stimulate tumor antigen-specific T cell responses in an in vitro human immunotherapy assay. These tumor-antigen specific T cells lysed tumor cell targets in an antigen-specific manner. PVSRIPO treated in vivo tumors showed increased innate and adaptive immune cell recruitment and were accompanied with increased mouse survival.

Conclusion: Our data suggest that along with destruction of the primary tumor, oncolytic poliovirus mediates immune events. Importantly, we demonstrate that human DCs co-incubated with PVSRIPO-induced tumor cell lysate stimulate tumor antigen-specific T cell responses in an in vitro human immunotherapy assay. We have also shown that oncolytic poliovirus mediates immune events in xenograft as well as syngeneic, immunocompetent murine models of brain, breast and prostate cancer. Introduction: The Suppressor of Cytokine Signaling (SOCS) family of proteins are critical negative feedback inhibitors of cytokine and growth factor signalling, helping to restore homeostasis and prevent excessive pathological responses. Whilst the biological role and molecular function SOCS3 and other members have been well resolved, SOCS5 remains poorly characterised. SOCS5 has been shown to regulate the EGFR in over-expression studies and may contribute to EGFR/Ras driven cellular transformation. The Drosophila homologue, Socs36E is also known to regulate both EGFR and JAK signalling in vivo.

Methods: Mouse models of breast cancer have been used to identify and dissect the role of SOCS5 in this disease. We have performed bioinformatic analysis of SOCS5 gene expression from Oncomine, Kmplot, bcgenex and independent analysis of the TCGA breast data sets. Mass spectrometry was used to stringently identify candidate SOCS5 interacting proteins. The function of SOCS5 is currently been investigated in human and mouse breast cancer cell lines using siRNA and CRISPR/Cas9 technologies.

Results: We show for the first time that in the Polyoma Middle T antigen (PyMT) driven mouse model of breast cancer, SOCS5deficient mice exhibit accelerated tumour onset relative to wildtype mice (53 d versus 70 d, respectively, p < 0.005), thus providing the first in vivo evidence that mammalian SOCS5 can act as a tumour suppressor. Analysis of expression databases indicates that SOCS5 is down regulated in a majority of patients with invasive ductal breast carcinoma (TCGA dataset, top 7% under-expressed genes, p = 2.53E-23) and SOCS5 expression level positively correlates with patient outcome. Using mass spectrometry, we have identified a number of known and novel SOCS5 interacting proteins, many of which include known drivers of human breast cancer.

Conclusion: Breast cancer remains the leading cause of cancer related death of women. Whilst significant advances in disease treatment and diagnosis have improved patient outcome, resistance to therapy, metastasis and patients who do not respond to therapies have highlighted the complexity and heterogeneity of this disease. Further understanding of the signaling pathways and molecular events that lead to oncogenic transformation of the mammary gland is required. We have discovered that the poorly studied SOCS5 protein plays an important protective role in the PyMT mouse model of breast cancer. The number and type of interacting proteins identified by mass spectrometry suggests that SOCS5 is likely to form a signaling scaffold at via its largely unstructured N-terminal region. Dissection of the signaling complexes formed by SOCS5 and the mechanisms by which it regulates these targets to prevent excessive signaling in tumour cells is currently the subject of further investigation. mechanisms underlining a hypothetical mutual correlation between autophagy and proliferative processes in melanoma cell lines.

Methods: M14 and 793 melanoma cell lines (ATCC; Manassas, VA, USA) were grown in DMEM and RPMI 1640, respectively, supplemented with FBS, L-glutamine and Pen-Strep at 37°C in 5% CO 2 . In this study, M14 and 793 cells were starved by low serum treatment (0.1% FBS for 4, 8, 12 and 24 h), or by Rapamycin treatment at different concentrations (10, 50 , 75 and 100 nM) or Cloroquine (10 nM for 2, 5, 8 and 12 h). We first evaluated cell viability by MTT, and subsequently p-ERK, p-P70S6 kinase and LC3BII modulation were analyzed by Western Blot and/or IP assays. In addition, confocal and fluorescent microscopy were used to assess the LC3BII expression on M14 and 793 melanoma cells, undergoing 24 h of starvation or 2 h Cloroquine treatment.

Results: On M14 and 793 cells undergoing starvation, Rapamycin or Cloroquine treatments showed a significant decrease of viability compared to control group. p-ERK western blots showed a significant modulation with strong ERK activation on M14 cells undergoing 12 h of starvation (p = 0.008) and on 793 melanoma cells after 8 h of starvation (p < 0.001) or 2 h cloroquine (p < 0.001). Western blot analysis didn't show a p-ERK modulation on M14 and 793 cells treated with Rapamycin. Furthermore, adopting as a positive control, M14 and 793 cells treated with EGF showed a significant increase of p-ERK expression comparable to the control group (p < 0.001), in a time-independent manner. Surprisingly enough, we observed a strong p-p70 expression both on M14 cell line undergoing 12 h and 24 h (p < 0.001) of starvation or treatment with 75 nM Rapamycin (p = 0.01) and with Cloroquine at different time course, 2 h (p = 0.02), 5 h (p = 0.01), 8 h (p = 0.003), and on 793 cells undergoing 12 h and 24 h of starvation (p = 0.01) or treatment with Cloroquine at 8 h and 12 h (p = 0.01) comparable to the control group. Regarding autophagic activation, western blot and confocal microscopy showed a slightly increase of LC3BII in M14 cells undergoing starvation or treated with 2 h of Cloroquine. Conversely 793 cells showed a LC3BII strong modulation, more evident on cells undergoing 24 h of starvation.

Conclusion: These findings evidenced a correlation between proliferative and autophagic processes. In fact, we evidenced a higher activation for the following mediators: p-ERK on cells treated with 8 h of starvation and 2 h Cloroquine; p-p70 on cells treated with 12 and 24 h of starvation and with 8 and 12 h Cloroquine; LC3BII on cells undergoing to 12 and 24 h of starvation. These findings evidenced a key role of proliferative pathway in controlling autophagic process. Introduction: Chronic Obstructive Pulmonary Disease (COPD) is a major global health problem affecting tens of millions of people [1] . It is a chronic irreversible lung disease characterized by abnormal enlargement of airspaces (emphysema), obstruction of the airways and chronic bronchitis, and persistent macrophage-rich inflammation. Progressive and permanent destruction of the lung parenchyma and airway thickening leads to unremitting breathlessness and disability. The presence of disease comorbidities, including infection susceptibility, metabolic syndrome, muscle wasting and osteoporosis, further reduce quality of life [2] . Alveolar macrophages are believed to play a key role by secreting factors that exacerbate inflammation and cause irreversible tissue damage. Treatment of COPD involves the use of bronchodilators and potent immunosuppressive drugs, which are only partially effective at managing symptoms and is not curative, thus there is a significant unmet clinical need to develop novel treatments [3] . Cytokine inhibition is an attractive target in COPD, as a multitude of cytokines have been linked to disease severity.

We have used a well-characterized animal model of COPD to determine the role of granulocyte-colony stimulating factor (G-CSF) in lung inflammation and associated co-morbidities by genetic ablation of this factor. Bronchoalveolar lavage (BAL) was used to isolate cells from the lung and flow cytometry was used to determine cellular populations and activation. Histopathology was performed on lung tissue and airspace size was measured by the mean linear intercept method. Luminex technology was used to determine levels of pro-inflammatory mediators in BAL fluid. Comorbidity assessment was performed by micro-CT analysis of bones, muscle measurements and histopathology of heart. Results: Using CLL patient samples and a mouse model of CLL, we have detected reduced levels of interferon alpha (IFNa) in the serum of patients with CLL or serum samples from a mouse model of CLL. We discovered that plasmacytoid dendritic cells (pDCs), which underpin the activity of effector immune cells critical for anti-viral immunity and anti-tumor responses, are reduced in number and functionally impaired in progressive CLL. This defect correlated with low levels of IFNa, a cytokine critical for immunity. Lower pDC numbers with impaired IFNa production was due to the decreased expression of FMS-like tyrosine kinase 3 receptor (Flt3) and Toll-like receptor 9 (TLR9), respectively. Reduced Flt3 expression was reversed using inhibitors of TGF-b and TNF, an effect correlating with a reduction in tumor load.

Conclusion: Defects in pDC numbers and function offer new insight into mechanisms underpinning the profound immunodeficiency affecting CLL patients and provide a potentially novel avenue for restoring immunocompetency in CLL.

Disclosure of Interest: None declared. Introduction: The ability to generate interferon lambda 4 (IFN-k4), the most recently discovered member of the type-III interferon family, has been associated with impaired clearance of hepatitis C virus (HCV) infection. IFN-k4 signaling through an IFN-k receptor complex (IFNLR1 and IL10R2) leads to activation of interferonstimulated genes and antiviral effects as a part of the JAK-STAT pathway. However, the possible role of IFN-k4 in carcinogenesis remains unclear. Here, we investigated IFNLR1-dependent and independent functions of IFN-k4 in a hepatoma HepG2 cell line.

We used the CRISPR-Cas9 system to eliminate IFNLR1 in our previously established stable hepatoma cell line (IFN-k4-HepG2 cells) where IFN-k4 expression was induced by doxycycline treatment. Successful IFNLR1 knockout cell line (IFN-k4-IFNLR1KO-HepG2) was confirmed by DNA sequencing, Western blotting and the interferon stimulated response element luciferase reporter (ISRE-Luc) assays. The effects of IFN-k4 in IFN-k4-IFNLR1KO-HepG2 and its parental IFN-k4-HepG2 cells were assessed by cell viability, apoptosis, transwell migration and wound healing assays. RNAsequencing was used to investigate the global transcriptome changes in these cell lines and conditions.

Results: We successfully generated a stable IFN-k4-IFNLR1KO-HepG2 cell line. Compared to the parental IFN-k4-HepG2 cells, these cells were impaired in the ability to induce ISRE-Luc reporter after treatment with recombinant IFN-k4 or induction of IFN-k4 expression by doxycycline. In addition, the antiproliferative effects of IFN-k4 were eliminated by IFNLR1 depletion, which means these phenotypes are IFNLR1-dependent. However, promotion of cell migration by IFN-k4 was observed in both IFN-k4-HepG2 and IFN-k4-IFNLR1KO-HepG2 cells, suggesting a possibility of an alternative, IFNLR1-independent pathway of IFN-k4 function.

Conclusion: In summary, we found that IFN-k4 exhibits antiproliferative effects through IFNLR1-dependent mechanisms, whereas it might promote cell migration through IFNLR1-independent pathways. Further RNA-sequencing is ongoing to investigate the possible IFNLR1-dependent and independent mechanisms of IFN-k4 signaling. These results will contribute to better understanding of IFN-k4 functions that are important for carcinogenesis.

Disclosure of Interest: None declared. Dept Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy; 2 Istituto Superiore di Sanità; 3 

Introduction: The connection between chronic inflammation and risk of cancer has been supported by several studies. The development of cancer might be a process driven by the presence of a specific combination of inflammatory mediators, including cytokines, chemokines and enzymes, in the tumor microenvironment. Virus-induced tumors, like HPV-induced Squamous Cell Carcinomas, represent a paradigmatic example of the interplay between inflammation, as integral part of the innate antiviral response, and malignant transformation.

Methods: To study the tumorigenic role of inflammatory mediators in HPV+ cells, it has been analyzed by real time RT-PCR the expression of selected inflammatory cytokines, chemokines and related molecules in human foreskin keratinocytes transduced by E6 and E7 from mucosal (HPV-16) or cutaneous (HPV-38) genotypes comparing them to primary Human Foreskin Keratinocytes (HFK). It has been evaluated the contribution of the single E6 or E7 from HPV-16 by using human foreskin keratinocytes transduced by E6 or E7. Introduction: While links between obesity and inflammation have been reported, little is known about the immune impact of dietary fibres and fermentable materials (DFM) and accompanying changes in the gut microbiome in this context. To this end, the effects of a high-energy diet supplemented with different DFM on obesityassociated biomarkers and cytokines in Sprague-Dawley (SD) rats were examined.

Methods: Male rats were fed an AIN93G high-energy diet containing 16% fat for 2 weeks, then segregated into obese-prone and obeseresistant phenotypes, and shifted to high-energy diets supplemented with varied DFM sources (7% wheat bran, oat bran, resistant starch type II, or fructooligosaccharide (FOS)) for an additional 11 weeks. Rats displaying the highest and the lowest weight gain (n = 10 per diet group) were examined for potential differences in concentrations of immune biomarkers, including cytokines associated with inflammation and obesity. Cytokine or adipokine profiles of serum, mesenteric lymph nodes (mln), adipose, mucosal (ileum, ileal Peyer's patches, cecum, proximal and distal colon), and systemic (liver, spleen) tissues were comparatively analyzed for cytokine or adipokine profiles using ELISA or Luminex technology. Immune cell populations in spleen, ileal Peyer's patches and mln were analyzed by flow cytometry to determine the impact of diet and phenotype.

Results: Obese-prone rats had higher concentrations of insulin (906 pg/mL vs 631 pg/mL, P < 0.02) and leptin (3481 pg/mL vs 2631 pg/ mL, P = 0.033) in serum than obese-resistant rats, however no differences were apparent between phenotype or diet in glucagon, adiponectin, glucose or serum endotoxin concentrations. Obeseprone rats displayed lower concentrations of cytokine-induced neutrophil chemoattractant-3 (CINC-3/CXCL2; 37 pg/g vs 69 pg/g, P < 0.01) in the mln, with higher concentrations of cecal total transforming growth factor-beta (TGF-b; 6350 pg/g vs 5878 pg/g, P < 0.05) and CINC-2ab/CXCL3 (25 pg/g vs 20 pg/g, P < 0.01) in the ileal Peyer's patches than obese resistant rats. However, these differences were not attributable to DFM. FOS-fed rats had decreased splenic percentages of CD3 + and CD3 + CD62L + T cells, CD8a + cytotoxic T cells, CD45 + leukocytes, CD45RA + B cells and CD3 -CD4 -CD161a + natural killer cells, and increased percentages of CD4 + T helper cells relative to rats fed the control diet, with no significant differences between obese-prone and obese-resistant phenotypes.

Conclusion: High-energy diets had differential impacts on systemic adipokines and mucosal proinflammatory and regulatory cytokines in obese-prone and obese-resistant SD rats. High-energy diets supplemented with FOS altered proportions of splenic immune cell populations. However, DFM had no significant effects on cytokine and adipokine profiles associated with the diet-induced obese phenotype.

Disclosure of Interest: None declared. Introduction: Cytokines are small secreted proteins which mediate and regulate Immunity and Inflammation. These molecules are produced de novo in response to an Immune stimulus. These small protein molecules do exhibit autocrine or paracrine properties. Cytokines have been well studied to play important role in infections in general. However there is not much information on their role in CNS infections. The present study is focused on cytokine analysis in chronic CNS infection tuberculosis (tuberculous meningitis).

Methods: Cytokines are detected by using double antibody sandwich ELISA where anti-cytokine antibody is coated on plate, test sample, cerebrospinal fluid (CSF) is added to the well which will capture the cytokines. The captured cytokine is detected by an enzyme conjugated antibody through a chromogen OPD. The cytokine tested were TNF alpha, IFN-gamma, IL-4 and IL-6. The protocol followed was as per the manufacturers instructions. The standard curve was constructed using known concentration of cytokine which correlated well with the nanogram levels and the optical density.

Results: The standard graph for the above four cytokines was a fairly accurate curve. Eighteen of the 37 CSF samples showed gamma interferon levels in the CSF of TBM cases. TNF alpha levels were seen in 20 of the 37 cases and IL-6 levels were seen in 36 of the 37 cases. However, IL-4 the TH2 type of cytokine was seen in only 7 of the 37 subjects.

Conclusion: Cytokines could be detected by ELISA in the CSF of TBM cases. The presence of cytokines obviously indicate that these molecules are contributing to the immune reactions in the CNS. It is interesting to note that gamma interferon and TNF alpha are detected to the extent of half of the cases whereas IL-6 was detected in almost all the cases. The precise significance of these molecules in the CSF needs to be evaluated. Further it needs to be seen whether the HIV infection in these subjects alters the pattern of these cytokines. The details of the findings and preliminary observations of cytokine expression in cases with and without HIV infection would be presented and discussed.

Introduction: Breast cancer is the most common cause of cancerrelated death among women. CCL5 and CCR5 are overexpressed in basal and HER-2+ breast cancer subtypes, with CCL5 expression associated with cancer progression and metastasis. We have shown that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells through mTOR-dependent mRNA translation of proteins associated with cell cycle progression and survival. mRNA translation is an energy dependent process. Moreover, the TORC1 complex in the mTOR pathway functions to integrate different signals beyond mRNA translation, including those associated with nutrient sensing. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, is associated with tumor progression.

Methods: Using human breast cancer cell lines and primary mammary tumors we undertook a comprehensive analysis of the effects of CCL5 activation of CCR5 both in vitro and in vivo on the regaulation of metabolism and how this influneces tumor proliferation and invasion.

Results: We provide further evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumor cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show CCL5 increases cell surface expression of the glucose transporter, GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomic profiling we show the metabolic signature of CCL5-treated primary mouse mammary tumor cells reflects increased levels of intermediates in multiple anabolic pathways, including glycolysis, the pentose phosphate pathway, amino acid synthesis and lipid metabolism. This accumulation of metabolites suggests the potential for diversion of intermediates into alternate pathways for macromolecule synthesis, a metabolic signature consistent with requirements for cell proliferation and invasion. In mouse studies we show that there is a delay in tumor onset and smaller tumor volume for breast tumors that lack CCR5, associated with reduced glucose uptake, reduced GLUT1 expression and diminished metabolic activity. Using FTMS tissue imaging of mammary tumors we provide evidence that CCL5-CCR5 interactions enhance glycolysis, amino acid synthesis and lipid metabolism.

Conclusion: Viewed altogether, these data indicate that CCL5-CCR5 interactions in the breast cancer microenvironment regulate metabolic events, specifically glycolysis, amino acid synthesis and lipid metabolism that support the energy and biosynthetic demands of tumor cell proliferation and invasion. Introduction: Type I IFNs, IFN-alpha and IFN-beta, possessing antiviral and immunomodulatory activities are widely used for the treatment of chronic hepatitis C and multiple sclerosis, respectively. However, it has been reported that these proteins impact adversely on the microvasculature causing thrombotic microangiopathy, which is also observed with the anti-VEGF mAb, bevacizumab. This suggests a causal link between IFN and interference of vascular endothelial functions. In-vivo studies in animal models of subcutaneous tumours have shown that IFN-beta inhibits tumour angiogenesis, while in-vitro effects of IFNs on endothelial cell proliferation remain controversial. In the present study, we investigated the role of different IFNs in the regulation of endothelial cell activities and angiogenesis in vitro.

Methods: Cell surface expression IFNAR and IFNLR was detected by flow cytometry. Cell proliferation was measured by BrdU (pyrimidine analogue) incorporation of growing cells. Cell viability was determined by alamarBlue assay incorporated REDOX indicator relating to cellular metabolic reduction. Apoptosis was examined by flow cytometric analysis of annexin-V binding to externalized phosphatidylserine. Angiogenesis was evaluated by an in-vitro human primary cell co-culture assay of endothelial-derived tubule formation.

Results: We revealed that primary human umbilical vein endothelial cells (HUVECs) express both subunits, IFNAR1 and IFNAR2, of type I IFN receptor, which is shared by IFN-alpha and IFN-beta. Type III IFN-lamda receptor both chains, IFN-lamda-R1 and IL-10R2, for IFNlamda-1 (also known as IL-29) are also present on HUVECs. IFN-beta-1a, IFN-beta-1b and IFN-lamda-1 inhibited BrdU incorporation of primary cultures of HUVECs in a concentration-dependent manner, but IFN-lamda-1 showed a less inhibitory effect than IFN-beta-1a. However, both IFN-alpha-2a and 2b at the same doses to other IFNs did not inhibit growth of HUVECs. IFN-beta and IFN-lamda-1 also reduced cell survival of HUVECs evidenced by dramatic reduction of metabolic activity of viable cells, but this was not apparent with IFNalpha. Both types I and III IFNs had little effects on cell apoptosis induced by serum deprivation. Furthermore, we evaluated the ability of IFNs to interfere with VEGF endothelial function as this growth factor is essential for endothelial cell survival, differentiation and angiogenesis. We found that IFN-beta treatment strongly inhibited VEGF-induced capillary development in an in-vitro model of angiogenesis in a manner comparable to bevacizumab antagonism of VEGF-induced angiogenesis. IFN-alpha also decreased VEGFinduced angiogenesis, but to a lesser extent. Interestingly, IFNlamda-1 had little impact on VEGF-induced angiogenesis. Data from experiments on animals suggest that special water drinking behavior may promote the activity of the immune system and improve health. However, responses to treated water in humans are unknown. This study tested associations between different water drinking behavior and the condition of oral immune protection over 3 months.

Methods: In a randomized placebo controlled cross-over fashion, 85 healthy young people (18 years of age) were divided into 3 representative groups: trial group (30 persons consumed 30-35 ml per kg of physically treated water); placebo group (30 persons with the above described mode but with the placebo-device); control group (25 persons with a usual water drinking behavior). We examined saliva IL-1, IL-4, IL-6, TNF-a, k-INF, amylase, Ca, P and compared them using the Mann-Whitney U test and the Kruskal-Wallis test. We monitored cytokines by ELISA method. The activity of amylase and the quantity of Ca were calculated using the calorimetric method.

Results: All groups participants showed stable IL-1b level (p > 0,05). The IL-4 level of the trial group raised (p > 0,05). The similar trend was noticed with k-INF and TNF-a, IL-6 (p > 0,05). The same change was observed in two other groups except for IL-6 and TNF-a (p > 0,05).

The significant changes of IL-1/IL-6 level and Ca/P level were found (p < 0,05).

Conclusion: Treated water intake is associated with a specific immune response. The strategy of this mechanism will help to understand that healthy people should consume 30-35 ml per kg of drinking water.

[1] Bao, Y., Q. Wei, Water for preventing urinary stones (Review)// The Cochrane Library. 2012 CD004292. [2] Prüss-Üstün A Bos R, Gore F, Bartram J. Safer water, better health: costs, benefits and sustainability of interventions to protect and promote health.-Geneva : World Health Organization, 2008. [3] Signoretto C Bianchi F, Burlacchini G, Sivieri F, Spratt D, Canepari P. Drinking habits are associated with changes in the dental plaque microbial community.// Journal of clinical microbiology. Introduction: Protein Kinase R (PKR) is an interferon-induced serine-threonine kinase that plays a key role in regulating the cellular stress response to viral infection, ER stress, oxidative stress, and serum deprivation through protein synthesis inhibition.

During these conditions, PKR is activated through its interactions with double stranded RNA (dsRNA) generated during viral infection or its cellular activator, PACT (Protein Activator of PKR). PKR subsequently phosphorylates the a subunit of the translation initiation factor eIF2, leading to the cessation of general cellular protein synthesis. Translation attenuation allows the cell to mount an appropriate response to the stressful conditions, which if sustained, eventually results in apoptosis.

In addition to its regulation by its two activators, PKR is also subject to negative regulation by various viral factors, as well as the RNAinduced silencing complex protein, TRBP (TAR RNA Binding Protein). TRBP inhibits PKR's activation during stressful cellular conditions by direct interaction, as well as by direct association with PKR's activators (dsRNA and PACT), thereby preventing subsequent inhibition of protein translation. Several studies have linked prolonged PKR activation to various neurodegenerative and neuromuscular disorders. Recent findings also indicate that TRBP inhibits PKR in other cellular contexts, but strongly suggest that TRBP's ability to efficiently inhibit PKR is affected by post-translational modifications.

Methods: The goal of this study was to elucidate the functional importance of TRBP phosphorylation on PKR inhibition and cell fate during cell stress. We observed changes in TRBP phosphorylation status in response to stress and tested the direct effects of such modifications on PKR activation and apoptosis. We also specifically tested for changes in PKR-TRBP interactions as well as TRBP homodimer formation after TRBP phosphorylation using various biochemical assays and phospho-mimic and phospho-defective TRBP mutants.

Our results indicate that TRBP is phosphorylated in response to cell stress, and this change in phosphorylation status significantly enhances TRBP's ability to efficiently inhibit PKR. This increase in TRBP's inhibition of PKR is further supported by our results which show that TRBP phosphorylation directly enhances TRBP's interaction with PKR while weakening TRBP-TRBP homodimer formation. , the effect of IL-3 on the  differentiation of preosteoblasts is unclear and we have investigated  the same using preosteoblastic cell line, MC3T3 -E1. The current study will help us to understand the role of IL-3 in regulating osteogenic differentiation, and also its anabolic potential for the treatment of bone disorders like osteoporosis and arthritis.

Methods: The effect of IL-3 on proliferation of preosteoblasts was checked by MTT assay. Osteogenesis was assessed by matrix mineralization assay followed by Alizarin Red S quantitation. The expression of various osteogenic genes was evaluated by both Reverse Transcriptase-(RT) and Real-Time PCR (qRT-PCR). Western blotting was performed for the assessment of signaling pathways involved in action of IL-3. Statistical significance was calculated by one-way ANOVA with a subsequent post-hoc Tukey's test for multiple comparisons.

Results: We observed that MC3T3-E1 cells express IL-3 receptor at both transcript and protein levels. IL-3 did not affect the proliferation of MC3T3-E1 cells and it was not toxic to the cells even at higher concentration. Interestingly, we found that IL-3 enhances the osteogenic differentiation of MC3T3-E1 cells in a dose-dependent manner. In addition, IL-3 upregulated the expression of osteoblast specific genes such as alkaline phosphatase, osteocalcin, osteopontin, runt-related transcription factor 2 and osterix. We have also observed the induction of phosphorylation of Stat5, Erk1/2 and Akt upon treatment of cells with IL-3.

These results suggest that IL-3 enhances osteogenic differentiation of MC3T3-E1 cells and thus could be a potential therapeutic candidate in the treatment of various bone pathologies such as osteoporosis and arthritis involving impaired osteoblast differentiation and function. Introduction: TNFa is actively involved in both acute and chronic phases of atopic dermatitis (AD), but role of membrane receptors and their regulatory function in AD remains unclear.

The aim of this study was to investigate changes in the expression of TNFa receptors in patients with atopic dermatitis by both percentage of cells with receptors in immunocompetent cells subsets and the absolute number of membrane-bound receptors themselves.

Methods: To reveal linear relationships among SCORAD and studied parameters of intact T-cells, B-cells and monocytes (evaluated by flow cytometry) in AD patients (n = 20) in acute stage) and within clinical improvement and parameters of mediators soluble content (evaluated by ELISA) building of multiple linear regression model (MLRM) with a standard assessment of the regression coefficients by least squares method were used. To determine receptor number on the cells QuantibritePE Beads (BD) were used ease severity index SCORAD in AD patients depending on the phase of the disease.

Results: Intact cells of PBMC subsets were found to differ both in the relative percentage of cells expressing type 1 and 2 TNFa receptors and in the absolute number of these receptors. An increase or decrease in the percentage of cells expressing the receptors in subsets of immune cells in patients with atopic dermatitis was not associated with a change in the number of receptors on the cell surface. Changes in the parameters of TNFa receptor expression are associated with disease severity index SCORAD in atopic dermatitis.

Conclusion: Different indicators of TNFa receptor system are associated with disease severity index SCORAD in AD patients depending on the phase of the disease. Introduction: Since its discovery more than twenty years ago, Signal Transducer and Activator of Transcription 3 (STAT3) has been tied to a diverse set of physiological and pathological functions. The pleotropic actions of STAT3 as a nuclear transcription factor activated by tyrosine phosphorylation have been thoroughly characterized. In addition to regulating nuclear transcription, a small pool of STAT3 resides in the mitochondria where it serves as a sensor and signaling hub for a number of metabolic stressors including reactive oxygen species (ROS). Mitochondrial-localized STAT3 (mitoSTAT3) largely exerts its effects through direct or indirect regulation of the activity of the electron transport chain (ETC). In contrast to cytokine-induced import of STAT3 into the nucleus, it has been assumed that levels of STAT3 in the mitochondria are static.

Methods: Various cell lines were incubated with cytokines or hydrogen peroxide and mitochondria were isolated, and treated with trypsin to remove nonspecific binding of proteins to the outer membrane. Mitochondrial extracts were probed for the presence of STAT3 or cyclophilin D. GST-cyclophilin D pull down assays were used to measure association of cyclophilin D with STAT3.

Results: We show that various stimuli, including oxidative stress and cytokines, trigger a novel signaling cascade that results in a rapid loss of mitoSTAT3. Recovery of mitoSTAT3, with time, is dependent upon S727 phosphorylation of STAT3 and new protein synthesis. Under these conditions mitoSTAT3 also becomes competent to bind to cyclophilin D (CypD). Binding of STAT3 to CypD is surprisingly mediated by the N-terminus of STAT3, thereby defining a site outside of S727 that may be important in regulating mitoSTAT3.

Conclusion: These results outline a previously unappreciated role for mitoSTAT3 in sensing external stimuli and provide evidence of a novel mitochondrial pathway. Introduction: Interleukin (IL)-6 is a pleiotropic inflammatory cytokine associated with the regulation of T-cell differentiation, proliferation and survival. While activation of the latent transcription factor STAT3 is critical to the role IL-6 performs in many of these processes, the impact of IL-6 signalling through STAT1 is less obvious. To address this issue, we have now profiled IL-6 responsiveness in CD4 T-cells and examined the relationship between IL-6 activation of STAT1 and STAT3. Our data shows that prior T-cell activation leads to a retuning of IL-6 signalling that is associated with a loss in STAT1 activity.

The signalling properties of IL-6 were examined in purified CD4 T-cell cultures and through ex vivo analysis of defined CD4 T-cell subsets. Temporal changes in the IL-6 activation of STAT1 and STAT3 were monitored by intracellular flow cytometry and accompanied by a transcriptomic analysis and epigenetic profiling of STAT1 and STAT3 involvement using Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq).

Results: Our data shows that IL-6 control of gene expression in CD4 T-cells is highly dynamic and influenced by the activation status of the CD4 population. Importantly, activation of CD4 T-cells by anti-CD3/CD28 co-stimulatory antibodies altered the signaling properties of IL-6 through regulation of protein tyrosine phosphatase activity, which blockade IL-6 control of tyrosine phosphorylated STAT1. While IL-6 control of STAT3 was comparable in naïve, central memory, effector and effector memory CD4 T-cells, the activation of STAT1 was a prominent feature of IL-6 signaling in naïve CD4 T-cells. We examined the differential STAT1 and STAT3 binding profiles of naïve and effector memory CD4 + T-cells to identify potential genetic markers of IL-6 driven disease. In naïve CD4 T cells ChIP-seq analysis identified 224 peaks induced by IL-6 for STAT1 and 162 peaks for STAT3 where 29.4% and 43.2% respectively are in the promotor region. Bioinformatic analysis of these datasets using the MEME-ChIP web service mapped the enrichment of defined consensus motifs. Three DNA consensus motifs were associated with STAT1 involvement -corresponding to Interferon-regulatory factors response element, the GAS motif and a third not centrally enriched motif. Conversely, STAT3 ChIP-seq peaks mapped predominantly to the GAS motif, a C2H2 zinc finger factor binding site and a consensus sequence associated with the binding of ETS transcription factors. Thus, indicating potential associations and interactions with other transcriptional complexes. Relative enrichment for STAT1 and STAT3 control genes was reduced in the effector memory samples. Introduction: X-linked inhibitor of apoptosis protein (XIAP)-associated factor-1 (XAF1) is a cytokine-regulated, tumor necrosis factor (TNF) receptor associated factor (TRAF) domain-containing protein that has a poorly defined cellular function [1] . It was originally implicated as a tumor suppressor due to the putative inhibitory effects toward XIAP [2] . Here we report a possible role of XAF1 in the TNFa/TNF receptor (TNFR)-mediated signal transduction.

Methods: Activation of nuclear factor kappa B (NFjB) by TNFa in XAF1-knockdown ACHN cells was assessed by luciferase assays using an NFjB response element-regulated luciferase reporter construct, or by real-time RT PCR analysis of IL-8 gene expression. The effect of XAF1 on RIP1 or TRAF2-induced NFjB activation and the interaction between XAF1 and TRAF2 or RIP1 were investigated by using luciferase assays and co-immunoprecipitation assays, respectively, in HEK293 cells ectopically expressing the proteins of interest. XAF1's impact on TRAF2 ubiquitination was assessed by immunoblot analysis. The effect of XAF1 on TRAF2/TRADD/RIP1 complex formation was also studied by immunoblot analysis of individual proteins following immunoprecipitation of endogenous TRAF2 in XAF1 knockdown ACHN cells, with or without TNFa stimulation.

The results demonstrated that shRNA silencing of endogenous XAF1 augmented TNFa induced NFjB activation and IL-8 expression. XAF1 interacted with TRAF2 and inhibited TRAF2-dependent NFjB activation, in part by blocking TRAF2 polyubiquitination. XAF1 also bound RIP1 but did not directly inhibit RIP1-dependent NFjB activation. However, XAF1 knockdown promoted TRAF2/RIP1 association with TRADD in response to TNFa stimulation.

Conclusion: These data suggested that XAF1 inhibited TNFa-induced NFjB activation via disruption of TRADD/TRAF2/ RIP1 complex function, possibly by physically blocking interaction between the components of the complex, thereby preventing them from receiving or retaining K63-ubiquitination that is essential for complex formation and signal propagation. Introduction: TNFR1, upon binding TNF-a, can induce activation of either NF-kB pathway or apoptosis by forming signalosome complex 1 and complex 2, respectively. Besides, TNF is known to activate ERK, p38, JNK and Akt pathways [1] . Although TNF mediated apoptosis and NF-kB signaling have been extensively studied, the signalosome responsible for activation of ERK, p38, JNK and Akt pathways has not characterized yet. TNFR1 is known to interact with JAK2 [2] and thus, we hypothesized that, TNFR1 could be tyrosine (Tyr) phosphorylated, triggering the activation of proliferative or stress-induced pathways.

Methods: In vitro kinase reaction was performed to demonstrate JAK2 mediated TNFR1 phosphorylation. Following confirmation, two putative Tyr phosphorylation sites (Y360 and Y401) were substituted by site-directed mutagenesis with alanine (A) residue to inhibit phosphorylation, or with aspartic acid (D) to mimic constitutive phosphorylation. Mutated or wild type TNFR1 were transfected into HEK293T cells. The effect of these mutations, following TNF treatment, on ERK, JNK, p38, Akt, and Stat3 activation was determined by western blot experiments. TNFR1 interaction with c-Src, p85, Stat3, Grb2, JAK2, FADD, TRADD, and RIP was examined by co-IP. NF-kB activity was measured by luciferase assay and STAT3 DNA binding activity was explored by EMSA. MTT and colorimetric caspase 8/3 assays were utilized for quantification of proliferation and apoptosis, respectively.

Results: JAK2 and TNFR1 interact with each other in a TNF inducible manner, resulting in TNFR1 Tyr phosphorylation. Y360A and Y401D mutants led to activation of Stat3, Akt, and ERK pathways, while they suppressed JNK pathway. These mutants improved the interaction of TNFR1 with c-Src, p85, and Stat3; but had no effect on Grb2 binding. NF-kB activation was shown to be correlated with p85-TNFR1 interaction. Double mutation of Y360D and Y401D led to maximum p38 activation, while double A mutants resulted in increased apoptosis and FADD-TNFR1 interaction.

Conclusion: In a nutshell, TNFR1 is Tyr phosphorylated by JAK2. Phosphorylation of Y401 augments TNFR1 mediated proliferative pathways, as well as complex 1 formation; while inhibition of phosphorylation at both Y360 and Y401 residues resulted in increased complex 2 formation and apoptosis. Introduction: HCC is the fifth most common and the third deadliest cancer worldwide. There is an urgent need to find sensitive markers for early diagnosis and to monitor postoperative recurrence in order to provide treatment for HCC. Cytokines play an important role in liver physiology, i.e., IL-6-type cytokines as mediators of the acute phase response. In addition, they contribute to cancer development and progression. Thus, HCC-associated cytokines as well as miRNAs are candidates for diagnostic and prognostic biomarkers of HCC. Results: In a first step, we analyzed and correlated the expression levels of inflammatory cytokines in HCC and NASH patients, being at high risk to develop HCC. Herein, serum levels of IL-6 and of HGF were higher in HCC patients than in healthy controls and in advanced NASH patients, as confirmed by Bio-Plex cytokine immunoassays. Next, we wanted to study a possible correlation between cytokine levels and the presence of the PNPLA3 p.148M risk variant in patients with NASH, thereby contributing to biomarker research in liver diseases.

Next, we have studied the effect of IL-6 on the miRNome in hepatoma cell lines and primary hepatocytes. Surprisingly, IL-6 caused thousands of mRNAs to be differentially regulated in HepG2 and HuH-7 hepatoma cell lines, whereas levels of only few miRNAs were significantly changed (FDR < 0.05, logFC 0.5). In contrast to HCC cell lines, a stronger response of the miRNome following IL-6 stimulation was observed in primary hepatocytes (68 and 27 differentially expressed miRNAs in two samples, respectively). To further address this possible cell type and tissue specificity, we tested the effect of STAT-1 or STAT-3-activating cytokines (IL-27, IFN-k, OSM and H-IL-6) on both the miRNome and the mRNA transcriptome of healthy and cancerous cell lines, derived from human liver, colon and skin cells. Moreover, the regulation and roles of selected IL-6/Jak/STAT3-related miRNAs with possible relevance for HCC are currently further analyzed. Introduction: Interleukin-17A (IL-17) is a proinflammatory cytokine essential in host defense against extracellular pathogens but also promotes inflammation in many autoimmune disease settings. IL-17 induces inflammatory signaling through de novo transcription and post-transcriptional regulation of downstream genes. One major mechanism of IL-17-mediated post-transcriptional gene regulation occurs through alterations in mRNA stability of target gene transcripts. Here, we report a new mechanism by which IL-17 increases the mRNA half-life of downstream target genes through the RNA binding protein known Arid5a (AT-rich interactive domaincontaining protein 5A). Arid5a was previously shown to stabilize Il6 (Interleukin-6) and Stat3 (Signal transducer and activator of transcription 3) transcripts by direct binding to the 3'UTR, thus counteracting the destabilizing effect of the endoribonuclease MCPIP1 (also known as Regnase-1).

Methods: To assess the role of Arid5a in IL-17 signaling, we performed gene knockdown using siRNA in the murine stromal cell line ST2. Following IL-17 treatment, expression of IL-17 target genes was analyzed using qRT-PCR and RNA-seq. mRNA half-life of IL-17 target genes was assessed after actinomycin D treatment. Arid5abinding transcripts were identified via RNA immunoprecipitation. Co-immunoprecipitation assay was performed to study adaptor proteins that associate with Arid5a. Introduction: Tumor necrosis factor alpha (TNFa) is an inflammatory cytokine regulating proliferation and/or apoptosis in a wide variety of cells. TNFa elicits its biological effects by binding to its cognate Type I (TNFR1) and Type II (TNFR2) receptors. Although pathways activated by TNFR1 have been extensively studied, factors or pathways regulating TNFR1 signaling remain elusive. cAMP has been previously implicated in regulation of TNFR1 signaling [1] , however the mechanism of this regulation is not documented. TNFR1 death domain bears two threonine residues (T411 and T417), which lie within putative PKA consensus motif (RxxT/S). Thus, we hypothesized that TNFR1 might be targeted by PKA, as a possible regulation mechanism.

Methods: In order to confirm PKA-TNFR1 interaction, we performed reciprocal co-immunoprecipitation (co-IP). We further validated phosphorylation by in-vitro kinase reaction. Thereafter we sitedirectionally mutated TNFR1 T411 and T417 residues to alanine (A), which inhibits phosphorylation, and to aspartic acid (D), which mimics phosphorylation. Plasmids carrying wild type or mutant TNFR1 were transiently transfected into HEK293T cells. By western blot, we determined p38, ERK, CREB, and Stat3 activations, while NF-kB activity was examined by means of luciferase assay. We explored STAT3 DNA binding ability using EMSA technique, and determined the extent of TNFR1 shedding by ELISA. Finally, rate of apoptosis induction was measured by caspase3/8 activation assays.

Results: Both T411/417A and T411/417D mutants decreased TNFinduced p38 activation, while they both increased TNF-induced ERK activation. Following TNF treatment, A mutants positively stimulated CREB phosphorylation, whereas D mutants failed to elicit the same effect. TNF-induced STAT3 activation was decreased for A mutants, increased for T411/417 double D mutant. In contrast to STAT3 results, TNF-induced NF-kB activity was upregulated in A mutants, and downregulated in A mutants. Apoptosis was increased with T411A mutation, and decreased with T411D mutation.

In conclusion, TNFR1 is apparently a substrate for PKA, and its phosphorylation by PKA has a differential effect upon signaling. Results: IL-22BPi2 and IL-22BPi3 differentially modulate IL-22 activity: IL-22BPi3 has lower inhibitory activity than IL-22BPi2, but is expressed more abundantly throughout the body during homeostasis. IL-22BPi2 and IL-22BPi3 also differentially alter the cooperative actions between IL-22 and IL-17, which are commonly co-expressed in autoimmune settings. In addition, we demonstrate that IL-22BPi1 is inactive because it is not secreted. Importantly, while IL-22BPi3 is the dominant isoform expressed constitutively, only IL-22BPi2 is modulated when myeloid cells are activated by TLR2 or retinoic acid. kinetics and magnitude of gene expression differs following  interferon sensing, the molecular mechanisms for the distinct  responses to type I IFN and type III IFN, and the functional  consequences of these differences are not fully understood. Methods: Here, we provide a detailed investigation on the distinct receptor-ligand interactions, the temporal recruitment and activation of adaptor molecules and transcription factors that guide interferon-stimulated gene expression in human epithelial cells. Furthermore, we examine the unique translational program that distinguish the biological activities of type I and type III IFN through ribosomal profiling. Introduction: Interferon-a (IFN-a) belongs to the type I interferon family of cytokines and is central to regulating host antiviral and immunoregulatory responses. However, dysregulation of IFN-a production can lead to disease. In the central nervous system (CNS), chronic production of IFN-a is the cause of a number of neurological diseases, collectively termed ''cerebral type I interferonopathies". They include chronic viral infections of the CNS, systemic lupus erythematosus-associated neurologic manifestations and genetic disorders exemplified by Acardi-Goutières syndrome. However, the effector cells in the CNS and the molecular mechanisms underlying IFN-a mediated CNS disease are unknown.

The intracellular effects of IFN-a are mediated through a number of signalling pathways that are regulated by phosphorylation of signalling proteins and transcription factors. Therefore, we hypothesise that the neurological disease phenotype caused by IFN-a is the consequence of the differential activation of signalling pathways that primarily involve protein phosphorylation cascades (the 'phosphoproteome') ultimately leading to the regulation of transcription factor activity.

Methods: High-throughput phosphoproteomics was used to determine the global and cell-type specific phosphoproteome in the murine CNS and glial cells, respectively. To determine the effects of IFN-a on the global phosphoproteome in the CNS, we used transgenic mice that chronically produce IFN-a in the CNS (GIFN mice). These mice recapitulate many of the key features of IFN-a related neuropathologies and symptoms. To assess cell-type specific changes, we analysed the phosphoproteome of IFN-a treated primary astrocytes and microglia.

Results: Preliminary results showed a significant change in the phosphorylation of more than 800 proteins in the brain of GIFN mice. Gene ontology revealed that increased phosphorylation was associated with protein complex assembly, vesicle transport, antiviral response and ossification, whereas proteins with decreased phosphorylation related with cytoskeleton regulation, homeostasis and neurogenesis. Surprisingly, analysis of the phosphorylation sites revealed that members of the mitogen-activated protein kinase (MAPK) family are major regulators of protein phosphorylation in the CNS of GIFN mice.

The results demonstrated links between the IFN-a mediated disease phenotype and changes in a number of different biological processes. The findings also show that phosphoproteomics is a highly suitable technique to explore changes in signalling in the CNS and the cellular response to IFN-a. This method may help answer the mechanisms of how IFN-a causes disease and reveal therapeutic targets of type I cerebral interferonopathies. Introduction: Interferons play a major role in controlling viral infections. They are key elements in innate immunity and are needed for efficient adaptive immunity. Nevertheless, they are associated with inflammation, immune suppression and chronic-immuneactivation. Persistent up-regulation of IFN regulated genes (ISGs) is a molecular signature of chronic immune-activation and progression in HIV and SIV infections but the respective contribution of type I and type II IFN remains unclear.

IFNs/IFN-receptors interactions induce ISGs by targeting specific sequences upstream promoters (GAS, ISRE) through signal transduction (phosphorylation of latent transcription factors belonging to STAT family mediated by Janus kinases). STAT dimers directly activate ISGs containing GAS-sequences. The association of STATs with interferon regulatory factor (IRF9) activates genes by interacting with ISRE-sequences.

Our aim was to better understand the respective involvement of type I and type II IFNs in ISG induction in both acute and chronic infection, and the respective functional enrichments of genes regulated by GAS or ISRE or both (GAS+ISRE) in specific biological functions.

Methods: Six macaques were exposed to SIVmac251 intravenously and different tissues, including PBMCs, lymph-nodes and rectum were subjected to Agilent Rh DNA array, both before infection, in acute and in chronic infection. In addition, virus loads and IFN expression were monitored. We analyzed differential expression of IFNs and 1,638 ISGs that were splitted into three distinct groups, GAS-, ISRE-and GAS+ISRE-regulated-ISGs.

Results: Increased IFNa,b-expression was detected in lymphoid tissues during acute infection but was barely detectable during the chronic stage. In contrast, IFNg production was detected during both phases. Among 297 differentially up-regulated ISGs, 72 displayed ISRE-, 113 GAS-and 112 both ISRE and GAS-sequence(s) within 5,000bp upstream promoter. The induction of ISGs was higher in acute than in chronic phase. ISRE/GAS-ISGs were more induced than ISRE and GAS genes. Some were induced in acute or chronic or both phases (MX1, ISG15, OAS1, IRF9). ISRE+GAS ISGs were overrepresented in inflammation and anti-viral canonical functions than were GAS-ISGs or ISRE-ISGs. Introduction: Macrophages are important players in host defence responses against pathogens and produce protective proinflammatory cytokines along with interleukin (IL)-10 in response to Toll-like receptor (TLR) ligation. The proinflammatory cytokines IL-12, tumor necrosis factor alpha (TNF-a) and IL-1b are essential for resistance against infection but, if produced at high levels, may contribute to immunopathology. In contrast, IL-10 is an immunosuppressive cytokine, which dampens these responses, but can also lead to defective pathogen clearance. The regulation of these cytokines is therefore central to the generation of an effective but balanced immune response.

Methods: In this study, we stimulated macrophages derived from two different mouse strains, C57BL/6 and BALB/c mice with either LPS, Pam3CSK4 or heat-killed Burkholderia pseudomallei. To investigate the different TLR activation responses a microarray approach, RT-PCR, ELISA and Western blot were used.

Results: We show that macrophages from C57BL/6 mice produce low levels of IL-12, TNF-a and IL-1b, but high levels of IL-10 in response to TLR4 and TLR2 ligands. In contrast, macrophages derived from BALB/c mice show a reciprocal pattern of cytokine production. Differential production of IL-10 in TLR4 stimulated C57BL/6 and BALB/c macrophages was due to a type I IFN-dependent mechanism. We demonstrate that type I IFN acts as a transcriptional regulator of Il10 mRNA potentially via activation of ERK1/2 and additionally stabilises Il10 mRNA transcripts in TLR4 stimulated macrophages.

Further, type I IFN contributed to differential IL-1b and IL-12 production in TLR4 stimulated C57BL/6 and BALB/c macrophages, via both IL-10-dependent and independent mechanisms.

Conclusion: Our data show distinct profiles of pro-and antiinflammatory cytokine production in C57BL/6 and BALB/c macrophages and highlights the important role of type I IFN in the regulation of innate immune responses.

Disclosure of Interest: None declared. Introduction: Recently, we identified the autophagy-related Unc-51-like kinase 1 (ULK1) as a novel kinase required for type I interferon (IFN)-induced activation of p38 MAPK, transcription of IFN-stimulated genes (ISGs), and antiproliferative effects on normal hematopoiesis and myeloproliferative neoplasms. In light of these new findings, we questioned whether ULK1 could also be activated downstream of the type II IFN receptor (IFNR) and be required for IFNk-mediated biological responses.

In vitro studies were conducted to examine the role of ULK1 in IFNk-mediated signaling pathways using LC-MS/MS, coimmunoprecipitation and western blot analyses. IFNk-induced gene transcription was evaluated by qRT-PCR analyses and by measuring luciferase activities of ULK1/2 wild type (WT) and double knockout (DKO) cells stably expressing IFNk activation site (GAS) elements-Luciferase cDNA. IFNk-antiviral responses were determined in antiviral assays using encephalomyocarditis virus as the challenge virus. Mann-Whitney U test was used to assess differences between two groups. One-way ANOVA was used to compare more than two groups followed by Tukey's multiple comparison test. Differences were considered statistically significant when p values were less than 0.05.

We initially immunoprecipitated ULK1 from untreated and IFNk-treated human KT-1 cells and performed LC-MS/MS analysis. Our results showed that ULK1 binds 33 proteins upon IFNk treatment. From the proteins identified, we confirmed the binding of ULK1 to a protein kinase previously shown to be phosphorylated upon engagement of type II IFNR. Next, we demonstrated that ULK1 expression is required for activation of downstream IFNk signaling pathways. Moreover, IFNk-induced transcriptional activation via GAS elements as well as the transcription of several ISGs was significantly reduced in the absence of ULK1/2 expression. In subsequent studies, we observed that ULK1/2 DKO cells are less responsive to the antiviral effects of IFNk compared to WT cells. Similarly, target-specific inhibition reduced the IFNk-mediated antiviral effects in human 2fTGH fibrosarcoma cells.

Here we define a novel IFNk signaling pathway that requires ULK1 activity and leads to transcription of ISGs and antiviral responses. Together, our results support the clinical modulation of ULK1 kinase activity as a potential approach to selectively enhance IFNk-induced biological responses. Introduction: Sjogren's syndrome (SS) is a chronic autoimmune disorder that results in dysfunction of exocrine glands, particularly the salivary and lacrimal glands. The pathogenesis of SS involves many components, including deregulated salivary glandular epithelial cells (SGECs) that present antigen presenting cell (APC)-like phenotype and increased production of proinflammatory cytokines/ chemokines. We hypothesized that oral bacteria may have a role in inducing the APC-like phenotype in SGECs.

The communities of oral bacteria collected by mouth rinse from healthy controls (HC, n = 15), patients with dry mouth due to medication as sicca controls (SC, n = 10) and SS patients (n = 25) were analyzed by pyrosequencing. Based on the results of microbiota analysis, Streptococcus salivarius, Rothia mucilaginosa, Fusobacterium nucleatum, Prevotella melaninogenica, and P. histicola were chosen for further study. Human salivary gland (HSG) cells were infected with the selected bacteria in the presence or absence of IFNg for 7 h. After killing the bacteria with gentamicin, HSG cells were further cultured for 65 h. Expression of major histocompatibility complex class I (MHC I), MHC class II (MHC II), CD80, and CD86 was then examined by flow cytometry.

Results: Pyrosequencing analysis revealed drastic changes in the bacterial communities associated with the dryness of mouth. Although the bacterial communities of SC and SS were quite similar to each other, the diversities of SS microbiota were significantly higher than those of SC, and differences in the relative abundance of 16 species/phylotypes were observed between the SC and SS groups. In particular, an increase in P. melaninogenica was specifically associated with SS risk by logistic regression analysis (Odd Ratio 5.4, p = 0.003). F. nulceatum, a highly immune stimulatory species used as a positive control, and P. melaninogenica up-regulated MHC I and CD86 in the absence of IFNg and further upregulated the IFNg-induced upregulation of MHC I and CD80. P. melaninogenica also up-regulated the IFNg-induced expression of MHC II. In contrast, S. salivarius, the most abundant species used as a negative control, down-regulated CD80 expression in the absence of IFNg as well as the IFNg-induced upregulation of MHC I. R. mucilaginousa, a species that increased in both SC and SS compared to HC, also showed suppressive effect on the IFNg-induced upregulation of MHC I and CD86.

Conclusion: Specific bacteria associated with SS in oral microbiota may play roles in deregulation of SGECs and the pathogenesis of SS.

Disclosure of Interest: None declared. Introduction: Type I and II IFNs are concurrently produced during infections. Type II IFN (IFNk) acts on macrophages to induce both pro-inflammatory and anti-microbial responses and is critical for resistance to bacterial infections. Conversely, type I IFNs promote antiviral immunity and exert anti-inflammatory effects associated with increased host susceptibility during Listeria monocytogenes (Lm) and other bacterial infections. Type I IFNs are also known to suppress ifngr1 transcription in myeloid cells to down regulate the receptor for IFNk (IFNGR). We hypothesized this suppression of IFNGR contributes to type I IFN-mediated susceptibility during bacterial infections.

Methods: To test our hypothesis, we developed a transgenic mouse model (fGR1) in which a functional flag-tagged IFNGR is expressed in macrophages at low levels using a promoter that is not silenced by type I IFNs.

Results: Macrophages from fGR1 mice express both endogenous and flag-IFNGR, but overall surface IFNGR expression is similar to that of WT congenic C57Bl/6 cells. When treated with type I IFNs WT macrophages lose cell surface IFNGR while fGR1 macrophages largely retain flag-IFNGR. fGR1 and WT macrophages respond similarly when treated individually with type I IFN or IFNk but in the presence of type I IFNs responsiveness of fGR1 macrophages to IFNk is greater. Consistent with this, fGR1 mice have heightened resistance to systemic Lm infection. Enhanced resistance of fGR1 mice is dependent on IFNk and correlates with increased expression of pro-inflammatory activation markers and reduced frequency of Lm-infected macrophages.

Conclusion: These data demonstrate that IFNGR down regulation in myeloid cells impairs macrophage activation and increases host susceptibility during infection by an intracellular bacterial pathogen. [1] . The IFN-b induction pathway activates specific transcription factor binding to interferon regulatory factor (IRF) DNA elements called IRF-E and stimulate the transcription of several genes [2] . Type I IFNs, including IFN-b, bind IFN-I receptor and trigger the IFN-I signaling cascade which triggers transcription factors that bind to DNA elements named IFN stimulated response elements or ISRE [3] , triggering transcription of IFN-stimulated genes (ISGs). The similarity between IRF-E and ISRE sequences [4, 5] supports the fact that many genes can be activated by both signaling pathways [6, 7] . Several works describe transgenic mice expressing reporter genes under the control of IFN-b [8, 9] , IFN-a6 [10] or Mx2 promoters [6] or create IFN-I specific responsive promoter Mx1 to control the expression of Cre recombinase [11] .

Methods: A recombinant adeno associated vector (AAV) containing multiple copies of an optimized regulatory sequence containing IRF-E and ISRE (IRF-ISRE) controlling the expression of the firefly luciferase reporter gen was generated. Intranasal or intravenous administration of the AAV vector into C57BL/6 mice or Syrian hamsters showed a specific response. Two weeks after administration, animals were infected with different doses of viruses such as Newcastle disease virus (NDV) or influenza A/PR8/34 virus and reporter activity was monitored in vivo after intravenous administration of luciferin substrate.

Results: In vivo luciferase activity detection was dependent of the presence of the AAV vector after the administration of different IFN-I agonist. The reporter vector can be accommodated to study both the strength and the kinetics of type I IFN signature.

Conclusion: With no need of animal transgenesis, we have developed an AAV reporter vector that can be delivered into different organs of different animal models in order to detect and quantify the IFN-I signature in vivo.

Introduction: Genotoxic stresses, such as that induced by DNA damage agents or DNA recombination, have the potential to generate DNA by-products detected by the innate immune system.

Methods: Relying on the study of non-cytotoxic, mild DNA damage in mouse embryonic fibroblasts, bone marrow derived macrophages, human fibroblasts and lung cancer cells (A549), we investigated the involvement of the cytosolic cyclic GMP-AMP (cGAMP) synthase (cGAS)-STING in the detection of DNA damage products. Cells deficient in STING, cGAS or gap junctions were utilized to address the direct contribution of the pathway to the protection against Semliki Forest virus infection.

Here we show that DNA damage caused by Cre recombination or by the use of select genotoxic agents induces a robust antiviral response in mammalian cells. This is due to the accumulation of cytoplasmic DNA, which recruits cGAS to promote cGAMP synthesis and downstream STING activation in target and adjacent cells. Interferon gamma (IFN-k) is a cytokine that is critical to innate and adaptive immunity. IFN-k binds as a dimer to the IFN-k receptor 1 and is involved in activation of Th1 cells, monocytes, and macrophages. This cytokine also up-regulates antigen presentation molecules and promotes immunoglobulin class switching in B cells. IFN-k can induce both pro-and anti-inflammatory responses and is involved in many autoimmune disorders. Increased detection sensitivity of IFN-k will allow for improved understanding of disease mechanisms and is necessary to better understand this cytokine as a biomarker and therapeutic target.

An ultrasensitive electrochemiluminescence assay format, S-PLEX TM , based on MSD's MULTI-ARRAY Ò technology, was developed for IFN-k and its performance was characterized. Monoclonal antibodies were evaluated and selected based on sensitivity, specificity, affinity, and performance characteristics against a WHO anchored IFN-k calibrator.

The detection limit for this novel IFN-k immunoassay is 10 fg/mL, 1000 fold more sensitive than current IFN-k immunoassays. The lower and upper limits of quantitation are 17 fg/mL and 50,400 fg/ mL respectively. Typical intra-plate coefficients of variation (CVs) ranged from 9% to 12%. Inter-plate CVs were 7%, 10%, and 9% for low, mid, and high Quality Control (QC) sample levels, respectively (n = 20 per QC level). The average percent recovery from spike experiments was 105% (n = 12 specimens) and dilution linearity average recovery was 103% (n = 12 specimens). The assay was optimized to minimize serum and plasma matrix effects and interferences. Specificity of the assay was demonstrated by analyte depletion using several anti-IFN-k specific antibodies, indicating the assay is specific for the IFN-k homodimer.

IFN-k was detectable in serum and plasma of all apparently healthy specimens (n = 55) with a median value of 315 fg/mL in serum (n = 31), 429 fg/mL in EDTA plasma (n = 10), and 428 fg/mL in heparin plasma (n = 14). IFN-k did not appear to be elevated in a small number of rheumatoid arthritis specimens tested, but appeared to be elevated in some systemic lupus erythematosus and Crohn's specimens.

MSD developed an ultrasensitive IFN-k immunoassay that is 1000 times more sensitive than the current limits of IFN-k immunoassays. MSD's S-PLEX immunoassay will aid in improved characterization of IFN-k in disease states and in development of more targeted therapeutic interventions. Introduction: Primary biliary cholangitis (PBC) is a liver autoimmune disease predominantly affecting women (90%); however the sex-biased mechanism in this disease is not understood. We have reported that deletion of the AU rich element in the IFN-g 3'UTR (ARE-Del-/-), leads to chronic expression of IFN-g. As a consequence, we have found that the mice develop primary autoimmune cholangitis with a strong female bias.

We have analyzed liver gene expression data utilizing RNA seq and pathological features to further understand disease development and progression.

Results: As a consequence, we have found that the mice develop primary autoimmune cholangitis with a strong female bias. Serological/pathological features and the gene expression signature in this mouse model are comparable with data seen in human PBC. RNA-sequencing of liver gene expression revealed that differentially expressed genes in female ARE-Del-/-mice are consistent with stronger Type I and II interferon signaling and T helper cell-mediated immune responses in female mice that serves as the basis of the sex bias observed in this disease. Female ARE-Del-/-mice have significantly increased CD4 T cells but decreased CD8 T cells compared to control littermates, resulting in an elevated CD4/CD8 T cell ratio. Consequently, transfer of CD4+ T cells from ARE-Del-/mice to B6/Rag1 -/mice induced moderate portal and parenchymal inflammation, whereas CD8+ T cell transfer did not induce any pathological phenotypes. Moreover, profiles of autoantigen reactivities using peptide arrays indicated that female ARE-Del-/-mice had a high induction of IgM reactive autoantigens associated with autoimmune diseases as compared to male mice, while the levels of IgG reactive autoantigens were not significantly different in females vs. males. To further assess the role of Type 1 interferon in the disease process ARE-DEL/IFNa/b receptor double knockout mice were generated. Despite the inability to measure any circulating Type 1 IFN, these mice had much lower female-prevalent pathological phenotypes such as portal and lobular duct inflammation, granuloma formation, bile duct damage, and induction of total bile acids.

In conclusion, chronic IFN-g expression is critical for the female-biased pathogenesis of PBC and involves CD4+ T cells as well as type I and type II interferon pathway interactions. Introduction: There are emerging evidences that IFN-k was more highly induced after respiratory viral infection than IFN-b and that deficiency of IFN-k is related to an exacerbation of respiratory infections in pneumonia and asthma. Our goal of this study is to determine the therapeutic effect of intranasal IFN-k treatment against influenza A virus (IAV)-induced lung infection in mouse model.

Methods: 20 Mice (C57BL/6J, male, 7-10 week) were anesthetized and challenged by intranasal administration of 213 pfu/30 ul of IAV (WSN/33/H1N1) and IAV-inoculated mice were euthanized at 3, 7, 10, 14 days post of infection (dpi). Recombinant IFN-k (1 ug/mice) was inoculated to mice (N = 5) by intranasal delivery and lung tissue and BAL fluid were collected.

Results: Our preliminary results showed that infected mice exhibited significant weight loss values which were observed from 6 dpi until 12 dpi days and the highest IAV mRNA levels from lung tissue and viral titer from BAL fluid were observed at 7 dpi. Correspondingly, histologic findings related with acute lung infection were dominantly observed at 7 dpi. IAV mRNA levels and viral titer were much lower in mice which were inoculated with recombinant IFN-k through nasal cavity at the same time with IAV infection. Also severe pathologies in IAV-infected lung tissue were not observed in IFN-k-treated mice. IFN-stimulated genes were induced in lung tissue of IAV-infected mice and these genes' transcription levels were considerably elevated in IFN-k-treated mice.

Conclusion: Intranasal IFN-k treatment contributes to enhance innate immune response in mice lung against IAV infection and may be a important biomaterial for the therapy of acute IAV-induced lung infection with a different biological activity from the existing antiviral agents.

Introduction: Interferon lambdas are critical antiviral effectors in hepatic and mucosal infections. While IFNk1, IFNk2 and IFNk3 act antiviral, genetic association studies have shown that expression of the recently discovered IFNL4 is detrimental to hepatitis C virus (HCV) infection through a yet unknown mechanism. Intriguingly, human IFNL4 harbors a genetic variant that introduces a premature stop codon. We performed a molecular and biochemical characterization of IFNk4 to determine its role and regulation of expression.

Methods: We amplified IFNL4 mRNA from stimulated hepatocytes and generated a cDNA library of the different isoforms. We assessed expression, secretion and antiviral activity of the cloned isoforms in an overexpression system. Furthermore, we generated human recombinant full-length IFNk4 protein in a Drosophila S2 cell system to validate our overexpression data. Finally, we determined translational efficiency of IFNL4 isoforms by ribosome profiling.

Results: We found that IFNk4 exhibits similar antiviral activity to IFNk3 without negatively affecting antiviral IFN activity or cell survival. We show that humans have evolved mechanisms to limit expression of functional IFNk4 through non-coding splice variants and non-functional protein isoforms. Furthermore, protein-coding IFNL4 mRNA are not loaded onto polyribosomes and lack a strong polyadenylation signal, resulting in poor translation efficiency.

This study provides mechanistic evidence that humans have sustained adaptations to suppress IFNk4 expression, suggesting that immune function is dependent on other IFNL family members.

Disclosure of Interest: None declared. Methods: C57BL/6 mice and TLR/RLH -/mice deficient for TLR and RLH signaling were either infected with 2 Â 10 4 pfu VSV and treated with a short-term regimen at 4, 8, 12, 18, and 24 h post infection (hpi) with recombinant IFN-a (rIFN-a) or treated with rIFN-a -4, 0, 4, 8, 12, 18, and 24 hpi and infected with VSV 0 hpi. For long-term treatment the above described regimen initiated 4 h before infection was followed by rIFN-a injections every 8 h for 9 days. Survival as well as the induction of VSV-specific cytotoxic lymphocytes and neutralizing antibody responses was monitored. Furthermore, the spleen of VSV-eGFP infected TLR/RLH -/mice subjected to short-term rIFN-a treatment initiated 4 h before or 4 h after infection was analyzed by immunohistochemistry.

To study the impact of the kinetics of IFN-I responses on the induction of adaptive immunity, we infected TLR/RLH -/mice with VSV and started short-term treatment with rIFN-a 4 hpi. Under such conditions TLR/RLH -/mice mounted normal adaptive immune responses and 76% of the mice survived. Re-infection experiments proved that the surviving mice developed effective memory responses against VSV. Initiation of the rIFN-a treatment 4 h before VSV infection significantly prolonged survival, whereas reduced VSV-specific cytotoxic T cell responses and basically no VSV neutralizing antibody responses were induced and eventually 100% of the mice died.

Long-term rIFN-a treatment for 9 days initiated 4 h before VSV infection promoted survival of 100% of the mice, however, no adaptive immunity developed and 100% of the animals succumbed to re-challenge.

In conclusion, long-term rIFN-a treatment initiated before virus infection prevented induction of protective adaptive immunity, whereas initiation of rIFN-a treatment 4 h after infection supported the establishment of normal adaptive immunity and protective memory. These observations have implications for patients under long-term IFN-I therapy who are supposed to be treated with live attenuated vaccines. Introduction: Phototherapy with ultraviolet (UV) light is a standard treatment for psoriasis, yet the mechanisms underlying the therapeutic effects are not well understood.

Methods: By using human and mouse keratinocytes and the skin tissues from human patients and mice, we identified the critical role of type I interferon (IFN) receptor chain IFNAR1 regulation in the beneficial effect upon UV treatment in psoriasis.

We found that UV treatment triggers ubiquitination and downregulation of IFNAR1, leading to suppression of IFN signaling and an ensuing decrease in the expression of inflammatory cytokines and chemokines. The severity of imiquimod-induced psoriasiform inflammation was greatly exacerbated in skin of mice deficient in IFNAR1 ubiquitination (Ifnar1 SA ). Furthermore, these mice did not benefit from UV phototherapy. Pharmacologic induction of IFNAR1 ubiquitination and degradation by an antiprotozoal agent halofuginone also relieved psoriasiform inflammation in wild type but not in Ifnar1 SA mice.

Conclusion: These data identify downregulation of IFNAR1 by UV as a major mechanism of the UV therapeutic effects against the psoriatic inflammation and provide a proof of principle for future development of agents capable of inducing IFNAR1 ubiquitination and downregulation for the treatment of psoriasis. Methods: To obtain basic knowledge about the direct antiviral as well as the immunomodulatory effects of IFNa subtypes, we used the HBV hydrodynamic injection (HI) mouse model to stably transfect mouse hepatocytes with an HBV expression plasmid which leads to the induction of host immune responses against HBV in the liver and thus mimicking HBV infection in vivo. Mice were treated daily with different IFNa subtype proteins or IFNa-expression plasmids for long-term endogenous IFN production was applied by HI. Viral antigens, ISG expression and innate and adaptive immune responses were monitored in serum, liver and spleen.

Results: Application of most IFNa subtype proteins inhibited HBV replication in vivo, with IFNa4 and IFNa5 being the most effective subtypes. Decreased viral loads after therapeutic application of IFNa4 and IFNa5 correlated with expanded effector cell populations of NK cells and T cells in both liver and spleen. Hydrodynamic injection of plasmids encoding for the effective IFNa subtypes (pIFNa) was even more potent against HBV than injecting IFNa proteins. The combination of pIFNa4 and pIFNa5 showed a synergistic antiviral effect on HBV replication, with a strong increase in NK cell and T cell activity as well as ISG induction in the liver.

The results demonstrate distinct anti-HBV effects of different IFNa subtypes against HBV in the mouse model, which may be relevant for new therapeutic approaches. Introduction: The Great East Japan Earthquake and the subsequent Fukushima Daiichi Nuclear Power Plant incident resulted in the mass evacuation and relocation of residents, primarily due to environmental radiation-contamination and a few due to their homes being destroyed. In response, the government built communities of fully subsidized temporary houses of less than 30 m 2 per family. While most reports focus on radiation effects in Fukushima residents, this report looks at the physical and psychological health of residents in temporary housing many of whom are afflicted with adverse health issues not derived from radiation-effects. As an initiative to provide health support to evacuees, doctors at Minami-soma Municipal General Hospital holds health forums in each of nine temporary housing communities once every two months. As a part of this program, evacuees underwent medical and other tests so doctors could get a clearer understanding of the health status and risks of temporary housing residents at July, 2015.

Methods: A total of 78 females and 27 males who have been residing in temporary housing were enrolled in this study. Evacuees' general health status was determined by questionnaires, various blood tests and interferon production-test (a measure of immune-ability).

Results: We discovered that evacuees had lower logIFN value of 3.78 ± 0.42 (mean ± SE) versus the controls. Although blood tests revealed that a substantial number of evacuees were afflicted with illness including hypertension, diabetes mellitus, and kidney disease, approximately half were unaware of their condition and had not sought medical attention. In male evacuees, unlike in female evacuees, IFN production and physical activity level were co-related. We also found a correlation between immune ability and psychological well-being; this correlation differed between males and females. For example, female evacuees from larger families and those living with their grandchildren tended to have higher IFN production ability. Male evacuees, who had a spouse to talk to about their concerns or troubles, had higher IFN production abilities than those who did not. Conversely, in females, not having anyone to confide in did not seem to negatively affect their IFN levels. Females who give or receive emotional support from their children who live away from home tend to have lower IFN production than those who do not.

Conclusion: Lower IFN production in evacuees in temporary housing suggest that although they are safe from radiation their physical and psychological health are still at risk. Our study also highlights areas where residents need more individualized support, in particular, assistance in seeking medical attention when physical symptoms appear and counseling/support for female evacuees from small families and males who feel they cannot confide in their spouse. While such initiatives are by no means a panacea we believe they can reduce the stress of living in temporary housing and therefore deserves attention. Methods: Our studies make use of the lymphocytic choriomeningitis virus (LCMV) model of persistent viral infection, wherein the viral variant Clone 13 elicits a persistent infection by quickly outcompeting the immune response and inducing immunosuppression. Naive transgenic LCMV-specific CD4 T cells were transferred into mice at the onset or in the midst of persistent viral infection demonstrating a severe inhibition of de novo Th1 cell priming in the midst of persistent viral infection. In vivo antibody blockades of various immunosuppressive factors prior to CD4 T cell priming, as well as various sorting and in vitro culture strategies tease out the mechanisms, molecules and cell types driving inhibition of CD4 Th1 priming. Further, by in vitro polarizing LCMV-specific Th1 cells and introducing them into mice with established persistent viral infection we can reconstitute the lost Th1 responses and assess the outcome of this on virus-specific CD8 T cell responses and ultimately viral control.

Results: We demonstrate that the loss of and inability to generate new virus-specific Th1 cells impedes control of persistent virus. Mechanistically chronic type I interferon (IFN-I) signaling drives the dual upregulation of PD-L1 and IL-10 on distinct populations of suppressive DCs to inhibit CD4 Th1 priming during persistent viral infection. Therapeutic blockade of IFN-I or both IL-10R and PD-L1 restored the differentiation of new CD4 Th1 cells in the midst of infection, and specifically those polyfunctional cytokine and granzyme B producers lost upon viral persistence. Interestingly, PD-L1 and IL-10 signaling impedes CD4 Th1 cell generation at the onset of both persistent and acute infection as well. Importantly, the ability to reconstitute and maintain Th1 cells during persistent viral infection enhances exhausted CD8 T cell responses and accelerates viral clearance.

Conclusion: Our studies demonstrate IFN-I driven PD-L1 and IL-10 as a novel combinatorial mechanism of immunosuppression to limit Th1 responses during persistent viral infection, and importantly impede viral control. Therapeutic transfer and maintenance of Th1 cells alongside Tfh cells restores a more diverse repertoire of CD4 helper T cell subsets and accelerates the clearance of persistent virus. Introduction: Type I and II interferons (IFNs) divergently impact host resistance to bacteria. Type II IFN (g) activates macrophages to promote effective anti-bacterial immunity, while type I IFNs (ab) suppress macrophage responsiveness to IFNk and increase susceptibility to several bacterial infections. The suppressive effects of type I IFNs correlate with suppression of macrophage cell surface IFNk receptor (IFNGR). Here, we show this suppression is independent of canonical signal transducer and activator of transcription (STAT) protein-dependent signaling and instead requires activity of the CK2 kinase.

Methods: The impact of CK2 inhibitors or knockdown of CK2 subunits was investigated in cultured macrophages and during systemic and lung bacterial infections.

Results: IFNb stimulated STAT-protein phosphorylation and stimulated inflammatory and antiviral responses were independent of CK2 activity, but CK2 inhibitors blocked repression of ifngr1 transcription in IFNb-treated mouse and human macrophages. A therapeutic administration of two distinct CK2 inhibitors significantly reduced bacterial burdens. The inhibitors did not directly impact bacterial viability. Therapy instead required host IFNk production and correlated with increased IFNk responsiveness in macrophages.

Conclusion: Our findings demonstrate that certain suppressive effects of type I IFNs can be segregated from their ability to induce canonical inflammatory and anti-viral responses. Further, our findings implicate CK2 as a target for host-directed therapy of bacterial infections. Introduction: Interferon regulatory factor 3 (IRF3) and 7 (IRF7) are key transcription factors for the expression of antiviral cytokines and interferon stimulated genes (ISGs). While cellular mechanisms that activate IRF3 and IRF7 are well-known, how the cell dampens IRF3 and IRF7 activation is less studied. Several studies show that cFLIPL inhibits type I IFNb production. Since this gene is controlled by IRFs, the question we asked is if cFLIPL affects IRF3 or IRF7 activation.

Methods: We use cell lines, including irf3 x irf7 -/-MEFs for our studies. We detected IRF3 activation in an IRF7 independent manner by examining the transcription of genes controlled only by IRF3. To detect IRF7 activation, we examined the transcription of genes controlled only by IRF7 in cells lacking IRF3.

Results: Here, we show that the cellular FLICE inhibitory protein (cFLIP L ) inhibits IRF3 activation. This mechanism is independent of caspase-8 activity and IRF3 degradation, which until now were the best-described cellular mechanisms to inhibit IRF3. Instead, cFLIP L binds to IRF3 to inhibit IRF3-DNA interactions. We mapped the Cterminal region 178-430 of cFLIPL as important for this function. IRF3 is most similar to IRF7. Here, we show that cFLIPL also inhibits IRF7 activation, using systems that specifically trigger IR7-IRF7 homodimer activation or systems that are devoid of IRF3. Surprisingly, cFLIP L does not co-immunoprecipitate with IRF7, implying that cFLIP L uses a different molecular mechanism to antagonize IRF7.

Conclusion: These data show that cFLIPL antagonizes both IRF3 and IRF7, but uses different molecular strategies to inhibit each transcription factor. These data provide a jumping off point for future areas of research, including how cFLIPL inhibits IRF3 activation. For studies with IRF7, our future approach is to use mass spectrometry to identify potential cFLIPL binding partners that would be responsible for cFLIPL inhibition of IRF7 activation. Understanding how this protein modulates this pathway can be very useful when thinking about modulating its expression in applicable clinical settings.

Disclosure of Interest: None declared. Introduction: A key component of the innate immune response are dendritic cells (DCs), which are the most powerful antigen-presenting cells and are uniquely able to recognize, via pattern recognition receptors (PRR), pathogen-associated molecules (PAMPs) and activate qualitatively different adaptive T-helper (Th) cell responses. In particular, a Th1-based response is considered protective against Paracoccidioides brasiliensis (Pb), the causative agent of Paracoccidioidomycosis. The development of Th1 cells relies on the production of IL-12p70 (subunits p40 and p35) by DCs and we show here that Pb inhibit the production of IL-12.

Methods: MiceC57BL/6 mice deficient in dectin-1 and TLR2 and strain-matched WT controls were used in this investigation.Fungus The yeast form of P. brasiliensis isolate 18 was grown on Fava-Netto medium Generation of BMDCs bone marrow cells were seeded and cultured for 8 days at 37°C in RPMI-1640 medium that contained 50 lM 2-mercaptoethanol, 20 ng/ml GM-CSF to obtain BMDCs. On day 8, non-and loosely adherent BMDCs were harvested and plated for experimental use Infection of BMDC with P. brasiliensis and treatments BMDC were infected with a yeast-to-cell ratio of 1:2. Incubation with the fungus was conducted for 24 h for cytokine quantification. When indicated, the cells were treated with LPS (100 ng/ml), PAM3CSK4 (300 ng/ml), zymosn (20 ug/ml), zymosan depleted (100 ug/ml) or antibody antiMincle (10 ug/ml). Cytokine measurement The cell-free supernatants of BMDC cultures were harvested for the determination of IL-1b, TNF-a, IL-6, IL-12p70 and IL12p40 concentrations using ELISA.

Results: BMDC were infected with Pb yeast cells in association with (or not) PRRs agonists. After 24 h the supernatant was collected for cytokines dosage. In general, IL-1b, TNF-a and IL-6 were produced in response to the fungal infection and an amplified secretion was observed when agonists were added. In contrast, IL-12p70 were not significantly produced by both infected and infected plus agonist treated-DCs, despite the fact that agonists alone induced significantly IL-12 secretion. Furthermore, Pb drastically reduced the production of IL-12p70 induced by LPS. Interestingly, fungal induced inhibition was not observed for the p40 subunit of IL-12, suggesting that Pb inhibits the specific p35 IL-12 subunit. In fact, mRNA IL-12p35 expression was severely inhibited by Pb in LPS-treated cells. It is known that fungus Fonsecaea monophora suppresses IL-12p35 via engagement of the PRR Mincle. To assess the role of PRRs in the inhibition of IL-12 production in our fungal model we used mice lacking the PRRs Dectin-1 and TLR2, and blocking antibody (Ab) against Mincle. BMDCs from TLR2 KO mice or treated with Ab had no effect in the production of IL-1b, TNF-a, IL-6, and IL-12p40 infected with Pb. In contrast, Dectin-1 deficiency caused reduction in the production of these cytokines. Regarding IL-12p70, neither the lack of Decitin-1 or TLR2 or Ab anti-mincle reverted the inhibition of this cytokine production. In addition, BMDCs treated with Ab plus LPS and infected with Pb did not produce significant IL-12p70 levels.

Conclusion: Our preliminary results show: Pb prevent IL-12p70 production via specific inhibition of p35; Dectin-1, TLR2 and Mincle engagement are not associated with this inhibition; Dectin-1 is important for the activation of DCs infected with Pb.

Disclosure of Interest: None declared. Introduction: Pathogens are sensed by innate immune receptors that initiate an adaptive immune response upon activation. Sporothrix schenckii is one of the causative agents of sporotrichosis, an opportunistic mycosis affecting humans and other mammals. The Nlrp3 (Nod-like receptor family piryn domain containing 3) receptor, together with the ASC (Apoptosis-associated Speck-like protein containing a Caspase recruitment domain) adaptor and caspase-1 forms the Nlrp3 inflammasome. This protein complex has been reported as the most versatile due to its broad specificity, being able to trigger the immune response against a diverse group of pathogens. The end point of the Nlrp3 inflammasome activation is the production of fully mature inflammatory cytokines (IL-1b and IL-18), as well as cell death through pyroptosis.

Methods: S. schenckii-infected and non-infected WT and Nlrp3 -/-C57BL/6 mice were followed for 7, 14 and 21 days post-inoculation. Peritoneal macrophages and total splenocytes were challenged with a S. schenckii alkali-insoluble fraction (F1) for assessment of the ex vivo release of nitric oxide (NO), IL-1b and IL-17. Peritoneal macrophages were also used to assess pyroptosis and the frequency of Th17, Th1/Th17 and Treg cells in the spleen was assessed by flow cytometry.

Results: Nlrp3 -/mice were generally more susceptible to the S. schenckii infection, as shown by higher spleen fungal loads and lower splenic index and weight when compared to their WT counterparts. Furthermore, with the exception of the Th17 cell frequency on day 14 post-infection, the knockout mice had diminished Th17 and Th1/ Th17 mixed cell frequencies during the entire studied period and their cells were less prone to release pro-inflammatory mediators (i. e., NO, IL-1b and IL-17) following ex vivo challenge with F1. Interestingly, the knockout mice had a higher Treg cells frequency than the WT mice on day 14 post-infection, which coincided with the time of higher Th17 cell frequency in Nlrp3 -/mice.

Conclusion: An intact Nlrp3 inflammasome is essential for optimal S. schenckii control, supposedly owning to the triggering of proinflammatory mediators and T helper cell responses, thus linking the S. schenckii innate recognition to the adaptive immune response. The data presented here are paving the way for future preventive and therapeutic strategies against sporotrichosis. Introduction: Dendritic cells (DC) not only present antigens to T cells but also communicate pivotal information about the environmental context in which antigen uptake has occurred. Precisely how these signals are integrated by DC and then decoded by T cells remains unresolved.

We have addressed this question using the doublestranded DNA pathogen Herpes Simplex virus (HSV) in an in vivo model of cutaneous infection. By applying this model to mice deficient in different pattern-recognition receptor (PRR) pathway molecules (e.g., TRIF, MyD88, Sting, MAVS, etc.), we have begun to disentangle the role different PRR signalling pathways play in driving HSV-specific CD8 + T cell priming.

Results: Efficient HSV-1-specific CD8 + T cell priming relied heavily on the provision of type I interferon (IFN). Unexpectedly, we found that prominent DNA sensing pathways leading to type I IFN production, such as those depending on MyD88, STING, and/or Caspase-1/11 played redundant roles in HSV-specific CD8 + T cell priming. However, pathways detecting RNA intermediates, such as the TLR3-TRIF axis as well as molecules signalling via MAVS, were essential for this process. Moreover, we have elucidated the subtypes of type I IFN induced, the transcription factors that were required and the effects those factors have on IFN-stimulated genes, such as IL-15.

Conclusion: These findings demonstrate that DC utilize several different mechanisms to translate the complex sensing of pathogens into effective adaptive immune responses and that the detection of RNA intermediates in HSV infection is key for the efficient priming of CD8 + T cells. Introduction: Two polarized states of macrophage functional activity, known as M1 and M2, have been identified in different temporal stages of inflammatory response. We have previously reported that cellular stress responses can dramatically enhance TLR4-induced inflammatory cytokine production in cultured bone marrow derived macrophages (BMDM) by increasing the magnitude and duration of gene transcription.

Methods: Real time PCR was used to measure cytokine expression levels. Flow cytometry was performed to identify the cell populations.

Results: Using GM-CSF or M-CSF cultured BMDMs to induce M1 or M2 like macrophages, resepectively, we now report that sensitivity for stress-induced enhancement of TLR4-mediated cytokine expression varies with the state of macrophage polarization. While M2-like M-CSF cultured BMDMs exhibit lower cytokine production that can be markedly increased by coincident cell stress, GM-CSF treated M1like cell populations exhibit strong cytokine production with very limited stress sensitivity. Resident tissue macrophages in the liver exhibit M2-like low cytokine expression while monocytes infiltrating the liver following acetaminophen (APAP)-induced acute liver injury show an M1 phenotype that transitions from M1 to M2 phenotype over 48-72 h. Exposure to cell stress (by ip injection of Tunicamycin) dramatically elevates LPS-induced cytokine production in resident liver macrophages and restores M1 phenotype to infiltrating cells that have transitioned to M2 status at late times following injury. Introduction: West Nile virus (WNV) infection in humans leads to a range of disease outcomes, varying from no clinical signs of disease through severe meningitis, encephalitis, and death. The inbred mouse models used for most WNV disease studies fail to capture the breadth of human disease outcomes, limiting the conclusions which can be made in terms of the genetic and transcriptomic underpinnings of viral susceptibility and disease progression. The Collaborative Cross (CC) mouse model has been developed as a recombinant inbred population which encompasses >90% of the diversity within the mouse genome. The CC mouse model effectively presents the full spectrum of disease associated with human WNV infection. Here, we apply the CC model toward understanding the transcriptional response to WNV over a range of disease outcomes to reveal the immune and inflammatory responses to WNV infection.

Methods: Total RNA from the brain and spleen of three CC lines each which are asymptomatic or symptomatic following WNV infection were analyzed by genome-wide microarray at multiple samples post-infection. The signatures of differential gene expression linked with genotype differences to reveal genes whose expression correlated with particular infection outcome phenotypes such as weight loss, viral neuroinvasion, and clinical score.

Results: We identified sets of genes which track with susceptibility to WNV infection, including a number of genes within known innate signaling pathways, along with potentially novel immune molecules.

To determine whether the genes which track with susceptibility to WNV infection in the CC mouse model are relevant to human disease development, we compared the differential gene signatures found in the CC mice to RNA-sequencing samples of a brain autopsy sample from a human patient who had died of WNV infection. We identified viral sequence reads with in the infected human brain, albeit differentially among brain regions. The human WNV brain exhibited an innate immune signature consistent with CNS immune activation, linking this response to aspects to the CC CNS infection/response profile.

Conclusion: Our study demonstrates the utility of the CC model for understanding complex WNV disease outcomes and the host response, including tissue-specific response, to infection. Introduction: Viral infection triggers a fast and effective cellular response mediated primarily by the production of INFb that induce an anti-viral state through complex signal cascades. Therefore, the regulation of its induction and subsequent IFNb signaling needs to be tightly controlled.

There is growing evidence implicating the members of Tripartitemotif (TRIM) protein family of E3 ligases as critical players in this regulation. However, the exact role, mechanism of action, and the physiological relevance of their activity in vivo still remain poorly investigated. Previous work in our lab revealed that an unprecedented large number of TRIMs play critical roles as enhancers in the regulation of innate immune signaling pathways.

To study the role of TRIM65 in innate immune signaling we have used luciferase assays, overexpression in A549 and 293T cells, transient knock down using siRNAs in 293T cells, Western blots, RT-qPCR, PR8-GLuc antiviral assays.

Results: Our recent studies focused on TRIM65 have revealed that its overexpression strongly increased the 2CARD-RIG-I-dependent activation of the INFb and ISRE promoters. Consequently, IFNb, ISG54, Mx1, IL-6 as well as OAS1 mRNA levels are decreased in TRIM65 knock down cells upon infection compared to infected/ treated control cells. These data suggest a stimulatory role for TRIM65 in innate immune signaling. Besides, TRIM65 showed antiviral activity comparable to TRIM25 in a PR8-GLuc antiviral assay.

Conclusion: Since the E3 ubiquitin ligase activity of many TRIMs has been linked to their antiviral functions, our future studies are aimed to identify TRIM65 interacting factors as potential substrates and delineate the molecular mechanism by which TRIM65-mediated ubiquitin or ubiquitin-like modifications could regulate the response to viral infection.

A better understanding of positive regulatory networks of the IFN response will provide new knowledge that will help to design more effective therapeutics.

Disclosure of Interest: None declared. Introduction: Induction of cell-intrinsic antiviral defense via type 1 interferon (IFN-I) requires recognition of viral signatures of infection. Cellular recognition of extracellular or cytosolic nucleic acid is considered the primary means of virus detection. However, avoiding recognition of self nucleic acids is also important to prevent autoimmunity. The threshold of this response can be modulated by disruptions to homeostasis, such as membrane perturbation, suggestive of a viral infection. All enveloped viruses must fuse with the cell membrane during entry, and there is evidence to suggest that membrane fusion is sensed by the cell and impacts antiviral gene induction.

Methods: To examine the response to enveloped virus entry in the absence of replication we used ultraviolet (UV) light inactivated virus or the fusion associated small transmembrane (FAST) protein p14 resuspended in lipid vesicles. We examined the role of calcium and nucleic acid signalling pathways upstream of IFN-stimulated gene (ISG) induction using a combination of fluorescence microscopy, chemical inhibitors, and genetic knockouts.

Results: We found that both calcium signalling and nucleic recognition pathways were required for a full antiviral response to enveloped virus. Inhibition of calcium signalling reduced the antiviral response to UV-inactivated virus infection and prevented complete activation of the STING-IRF3 signalling complex. Lipid vesicles containing p14 FAST protein, but not a fusion-defective mutant, were sufficient to induce an antiviral response on their own despite lacking any nucleic acid. p14-lipid vesicles appear to induce IFN-I through a pathway involving calcium, MAVS, and IRF3.

The calcium signalling response to various fusogenic lipid-based particles appears to be triggered by envelope-membrane fusion and plays a role in the cell-intrinsic antiviral response. The response to enveloped RNA or DNA viruses requires calcium signalling and nucleic acid sensing, and both appear to be required for assembly of the IRF3 activation complex. p14-lipid vesicles were used as a model of virus entry and appear sufficient to induce IFN-I through a MAVS-dependent pathway in the absence of exogenous nucleic acid. Further work will elucidate the nature of the antiviral response to p14 and the role of calcium in IRF3 activation.

Disclosure of Interest: None declared. Introduction: Cell intrinsic innate immunity provides a rapid first line of defense to thwart invading viral pathogens through the production of antiviral cytokines and inflammatory chemokines. However, the presence of many of these signaling pathways in the liver and their role in HBV pathogenesis is unknown. Recent identification of intracellular DNA sensing pathways and involvement in numerous diverse disease processes including viral pathogenesis and carcinogenesis suggests a role for these processes in HBV infection that has a DNA genome.

Methods: In order to characterize HBV intrinsic innate immune responses and the role of DNA and RNA sensing pathways in the liver during recognition of HBV, we utilized in vivo and in vitro models including analysis of gene expression in liver biopsies from HBV infected patients. In addition, mRNA and protein expression was measured in HBV stimulated and DNA treated hepatoma cell lines (HepG2, HepG2.2.15, HepG2-NTCP and HepaRG) and primary human hepatocytes (PHHs). Specific RNA and DNA dependent innate and inflammatory pathways, such as RIG-I/MDA5 and IFI16/cGAS signaling, were studied as well as additional pathways involving the production of interferon and chemokines such as IP-10 and CCL5 that may drive subsequent liver disease. siRNA experiments were utilized to determine the signaling pathways involved in HBV recognition. Microarray analyses was used to characterize global transcriptome changes in HBV infected primary human hepatocytes. UV inactivation of HBV was utilized to determine effects observed from viral replication.

Results: HBV and foreign DNA results in a rapid innate immune responses in hepatocytes characterized by the production of inflammatory chemokines at the mRNA and protein levels. Analysis of liver biopsies from HBV-infected patients supported a correlation among hepatic expression of specific antiviral genes including IP-10 and CCL5. In addition, HBV elicits a much broader range of gene expression alterations as demonstrated by microarray analysis. The induction of chemokines, including IP-10, was mediated predominantly by MDA5 and NF-kB-dependent pathways following HBV stimulation.

Conclusion: HBV stimulated pathways predominantly activate an inflammatory response that would promote the development of hepatitis where as HCV stimulates a much stronger Interferon response. Understanding the mechanism underlying these inflammatory and antiviral responses may provide new strategies to trigger noncytopathic clearance of cccDNA to ultimately cure patients with HBV infection.

Disclosure of Interest: None declared. Introduction: Cell autonomous immunity is mediated by both the innate and adaptive immune system. Correct coordination of these systems is critical for the control of intracellular pathogens. Proinflammatory interferons (IFNs) play a key role in combating intracellular pathogens. IFNs are capable of stimulating several hundred genes, including the IFN-inducible GTPases the Guanylatebinding proteins (Gbps), which have been shown to be important for the control of intracellular pathogens. In order for the Gbps to exert their antimicrobial function, they must first correctly recognize and target to intracellular pathogens. Here we show that targeting to intracellular pathogens by the Gbps is in part controlled by galectin 3. Cytosolic galectin 3 is a known guardian of membrane integrity: galectin 3 can bind to galactosides on the luminal surface of endosomal membranes upon loss of membrane integrity.

Methods: Bone marrow derived macrophages from wild type and Galectin 3 knockout mice were stimulated with interferon gamma (IFNk) and infected with virulent and avirulent vacuolar pathogens. We assessed the ability of Gbps to target to the pathogen vacuole by immunofluorescence. Host endomembranes were disrupted by hypotonic shock or calcium phosphate precipitates. We measured the recruitment of Gbps to damaged vesicles by immunofluorescence.

The presence of bacterial secretion systems present a ''pattern of pathogenesis" associated with bacterial PVs that can be recognized by the host protein galectin 3. We show here that the presence of bacterial secretion systems leads to the recruitment of galectin 3 to PVs, likely indicating the presence of membrane lesions associated with the insertion of bacterial secretion systems into PV membranes. In addition, sterile disruption of host endomembrane vesicles with chemical agents results in Gbp recruitment to the site of damage. This recruitment is dependent on galectin 3.

Galectin-3 directs the delivery of Interferon (IFN)inducible, antimicrobial Guanylate binding proteins (Gbps) to PVs as well as sterilely damaged host vesicles. Our study thus identifies galectin 3 as a danger sensor that controls the recruitment of Gbps to PVs and damaged vesicles. Introduction: Human MxA protein belongs to the family of dynamin-like large GTPases and exerts antiviral activity primarily against negative-stranded RNA viruses, including influenza A (IAV). MxA is able to form higher order oligomeric structures. However, the mode of action of MxA remains unknown. There is increasing evidence that MxA targets the IAV nucleoprotein (NP) in conjunction with UAP56, a cellular DEAD-box RNA helicase required for efficient IAV replication. In order to better understand the molecular mechanism of action of MxA we analyzed in detail the interplay between MxA, UAP56 and the viral target NP.

Methods: To assess the potential interactions of MxA, NP and UAP56 we employed co-immunoprecipitation (Co-IP) as well as a tripartite split-GFP system. In particular, we investigated the effect of various NP mutants on binding and sensitivity to MxA.

Results: Our data clearly showed that only the dimeric variant of MxA (R640A) is able to form stable complexes with NP and UAP56. In addition binding of UAP56 to MxA is greatly enhanced in the presence of NP. Moreover, we observed that the dimeric form of MxA is able to sequester the UAP56 from a UAP56-NP complex, while the catalytically inactive form of MxA (T103A) fails to do so. Mutations in NP reducing its nucleo-cytoplasmic shuttling activity strongly increased binding to MxA, while mutations abrogating oligomerization of NP strongly reduced binding.

Conclusion: These data demonstrate that the dimeric form of MxA plays a critical role for its antiviral function. Hence, the oligomeric MxA (presumed storage form) has first to disassemble into dimers in order to sequester UAP56 from the UAP56-NP complex.

Disclosure of Interest: None declared. Introduction: Sporotrichosis is a mycosis caused by fungi from the Sporothrix schenckii species complex affecting humans and other animals. Sporotrichosis therapy can be difficult, mainly because of complications arising from the long duration and high toxicity of the conventional drug. The drug of choice for the cutaneous and lymphocutaneous forms of sporotrichosis in humans and small animals is itraconazol, a broad spectrum antifungal agent. Considering the research reporting the association between antifungals and immunomodulators is able to reduce therapy duration, we decided to assess the possibility of using imiquimode in association with itraconazol to increase therapy efficacy in sporotrichosis. Imiquimode is a synthetic imidazoquinoline with immunostimulant properties owning to a toll-like receptor (TLR) 7-dependent activation of innate and adaptive immune cells, which results in secretion of pro-inflammatory cytokines and chemokines.

Methods: Male Balb/c mice were infected with a suspension containing 1,6 Â 10 7 S. schenckii ATCC 16345 yeast cells in 20lL of PBS through the subcutaneous route in the metacarpal pad of the left back paw. Imiquimode therapy was initiated 48 h post-infection through the topic route, alone or in association with itraconazol (10 mg/kg) through the oral route, once a day. On day 14 post-infection mice were euthanized and had the popliteal lymph node and spleen removed for, respectively, CFU determination and assessment of the IL-17 and IL-10 ex vivo release; cytokines were quantified in the culture supernatant of total splenocytes by ELISA. Additionally, nitric oxide (NO) production by imiquimode-stimulated thioglycolateelicited peritoneal macrophages from healthy mice was determined by the Griess assay.

Results: The number of CFU recovered from imiquimode-treated mice was significantly greater than that of itraconazol-or itraconazol plus imiquimode-treated mice, and more than seven times that of the untreated control mice. It is noteworthy that treatment with itraconazol alone was ineffective in reducing the fungal burden. Assessment of the ex vivo release of cytokines showed that IL-17 levels were much higher in the culture supernatant of total splenocytes from untreated and imiquimode-treated mice, whereas IL-10 was only increased from the cells of imiquimode-treated mice. Regarding NO production by imiquimode-stimulated macrophages, only the higher concentrations (4 and 1 mg/mL) were able to induce expressive amounts of this molecule.

Our results indicate that imiquimode, alone or in association with itraconazol, is not an efficient alternative for sporotrichosis therapy in the model used by us. Introduction: Community acquired pneumonia is caused primarily by Streptococcus pneumoniae, which is responsible for 4 million illness episodes, 445,000 hospitalizations and 22,000 deaths annually in the US. Children younger than 2 years old and individuals older than 65 years are the most vulnerable for invasive disease. Interleukin 22 (IL-22) is a secreted cytokine produced mainly by cells of the lymphoid lineage and innate lymphoid cells. Ligation of IL-22 to IL-22RA1 and IL-10R2 results in activation of JAK1/TYK2 kinases and downstream phosphorylation of STAT proteins, in particular STAT3. IL-22RA1 is characteristically expressed in epithelial cells, and its activation by IL-22 is involved in the pathogenesis of extracellular organisms including Klebsiella pneumoniae lung infection. IL-22 signaling is related to maintaining barrier integrity, mediating recruitment of leukocytes, and induction of antimicrobial peptides. We hypothesized that enhancement of IL-22/IL-22RA1, either through recombinant IL-22 or absence of IL-22RA2 translates into decrease pneumococcal lung burden and dissemination.

Methods: C57BL/6, Il22 -/-, Il22ra1 fl/fl Albumin-Cre mice were used to assess the endogenous IL-22 production upon S. pneumoniae lung infection. Recombinant IL-22:Fc or Isotype control were given by intraperitoneal route to C57BL/6 mice, before pneumococcus infection to evaluate exogenous IL-22 any effects in bacterial dissemination.

Results: IL-22 was rapidly induced in the lung during pneumococcal infection in wild type mice and Il22 -/mice had higher pneumococcal burdens compared to controls. Additionally, mice with hepaticspecific deletion of Il22ra1 also had higher bacterial burdens in the lung and liver compared to littermate controls after intrapulmonary pneumococcal infection. Thus, we hypothesized that enhancement of IL-22 signaling would control pneumococcal burden in lung tissues in an experimental pneumonia model. Administration of recombinant IL-22 systemically to infected wild type mice decreased bacterial burden in lung and liver at 24 h post infection. Our in vitro studies also showed that serum from mice treated with IL-22 improved opsonic capacity of complement by increasing Complement 3 binding on S.pneumoniae. Pre-treatment with IL-22Fc in wild type increased C3 transcript and protein expression in liver. Furthermore, pre-treatment with IL-22Fc did not increase neutrophil recruitment but there was higher CpsA transcript in lung neutrophils from mice pretreated with IL-22Fc, suggesting that there may be increased neutrophil phagocytosis due to IL-22 dependent C3 bacterial binding. The University of Melbourne, Parkville, Australia; 2 Hokkaido University, Sapporo, Japan; 3 Gifu University, Gifu, Japan Introduction: Lyssaviruses including rabies virus comprise a globally distributed genus of zoonotic pathogens that cause incurable rabies disease in humans and animals, with a case-fatality rate of 100% (60,000 human deaths/year). Central to lyssavirus pathogenesis is the capacity to shut down inflammation in the central nervous system, which involves intricate mechanisms for immune evasion.

Lyssavirus P protein is the viral interferon (IFN) antagonist, which interacts with STAT1/2 to subvert type-I interferon signalling [1, 2] . We recently found that P also targets other STATs including STAT3, thereby modulating responses to multiple cytokines including IL6, suggestive of broad roles in modulation of innate immune signalling [3] . We further showed that by preventing P-STAT interaction, we can prevent subversion of IFN signaling to render a normally 100% lethal rabies virus non-lethal [4] . Thus, P-STAT interaction is critical to disease and a potential target for the design of vaccines or therapeutics. However the structural basis of P binding to STATs and consequent mechanism of STAT antagonism are unresolved. Furthermore the precise impact of P on the complex signalling networks initiated by cytokines remains poorly understood.

Methods: To elucidate the structure of the P-STAT interaction and delineate its effects on cytokine signaling, we are using a combined approach incorporating immune signalling assays and functional genomics techniques, viral reverse genetics and disease models, and biophysical and structural biology approaches.

We have generated the first direct structural data for the P-STAT complex. The data indicate that this involves novel surfaces of P not previously implicated in STAT-binding. We are now using these approaches to define the regions of STAT bound by P to provide insight into the mechanism of P-dependent STAT shut down, as well as to discover how P-protein coordinates and regulates STAT interaction with respect to other critical interactions with viral/host proteins. We are also defining in detail the relationship of P with members of the STAT family; our data now indicate that specific interactions with distinct STATs enable precise regulation of cytokine signaling.

Conclusion: Our data provide the first insights into the regions of P that mediate interaction with STATs, and how P coordinates its interactions with STAT and other important interactors at the virushost interface. Together with functional data on P-protein's modulation of cytokine signaling, this is providing important new insights into the fundamental molecular events by which this lethal virus efficiently disables inflammation. In addition, the structural data is identifying new targets for antiviral approaches aimed at rendering virus sensitive to innate immunity. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key cytokine in the adaptive and innate immune system. Infection and inflammation cause macrophages, mast cells, and lymphocytes to secrete GM-CSF, which stimulates differentiation of myeloid progenitor cells into immune cells. Chemotherapy patients are sometimes given recombinant GM-CSF (molgramostim or sargramostim) to increase production of white blood cells and prevent neutropenia. The basal levels of GM-CSF in blood are in the fg/mL range, and are therefore too low to be measurable with standard ELISAs.

To enable accurate measurements of GM-CSF, MSD developed an ultrasensitive S-PLEX TM immunoassay and evaluated its performance with clinical samples. Anti-GM-CSF monoclonal antibodies were screened and selected based on their sensitivity, specificity, and affinity for GM-CSF secreted by a differentiated THP-1 cell line. Development and characterization of the S-PLEX immunoassay was performed using a recombinant GM-CSF expressed in E. coli and anchored to the GM-CSF WHO International Standard (NIBSC code: 88/646). Normal sera and plasma specimens and several cell models were screened to assess performance of the ultrasensitive assay.

Results: The S-PLEX GM-CSF assay provides a limit of detection (LOD) of <1.0 fg/mL with a dynamic range of four orders of magnitude. The lower and upper limits of quantitation are 2.5 fg/mL and 8000 fg/mL, respectively. The intra-plate coefficients of variation (CVs) for Quality Control (QC) samples spanning the assay range were 4-16% and the inter-plate CVs were 7-14% (16 replicates on 4 plates). Assay accuracy was assessed in a spike recovery experiment; the average spike recovery of GM-CSF was 96% (n = 9 specimens). The dilution linearity average recovery was 92% (n = 9 specimens). Analyte specificity was demonstrated by depleting eight specimens (sera and plasma) with commercial anti-GM-CSF antibodies not included in the S-PLEX assay. Depletion results showed that the assay measures the true analyte, rather than some other non-specific interaction.

GM-CSF was detectable in 100% of apparently healthy specimens (n = 42) with a median value of 51 fg/mL in serum (n = 28) and 27 fg/ mL in EDTA plasma (n = 14). GM-CSF did not appear to be elevated in a small set of autoimmune specimens or sepsis specimens tested. Further validation of assay specificity was performed by measuring changes in GM-CSF secretion in several cell models, pre-and postdifferentiation. An undifferentiated monocytic cell line, THP-1, was shown to secrete GM-CSF only after differentiation into a mature macrophage. Also, as expected, GM-CSF secretion in a HaCaT cell line decreased substantially after differentiation into mature keratinocytes.

Conclusion: MSD developed an ultrasensitive GM-CSF immunoassay with a <1.0 fg/mL limit of detection, improving on current ELISAs by approximately 1000 fold. This will improve current measurements and understanding of GM-CSF in the immune/inflammation cascade. Div. Immunol. Dept. Biomol. Sci., Saga Univ. Faculty Med., Saga, Japan; 2 Div. Immunol., Dept. Mol. Microbiol. Immunol., Nagasaki Univ. Grad. School Biomed. Sci., Nagasaki, Japan; 3 Immunology, Kagoshima Univ. Grad School Med. Dent. Sci., Kagoshima, Japan

Introduction: IL-27 is a heterodimeric cytokine of IL-12 family consisted of p28 and EBI3, and is reportedly produced by innate immune cells of myeloid origin, such as macrophages and dendritic cells, in response to TLR stimulation. It is well known that, during maralia infection, various types of immune suppression occur.

Methods: Mice were infected with Plasmodium berghei ANKA (PbA) and T cells (PbA-specifically activated) were examined for their regulatory functions, cytokine production, and surface markers.

We found that CD4 + T cells from mice infected with Plasmodium berghei ANKA (PbA) produce IL-27 in response to malaria antigen or anti-TCR mAb. IL-27-producing CD4 + T cells were foxp3 -CD11a hi CD49d hi , expressed PD-1 and LAG-3, and inhibited IL-2-production and proliferation of other CD4 + T cells. TCRb KO mice transferred with CD4 + T cells from either p28 KO or EBI3 KO mice showed increased frequency of IFN-g-producing CD4 + T cells and improved resistance to PbA-infection when compared with those transferred with wild-type CD4 + T cells, suggesting that these CD4 + T cells regulate protective host immune responses in IL-27-dependent manner.Intracellular cytokine staining indicated that IL-27-producing CD4 + T cells were distinct from IL-10-producing Tr1 or IFN-gproducing Th1 cells. In addition, anti-IL-27 mAb profoundly neutralized the inhibitory activities of the culture supernatant of CD4 + T cells from PbA-infected mice, while anti-IL-10 mAb had little effect, suggesting that IL-27 is a major inhibitory cytokine produced by CD4 + T cells during infection with PbA.

Conclusion: These studies imply that IL-27-producing CD4 + T cells are novel foxp3regulatory cells that are induced during infection, and we would like to propose naming these IL-27-priducing CD4 + T cells as Tr27 cells. Introduction: Toll like receptors (TLRs) are essential components of pathogen detection system of innate immunity. In comparison to cell surface TLRs that recognize external and specific pathogen-derived structures, endosomal TLRs recognize broadly nucleic acids to activate immunity. While recognition of pathogen-derived nucleic acids is beneficial, host-derived nucleic acids recognition can be highly detrimental for the host. Therefore, activation of endosomal TLRs must be subject to tight regulation.

Methods: We used mice deficient for IRAP (insulin responsive aminopeptidase) to study the impact of in vivo immunological response to TLR ligands and pathogens. We studied in vitro generated bone marrow derived dendritic cells and various subclass of ex vivo dendritic cells and macrophages. Proinflammatory cytokines and Interferon responses were studied by ELISA in supernatant and fluids and by qRT-PCR. TLR9 cleavage and IRAP interaction with FHOD4 were studied by immunoprecipitation and western blot. We studied IRAP, VAMP3, RAB14, STX6, LAMP, TLR9-GFP and TLR3-HA trafficking by confocal microscopy. We generated lentivirus containing IRAP specific ShRNA and FHOD4 specific ShRNA to study the impact of this cytoskeleton anchor on TLR9 responses and trafficking.

Results: Here, we describe a new role for IRAP-containing endosomes as a cellular compartment that regulates TLR9-dependant inflammation by modulating its trafficking and activation. In IRAPdeficient dendritic cells (DCs), the active TLR9 fragment abnormally accumulated in signaling compartment instead of being stored as inactive proreceptor in endoplasmic reticulum (ER). In vitro experi-ments demonstrated exacerbation of inflammatory and interferon responses in IRAP-deficient DCs and macrophages upon TLR9 ligands. IRAP-deficient mice consistently demonstrated uncontrolled inflammation culminating in high level of proinflammatory cytokines secretion responsible for increased death of animals following infection with Pseudomonas aeruginosa. We additionally identified a molecular interaction of IRAP with Formin-like protein 1 (FHOD4), which mediates the anchoring of IRAP vesicles to the actine cytoskeleton network. Accordingly, FHOD4 deletion leads specifically to TLR9 mislocation and exacerbated inflammation, confirming the role of IRAP-containing endosomes as a compartmentalization system, regulating TLR9 trafficking to lysosomes and its subsequent activation.

Conclusion: IRAP-containing endosomes regulate TLR9 activation, being a link between cytoskeleton remodeling and the shaping of inflammatory response driven by TLR9. This newly identified function of IRAP, together with the previously described role of IRAP in cross presentation of antigens by DCs and priming of adaptive TCD8 cells [1] , reveals IRAP-containing endosomes as a regulator at the crossroads between innate and adaptive immunity. Introduction: RIG-I is a key cytosolic sensor that detects RNA viruses through its C-terminal region and activates the production of antiviral interferons (IFNs) and proinflammatory cytokines. While post-translational modification has been demonstrated to regulate RIG-I signaling activity, its significance for the sensing of viral RNAs remains unclear.

Methods: For that HDAC6 -/mice are susceptible to lethal RNA virus infection and show decreased cytokine production, the survival of age-and sex-matched HDAC6 +/+ mice and HDAC6 -/mice was monitored for 9 days after intravenous VSV infection. For the confirming that HDAC6 positively regulates the innate antiviral response in bone marrow derived macrophages and peripheral blood mononuclear cells, virus replication at 12 and 24 hpi, in HDAC6 +/+ and HDAC6 -/-BMDMs in response to VSV-GFP (MOI = 10) infection and PR8-GFP (MOI = 5) infection. For the confirming that HDAC6 interacts with and deacetylates RIG-I in response to RNA viral infection, 293T cells were transfected with poly(I:C) and cell lysates were prepared at the indicated times for co-immunoprecipitation analysis of endogenous HDAC6 and RIG-I. For the checking that HDAC6-dependent RIG-I deacetylation regulates RIG-I binding to double stranded RNA, 293T cells were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were subjected to dsRNA pull-down assays. For the confirming that acetylation of 909K interacts to bind with 907K, but K907 not to bind with viral RNA, The structural modeling result of RIG-I/dsRNA complex and positions of the lysine residue were presented.

Results: Here, we first show that the RIG-I C-terminal region undergoes deacetylation to regulate its viral RNA sensing activity and that the HDAC6-mediated deacetylation of RIG-I is critical for viral RNA detection. HDAC6 transiently bound to RIG-I and removed the lysine 909 acetylation in the presence of viral RNAs, promoting RIG-I sensing of viral RNAs. Depletion of HDAC6 expression led to impaired antiviral responses against RNA viruses, but not against DNA viruses. Consequently, HDAC6 knockout mice were highly susceptible to RNA virus infections compared to wild-type mice.

Conclusion: These findings underscore the critical role of HDAC6 in the modulation of the RIG-I-mediated antiviral sensing pathway. Methods: To gain more insight into the complex regulation of this pathway a kinome-wide siRNA screen was performed. The primary screen revealed over 100 siRNAs that significantly altered the translocation of IRF3 to the nucleus upon RIG-I stimulation. The top 58 candidates were further analyzed in three independent validation screens based on IRF3-sensitive promoter reporter assays or Rift-valley-fever virus replication. Taking all three validation screens into account, 21 novel regulators of the RIG-I signaling pathway could be identified. Relevance of the identified hits in regulating the host-cell antiviral defense was demonstrated by analyzing their impact on Influenza A virus replication.

Results: In the course of this screen, DAPK1 was identified as an inhibitor of RIG-I mediated IRF3 activation. DAPK1 was further shown to be activated during RIG-I stimulation, therefore constituting a bonafide negative feedback loop. Extensive mapping experiments revealed a minimal construct, including the kinase domain, to be sufficient for inhibiting IRF3 reporter activation in over-expression experiments. Furthermore, interaction studies revealed binding of DAPK1 to RIG-I, suggesting that a DAPK1 mediated phosphorylation of RIG-I inhibits its activity. In fact, by means of an in vitro kinase assay we could demonstrate that RIG-I is a substrate of DAPK1. Subsequent phospho-mass spectrometry analysis identified specific sites in RIG-I that are phosphorylated by DAPK1. Using phosphomimetic mutants of the identified sites we could show that phosphorylation at a single amino acid completely abrogated RIG-I signaling.

Conclusion: By means of high throughput screening we identified a novel feedback regulation of the antiviral RIG-I signaling pathway in which DAPK1 gets activated and subsequently inhibits signaling by phosphorylation of the sensor RIG-I. Introduction: The respiratory tract is populated with innate immune cells and represents a unique compartment in pathogen entry and protective immunity, but the control of local innate immunity is poorly defined.

Methods: We used Trim29 knoxkout mice, in vivo virus infection and bacterial infection, histology, luciferase reporter gene assay, in vitro pull-down and immunoblot analysis, in vitro ubiquitination assay, confocal microscopy assays to investigate the role of TRIM29 in control of local innate immunity in the respiratory tract.

Results: Here we report that the alveolar macrophages selectively expressed the E3 ubiquitin ligase TRIM29 serves as a key regulator of type I interferons (IFNs) as well as proinflammatory cytokines production upon infection. We found that genetic deletion of TRIM29 (Trim29 -/-) enhanced type I IFNs production (>5 folds) in bronchoalveolar lavage fluid (BALF) in response to intranasal influenza A virus infection. Importantly, challenge of wildtype mice with influenza A virus led to lethal pulmonary inflammation and injury. In contrast, duo to the enhanced type I IFNs production, none of the Trim29 -/mice succumbs to this infection. Histologically, the lung of Trim29 -/mice exhibited attenuated viral loads, reduced edema, alveolar hemorrhage, alveolar wall thickness and neutrophil infiltration. Similarly, deletion of TRIM29 markedly enhanced production of proinflammatory cytokines (up to 9 folds) in response to intranasal lipopolysaccharide (LPS) challenge. Consequently, all Trim29 -/mice died to this uncontrolled production of proinflammatory cytokines, whereas 75% of wildtype mice survived from LPS induced endotoxin shock. Mechanistically, we demonstrated that TRIM29 induced Lys48 (K48)-linked ubiquitination and degradation of NEMO, the key adaptor protein in both interferon regulatory factors (IRFs) signaling and NF-jB signaling. TRIM29 used the OmpH-OmpH domains to bind to NEMO and ubiquitinated NEMO at the Lys183 site.

Conclusion: These data demonstrate that TRIM29 functions NEMO to control lung innate immunity, and this effect is especially important in the maintenance of local immune homeostasis.

Disclosure of Interest: None declared. Circulating nucleic acids are associated as causative agents for autoimmune diseases such as lupus. We previously discovered that class A scavenger receptors (SRAs) are ubiquitously expressed and are essential for type I interferon production in response to extracellular dsRNA [1] . Moreover, while SRAs are thought to mediate uptake via clathrin-mediated endocytosis, we found that full interferon production requires signaling through both the endosomal TLR3 and cytosolic RLR pathways [1] . While the short, cytosolic tail of SRAs is predicted to have signaling capacity, we recently showed that SRAs cannot mediate interferon production in the absence of TLR3 or RLR pathway involvement [2] . Although our previous studies focused on extracellular dsRNA, as this is a prototypic pathogen associated molecular pattern, a hallmark of many autoimmune diseases is the presence of autoantibodies against circulating DNA. Thus, in this study we compare the signaling capacity of extracellular RNA versus DNA.

Purified collagenous or cysteine-rich domains of SRA-I were produced for in vitro binding assays. To monitor trafficking of extracellular RNA and DNA following uptake, we employed both confocal microscopy and differential centrifugation. Finally, to identify proteins that interact with the cytosolic tail of SRA-I, we employed the bio-ID technique. Here, a biotin ligase is fused to the cytosolic tail of SRA-I, leading to biotinylation of proteins that come into close contact. Biotinylated proteins are precipitated using streptavidin and identified by mass spectrometry.

Results: We found that the purified collagenous domain, but not the cysteine-rich domain, binds DNA and RNA will equal efficiency in a length and sequence independent fashion. Similarly, SRA-containing fibroblasts bind and internalize extracellular DNA and RNA with similar efficiency and kinetics. However, extracellular DNA fails to elicit type I interferon production, despite the presence of competent cytosolic DNA sensing pathways, as determined by DNA transfection. These results suggest that either internalized DNA and RNA are differentially trafficked upon binding and uptake or that cross-talk between endosomal and cytosolic sensing pathways differ between DNA and RNA. Current experiments are underway to investigate these possibilities.

Conclusion: Understanding how cells recognize and respond to circulating foreign and self nucleic acids is critical to develop clinical tools to fight infectious and autoimmune diseases. Here we find that despite SRAs binding DNA or RNA indiscriminantly, the subsequent cellular outcome is very different. Little is understood how extracellular nucleic acid is trafficked within the cell upon its uptake. Thus, these studies will shed important light on the differential trafficking or signaling of extracellular DNA and RNA in fibroblasts, important sentinel cells of the innate immune system, given their expression of all SRA family members and robust induction of type I interferon in response to both pathogen and danger associated molecular patterns. Introduction: Toll-like Receptors (TLRs) detect pathogen molecules to mount immune responses required for microbial resistance. However, their inappropriate activation can also contribute to inflammation-driven diseases such as atherosclerosis and inflammatory arthritis. Here we uncover the mechanism by which a range of TLRs trigger non-canonical NF-kB to initiate inflammatory responses against pathogen molecules. We also explore how this same mechanism might also promote auto-inflammatory disease that results from the mutation of the IAP family member, XIAP.

Methods: Bone marrow derived macrophages were stimulated with a range of TLR ligands and signalling and cytokine induction examined by Western blot and ELISA assays. Mice were challenged with endotoxin and serum cytokine levels assayed.

Results: We document that a range of different TLRs that signal via the adaptor protein Myd88 induce the degradation of the IAP protein cIAP1, and the cIAP1 binding partner TRAF2, resulting in two distinct cellular outcomes. Firstly, we show how TLR-induced loss of cIAP1-TRAF2 triggers non-canonical NF-jB activity to induce a sub-set of pro-inflammatory cytokines and chemokines. Secondly, we demonstrate that in the absence of the related IAP protein, XIAP, TLR-induced cIAP1 degradation triggers activation of the NLRP3-caspase-1 inflammasome, which cleaves and thereby activates precursor IL-1b and IL-18. Consistent with this, the genetic deletion of XIAP and cIAP1 in myeloid cells, although resulting in no overt phenotype, sensitizes to endotoxin induced IL-1b production, both in vitro and in vivo. This mechanism of TLR-induced cIAP1 loss may explain why, in response to pathogen infection, XIAP mutant patients can develop severe autoinflammatory disease characterized by enhanced inflammasome activity. Finally, we identify a novel mechanism that can prevent TLR-Myd88 mediated cIAP1 degradation, which requires type I Interferon (IFN) production, and is able to repress TLR-induced inflammasome responses.

Conclusion: TLR-induced cIAP1-TRAF2 loss represents a universal mechanism for the activation non-canonical NF-KB from a range of different TLR family members. This pathway is likely to be important for activating appropriate immune responses against microbial infections, or in the case of XIAP loss, can trigger pathological auto-inflammation mediated by excessive inflammasome and IL-1 activity.

Disclosure of Interest: None declared. Methods: We tested the hypothesis that exogenous IL-15 enhances ACT in the absence of lymphodepletion. For this purpose, we selected the B16 melanoma mouse model where tumor cells express the melanoma cell-associated antigen gp100. For ACT therapy, C57BL/6-pmel-1-Thy1.1 transgenic mice were used as a source of Pmel-1 cells, which recognize gp100. Pmel-1 cells were transferred into B16 melanoma-bearing C57BL/6 mice comparing 3 strategies: (i) cell transfer without lymphodepletion (ACT), (ii) cell transfer in irradiated mice (ACT+XRT) and (iii) cell transfer plus exogenous hetIL-15 administration (ACT+hetIL-15) in lymphoreplete mice. We performed analysis of tumor-infiltrating lymphocytes by flow cytometry and multi-color immunohistochemistry.

Results: hetIL-15 treatment promoted infiltration and persistence of both adoptively transferred Pmel-1 cells and endogenous CD8 + T cells in the tumor. Following irradiation, Pmel-1 cells also localized to tumor sites efficiently, but their persistence was severely reduced in comparison to mice treated with hetIL-15. Importantly, we found that hetIL-15 treatment led to preferential enrichment of Pmel-1 cells in the tumor sites in an antigen-dependent manner. hetIL-15 treatment also increased proliferation and cytotoxic ability of tumorinfiltrating Pmel-1 cells, while reducing their PD-1 level and resulting in improved tumor control and survival benefit. Introduction: Type 1 diabetes (T1D) is characterized by a progressive immune-mediated destruction of pancreatic b-cells and associated metabolic dysfunction caused mostly by the loss of regulatory T cells (Tregs). Recent studies have shown deficiencies in both the interleukin-2 (IL-2) receptor and its signaling pathway. In fact, decreased responsiveness to IL-2 measured by STAT5 phosphorylation (pSTAT5) is observed in T1D patients. Originally, IL-2 was developed as an immune stimulator, able to enhance effector T cell (Teff) and natural killer (NK) cell function. It is now well known that another function of IL-2 is the generation and survival of Tregs. Several studies in both mouse and human have demonstrated that IL-2 therapy can suppress autoimmunity. The major goal for immunotherapies would be to develop IL-2 that would selectively activate and boost the tolerogenic immune response.

Methods: In order to achieve this result, we selectively generated some high-quality anti human IL-2 antibodies using a phage display scFv library. The antibodies screened using this technology were later selected on the relative inhibition of pSTAT5 versus pAKT. After this selection, the antibodies were tested in vitro on purified human immune cells as CD4, CD8 and Tregs, evaluating their ability to affect cell proliferation.

: Their function was also tested in a human PBMC assay, in which few antibodies show a total inhibitory activity on all the lymphocyte populations while others are able to increase Treg proliferation, inhibiting at the same time CD4 and CD8.

Conclusion: Current efforts are devoted to understanding the correlation between the different activities and the antibody characteristics, and in vivo testing in humanized mouse models is underway.

Disclosure of Interest: None declared. Introduction: Interleukin-33 (IL-33) is a cytokine that plays a key role in several inflammatory diseases, including asthma and atopic dermatitis. It is released by damaged cells and stimulates proinflammatory signaling pathways in various cell types such as T cells, innate lymphoid cells, mast cells and macrophages. IL-33 signals through a receptor complex at the cell membrane consisting of the IL-33 receptor (ST2) and the co-receptor IL-1RAcP. Blocking the IL-33/ST2 signaling axis with the soluble (extracellular) form of the IL-33 receptor (sST2) has been shown to be beneficial in experimental models of asthma. However, the effect is rather weak due to the low ligand binding affinity of sST2, implicating a need for better and stronger antagonists. We show here that a fusion protein consisting of the extracellular domains of mouse ST2 and IL-1RAcP, further referred to as IL-33trap, is much more potent than sST2 in inhibiting mouse IL-33 activity in vitro and in vivo.

Methods: Isothermal titration calorimetry was used to study the affinity of sST2 or IL-33trap for IL-33. To further compare sST2 and IL-33trap in vivo, mice were injected intra-peritoneally for 5 consecutive days with IL-33trap or sST2 followed by IL-33. Peritoneal eosinophil infiltration, as well as IL-5 and IL-13 production by splenocytes upon in vitro re-stimulation with PMA, were analyzed at day 6. Finally, the effect of the IL-33trap was analyzed in the house-dust mite (HDM)-induced asthma model in mice.

Results: Isothermal titration calorimetry showed that IL-33trap has at least 30-fold higher affinity for IL-33 compared to sST2. We further studied the ability of sST2 and IL-33trap to neutralize the activity of recombinant IL-33 in vivo. Again treatment with IL-33trap showed considerably bigger potential to inhibit IL-33 activity when compared to sST2. Furthermore, IL-33trap delivery at the sensitization phase of HDM-induced asthma model strongly prevented infiltration of eosinophils and type II innate lymphoid cells in the lungs of sensitized mice challenged with HDM. Currently, we are further testing the therapeutic potential of IL-33trap when administered at later time points.

Conclusion: Together, our studies demonstrate the improved potential of IL-33trap cytokine blockers for therapeutic use. Introduction: In response to viral infection, type I and type III interferons (IFNs) exert common antiviral activities through distinct receptor complexes, both of which activate shared IFN-triggered Jak-STAT pathway. Although the contributions of type I IFNs to antiviral defense have been well characterized, understanding of a distinct role for type III, or -k, IFNs for overall antiviral protection is still emerging, and their functions beyond antiviral protection are largely unknown.

Methods: Mice lacking mature lymphocytes (Rag2 KO) and mouse strains with total or cell type-specific deficiency in IFN-k signaling were used to investigate contributions of adaptive immunity and type III IFNs to protection against heterologous simian rotavirus (RRV) infection in suckling mice.

Results: We recently demonstrated that both type I and type III IFNs serve as important mediators for limiting replication of heterologous simian rotavirus RRV in suckling mice, and cooperate to engage antiviral defenses in the gastro-intestinal (GI) tract. However, we also demonstrated that levels of RRV replication in the GI tract were not directly associated with diarrheal disease. Rag2 KO mice developed chronic infection but resolved RV-triggered diarrheal disease with the same kinetics as wild type mice, which clear RV infection. We also unexpectedly observed that, while RRV replicated to higher titers in Ifnlr-deficient animals, diarrhea was better controlled in the absence of IFN-k signaling. Reduced diarrhea with elevated viral titers was also detected in mice lacking IFN-k receptor only in intestinal epithelial compartment (villin-Cre-Ifnlr fl/fl mice), revealing dependency of the observed effect on the presence of IFN-k receptor in intestinal epithelial cells.

Conclusion: Therefore, type III IFNs may exert a double-edged action in the GI tract by restricting of RV replication on the one hand, but promoting the intestinal pathology on the other. Introduction: Immunotherapy is currently used in combination with radio-and chemotherapy in order to improve the clinical outcome after surgical resection. IL-15 is of particular interest, as it targets and stimulates the immune system, but the optimal dosing regimen still needs to be determined. The aim of the present study is to define these treatment modalities with an IL-15 agonist.

Methods: In order to study different tumor types, we used two models in two different mouse strains. We then compared two dosing regimens, depending on whether it was a metastatic or solid tumor model. For comparison, Kruskall Wallis and Kaplan Meier analyses were performed. We also looked at the role of CD8 T cells and NK cells, and their interaction within in vivo depletion studies and ex vivo proliferation assays. In each case, expansion of immune cells was tested by flow cytometry.

Results: It appears that in the metastatic model, two injections of the IL-15 agonist are sufficient to reach the maximal therapeutic effect, whereas several injections are needed to get a therapeutic benefit in solid tumors. A pharmacological analysis showed that this difference was correlated to a decrease in NK cell response to several injections of the IL-15 agonist, whereas the CD8 T central memory cell response remained high, even after multiple injections. Interestingly, the lack of NK cell response was restored by CD8 T cell depletion, suggesting a close relationship between NK and CD8 T cells.

The use of an IL-15 agonist in cancer immunotherapy has much promise, as it enables a significant reduction of metastases, both in metastatic or solid tumor models. However, the dosing regimen has to be chosen very carefully in order to maximize the therapeutic effect of the IL-15 agonist. Moreover, the understanding of the IL-15 mechanism in a tumoral context is crucial for combination treatment studies in possible future human trials.

Disclosure of Interest: None declared. Introduction: The function of overlapping cytokines from the c-chain (c-c) family are linked to a variety of inflammatory and autoimmune diseases. Therapeutic agents that exhibit a selective c-c cytokine inhibitory profile would display an optimal balance between efficacy and toxicity in vivo, and fill a critical therapeutic void that exists between single mAb therapy, which lacks optimal efficacy when multiple cytokines are disease modulators, and small molecule JAK inhibitors, which display pan-cytokine inhibition and unwanted side-effects. We have developed BNZ132-1, an antagonistic peptide specific for blocking the signaling of the c-c cytokines IL-2 and IL-15, without displaying significant inhibitory activity against the remaining cytokines in the c-c family. We hypothesized PEGylation of BNZ132-1 (BNZ132-1-40) would increase the half-life and clinical potential of the peptide. BNZ132-1-40 was monitored following long-term administration in both mouse and monkey, and further evaluated in a rheumatoid arthritis mouse model -a disease condition involving IL-2 and IL-15 signaling.

Methods: BNZ132-1-40 was evaluated at multiple concentrations in a 13-week repeat-dose study in C57BL/6 mice and cynomolgus monkeys. Blood samples were utilized for flow-cytometric analysis on the potential effects of BNZ132-1-40 on lymphocyte subsets at the study mid-point and upon study completion. To evaluate one aspect of BNZ132-1-40 efficacy, the agent was compared to the steroid dexamethasone and the small molecule JAK inhibitor Tofacitinib in mice with semi-established type II collagen-induced arthritis (CIA).

Results: BNZ132-1-40 displays highly targeted effects against lymphoid/immune cell lineages expected following an effective blockade of IL-2 and IL-15 in both mouse and monkey. The compound did not exhibit any adverse effects thus highlighting the agent's selective cytokine inhibitory profile without the strong off-target activity often observed following the administration of small molecule JAK inhibitors. BNZ132-1-40 showed moderate yet equipotent efficacy as compared to the small molecule JAK inhibitor Tofacitinib in the CIA model as determined by evaluation of clinical and histopathology parameters.

Conclusion: BNZ132-1-40 is effective at targeting IL-2 and IL-15 in vivo, and is well-tolerated following long-term administration. The compound displays a high selectivity for IL-2 and IL-15, without significant impact on the remaining c-c cytokines or other cytokine families. The lack of off-target effects of BNZ132-1-40 is in stark contrast to small molecule JAK inhibitors, which act to block signaling of the entire c-c cytokine family and thereby elicit cytotoxic effects following in vivo administration. BNZ132-1-40 represents a well-tolerated and effective alternative for therapeutic strategies that employ small molecule JAK inhibitors. Methods: As in vitro and in vivo model of HBV infection, we used human hepatoma cell line or chimeric mice with humanized liver, respectively. The antiviral effect of test article was assessed by measuring HBV core antigens and HBV nucleotides via immunoassays or quantitative polymerase chain reaction assays. To investigate the profile of antiviral effect, DNA microarray technology was employed. The level of chemokine secretion was determined with an enzyme-linked immunoassay.

Results: In T23 cells, a HBV-producing human hepatoma cell line, TRK-560 remarkably inhibited production of HBV replicative intermediates. It is notable that TRK-560 is over 100 times more potent than PEGylated-IFNa 2a. In addition, TRK-560 can reduce the amount of released HBsAg, a strong predictor of SVR, whereas PEGylated-IFNa 2a cannot. These superior antiviral effects of TRK-560 are probably due to a relatively greater induction of IFNstimulated genes (ISGs) with direct antiviral activities.

The administration of TRK-560 in chronically HBV-infected chimeric mice led to significant decreases in serum HBV-DNA, HBeAg and intrahepatic cccDNA. Lower level of serum HBsAg was also observed compared with that in control mice, although the change was not statistically significant. At the end of the examination, global gene expression profiles in the TRK-560 treated livers were analyzed by DNA microarrays. Although ISGs upregulation had largely disappeared at this point, gene set enrichment analysis revealed that a variety of process networks are remarkably altered in TRK-560 treated livers, including those related to chemotaxis. We therefore examined the effect of TRK-560 on chemokine secretion using fresh human hepatocytes isolated from chimeric mice. Compared with PEGylated-IFNa 2a, TRK-560 evoked a remarkable increase in secretion of chemokine (C-X-C motif) ligand 10 (CXCL10), which drives immune cell chemotaxis. These findings indicate that the in vivo antiviral effect of TRK-560 is probably accounted, at least partially, for by immune cell-mediated viral clearance.

Conclusion: In this study, we show that TRK-560 has potent antiviral effect due to its prior activities in not only induction of ISGs but also stimulation of immune cell chemotaxis. Taking Introduction: Many cytokines have potential therapeutic applications and several, including interferon (IFN)a and IFNb, have been approved for the treatment of cancer and/or autoimmune disease. While these agents are effective, they are associated with significant dose-limiting toxicities that prevent their use at levels sufficient to promote optimal therapeutic benefit. The toxicity of therapeutic cytokines is likely mediated via cognate receptor activation on cells other than those that mediate their therapeutic effects. For example, IFNa, which is indicated for the treatment of multiple myeloma (MM), has a narrow therapeutic index. Approaches that seek to minimize dose-limiting systemic toxicity while maximizing tumorspecific cytokine exposure could greatly enhance the therapeutic index and effectiveness of class I IFNs and other cytokines.

Methods: A moderate degree of tumor-specificity may be achieved by attaching a cytokine such as IFNa to a tumor-targeting antibody; such antibody-targeted cytokines (immunocytokines) are highly active but show only moderate tumor-specificity since the cytokine can still potently stimulate its receptor on antigen-negative cells. We sought to improve the ''therapeutic index" of antibody-targeted cytokines by mutating the cytokine portion to significantly reduce its affinity for its receptor, thereby making it more dependent on antibody-based cell-targeting.

Results: Here we demonstrate that such fusion proteins, consisting of mutant or attenuated cytokines (Attenukines TM ) attached to tumortargeting antibodies, are 1000-100,000-fold more potent on targetpositive cells compared to target-negative (normal) cells. This is shown for antibody-Attenukine TM fusion proteins based on multiple tumor antigens (CD20, CD38, CD138, HMW-MAA, HLA) and multiple attenuated mutants of IFNa, IFNb, IL-4 and IL-6. Furthermore, we have evaluated an anti-CD38-attenuated IFNa molecule (anti-CD38-Attenukine TM ) in various CD38 + MM xenograft models and found that this molecule retains potent tumor-targeting activity and anti-tumor efficacy. Our recent studies presented here indicate multiple mechanisms are involved in the robust anti-tumor responses.

Conclusion: Taken together, our findings suggest that the administration of antibody-attenuated cytokine fusion proteins to cancer patients may promote robust cytokine-dependent tumor-killing while minimizing systemic toxicity. 

Introduction: There is an increasing list of human necrotizing diseases in which cytokines play pathogenic role. These diseases often involve more than two cytokines that share receptor components and hence display functional redundancy. A targeted monoclonal antibody approach only specifically neutralizes one cytokine and often fails to suppress the in vivo activation caused by such cooperation. To address this issue, the Tagaya lab and BIONIZ Therapeutics have developed new technology based on the structural information available. In short, we utilized the gc (common gamma) using cytokine family as our target and designed our first peptide BNZ132-1 to block the access of IL-2 and IL-15 to the gcmolecule. The detail and the proof-of-concept demonstration of our technology can be seen in our recent publication (Nata et al. 2015). BNZ132-1 is a novel inhibitor of IL-2 and IL-15 that blocks the actions of each cytokine efficiently in vivo. We next switched our focus on blocking the interplay between IL-15 and IL-21, another gcfamily cytokine, because these two are now being implicated in the pathogenesis of Celiac disease. We hypothesize that IL-15 and IL-21 may show functional synergism on CD8 T cells that are the primary pathogenic cells in Celiac Disease.?

Methods: We purified human CD8 T cells from blood by FACS, or used IEL CD8 T cell lines established from Celiac Patients. Target cells were then stimulated by various doses of IL-15, IL-21 or their combination and we then assessed the intracellular signaling events by conventional immunoblotting as well as examining the induced genes by RNA-seq.

Results: As reported, only IL-15, but not IL-21, induced robust proliferation of these cells ex vivo. IL-15 caused strong phosphorylation of STAT5, Akt, Erk1, and p38 MAPkinase whereas IL-21 acted more preferentially on STAT3 and showed rather weak activation of non-STAT intracellular signaling components. Curiously, when suboptimal doses of IL-15 and IL-21 were combined, we saw synergic phosphorylation of major signaling molecules such as STATs, Akt, and Erk. This was also confirmed by RNA-Seq of the induction of Granzyme B which is the key component of CD8 mediated cytotoxicity that presumably causes intestinal-tissue damages in the Celiac Disease environment.

Conclusion: Our data, although preliminary, suggests that IL-15 and IL-21 have distinct roles on CD8 T cells, but show combined and synergistic effects upon co-operation, which may give rise to a new mode of cellular activation that neither of these factors could accomplish alone. We thus believe that the co-inhibition of these two factors may have more efficient therapeutic effects than monotherapies targeting either IL-15 or IL-21. Our second peptide, BNZ132-2, shows potent inhibitory action on IL-15 and IL-21 in vitro without blocking other gc-or non-gc cytokines. BNZ132-2 thus can be a prime candidate to safely and efficiently treat refractory Sprue, a disease without an established treatment. BIONIZ Therapeutics will pursue the clinical development of BNZ132-2. Introduction: Interleukin 17A (IL-17A) is a pro-inflammatory cytokine produced by CD4 + T cells, CD8 + T cells, and a variety of innate immune cells. IL-17A transcription is mainly driven by retinoid-related orphan receptor gamma t (RORgt) and IL-17A production can be induced in vitro upon T cell receptor activation in the presence of certain cytokines. We investigated the relative contribution of different immune cell types to IL-17A production in human, rat and mouse whole blood, and compared the effects of several selective RORgt modulators to evaluate the relative contribution of RORgt in the immune cells from different species.

Methods: Whole blood from human, rat and mouse was stimulated in vitro, and IL-17A-producing cells were analyzed through FACS analysis. In addition, CD4 + and gd T cells were isolated from mouse spleen and stimulated for IL-17A production. The effects of RORgt modulators on IL-17A production by these cells was evaluated through FACS and ELISA analysis.

Results: We found that in human and rat whole blood, the predominant cells producing IL-17A were CD4 + T cells, but in mouse whole blood, it was the gd T cell population that were the major IL-17A-producing cells. IL-17A was also produced by CD8 + T cells in whole blood of all three species, with the highest percentage in human and the lowest in mouse. Selective RORgt modulators showed potent inhibition of IL-17A production by both CD4 + T cells and gd T cells. Interestingly, some RORgt modulators demonstrated differential inhibitory activities between CD4 + T cells and gd T cells.

Conclusion: For certain RORgt modulators, we have observed differences between pharmacological activity in whole blood from different species. Based on our findings, differences across species in predominant cell sources that produce IL-17A could be one factor contributing to these observations. The underlying mechanism(s) resulting in differential activities of RORgt modulators in CD4 T cells vs gd T cells is not clear and will require further investigation.

Disclosure of Interest: None declared. Chemokine receptors undergo a basal level of recycling that is enhanced by the binding of their appropriate ligand. In addition to canonical markers used to identify populations, many subsets of a particular cell population can be identified by the expression of chemokine receptors. Chemokine are secreted from activated cells, and bind to their specific chemokine receptor on nearby or distant cells. Cells expressing the appropriate chemokine receptor are then attracted by the chemokine to the site of infection. In this report, we evaluated the effects of incubation temperature on chemokine receptor antibodies (CD180 to CD200) and its outcome on the staining resolution.

Methods: Anti-coagulated peripheral blood (EDTA) was collected from three healthy volunteer donors, and was used within 2 h after collection. A cocktail of two chemokine receptors (CCR7 plus one of the following antibodies: CD180-CD200) was added to 100 ul of fresh whole blood and incubated for 10 min at either room temperature or 37°C in the dark. After incubation, a cocktail of antibodies (CD3; CD4; CD8; CD45RA; CD45RO; CD27; CD28; CD127; and CD25) was added to each sample and incubated for an additional 20 min at room temperature in the dark. Erythrocytes were then lysed and leukocytes were fixed using BD lysing solution. Samples were then washed and resuspended in staining buffer for analysis. Samples were acquired immediately on a BD LSRFortessa X-20 flow cytometer, and acquired using application settings derived for whole blood specimens. FMO controls were used to evaluate population boundaries and spectral spread. Acquisition and analysis were done on BD FACS Diva 8.0.1.

Results: Pre-incubation of anti-human CCR7 antibody at 37°C demonstrated enhanced resolution when compared to room temperature control. Additionally, many T cell populations that expressed a chemokine receptor (CD180-CD200) had an increased stain index, when that anti-chemokine receptor antibody was preincubated at 37°C compared to room temperature pre-incubation.

Conclusion: Using peripheral blood, and antibodies to identify the major subsets of CD4 and CD8 T cells, we observed that an incubation with anti-chemokine receptor antibodies for 10 min at 37°C led to improved resolution of certain chemokine receptors as measured by stain index, when compared to room temperature. The increase in resolution for these receptors is important when considering the low percentage for some of the subsets these help in identifying. Introduction: IL-23 is critically involved in induction of Th17driven pathological responses in the gut via an unknown mechanism. Previous studies used high throughput transcriptomics approaches to identify gene signatures at the mRNA level in mouse pathogenic Th17 cells, with only a few targets being proposed to be regulated by IL-23.

We have utilized high throughput methods, including microarray and phospho-proteomics, to define IL-23 regulated networks in in vitro differentiated mouse Th17 cells both at the RNA and protein levels. We have also employed quantitative PCR and Western blotting to test the expression of selected targets in wild type and IL-23 receptor-deficient Th17 cells in vitro. Using flow cytometry-assisted cell sorting we have finally characterised the expression of these genes in the steady state and inflamed gut using a T cell transfer model of murine colitis.

Results: Integrated omics approach employed in this study has shown a global impact of IL-23 signalling on gene expression and protein phosphorylation in in vitro differentiated mouse Th17 cells. The analysis of this data revealed novel genes and proteins regulated by IL-23 signalling and previously uncharacterised in this context. We have further confirmed IL-23-driven expression of the selected top hits at RNA and protein levels using low throughput methods. Furthermore, we isolated distinct cell populations from the steady state murine colons and confirmed the expression of the selected IL-23 targets in CD4 T cells. Some of the validated hits (e.g. TIMP1, FOSL2, LTB4R1, IL-17F) have been previously linked to pathogenic Th17 cells and shown to be involved in inflammatory bowel disease. To mimic the human condition we induced colitis in mice via an adoptive transfer of a mixture of wild type and IL-23receptor-GFP reporter CD4 T cells and have shown the enriched expression of the short-listed IL-23 gene targets in IL-23R + CD4 T cells isolated from murine spleens, mesenteric lymph nodes and inflamed colons.

The integrated omics approach used in this study revealed novel IL-23 targets which may be responsible for Th17driven pathology in the intestine.

Disclosure of Interest: None declared. Introduction: The expression of type I interferons (IFN) is the immediate host response during most viral infections. These cytokines induce an antiviral state in cells by the production of antiviral proteins. Apart from this, type I IFNs can also modulate immune cell functions as well as elicit antiproliferative responses. IFNa is composed of multiple IFNa subtypes (up to 14 in mice), and although they all bind the same receptor, they differ in their biological activity. Here, we are interested in the immunoregulatory functions of IFNa subtypes on CD8 + T cell responses to further use them as an immunotherapeutic agent during chronic retroviral infection.

Methods: To investigate the influence of different IFNa subtypes on CD8 + T cell proliferation, activation and effector functions we performed antigen-specific T cell proliferation assays in vitro. The impact of IFNa subtypes on the cytolytic capacity of CD8 + T cells was analyzed by using an in vitro kill assays. We further investigated the role of the IFNa/b-receptor (IFNAR) by the usage of IFNAR -/-CD8 + T cells and IFNAR -/dendritic cells (DCs) in these settings. CD8 + T cells and DCs were characterized by flow cytometry.

Results: We detected a direct antiproliferative capacity of all tested IFNa subtypes on CD8 + T cells. In particular, IFNa4 and IFNa9 strongly induced the expression of cytokines like IL-2, TNFa and IFNk in vitro. IFNa subtype-treated DCs were highly activated and maturated and showed increased production of TNFa and IL-6. The induction of cytokines in CD8 + T cells by IFNa subtypes required IFNAR expression on DCs. Furthermore, IFNa subtype stimulation (especially IFNa4 and IFNa6) induced highly activated CD8 + effector T cells which exhibit an improved cytotoxic phenotype (Granzyme B, Eomes) whereby killing of target cells by CD8 + T cells was enhanced.

Conclusion: These results demonstrate the distinct immunostimulatory properties of each IFNa subtype which can be used to develop future immunotherapies against retroviruses. Introduction: IL-22 is expressed by activated lymphocytes and is important in modulation of tissue responses during inflammation. The cytokine induces proliferative and anti-apoptotic pathways in epithelial cells allowing enhanced cell survival. This can have positive effects, such as in the maintenance of epithelial barriers in the gastrointestinal tract, but also negative effects, such as contributing to colorectal tumorigenesis. As IL-22 can be dualnatured, we hypothesized that its biological activity should be tightly regulated in order to limit IL-22 expression to the sites of inflammation. One such environmental cue could be low oxygen, which often accompanies inflammation.

Methods: Naïve mouse CD4 T cells (CD4+ CD25-CD62L high CD44 low ) were activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-23, IL-6, IL-1b and FICZ. For the last 24 h of culture, cells remained at normoxia (~17% oxygen) or were cultures at hypoxia (1% oxygen). Three days post-activation Introduction: A balanced immune response is required to protect the host from pathogenic microorganisms without causing host damage. Critical for host defense are CD4 + T-cells, which, depending on the local cytokine milieu, differentiate from naïve into effector T helper (Th)1, Th2 or Th17 cells defined by their cytokine production and function. Metabolism plays an essential role in Th cell responses including the production of their hallmark pro-inflammatory cytokines. However, Th cells can cause immunopathology unless restrained by anti-inflammatory mechanisms such as interleukin (IL)-10. However, the contribution of metabolism to Th cell proinflammatory hallmark cytokine production versus that of IL-10 is not known.

Methods: In this study, we use the glucose analogue 2-deoxyglucose (2DG) and Tunicamycin to restrict metabolic pathways during Th cell differentiation. Intracellular cytokine or phospho protein staining, RT-PCR, ELISA, U-13 C-glucose labeling followed by GS-MS, Western Blot and RNA-Seq were further used to determine their effects on the differentiation of Th cells.

Results: We show that 2DG specifically inhibits Th1 and Th2 cell differentiation and accompanying IL-10 production, suggesting that IL-10 production is tightly linked to the differentiation program of Th1 and Th2 cells. In contrast, 2DG enhances IL-17A production in Th17 cells, even in the presence of the known Th17-inhibiting cytokine, IL-2. However, this is also accompanied by a loss in IL-10 production. Mechanistically we show that, rather than inhibiting glycolysis, 2DG inhibits glycosylation, dampening IL-2Ralpha surface expression and thus reducing downstream IL-2 signaling. Strikingly, we demonstrate that IL-2 is essential for IL-10 production by Th17 cells, in contrast to its inhibitory effect on the production of IL-17A, and also for increasing the responsiveness to IL-10 through the upregulation of Il10rb expression.

Conclusion: Our studies reveal a novel, previously unappreciated, anti-inflammatory role for IL-2 in Th17 cell production of, and responsiveness to, IL-10. Furthermore, these data indicate that modulation of IL-2 signaling, be it directly or through glycosylation, may be exploited as a therapeutic target for the treatment of T cellmediated inflammatory diseases.

Disclosure of Interest: None declared. Methods: We analyzed peripheral blood mononuclear cells (PBMCs) from maternal and cord blood in patients with preterm labor (PTL) and healthy term controls and examined the cytokine profiles in the plasma of those samples. We also performed in utero fetal liver injections in pregnant females in order to address the fetal immune system activation in a mouse model.

: We found activation of cord blood myeloid and plasmacytoid dendritic cells, with concordant increases in inflammatory cytokines in preterm infants compared to controls. Preterm infants had higher levels of central memory cells with a Th1 phenotype, as well as increased expression of T cell activation genes compared to healthy controls. Importantly, fetal T cells proliferated robustly and produced IFN-k when stimulated with fetal antigen presenting cells and maternal antigens in patients with PTL, while such alloreactivity was low in healthy pregnancies. The levels of maternal microchimerism were increased among preterm infants, a possible source of priming. Finally, products of T cells immune activation, resulted in uterine contractility in vitro, and direct fetal liver injection of activated T cells in pregnant mice resulted in fetal resorption, a process that can be prevented by the injection of IFN-k KO and TNFa KO activated T cells.

Our findings indicate that the inflammatory environment in PTL results in activation of dendritic cells, a cascade that culminates in fetal T cell priming against maternal antigens and breakdown of maternal-fetal tolerance. Thus, aberrant fetal immune system activation should be considered in the pathogenesis of preterm birth and subsequent neonatal complications.

Disclosure of Interest: None declared. To induce EAE, mice were immunized with MOG 35-55 subcutaneously and scored as previously described [1] .

Results: HBCGM identified MINT alleles as an important effector of Th cell differentiation. This result was confirmed by the effect of a siRNA-mediated knockdown of MINT expression in CD4 T cells that diminished IFNk expression during Th1 differentiation. Analysis of MINT expression in different Th subsets revealed that MINT is most highly expressed in Th9 and Th17 cells; and CD4 T cells from MINT cKO mice exhibited defective Th9, Th17, and Treg differentiation, even though Notch1 and RBP-j expression were not affected in these cells. It was previously demonstrated that Notch/RBP-J signaling cooperates with the TGFb/SMAD pathway to induce IL9 expression and to regulate the autoimmune response in experimental autoimmune encephalomyelitis (EAE) [1] . In EAE, MINT cKO mice had reduced disease severity with a lower clinical score relative to control mice. There was also less CD4 + T cell infiltration in the spinal cords of MINT cKO mice, as well as a lower level of production of inflammatory cytokines. Since RBP-J and SMAD3 transactivate the Il9 promoter, it was possible that MINT could indirectly function through RBP-J or SMAD3 to regulate IL9 expression. However, ChIP assays showed that RBP-J and SMAD3 binding at the Il9 locus was intact in MINT KO Th9 cells.

Conclusion: MINT is a novel regulator of Th differentiation, which modulates IL9 expression via a novel RBP-J/SMAD independent mechanism. The ''innate-like" property of blood MAIT cells has been defined, where they respond to a range of cytokines and produce proinflammatory cytokines independent from the TCR signalling. We seek to understand whether gut MAIT cells, either in health or disease, possess the similar ''innate-like" property in response to cytokine triggering. Additionally we aim to dissect how these TCRdependent and TCR-independent signals may combine to induce activation of MAIT cells in the blood and the gut.

Methods: PBMCs were obtained from adults (whole blood leukocyte cones; NHS Blood and Transplant) following appropriate ethical review. They were stored in liquid nitrogen until required. Colonic lamina propria lymphocytes (LPLs) were obtained from either healthy adjacent resections of colon-rectal cancer patients, or from inflamed resections of patients with inflammatory bowel disease (IBD). Fresh gut cells were used for each experiment. Patients were approached for appropriate consent for their tissues to be used for scientific research. PBMCs and LPLs were then either stained for surface markers including CD3, CD4, CD8, CD161 and Va7.2 at resting stage, or stained intracellularly for the expression of IFN-k and TNF-a following overnight stimulation by TCR+/-cytokine triggering.

Results: Compared with blood T lymphocytes, those in the gut, either in health or disease have higher surface expression of CD161, notably in the CD4+ compartment, whereas the TCR-Va7.2 is less frequently expressed. Gut MAIT cells respond to cytokine stimulations in a similar way with blood MAIT cells, where IL-15 and TL1A synergise with a low dose of IL-12 and IL-18 and induce higher level of activation. In addition, the combinatorial trigger by the TCR and cytokine signalling potently activates MAIT cells from the blood and the gut (in both health and disease). However, gut MAIT cells show a slightly different functional profile in comparison to their blood counterparts, with CD8+ gut MAIT cells producing less IFN-k, and CD4+ cells more TNF-a.

Conclusion: Gut lymphocytes have higher surface expression of CD161 compared with healthy PBMCs. These gut CD161++/MAIT cells produce less IFN-k and more TNF-a in response to TCR+/cytokine triggering, suggesting that the gut environment (the cytokine milieu, the gut microbiota, etc) could have an impact on the phenotype and functionality of the gut CD161++/MAIT cells. Our study may be of translational value in developing novel therapies against IBD by targeting these CD161-expressing cells in the gut.

Disclosure of Interest: None declared. Introduction: Parasitic intestinal worms (helminths) often establish chronic infections within the host, in part due to their ability to actively modulate and/or down-regulate host immune responses and inflammation [1] . Paradoxically, it has also been proposed that molecules derived from helminths may be harnessed as novel immune-modulators to treat auto-immune diseases [2] . Here, we investigated whether products derived from the porcine helminth parasite Ascaris suum modulated inflammatory cytokine secretion in human dendritic cells (DCs).

Methods: A. suum body fluid (ABF) was harvested from adult worms collected from the intestine of infected pigs. Monocyte-derived human DCs were cultured with the TLR-agonist lipopolysaccharide (LPS) in either the presence or absence of ABF, and cytokine secretion measured in cell culture supernatants by ELISA. In addition, RNA was extracted from cells and microarray (Illumina HT-12 beadchip) and gene-set enrichment analysis (GSEA) was performed to elucidate the intracellular pathways modulated by A. suum. Selected genes that were shown to be differentially regulated by microarray were validated by qPCR and their corresponding protein levels further investigated by ELISA, flow cytometry and/or Western blotting.

Results: The response of DCs to LPS was profoundly affected by ABF. Secretion of the inflammatory cytokines IL-6, IL-12p70, IL-23 and TNF-a was strongly suppressed in ABF-treated DCs. Microarray analysis of ABF-treated DCs indicated a down-regulation of numerous genes encoding cytokines and chemokines, as well as molecules involved in intracellular inflammatory pathways and DC adhesion and migration. GSEA indicated that ABF treatment significant disrupted a number of pathways involved in inflammation and chemokine signalling. Notably, ABF strongly inhibited production of cyclooxygenase-2, abrogated secretion of CXCL1, and reduced expression of the alpha-1 adrenergic receptor ADRA1B. In contrast, expression of NRROS, a negative regulator of reactive oxygen production and inflammatory responses, was significantly enhanced.

Conclusion: The body fluid from the helminth parasite A. suum contains molecules that strongly inhibit inflammatory responses in human DCs. As aberrant inflammatory cytokine production from DCs is linked to autoimmunity and inflammatory disorders [3] , ABF may provide a source of immunomodulatory molecules with therapeutic potential. Our results also may shed light on the host-parasite interaction in Ascaris infections, and how A. suum and its human counterpart A. lumbricoides successfully colonise billions of pigs and people worldwide. Results: We first examined the expression of IL-3 receptor on the surface of CD4 + T cells in PBMCs. There was no IL-3Ra expression on peripheral blood CD4 + T cells, but its expression was detected after 48 h of stimulation with antiCD3/antiCD28. In the presence of IL-3, we found a significant increase in the differentiation of induced Tregs. Although there was a significant increase in the frequency of induced Tregs upon IL-3 treatment, but no difference was observed on the expression of CTLA-4 and TGF-b1 in both induced and natural Tregs. In a preliminary finding, it was found that IL-3 down regulated the percentage of Th17 cells generated from memory CD4 + T cells in the presence of Th17 polarizing factors.

The present study reveals that IL-3 could be a potential candidate in the treatment of inflammatory disorders like rheumatoid arthritis, where inflammation modulates Tregs/Th17 balance, resulting in the loss of immune tolerance. anti-IL-17A antibody only at the induction phase of EAE showed a similar attenuation of disease and reduction in Th17 responses as IL-17A -/mice, indicating a key role for IL-17A in the priming of autoreactive Th17 cells. Surprisingly, T cells from MOG-immunized IL-17A -/mice were capable of inducing disease following transfer into WT recipient mice, demonstrating that IL-17A production by autoreactive T cells is not essential for development of EAE. Previously, we have demonstrated that IL-17A induces IL-17A expression by T cells and also enhances TLR-induced IL-1b, IL-23 and IL-6 production by dendritic cells. Finally, expression of the Th17-promoting cytokines IL-1b, IL-23 and IL-6 was reduced in the lymph nodes of IL-17A -/mice compared with WT mice in the early stages of EAE, providing further evidence that IL-17A plays a role in promoting the development of Th17 cells.

Conclusion: The findings of this study suggest that in CNS autoimmunity IL-17A may function not primarily as an effector cytokine that promotes chemokine production and neutrophil recruitment, but as a T cell polarizing cytokine that directly and indirectly promotes the induction or expansion of pathogenic Th17 cells.

Disclosure of Interest: None declared. Introduction: The transcription factor interferon regulatory factor 5 (IRF5) is primarily expressed in human monocytes, B cells, and dendritic cells. Polymorphisms in the IRF5 gene have been significantly associated with susceptibility to the autoimmune disease systemic lupus erythematosus (SLE). In peripheral blood mononuclear cells (PBMC) from SLE patients, IRF5 expression is significantly elevated as compared to expression in healthy donor PBMC. The specific immune cell subset that IRF5 is (dys)functioning in to contribute to SLE pathogenesis is not known. SLE patients are characterized by increased levels of auto-reactive B cells and autoantibodies, and Irf5 -/mice were previously shown to have drastically reduced numbers of antibody secreting cells. Conclusions made from these mice, however, were compromised by the discovery of a secondary mutation in DOCK2. Important, multiple murine models of lupus show Irf5 -/mice are protected from pathogenic autoantibodies. To determine if IRF5 has similar function(s) in human primary B cells, we sought to characterize the role of IRF5 in plasmablast differentiation, antibody secretion, and B cell activation.

Methods: Knockdown of IRF5 was performed in primary human B cells using an Amaxa 4D nucleofector, followed by in vitro culture to assay differentiation of naïve B cells to plasmablasts. B cell receptor cross-linking antibody in combination with the TLR9 stimuli CpG-B was used to activate B cells. Identification of IRF5 transcriptional targets was done through chromatin immunoprecipitation combined with next generation sequencing. B cell proliferation was assessed through reactive dye dilution.

Results: Upon IRF5 knockdown in primary human B cells, a marked decrease in plasmablast differentiation was seen. Secreted levels of IgG1, IgG2, and IgG3 were severely decreased. Interestingly, upstream B cell activation pathways were also found to be decreased in B cells with the IRF5 knockdown. B cell proliferation was also found to be consistently decreased following IRF5 knockdown. ChIP-seq experiments identified IRF5 enrichment on the promoter regions of several plasmablast-associated genes as well as several genes known to impact B cell proliferation.

Conclusion: These findings demonstrate for the first time that IRF5 is a critical regulator of plasmablast differentiation. Furthermore, our work suggests that IRF5 knockdown impacts early stage B cell activation and proliferation. IRF5 ChIP-Seq results suggest that upon activation, IRF5 acts as a transcriptional regulator for a large group of genes which regulate both plasmablast differentiation and B cell function. Given that plasmablasts are over-represented in SLE, the conclusions of our work here demonstrate the therapeutic potential of IRF5 inhibition.

Disclosure of Interest: None declared. Introduction: Genetic variants of interferon regulatory factor 5 (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF5 regulates the expression of proinflammatory cytokines and type I interferons (IFNs) believed to be involved in the pathogenesis of SLE. The IRF5 risk genotype has been shown to associate with elevated IRF5 expression, high serum IFN-a activity and autoantibody production in SLE patients. However, elevated IRF5 expression may have been a result of disease-associated factors, such as elevated type I IFNs, rather than genotype. Furthermore, previous data from our lab has shown that IRF5 is constitutively activated in SLE immune cells. To avoid interference from the disease, we took advantage of the Genotype And Phenotype (GAP) Registry at the Feinstein Institute for Medical Research and examined IRF5 expression and function in healthy donors carrying the homozygous risk or non-risk IRF5 genotype, and intermediate haplotypes. The aim of this study was to identify the functional contribution of the IRF5 risk genotype to SLE onset.

Methods: IRF5 expression and cellular localization were examined in B cell subsets from peripheral blood mononuclear cells (PBMC) of healthy donors carrying risk and non-risk alleles at rs2004640, rs10954213, rs10488631 and the CGGGG promoter insertion/deletion using imaging flow cytometry. IRF5 activation was determined after ex vivo stimulation of PBMCs with R848. B cell subsets were gated on as follows: plasmablasts were characterized by high CD38 and low CD24 surface marker expression; naïve B cells by low CD38 and IgD expression; transitional B cells by high CD38 and high CD24 expression. The IFN gene signature was determined by reporter assay and anti-nuclear antibodies (ANA) detected by Hep2-ANA. Differences in expression, activation, subset profiles, gene signatures and ANA were determined by statistical analysis.

: Surprisingly, neither IRF5 expression nor activation showed any significant difference between risk and non-risk carriers. Furthermore, the percentage of naïve B cells and transitional B cells did not show significant differences. However, the percentage of plasmablasts was significantly higher in homozygous risk carriers as compared to non-risk carriers.

Conclusion: Results from the GAP Registry indicate alternate function(s) for IRF5 that is not directly related to expression or activation. Given that plasmablasts are over-represented in SLE, these findings support an early effect of the IRF5 risk genotype on SLE disease onset. Furthermore, the fact that autoantibodies occur in SLE patient blood prior to disease onset, data presented here suggest a contribution of the IRF5 risk genotype to multiple aspects of disease pathogenesis. In conclusion, this is the first finding of an IRF5 genotype-phenotype effect on SLE susceptibility in healthy donors. Introduction: Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunoregulatory enzyme which catalytic and non-catalytic effects are involved in the regulation of immunity and autoimmune diseases. Cytokines are important modulator of the expression of IDO1: interferon-c upregulates IDO1 in dendritic cells while interleukin 6 (IL-6) promotes proteasomal degradation of the enzyme. Current data show that approximately 25% of patients with type 1 diabetes (T1D) exhibit a post-translational (and not genetic) defect of IDO1 in a subset of PBMC which is characterized by a IDO1 high IDO1 low IL-6 high phenotype. Interestingly, this phenotype can be completely corrected by co-incubation of PBMCs with tocilizumab, a licensed IL-6 receptor blocker. Therefore, data indicate the existence of a subset of individuals with T1D who may gain clinical benefit in restoring IDO1 immunoregulatory mechanisms by treatment with tocilizumab.

Methods: We aim to verify whether the profile identified from PBMCs may also be demonstrated in pancreata by using tissue specimens obtainable from the network of pancreatic organ donors (nPOD). Pancreatic sections from T1D, T2D patients and non-diabetic were analysed for IDO1 and IL-6 expression and localization by mmunohistochemistry.

Results: Results show that IL-6 can be produced by alpha and beta cells in diabetics and non-diabetic donors. Compared to islets from non-diabetic controls, expression levels of IL-6 tend to be higher in T2D donors and lower in double autoantibody positive donors. On the other hand, IDO is predominantly produced by beta cells. Interestingly, IDO is not expressed in insulin deficient islet from T1D donors whereas it is still expressed in insulin containing islets. Introduction: Systemic lupus erythematosus (SLE) is often thought of as a disease driven by molecular pathways related to autoreactive B cells and anti-nuclear antibodies. However, therapeutics targeting of B cells, including BAFF blockade, have been only partially effective in SLE. NF-jB Inducing Kinase (NIK) mediates BAFF signaling and also noncanonical NF-jB signaling downstream of non-BAFF/BR3 TNFR family members. We thus hypothesized that a NIK inhibitor might be more efficacious than BAFF blockade in lupus.

Methods: IFNa-AdV treated NZB/W F1 lupus prone mice were used. Gene expression was monitored by RNAseq, ELISA and Fluidigm.

Results: To address this, we generated NIK small molecule inhibitors that are highly selective and potent. In cellular assays, these molecules inhibit noncanonical NF-jB signaling downstream of multiple TNFRSF family member signaling, including CD40, OX40, and TWEAK. In order to differentiate the effects of NIK inhibition and BAFF blockade in vivo, we compared NIK inhibition with BAFF blockade in the context of IFNa-accelerated NZB/W F1 lupus prone mice. As predicted, NIK inhibition recapitulated the pharmacological effects of BAFF blockade. Furthermore, we were also able to demonstrate a role for NIK in OX40 driven T cell costimulation in vitro, and T effector cell number in IFNa-NZB/W F1 mice. Finally, TWEAK inducible genes in renal tubulointerstitial cells were highly NIK dependent, and their expression correlated with disease associated pro-inflammatory genes in IFNa-NZB/W F1 lupus prone mice. Indeed, expression of these genes was suppressed in NIK SMI treated, but not anti-BAFF treated, IFNa-NZB/W F1 lupus mice.

Conclusion: Collectively, our data suggest that NIK inhibition affects multiple disease-relevant pathways and will therefore have superior efficacy compared to BAFF inhibition in SLE. Introduction: ABCF1 is an ABC transporter family protein that has been shown to regulate innate immune response and is a risk gene for autoimmune pancreatitis. Unlike other members of ABC transporter family, ABCF1 lacks trans-membrane domains and is thought to function in translation initiation through an interaction with eukaryotic translation initiation factor 2 (eIF2).

Methods: In order to study ABCF1 in development and disease, we used a single gene trap insertion in the ABCF1 gene in embryonic stem cells (ES cells) that allowed lineage tracing of the endogenous ABCF1 promoter by following the expression of a b-galactosidase reporter gene. From the ES cells, heterozygous mice (ABCF1+/-) were produced. No live born ABCF1-/-progeny were ever generated and the lethality was not mouse strain specific.

Results: ABCF1-/-mice were found to be embryonic lethal at 3.5 days post coitum (dpc), while ABCF1+/-mice appeared developmentally normal. ABCF1+/-mice were fertile and showed no significant differences in their anatomy when compared to their wild type littermates. The ABCF1 promoter was found to be active in all tissues in adult mice, with varying levels in different cell types. Furthermore, we observed high promoter activity in the blastocysts and embryos.

Conclusion: Overall, ABCF1 expression in embryos is required for development and its expression in adults was highly correlated with actively proliferating and differentiating cell types. Introduction: IL-17A drives the pathogenesis of autoimmune disease but promotes immunity to fungal infections. To date, the IL-17 signaling pathway is still not well understood. IL-17A binding to the IL-17 receptor (IL-17R) initiates recruitment of adaptor proteins such Act1 and TRAF6, leading to activation of the NF-jB and MAPK pathways. These pathways are negatively regulated by the A20/ TNFAIP3 ubiquitin editing protein. The TNIP1 gene is associated with autoimmune diseases in GWAS studies. Its gene product, ABIN1, is an A20-associated factor. Accordingly, we hypothesized that ABIN1 might function as an inhibitor in the IL-17 signaling pathway.

Methods: Using ST2 cells or murine fibroblasts, we performed siRNA knockdown, mRNA extraction for RT-PCR analysis, or protein extraction for western blot analysis, and analysis of the supernatant by ELISA. Additionally, HEK293T cells were used to analyze proteinprotein interaction by overexpression of proteins of interest, immunoprecipitation and WB. Promoter activation was measured by luciferase assays.

Results: First, knockdown of ABIN1 in the murine stromal cell line ST2 led to enhanced expression of IL-17-dependent genes such as lipocalin 2 (Lcn2). Reconstitution of ABIN1-deficient fibroblasts with the ABIN1 gene normalized Lcn2 expression. Surprisingly, A20 was dispensable for ABIN1-mediated inhibition, as ABIN-1 knockdown enhanced IL-17-dependent signals in A20-deficient cell lines. Similar to A20, TRAF6 deubiquitination was enhanced in the presence of ABIN1. Since many negative regulatory signals are part of ligandinducible feedback loops, we investigated whether expression of ABIN1 was regulated by IL-17R signaling. Interestingly, while mRNA expression of ABIN1 was upregulated following IL-17A stimulation of cells, ABIN1 protein levels decreased.

Conclusion: Collectively, these data indicate that ABIN1 inhibits the IL-17R signaling pathway independently of A20 through a feedback loop that differentially impacts ABIN1 mRNA and protein. Methods: Here, we have evaluated the proinflammatory role of OSM in a mouse model of skin inflammation induced by intradermal injection of murine OSM-encoding adenovirus (AdOSM) in comparison with AdIL-6. We then adressed its contribution in the model of imiquimod-induced psoriasis-like skin inflammation.

Results: Firstly, we showed that OSM potently regulates the expression of genes involved in skin inflammation and epidermal differentiation in murine primary keratinocytes. In vivo, intradermal injection of AdOSM in mouse ears led to skin inflammation with epidermal thickening and keratinocyte proliferation, while minimal effect was observed after AdIL-6 injection. OSM overexpression in the skin increased the expression of antimicrobial peptides, chemokines and Th1/Th2 cytokines, in correlation with neutrophil and macrophage infiltration. In contrast, OSM downregulated the expression of epidermal differentiation genes, such as cytokeratin-10 or filaggrin. However, OSM expression was not required in the murine model of psoriasis induced by topical application of imiquimod, as demonstrated by the inflammatory phenotype of OSM-deficient mice or wild-type mice treated with anti-OSM antibodies.

Conclusion: Collectively, these results support the proinflammatory role of OSM when it is overexpressed in the skin, but its expression is not necessary for the development of psoriasis-like skin inflammation after topical application of imiquimod. Methods: We constructed experimental autoimmune encephalomyelitis (EAE) model to detect whether CCDC134 expression is correlated with autoimmune disease. In EAE model, CCDC134 transgenic mice and rhCCDC134 protein were used. Moreover, the disease symptoms were observed and histopathology changes were analyzed. Inflammation reaction was detected by FACS, ELISA and real-time PCR.

Results: In this study, we found that CCDC134 level in mouse sera was increased in the onset and peak stage of EAE. Moreover, we demonstrated that CCDC134 was efficacious in a model of EAE that mirrors chronic progressive multiple sclerosis. A short-term systemic treatment with recombinant CCDC134 reduced clinical severity and incidence of EAE. In addition, we used CCDC134transgenic mice model and found that the onset and severity of EAE, the appearance of inflammatory infiltrates in spinal cord and the subsequent demyelination and axonal damage were significantly alleviated in CCDC134-transgenic mice compared with control mice. We further sought to elucidate the basis of CCDC134 protective effect on EAE by characterizing the T effector/regulatory responses. And the presence/activation of encephalitogenic Th1/Th17 cells and several inflammatory mediators in peripheral lymphoid organs and central nervous system were down-regulated in CCDC134-transgenic mice.

Conclusion: These findings indicate that CCDC134 plays a pivotal role in the pathogenesis of EAE and provide insights into the role of recombinant CCDC134 as a unique therapeutic agent for the treatment of autoimmune diseases.

Disclosure of Interest: None declared. Introduction: S100A8 functions as an essential factor in inflammatory response and it induces the migration and adhesion of neutrophils. S100A8 also triggers the differentiation of myeloid cells. In this study, we investigated the contribution of S100A8 to differentiation and chemotactic activity in the human eosinophilic leukemia cell line EoL-1.

We performed Proliferation assay, Chemotaxis assay and Western blotting.

Results: S100A8 induces morphological change such as production of vacuoles, indicating that it induced the differentiation of EoL-1 cells. S100A8 induced the migration of EoL-1 cells and the migration was inhibited by rottlerin, an inhibitor of protein kinase C delta (PKCä), PD98059, an inhibitor of ERK and SB202190, an inhibitor of p38 MAPK. S100A8 induced phosphorylation PKCä, ERK and p38 MAPK in a time-dependent manner.

Conclusion: S100A8 induces the differentiation of EoL-1 cells. The chemotaxis mechanism due to S100A8 is involved in PKCä, ERK and p38 MAPK pathway. This finding contributes to an understanding of S100A8 in eosinophil biology and to the development of novel therapies for the treatment allergy. Introduction: Allergic asthma is characterized by bronchoconstriction, allergen-specific IgE and bronchial inflammation. S100A8 is an important protein in the pathogenesis of allergic asthma. In this study, we examined that the pathogenic mechanism of asthma is associated with interaction of cytokine release in bronchial epithelial cells with constitutive apoptosis of neutrophils.

Methods: we performed ELISA assay, Western blot and FACS.

Results: S100A8 increased the secretion of IL-6, IL-8, and MCP-1 in a time and dose dependent manners. This secretion was suppressed by TLR4i, an inhibitor of TLR4, LY294002, an inhibitor of PI3K, AKTi, an inhibitor of Akt, PD98059, an inhibitor of ERK, SB202190, an inhibitor of p38 MAPK, and BAY-11-7085, an inhibitor of NF-jB. S100A8 induced both ERK and p38 MAPK activations in a time-dependent manner. Supernatants collected from bronchial epithelial cells after S100A8 treatment suppressed the apoptosis of neutrophils.

Conclusion: These findings improve our understanding of the role of S100A8 in interaction between bronchial epithelial cells and neutrophils in allergic asthma and will enable elucidation of asthma pathogenesis. Introduction: House dust mite (HDM) is an essential allergen in allergic diseases such as allergic rhinitis and asthma. The pathogenic mechanism of allergy is associated with cytokine release of lymphocytes and constitutive apoptosis of neutrophils. In this study, we examined whether HDM induces cytokine release of lymphocytes and if the secretion of cytokines is involved in modulation of neutrophil apoptosis.

Methods: We performed ELISA, Western blotting, NF-jB activity assay, and apoptosis assay.

Results: In normal and allergic subjects, extract of Dermatophagoides pteronissinus (DP) increased IL-6, IL-8, MCP-1, and GM-CSF secretion in a time-dependent manner. This secretion was suppressed by PAR2i, an inhibitor of PAR2, in a dose-dependent manner, as well as by LY294002, an inhibitor of PI3K, AKTi, an inhibitor of Akt, PD98059, an inhibitor of ERK, and BAY-11-7085, and an inhibitor of NF-jB. ERK activation was suppressed by PAR2i, LY294002 and AKTi, and NF-jB activation was blocked by PAR2i, LY294002, AKTi, and PD98059. Supernatants collected from normal and allergic neutrophils after DP treatment inhibited the apoptosis of normal and allergic neutrophils through suppression of caspase 9 and caspase 3 cleavage.

Conclusion: DP induces the release of cytokines through the PAR2/ PI3K/Akt/ERK/NF-jB pathway, which has anti-apoptotic effects on neutrophils of normal and allergic subjects. These results will facilitate elucidation of the pathogenic mechanism of allergic diseases. Methods: Neonatal pDC-diphtheria toxin receptor (pDC-DTR) knock-in mice and wild-type (WT) littermate controls were infected with low-dose pneumonia virus of mice (PVM, 10 pfu/i.n.), and challenged with 100 pfu 6 weeks later. DT (3 ng/g body weight/i.p.) was administered -1, +1, +3, and +5 days post infection to WT and pDC-DTR neonatal mice and pathologic features of bronchiolitis or asthma assessed. Tregs (3 Â 10 4 ) were isolated from virus-challenged pDC-DTR/FoxP3-RFP + or WT/FoxP3-RFP + donors and transferred to pDC-DTR or WT recipients.

Results: Temporary pDC depletion reduced antiviral cytokine production, increased viral load and induced severe bronchiolitis (airway epithelial cell sloughing, neutrophilic inflammation). This phenotype was associated with significantly lower levels of IL-10 and a failure to expand neuropilin-1+ Tregs. Ligation of neuropilin-1 was necessary for pDC-mediated proliferation of Tregs ex vivo. Viral challenge of pDC-DTR but not WT mice induced the hallmark features of asthma, including airway hyperreactivity and type-2 inflammation, and was associated with lower numbers of antigenexperienced Tregs. Adoptive transfer of antigen-experienced WT Tregs during primary infection or prior to viral challenge of pDCdepleted mice prevented the development of both severe bronchiolitis and postviral asthma. In contrast, Tregs from 'asthmatic' pDC-DTR mice were insufficient to suppress virus-induced asthma in pDC-DTR recipient mice. Introduction: Frequent viral lower respiratory infections (vLRI) and allergic sensitization in early life are independent risk factors for asthma onset, yet together significantly increase the development of persistent asthma.

We developed an experimental model of asthma to investigate this synergy. Neonatal BALB/c mice were inoculated with low dose pneumonia virus of mouse (PVM; 1 pfu) then exposed to low dose (1 lg) cockroach antigen or vehicle control at 3 days post infection (dpi). Some mice were re-infected 6 weeks later and exposed to weekly doses of allergen.

Results: Virus and allergen co-exposure was critical in both early and later life for disease onset and progression, including airway hyperreactivity, airway remodelling and type 2 inflammation. Allergen exposure during primary vLRI increased IL-33 release and impaired antiviral cytokine production, leading to increased epithelial viral burden, Th2-type inflammation and airway smooth muscle growth. Neutralisation of IL-33 in early life prevented type 2 inflammation, airway remodelling and reversed the dampened interferon response mediated by cockroach antigen. Substitution of allergen with exogenous IL-33 attenuated antiviral cytokines, elevated viral load and promoted airway remodelling. Mechanistically, we found that IL-33 degraded IRAK1 to dampen type I IFN production by plasmacytoid DC.

Conclusion: In summary, we identify a novel role for IL-33 in regulating antiviral immunity and as a target to attenuate the synergistic interplay between two important environmental insults in the onset and progression of asthma.

Disclosure of Interest: None declared. 

Introduction: Der p 2, which is a major allergen of house dust mite, plays an important role in the pathogenesis of allergic disease. There is controversy regarding whether Der p 2 binds to Toll-like receptor 4 (TLR4), and its inflammatory effect has not yet been elucidated. In the current study, we examined the interaction of Der p 2 with TLR4 and the effect of Der p 2 on cytokine release of THP-1 cells and lymphocytes. Among house dust mite extracts, recombinant TLR4 protein interacted with Der p 2.

Methods: we performed MALDI-TOF/TOF MS analysis, ELIS Assay and Apoptosis assay.

The overall structure of Der p 2 is characteristic of the immunoglobulin superfamily and contains ten b-strands, forming a b-cup fold with two anti-parallel b-sheets, and a short 310 helix. The two sheets can be separated, further allowing the formation of a large internal pocket, which is narrow and suitable for binding large flat molecules such as lipid-like molecules. Der p 2 caused increased secretion of IL-6, IL-8, and MCP-1, which are neutrophil survival factors, in human monocytic THP-1 cells in a time-dependent manner. Der p 2 also induces the release of cytokines in normal and allergic lymphocytes. Supernatant after treatment with Der p 2 inhibited neutrophil apoptosis. In coculture of lymphocytes with neutrophils, Der p 2 inhibited spontaneous apoptosis of allergic neutrophils.

Conclusion: In summary, Der p 2 binds to TLR4 and induces an inflammatory response such as cytokine secretion in immune cells. These findings may enable elucidation of allergy pathogenesis by specific allergen of house dust mite. Introduction: Necrotic cell death triggers inflammation, whereas apoptosis contributes to its resolution. Interleukin-1 (IL-1) family cytokines are key players in this interaction and are produced by necrotic cells to induce sterile inflammation. Release of IL-1 family proteins from apoptotic cells to regulate inflammation has not been described. The novel cytokine IL-38 shares homology with IL-1 family receptor antagonists and was therefore proposed as a negative regulator of IL-1 family receptor signaling.

Methods: IL-38 concentrations were measured by ELISA. Apoptotictumor cell conditioned medium (ACM) was used to stimulate human primary immune cells. The response to ACM was determined as cytokine release (Cytometric Bead Array) as well as transcription factor activity (Luciferase reporter assays). N-terminal processing of IL-38 was determined by mass spectrometry. Binding of IL-38 to its putative receptors was determined by receptor binding assays. The pathophysiological function of IL-38 was analyzed using the imiquimod (IMQ)-induced psoriasis mouse model in IL-38-deficient animals compared to WT controls. Statistical analysis was performed using ANOVA with Bonferroni's correction.

We show that IL-38 is produced selectively by human apoptotic cells to limit inflammation. Depletion of IL-38 in apoptotic cells provoked enhanced IL-6 and IL-8 levels and AP1 activation in co-cultured human primary macrophages, subsequently inducing IL-17-producing T cell activation. IL-38 was N-terminally processed in apoptotic cells to generate a mature cytokine with distinct properties. Both full-length and truncated IL-38 bound to X-linked interleukin-1 receptor accessory protein-like 1 (IL1RAPL1). However, we show higher affinity binding of mature IL-38. Likewise, we confirmed the previously reported low-affinity binding of the IL-38 precursor to IL1R1 and show an increased affinity of mature IL-38 to this receptor. Functionally, the IL-38 precursor induced an increase in IL-6 production by human macrophages, whereas truncated IL-38 reduced IL-6 production by attenuating the JNK/AP1 pathway downstream of IL1RAPL1. Ongoing studies show selective secretion of IL-38 from dying cells challenged with tolerogenic chemotherapy, but not with immunogenic chemotherapy. Moreover, strengthening the role of IL-38 as a tolerogenic factor, IL-38-deficient mice subjected to IMQ-induced psoriasis show an increased IL-17mediated immune response and a strong delay in the restoration of skin architecture.

We identified a mechanism of apoptotic cell-dependent immune regulation requiring IL-38 processing and secretion. IL-38 limits cytokine production in macrophages antagonizing the IL1-RAPL1/JNK/AP1 pathway and subsequently preserving a low T cell IL-17 production. We propose apoptotic cell-derived IL-38 as the counter-regulatory equivalent of necrotic cell-derived alarmins of the IL-1 family, which might be relevant in resolution of inflammation, autoimmunity, and cancer.

Disclosure of Interest: None declared. Introduction: HTLV-1 was the first human retrovirus to be discovered, but it is still enigmatic how it can cause both malignant and inflammatory disease. In contrast to HIV, most HTLV-1-infected carriers remain asymptomatic lifelong, while a minority progresses to either fatal hematologic malignancy (Adult T-cell Leukemia, ATL) or neuroinflammation (HTLV-1-associated myelopathy, HAM). A functional FAS -670 promoter polymorphism, corresponding to a STAT1 binding site, has been associated to increased ATL and HAM susceptibility. We have recently shown that the same Fas polymorphism links IFN-dependent apoptosis and circulating T stem cell memory (Tscm CD3 + CD45RA + CD45RO -CCR7 + Fas hi ) levels. Moreover, Fas has been identified as part of an IFN-inducible gene signature in HAM, but its role in pathogenesis remains unclear.

Methods: We comprehensively explored Fas expression (protein and mRNA) and function in lymphocyte activation, apoptosis, proliferation and transcriptome, using flow cytometry, [3H]-thymidine incorporation and microarray analysis in PBMC from a total of 39 HAM patients, 40 asymptomatic HTLV-1-infected individuals (AC) and 58 HTLV-1-uninfected healthy controls.

Results: CD4 or CD8 Tscm (CD3 + CD45RA + CD45RO -CCR7 + Fas hi ) levels did not differ between the three clinical groups. However, Fas surface expression followed a two-step increase, first upon HTLV-1 infection and second upon HAM/TSP disease progression (p < 0.001).

In HAM patients, Fas levels correlated positively to lymphocyte activation markers (HLA-DR/CD86, p < 0.05), but negatively to age of disease onset (p = 0.019, r = -0.69), suggesting higher levels of Fas predispose to earlier, more aggressive disease. The most surprising finding of this study is a selective defect in Fas-mediated apoptosis in HAM. First, ex vivo and in vitro Fas surface expression correlated negatively to in vitro apoptosis (p = 0.012, r = -0.63). Second, by fluorescence microscopy we document that Fas lo but not Fas hi cells preferentially undergo apoptosis in vitro. Conversely, in vitro treatment of PBMCs with agonist anti-Fas mAb, but not antagonist anti-Fas mAb, was able to trigger apoptosis and restore the selective defect in HAM patients, indicating that the receptor is functional but skewed towards other biological pathways, e.g. proliferation. Indeed, Fas expression was positively correlated (p = 0.018, r = 0.62) to lymphoproliferation upon in vitro culture, but not to proviral load or viral protein levels. A systems analysis of HAM in vitro and ex vivo gene expression showed that triggering Fas receptor induces a specific gene set with a central role for NFkB pro-survival signaling, and confirmed our hypothesis at the transcriptomic level: transcripts significantly correlated to FAS mRNA levels are enriched in proliferative but not apoptotic pathways.

Conclusion: Our comprehensive approach reveals: (1) a two-step increase in Fas hi , but not Tscm levels upon HTLV-1 infection and disease progression; (2) defective Fas-mediated apoptosis, linked to early age of onset as a possible factor in HAM pathogenesis; and (3) Introduction: Proteolytic processing of cytokine receptors is an important element that regulates their signalling capacity. It does not only control the amount of the receptors on the cell surface, but also creates soluble receptors with distinct biological functions. Interleukin (IL)-11 is a member of the IL-6 family which has been initially described to have anti-inflammatory properties. However, recent studies revealed that overshooting IL-11 activity is involved in inflammation and the progression of epithelial cancers. IL-11 binds to the membrane-bound IL-11 receptor (IL-11R), and this complex can then recruit two molecules of the b-receptor glycoprotein (gp)130, which predominantly leads to the activation of the JAK/STAT pathway. We analysed whether the IL-11R is a substrate for proteases and whether proteolysis might be involved in IL-11 signalling.

We activated or inhibited proteases in cells and analysed resulting fragments of the IL-11R as well as IL-11R cell surface expression. Furthermore, we used chimeric receptors of the IL-11R and IL-6R as well as receptor mutants to determine which structural traits are responsible for proteolysis. The biological activity of the soluble IL-11R (sIL-11R) was evaluated with the help of cytokinedependent Ba/F3-gp130 cells. Additionally, we investigated whether sIL-11R is present in human blood.

Results: We found that the IL-11R is subject to proteolysis and could detect sIL-11R in serum of healthy humans in concentrations around 400 pg/ml. Interestingly, the metalloprotease ADAM10, but not its close relative ADAM17, is able to release the soluble IL-11R ectodomain. We found that a small part of the stalk region is responsible for protease specificity. Furthermore, we show that a single amino acid mutation within the stalk is sufficient to generate an ADAM10-resistant IL-11R variant. Additionally, after ectodomain shedding, the IL-11R membrane stub is further processed by the intramembrane protease gamma-secretase.

The released sIL-11R is able to bind IL-11 and the resulting IL-11/sIL-11R complex can activate the signal transducer and activator of transcription 3 (STAT3) pathway via gp130 in cells that do not express the membrane-bound IL-11R. This trans-signalling pathway can be inhibited by the anti-inflammatory designer protein sgp130Fc.

Conclusion: Proteases regulate IL-11 signalling via the cell surface expression of the IL-11R and the generation of biologically active sIL-11R. The newly identified IL-11 trans-signalling pathway widens the number of cells that can be activated by IL-11. Sgp130Fc, which has previously been shown to inhibit IL-6 trans-signalling, also inhibits IL-11 trans-signalling, which opens the possibility that the beneficial effects of sgp130Fc in vivo could at least in part be due to blockade of IL-11 trans-signalling.

Disclosure of Interest: None declared.

K. Ozato * PGD, NICHD, National Institutes of Health, Bethesda, United States Introduction: We have been studying BRD4, a BET family of bromodomain protein that binds to acetylated core histones. Through the interaction with P-TEFb, BRD4 regulates transcription of numerous genes and is thought to influence epigenetic states of many cells. Recent development of small molecule inhibitors (JQ1, I-BET as first generation drugs) that impede histone-bromodomain interaction demonstrated that BRD4 and other BET proteins are critical for cell proliferation and immune responses. These inhibitors hinder growth of various leukemia, lymphoma, and ameliorate inflammatory and autoimmune diseases. Thus these inhibitors offer a new ''epigenetic" therapy, and are being tested for a wide range of cancer and chronic diseases. However, due to uncertainty in specificity and metabolism of these drugs, the role of BRD4 in innate immunity inflammatory responses is not fully understood.

We have constructed Brd4 conditional knockout mice and investigated the role of BRD4 in cytokine-stimulated macrophages and inflammatory and autoimmune conditions.

Results: RNA-seq data indicate that BRD4 regulates a highly selective set of cytokines, chemokines and inflammatory genes in macrophages and T cells, in addition to self-renewal of hematopoietic stem cell. ChIP-seq data showing global distribution of BRD4 in enhancers and super-enhancers, highlighting its role in shaping the chromatin landscape in macrophages.

Conclusion: Our data support the view that BRD4 affects epigenetic states of chromatin, and is a potentially promising target of chronic diseases. Introduction: Immunotherapy has considerable potential to improve disease outcome and survival of patients with cancer, as highlighted by recent approvals of checkpoint inhibitors for the treatment of melanoma, lung and renal cancer. Immune cells, such as T cells, can target and kill tumour cells. However, tumours have a number of mechanisms for evading or suppressing immune responses, such as secretion of anti-inflammatory cytokines, recruitment of suppressive cell types and expression of immune checkpoint ligands, and these are obstacles to the development of successful cancer immunotherapies. The aim of this study was to characterise immunosuppressive mechanisms elicited by tumour cells and to utilize this information to develop a cancer vaccine combination that blocks suppressive and enhances effector anti-tumour immune responses.

Methods: Balb/c mice were injected with CT26 tumour cells and tumour-infiltrating lymphocytes were analysed by flow cytometry. For the vaccine experiments, mice were immunized with heatshocked and irradiated tumour cells and the TLR7/8 agonist R848 on days 3, 10 and 17 post tumour induction and an anti-PD-1 antibody was injected one day prior to immunization.

Results: During the development of CT26 tumours in mice, we found an inverse correlation between the Th1-type cytokine production and the expression of co-inhibitory receptors, the so-called immune checkpoints on T cells. In tumour draining lymph nodes, T cells expressed low levels of the checkpoints PD-1 and LAG-3 and secreted high concentrations of TNF and IFNk. In contrast, tumourinfiltrating T cells expressed high PD-1 and LAG-3 and secreted low concentrations of TNF and IFNk, suggesting that co-inhibitory receptors negatively regulated T cell effector function. Tumour cells secrete TGF-b which promotes induction of Treg cell and creates a suppressive tumour microenvironment. Blocking PD-1 in vitro reversed the suppressive effects of TGF-b on T cells, resulting in enhanced T cell proliferation, IFNk secretion and cytotoxic activity. Moreover, blocking PD-1 reduced TGF-b-induced Treg conversion, possibly through enhanced secretion of IFNk.

Using the CT26 tumour model, we found that therapeutic administration of an anti-PD-1 antibody alone had little effect on tumour growth. In contrast, treatment of mice with anti-PD-1 one day prior to therapeutic immunization with the cancer vaccine led to the eradication of established tumours and significantly prolonged survival. Moreover, spleen cells from mice immunized with the vaccine and anti-PD-1 induced higher levels of CT26 cell death ex vivo and secreted significantly higher levels of IFNk compared with cells from mice immunized with the vaccine alone.

Conclusion: Our findings demonstrate that the PD-1/PD-L1 axis is a major control mechanism that dampens anti-tumour T cell responses and blocking this axis can significantly enhance effector T cell responses and the efficacy of a therapeutic vaccine in a murine cancer model. Introduction: Cardiovascular diseases (CVDs) including atherosclerosis are disorders of the heart and blood vessels. Key factors contributing to early stages of CVDs include the pro-inflammatory cytokines: Interferon (IFN)a, IFNk, Interleukin (IL)-6 and Toll-like receptor 4 (TLR4) stimuli. Together, they trigger activation of members of the Signal Transducer and Activator of Transcription (STAT) and Interferon Regulatory Factor (IRF) families. In particular STAT1, 2, 3, IRF1 and IRF8 have recently been recognized as prominent modulators of inflammation, especially in immune and vascular cells during atherosclerosis. Using RNAseq, we identified a specific set of STAT-and IRF-dependent pro-atherogenic genes that were synergistically affected by interactions between IFNk and LPS in primary aortic VSMCs, namely: multiple chemokines, adhesion molecules, antiviral and antibacterial response genes. Based on this, STATs and IRFs represent interesting therapeutic targets and their targeted inhibition could be a promising novel treatment strategy in CVDs.

Methods: In our research we combined comparative in silico docking of STAT-SH2 models with an in vitro multiple STAT activation inhibition assay. STATs models were generated using homology procedure followed by docking of STAT3 inhibitors: STATTIC and STX-0119 to the selected binding cavities in STAT-SH2 domain of all human STATs. Subsequently, we have performed the following in vitro experiments: Western Blot analysis to verify the specificity of tested compounds towards STAT3 and gene expression analyses by qPCR to show inhibition of expression of STAT and IRF specific genes induced by IFNk and LPS. Moreover, we carried out scratch assay to check proliferation and migration of human microvascular endothelial cells in presence of tested compounds and IFNs.

Results: Due to its importance not only in inflammation but also in cancer development, searches for STAT3-targeting compounds are numerous, and yielded many synthetic small molecules. From the most potent known STAT3-specific inhibitors we chose STATTIC and STX-0119 to increase our understanding of the molecular basis of STAT protein and small compound inhibitor interactions. For this purpose, we generated new 3D structure models for all human STATs. Subsequently, a comparative in silico docking strategy was applied which provided evidence suggesting STAT-SH2 cross-binding specificity of STATTIC and STX-0119. This was confirmed in HMECs in vitro, in which STATTIC and STX-0119 inhibited IFNa-induced phosphorylation not only of STAT3 but also of STAT1 and 2 at varying concentrations.

In a second set of experiments, we tested the effect of STATTIC and STX-0119 on the expression of above mentioned STAT and IRF dependent genes in response to IFNk and LPS in HMECs. Strikingly, both compounds completely inhibited CXCL9, CXCL10, CCL5, IFIT2, OAS2, SOCS3, and IRF1 at 6.25 ìM for STX-0119 and 2.5 ìM for STATTIC pre-treated for 24 h and 8 h respectively. In addition we observed that STATTIC and STX-0119 were able to inhibit proliferation and migration of HMECs in response to IFNs and LPS in a similar concentration range.

Conclusion: Collectively, our data prove that STATTIC and STX-0119 simultaneously block STAT1, 2 and 3 activity and pro-atherogenic STAT-and IRF-target gene expression, and may offer a promising avenue for treatment of CVDs. Introduction: Nucleotide-binding oligomerization domain 2 (Nod2) is a cytosolic sensor for bacterial peptidoglycan-derived muramyl dipeptide and plays important roles in the host defense against several bacterial and viral infections. Acinetobacter baumannii is a multi-drug resistant gram-negative bacteria and its infection is one of major causes of mortality in intensive care unit. Although Nod2 is known to mediate innate immunity against Acinetobacter baumannii in airway epithelial cells, its role in vivo against pulmonary infection of A. baumannii remains unknown. In this study, we sought to define the role for Nod2 in host defenses against pulmonary infection of A. baumannii.

Methods: Wild-type (WT), Nod2-, and Rip2-deficiency mice were infected intranasally with A. baumannii (3'10 7 CFU) and bacterial load in the bronchoalveolar lavage (BAL) or the lungs was determined at 6 h, 1 and 3 d after infection. Immune cell recruitment in the BAL fluid was also determined by cytospin. For in vitro study, alveolar macrophages were isolated and infected with A. baumannii for 24 h. Cytokine and chemokine levels in the BAL fluid and cell culture supernatants were analyzed using ELISA.

Results: As compared with WT mice, Nod2-deficient mice showed significantly higher pulmonary bacterial load. Nod2 deficiency did not influence immune cells recruitments into the BAL fluid. The level of IL-6, TNF-a, IL-1b, CXCL1, and CXCL2 was higher in the BAL fluid of Nod2-deficient mice, whereas IL-10 was lower, as compared with that of WT mice. In vitro assay revealed that the production of IL-6, TNF-a, CCL2, and IL-10 was impaired in Nod2-deficient alveolar macrophages. Finally, we showed that mice deficient with Rip2, an adaptor molecule for Nod2, exhibited impaired bacterial clearance in the lungs.

Conclusion: These results demonstrate that Nod2-Rip2 pathway plays an important role in host defense against pulmonary infection of A. baumannii. Introduction: Interleukin-6 (IL-6) is a pleiotropic cytokine produced by many cell types and can promote inflammation, immune response, differentiation, and proliferation. A principal signaling pathway of the IL-6/gp130 receptor activates Janus kinases and the STAT3 transcription factor. In this study, we sought to determine the impact of IL-6 on cellular survival and tumor development in MYCdriven tumorigenesis. We used the Eì-myc transgenic mouse, a model of Burkitt's B cell lymphoma, in which elevated levels of c-MYC are expressed in the B cell lineage.

Methods: Eì-myc mice were generated that either lacked the interleukin-6 (IL-6) gene, or lacked the STAT3 gene specifically in B cells. Introduction: Basophilia is a frequently observed hematological abnormality in chronic myeloid leukemia (CML), but its pathophysiological roles are undefined. We previously demonstrated that an inflammatory chemokine, CCL3, preferentially acts on normal hematopoietic stem/progenitor cells (HSPCs) and crucially contributes to the maintenance of leukemia initiating cells (LICs) in bone marrow (BM) during the initiation process of CML. However, the major cellular source of CCL3 in BM and the precise mechanism of CCL3-mediated maintenance of LICs remain to be investigated. To delineate the cellular process facilitating this CCL3-mediated crosstalk between normal and leukemic hematopoiesis, we precisely examined CCL3-expressing cells and their functions in both normal hematopoiesis and CML leukemogenesis.

We delineated CCL3-expressing cells in bone marrow of normal healthy mice and CML mice, by a flow cytometric analysis. We generated bone marrow chimeras by injecting hematopoietic progenitor cells (ten million) intravenously or into the right bone marrow cavity (200,000 cells). HSPCs were obtained from bone marrow of WT or MCPT8-diphteria toxin receptor (DTR) mice and were transuduced BCR-ABL gene by using a rectrovirus vector system. The resultant LICs (total 30,000 cells) were injected into the tibial bone. In some experiments, DT or mutant DT was administered to LIC-injected mice.

Results: Basophils in normal healthy mice can constitutively expressed CCL3 as revealed by a flow cytometric analysis. Moreover, CCL3 negatively acted on HSPCs expressing either CCR1 or CCR5, its receptors, and regulated the normal hematopoietic process, especially hematopoietic reconstitution after bone marrow transplantation. Moreover, CCL3-expressing basophil-like leukemia cells were found to accumulate in CML bone marrow. MCPT8-DTR mousederived LICs induced CML development when injected into the tibia bone marrow. However, the same LICs failed to cause CML when basophils were depleted by the treatment of DT. Moreover, CML development was prevented by maraviroc, a selectively inhibitor for CCR5, a specific receptor for CCL3, when maraviroc administration started immediately after LIC injection into bone marrow.

Conclusion: Intra-bone marrow basophil expansion observed in CML, can favor leukemia-tropic hematopoiesis in CML by providing CCL3, a potent inhibitor of normal hematopoiesis and therefore, that basophil-derived CCL3 may be a novel target molecule for the treatment of CML. Introduction: Interleukin-6 (IL-6) signaling is a key component of inflammatory responses. The complex recruits two molecules of the signal transducing subunit glycoprotein130 (gp130), which is ubiquitously expressed whereas IL-6R expression is restricted to hepatocytes and some leukocyte populations. Besides the membrane-bound form a soluble form of the IL-6 receptor (sIL-6R) is found, which binds IL-6 with the same affinity. This trans-signaling mechanism greatly expands the target cell spectrum of IL-6. It has been shown that this mechanism mediates most of the proinflammatory actions of IL-6 and is also involved in the recruitment of mononuclear cells during acute inflammation. We aimed to find the protease contributing to circulating sIL-6R in the blood and analyzed whether the sIL-6R is generated at the site of inflammation by proteolytic cleavage from infiltrating leukocytes.

We analyzed the proteolytic processing of the IL-6R on leukocytes in a mouse model of acute inflammation. In the air pouch model of acute inflammation we measured cellular migration of inflammatory cells and released cytokines in a defined microenvironment. To investigate the responsible protease for IL-6R cleavage in vivo we analyzed different protease knock-out mouse strains for sIL-6R levels in the serum and inside the air pouch.

Results: We could demonstrate that the sIL-6R is cleaved by ADAM17 from bone marrow-derived macrophages in vitro as well as during systemic inflammation in vivo. In line with this, hypomorphic ADAM17 (ADAM17 ex/ex ) mice show significantly (ttest p < 0.0001) less sIL-6R in the pouch lavage during acute inflammation compared to wildtype mice, although steady state sIL-6R levels in the blood are unchanged. However, these mice still exhibit normal cell migration into the air pouch cavity in contrast to IL-6R deficient mice. The sIL-6R, which orchestrates the cellular influx, rather reaches the site of inflammation from the circulation, since we found substantial amounts of serum proteins within the pouch lavage.

The results suggest that ADAM17 is one of the major IL-6R proteases under inflammatory conditions. While we demonstrate that although IL-6 and the trans-signaling mechanism are mandatory for cellular infiltration in this model, it is not local proteolysis of the IL-6R within the air pouch, which coordinates this inflammatory process. Instead, the sIL-6R is infiltrating from the circulation in an ADAM17 independent manner. Since we can exclude neutrophil serine proteases, ADAM8, ADAM10 and ADAM17 as potential proteases for the IL-6R, the responsible protease for the generation of circulating sIL-6R remains unknown. Introduction: Interleukin-6 (IL-6) is a potent pro-inflammatory cytokine that regulates the survival and proliferation of haematopoietic cells, yet its cell type and context-specific regulation remains unclear. We found that the NFjB family member NFjB1 (p105/p50) plays an essential role in preventing the uncontrolled production of IL-6 by mature follicular (Fo) B cells.

Methods: We utilised mice lacking NFjB1 (Nfjb1 -/-) to study the effect of increased IL-6 production in vivo, and bone marrow chimeric mice to demonstrate that the cause of dysregulated IL-6 was due to the intrinsic loss of NFjB1 in mature B cells. Extensive B cell in vitro assays and in vivo anti-IL-6 blocking experiments revealed the effect of IL-6 in B and T cells in an autocrine and paracrine specific manner.

Results: Ageing studies revealed that mice lacking NFjB1 (Nfjb1 -/-) develop a severe multi-organ autoimmune disease and succumb prematurely. Disease progression was primarily mediated through the effects of IL-6, which contributed to the enhanced differentiation and proliferation of Nfjb1 -/-Fo B cells and in turn resulted in the elevated production of immunoglobulins, autoantibodies and an immune cell infiltrate in several organs. Due to the excessive production of IL-6 by Nfjb1 -/-Fo B cells, the activation threshold in response to antigen receptor and TLR stimulation was significantly reduced in these B cells. While in a paracrine manner the higher levels of IL-6 increased the differentiation of follicular helper CD4 + T cells (T FH ). We showed that the p50 component of NFjB1 is a repressor of Il-6 transcription in Fo B cells, and the absence of this key regulator leads to the uncontrolled expression of Il-6 via RelA the major dimer partner of p50-NFjB1.

Conclusion: Our findings highlight a novel role for p50-NFjB1 in limiting autocrine IL-6 signaling in mature B cells and supports a role for NFjB1 in preventing multi-organ autoimmune disease through the negative regulation of the Il-6 gene. Aside from their potent anti-apoptotic properties, the IAPs are central to NF-jB signaling, immunity and in tumor evasion of immune attack. We have discovered a potent therapeutic strategy to target cancer in which we combine various immunotherapies with a class of IAP antagonists called Smac mimetics.

We assessed the efficacy and determined the mechanism of action of combining Smac mimetics with immune checkpoint inhibitors in orthotopic, syngeneic mouse models of glioblastoma.

Results: Smac mimetics synergize with immune checkpoint inhibitors to produce durable cures in mouse models of aggressive cranial glioblastoma in which single agent therapy is ineffective. In longterm survivors, tumor growth was not observed upon tumor rechallenge, suggesting that tumor-specific immune memory responses were generated. The combination of Smac mimetics and inhibitory checkpoint blockade increased activated CD8 + T-cells and decreased suppressive immune cells within the tumor microenvironment. Furthermore, the complementation of activities between these classes of therapeutics is dependent on cytotoxic CD8 + T-cell activity.

Conclusion: IAP antagonists incorporate both innate and adaptive immunity to kill tumor cells suggesting that this approach to combination immunotherapy will be highly effective in the treatment of cancer.

Disclosure of Interest: None declared. Introduction: Both inflammation and genetic mutations have been tied to tumor initiation but the interaction of both for promoting tumorigenesis was less investigated and the underlying mechanism was largely unknown. How inflammatory signals affect mutant p53 in a mouse model that mimics the mutations seen in a Li-Fraumeni mouse model (mutations at amino acid 172 of p53 corresponding to the human p53 hot spot mutation at amino acid 175) has not been investigated. Additionally, while IL27 possesses both the pro and anti-inflammatory properties in vivo in a context-dependent manner, how endogenous levels of IL-27 affect spontaneous tumor development or survival in these mice remains largely unknown. In this presentation, the interaction of IL27 deficiency and p53 mutation for causing sarcoma in the bone will be discussed.

Methods: Genetic models (p53 mutaion plus IL27 receptor deficiency) will be used to investigate the ineraction between p53 defect and IL27 signaling deficiency for causing tumor initiation in bones; Ras mutated mice fed with IL27 signaling will be used to study skin tumorigenesis. The current and conventional molecular and pathological analysis will be used to investigate tumor initiation.

Results: In the current study, we found that lack of IL-27 signaling significantly shortened the survival of mice with tumors expressing both copies of the mutant p53 gene (Li-Fraumeni mouse model).

Interestingly, in mice that were heterozygous for mutant p53, lack of IL-27 signaling not only significantly shortened survival but also doubled the incidence of osteosarcomas. These results suggest that IL-27 signaling modulates the oncogenic properties of mutant p53 in vivo and that lack of IL-27 signaling is closely associated with early mutant p53 stability in vivo. Interestingly, IL27 signaling may play an opposite role in skin tumorigenesis and tumorigenic stem cell proliferation as shown in a Ras mutation model. This descripency may be due to induction of inflmmatory cells via an opposite molecular mechanism in different tissues.

The authors feel that this topic would generate excitement from a broad readership and have shown for the first time as we know of a specific inflammation and genetic defect to cause tumor initiation in bones and skin. Introduction: More than 95% of chronic myeloid leukemia (CML) patients have the Philadelphia chromosome (Ph), which is generated by a reciprocal translocation between chromosome 9, which contains the ABL gene, and chromosome 22, which contains the BCR gene. The resultant BCR-ABL fusion gene is translated [1] [2] [3] , and the protein product is a constitutively active tyrosine kinase [4] . This protein is a tyrosine kinase, and tyrosine kinase inhibitors (TKIs) that target BCR-ABL are the standard treatment for CML. However, some patients who have received TKIs have started to exhibit resistant CML [5] . One of the resistance mechanisms is a BCR-ABL mutation in the kinase domain, which is effectively managed by newly developed TKIs. However, a BCR-ABL mutation-independent mechanism is also present, and the exact mechanism is not known.

Methods: To determine BCR-ABL mutation-independent mechanism, we generated a TKI-resistant CML cell line that had no mutations within the BCR-ABL fusion gene. In this line (called K562R), interleukin-1b (IL-1b) had higher expression than in the mother cell line (K562S).

Results: Imatinib efficiently induced apoptosis in K562S cells, but IL-1b treatment significantly inhibited this apoptosis. Moreover, the viability of the K562R cells decreased when the secreted IL-1b was neutralized. In addition to this autocrine effect on CML cell survival, secreted IL-1b from K562R affected stromal cell production of chemokines, such as CXCL11, which in turn promoted K562R cell migration into stromal cells.

Conclusion: Conclusively, our data indicate that imatinib-resistant cells acquire the ability to produce IL-1b, and that elevated levels of IL-1b affect imatinib resistance via an autocrine effect that promotes cell survival and a paracrine effect that controls migration into the stroma. These observations have ramifications for treatment of TKIresistant patients. Introduction: Constitutive activation of JAK/STAT signaling associated with progression of various tumor. In esophageal squamous cell carcinoma (ESCC), chronic inflammation was involved in carcinogenesis and cancer growth. Previous reports showed that IL-6 was significantly associated with treatment-resistant and prognosis of ESCC, and STAT3 also acts as an important transcriptional mediator of pro-inflammatory cytokine signaling pathways and contributes to oncogenesis by preventing apoptosis and enhancing cell proliferation. Suppressor of cytokine signaling-1 (SOCS-1) has been cloned as a negative regulator of various cytokine signaling including JAK/STAT pathway. However, the epigenetic silencing of SOCS-1 by methylation of the CpG island is detected and is implicated in cancer development in various cancer. This study was aimed to evaluate the antitumor effect of overexpression of SOCS-1 using adenovirus vetctor (AdSOCS-1).

Methods: We evaluated SOCS-1 methylation status in ESCC cell lines and human tumor/normal tissue by methylation-specific PCR. As in vitro, we evaluated the antiproliferative effect and mechanism in AdSOCS-1, and examined the antiproliferative mechanism by using JAK inhibitor and FAK-siRNA. As in vivo models, ESCC cell line (TE-14) xenograft mice and patient-derived tumor xenograft mice (PDX) using human ESCC were also assessed. Mice were intra-tumorally injection with AdSOCS-1 or control adenovirus vector (AdLacZ) twice a week for 4 weeks.

Results: SOCS-1 gene was methylated in human ESCC tissues and all ESCC cell lines, but not in normal tissues. By real-time PCR analysis, all ESCC cell lines failed to upregulate or expressed very low levels of SOCS-1 in response to IFN-k, whereas human PBMC remarkably upregulate SOCS-1 expression in response to IFN-k. AdSOCS-1 markedly suppressed proliferation of all ESCC cell lines in vitro regardless of activation level of STAT3 and induced apoptosis via inhibiting not only JAK/STAT pathway but also FAK/ERK signaling. Although TE14 which has low activation level of STAT3 was not significantly suppressed only by JAK inhibitor, the inhibition of JAK/ STAT and FAK/ERK signaling by using JAK inhibitor and/or FAK-siRNA showed the antiproliferation effect regardless of activation level of STAT3 as same as AdSOCS-1. As in vitro, the tumor volume and tumor weight with AdSOCS-1 was significantly lower than AdLacZ. Especially, PDX mice showed better antitumor effect by AdSOCS-1 therapy. We confirmed the expression of SOCS-1 and the inhibition of pSTAT3 and FAK/ERK signaling as same as in vitro by western blotting of AdSOCS-1 injected tumor. Introduction: Common gamma receptor cytokines IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, their receptors and signaling pathways that involve JAK1/JAK3 and STAT3/STAT5 play pivotal roles in control of Tlymphocyte immune responses [1] . Abnormal constitutive activation of this system was pervasive in diverse T-cell malignancies as assessed by constitutive STAT3 and STAT5 phosphorylation and nuclear translocation.

The following antibodies were purchased from Cell Signaling Technology: JAK1, JAK2, STAT3, pSTAT3, STAT5, and pSTAT5. Ruxolitinib was purchased from LC Laboratories. shRNA targeting JAK1, JAK2 and STAT3 were cloned into a doxycyclineinducible PRSMX-puro-GFP vector. Retroviruses containing the shRNA were produced by co-transfection of 293T cells with helper plasmids expressing gag-pol genes, an ecotropic pseudotyping env gene and a retroviral vector using Fugene6 (Promega). RNA-seq was performed on RNA samples extracted from T-cell leukemia/lymphoma lines. Mutations were confirmed by Sanger Sequencing. For NanoString analyses RNA from T-cell tumors were extracted with RNeasy FFPE mini kit (Qiagen). 200 ng of total RNA were used for analysis of human immunoglobulin gene expression following the protocol of NanoString technology. Fold increases were calculated comparing to gene expression of normal purified T cells.

Results: Activating mutations of STAT3 and STAT5 were defined in some but not all cases of constitutive activation of the signaling pathway. The defined JAK kinase inhibitor, ruxolitinib had a major inhibitory impact on leukemic T-cell line proliferation and expression of nuclear phospho-STAT3 and 5. Cell lines that manifested pSTAT3/ pSTAT5 were addicted to these STATs whether or not STAT elements were mutated. With the exception of leukemias with chromosomal translocations, activating STAT mutations were not sufficient to initiate leukemic cell proliferation but rather only augmented signals from the above cytokine-cytokine receptor pathway. Therefore, we searched for JAK mutations. JAK mutations were identified that synergized with constitutive phosphorylation or mutations of STATs. Using shRNA loss-of-function analysis with pSTAT malignant T-cell lines, both JAKs and STATs were required for proliferation and survival of T-cell lines whether or not JAK or STAT elements were mutated. Examining JAK mutations a cytokine receptor was required as a JAK activation scaffold and as a STAT docking site. Therefore, constitutive activation required the full pathway including cytokine-cytokine receptor and JAK/STAT elements. In addition to JAK/STAT mutations, there were abnormalities that indirectly augmented cc cytokine expression or JAK/STAT signaling.

The demonstration of pervasive constitutive activation of the cytokine-cytokine/receptor JAK/STAT-pathway in most forms of T-cell malignancies represents an exciting development. Activating mutations of STATs and JAKs were identified in many but not all cases. These mutations were not sufficient to support leukemic cell proliferation but only augmented signals from above in the cytokinecytokine receptor, JAK/STAT signaling pathway. These basic observations support the clinical evaluation of inhibitors of the JAK/STAT pathway as exciting candidates for inclusion in multidrug combination therapy of T-cell malignancies. Introduction: Vascular endothelial growth factor A (VEGF-A) is thought to play a pivotal role in lymph node expansion during inflammation. In humans, the cell type and the mechanisms regulating VEGF-A intranodal production remain elusive. Here, we investigate the ability of Dendritic Cells (DCs) to produce VEGF-A in human activated lymph nodes and unveil the molecular mechanisms responsible of the activation of VEGF-A transcription in inflammation.

Methods: Immunohistochemistry was performed on human reactive secondary lymphoid organs. DCs at day 6 of culture were stimulated with different TLR ligands and after 24 h, supernatants were collected and VEGF-A and PGE 2 production was evaluated by ELISA or EIA, respectively. The involvement of different transcription factors in VEGF-A production was assessed by WB, ChIP and inhibitor experiments. Results: Here, we reveal a pro-tumourigenic role for the key inflammasome adaptor ASC in GC. The genetic ablation of ASC reduced gastric tumour growth by augmenting cell death in the tumour epithelium, independent of hematopoietic-derived immune cells and chronic inflammation. Suppressed tumourigenesis was associated with reduced expression of IL-18, but not IL-1b, in gastric tumours, with targeted IL-18 deficiency in gp130 F/F mice also decreasing tumour burden characterized by increased tumour cell death. Antibody-mediated blockade of IL-18 reduced human GC cell growth in vitro. Elevated IL-18 protein and ASC mRNA levels were observed in human GC tumour biopsies.

Conclusion: Collectively, these findings reveal the ASC/IL-18 axis as a potential therapeutic target in GC.

Disclosure of Interest: None declared. Introduction: Stroma plays an important role in breast cancer occurrence and development, and fibroblasts are the main components of the breast cancer stroma. In order to investigate the molecular mechanisms of the effect of cancer stromal fibroblasts on breast cancer, in this study, FSP-1(fibroblast-specific protein-1) expression was investigated in human fibroblasts co-cultured in vitro or co-growth in vivo with human breast cancer cells and its impact on breast cancer growth.

Methods: Human fibroblast line ESF was co-cultured with human breast cancer cell line MDA-MB-231 using Transwell method. The level of FSP-1 mRNA was examined in the co-cultured ESF cells by RT-qPCR. The expression levels of FSP-1 protein were detected by immunofluorescence flow cytometry. The proliferation of cocultured and single cultured MDA-MB-231 cells was tested using Cell Counting Kit-8(CCK-8) method. FSP-1 expression in the breast cancer tissue of tumor-burdened nude mice was analyzed using immunofluorescence and laser confocal microscope, and the effect of FSP-1 on tumor growth of tumor-burdened nude mice was examined. Experimental data were analyzed using SPSS statistics software, version 17.0. Data were presented as mean ± standard deviation. The differences between two groups were analyzed by the Student's t-test. A probability value (P) of <0.05 was considered to indicate a statistically significant difference. Results: We demonstrate that in the absence of STAT1, the lethal virus-induced disease caused by intestinal MNV infection is associated with MNV dissemination beyond mucosal barrier surfaces and hyperactive cytokine production in virus-specific T cells. In a model of natural MNV exposure where STAT1-sufficient and deficient littermates acquire MNV as a part of their commensal microbiome, Stat1 -/mice accumulate elevated numbers of virusspecific effector T cells and have high viral burdens in multiple tissues that are associated with pathological tissue inflammation. Mechanistically, the combination of uncontrolled viral replication, heightened activation and impaired resolution of antiviral T cell responses contributes to widespread viral-induced disease.

Conclusion: Together, these data suggest that in addition to limiting early viral replication in infected cells, STAT1-dependent signaling is critical for establishing tolerance to persistent enteric viral exposure and limiting viral-induced disease.

Disclosure of Interest: None declared. Introduction: Tuberculosis (TB) causes~1.5 million deaths every year, thus remaining a leading cause of death from infectious diseases in the world. A growing body of evidence demonstrates that type I IFN plays a detrimental role in TB pathogenesis, likely by interfering with IFN-k-dependent immunity.

Methods: Using the murine aerosol model of TB and a virulent strain of Mycobacterium tuberculosis (BTB 02-171) that induces high levels of type I IFN, we reveal a novel mechanism by which type I IFN may confer protection to M. tuberculosis infection in the absence of IFN-k signaling.

Results: Absence of both type I and type II IFN receptors led to a strikingly increased level of arginase 1 (Arg1) gene expression and activity in infected lungs, characteristic of alternatively activated macrophages. This correlated with increased bacterial burden and pathology, and decreased survival, as compared to mice deficient in either receptor. Increased expression of other genes associated with alternatively activated macrophages, as well as increased expression of Th2-associated cytokines and decreased TNF expression, were also observed. Thus, in the absence of IFN-k signaling, type I IFN suppressed the switching of macrophages from a more protective classically activated to a more permissive alternatively activated phenotype.

Conclusion: Together, our data support a model in which suppression of alternative macrophage activation by type I IFN during M. tuberculosis infection, in the absence of IFN-k signaling, contributes to host protection.

Disclosure of Interest: None declared. Introduction: Similar to Type I IFNs (IFN-a/b), Type III IFNs (IFN-k1,2,3,4) exert antiviral and antiproliferative effects through a JAK-STAT mediated pathway that activates ISGF3 (STAT1/STAT2/IRF9) and consequently induces the expression of interferon-stimulated genes (ISGs). IFN-ks signal through a receptor complex distinct from that of IFNa/b, composed of IL-10R2 and IFN-kR, which appears to be preferentially expressed on epithelial cells. This suggests that IFN-k's primary role is to provide antiviral protection to mucosal compartments. IFN-kR-deficient mice have been developed, but due to the overlapping activities of type III and type I IFNs, a unique role for IFN-k in antiviral immunity has yet to be elucidated. Most viral infections induce the expression of both type I and type III IFNs, but it is unclear which cells are responsible for IFN-k production in vivo, and whether IFN-k production is induced by the same mechanisms which trigger type I IFN synthesis.

Methods: In order to identify the source(s) of type III IFNs in the course of an in vivo virus infection, we have generated a novel IFN-k reporter mouse in which the IFN-k2 coding sequence was replaced with eGFP by homologous recombination, while maintaining the IFN-k2 promoter regions and UTRs intact. While both IFN-k2 alleles have been replaced by eGFP, these mice retain the gene encoding IFN-k3 intact. Using the IFN-k reporter mouse we have now been able to identify IFN-k-producing cell populations following Poly I:C treatment and RNA virus infection in vivo.

: Results from our IFN-k reporter mice show that intravenously administered Poly I:C (a dsRNA mimetic) induces IFN-k expression predominantly from CD8a dendritic cells in the spleen. Infection of the gastrointestinal tract of the indicator mice with rhesus rotavirus (RRV), a dsRNA virus of the family Reoviridae, shows the epithelial cells of the small intestine as the predominant source of IFN-k induced by this pathogen. We also observed that viral titers between IFN-k reporter mice and non-reporter wild type mice of the same genetic background were equivalent following RRV infection, suggesting that IFN-k3 alone is sufficient to elicit adequate type III IFN-based antiviral response and, therefore, the reporter mice are not expected to show enhanced susceptibility to virus infections.

These experiments suggest that the subset of cell types responsible for IFN-k production in vivo is likely to be pathogenspecific. As the source of type I and type III IFNs in a given infection appear to differ, identifying the cellular sources of IFN-k may offer some insight into the IFN type-specific versus.

Disclosure of Interest: None declared. The importance of type I interferons (IFNs) in infection and immunity has been well-documented in vivo. They play important roles in regulating inflammation and are classically known for their immunomodulatory and anti-viral effects, elicited following binding to their cell surface receptors, Ifnar1 and Ifnar2. IFNs have also been shown previously to negatively regulate IL-1b responses. However, a role for IFN receptors and Ifnar2 specifically in regulating IL-1b responses has not been characterised.

We have utilised mice lacking one of the type I IFN receptors, Ifnar1 or Ifnar2, as well as the HKx31 (H3N2) IAV mouse model of infection.

We have identified that the IFN receptor Ifnar2 plays an important role in regulating IL-1b responses in vivo during IAV infection. In the airways of Ifnar2 -/mice following IAV infection, we observed increased neutrophil infiltration and elevated levels of IL-1b. To elucidate the mechanisms involved in Ifnar2-mediated regulation of IL-1b responses, we examined the expression of inactive precursor protein, pro-IL-1b, as determined by immunoblot in lung tissues from wild-type, Ifnar1 -/and Ifnar2 -/mice. The expression of pro-IL-1b was significantly increased in the lungs of untreated Ifnar2 -/mice, suggesting Ifnar2 limits pro-IL-1b protein expression in vivo. We have identified that Ifnar2-mediated regulation of pro-IL-1b is post-translational and the mechanisms involved are currently being investigated. Importantly, the dysregulation of pro-IL-1b expression in Ifnar2 -/mice correlated with increased susceptibility to IAV infection, in comparison to both Ifnar1 -/and wild-type mice. In particular, by day 7 post-infection mice lacking Ifnar2 but not Ifnar1 displayed severe weight loss, clinical signs of disease and the hallmark features of ARDS, namely pulmonary edema and vascular leakage. Methods: We stimulated human peripheral blood mononuclear cells (PBMC) and plasmacytoid dendritic cells (pDC) carrying either the responder or the non-responder genotype with synthetic TLR ligands, HCV and HCV-infected hepatoma cells.

Results: Interestingly, irrespective of whether human peripheral blood mononuclear cells (PBMC) carried the responder or the nonresponder genotype at rs368234815, stimulation with synthetic Toll-like receptor (TLR) ligands induced similar IFN mRNA as well as IFN-a protein responses. Furthermore, upon direct stimulation with the HCV variant Jc1 primary human plasmacytoid dendritic cells (pDC) homozygous for the responder or the non-responder genotype showed similar IFN-a protein expression. In contrast, co-culture of Jc1 infected hepatoma cells with pDC carrying the non-responder genotype resulted in reduced IFN-a as well as IFN-k1 protein responses. This effect was independent of whether pDC carried the normal or the hypo-functional IFN-k4 P70S variant. Unlike poly(I:C) stimulated primary human hepatocytes, HCV triggered pDC did not show IFN-k4 mRNA expression. In line with these experiments, and similar to pDC pre-incubated with recombinant IFN-k1-3, also IFN-k4 enhanced TLR7 ligand-induced IFN responses.

Conclusion: These data support the hypothesis that in pDC SNPs within the IFN-k locus can affected cell function independent of IFN-k4 secretion and extracellular IFN-kR triggering. We have previously shown that IFN-b can bind directly to IFNAR1, in the absence of IFNAR2, and can transmit a signal that contributes to lethality in a mouse model of sepsis 1 . In this previous study we also determined the crystal structure of IFN-b bound to the extracellular domain (ECD) of IFNAR1, showing unique interaction interfaces not previously defined for other IFN-receptor complexes and enabling us to hypothesise a molecular basis for the higher binding affinity of IFN-b for IFNAR1 compared to other IFNs 1 . In light of the structural and functional insights we have into this unique signaling complex, herein we sought to investigate the molecular determinants that influenced stability of the IFN-b/IFNAR1 complex and contributed to IFN-b driven signaling in an effort to inform drug discovery to antagonize this ligand-receptor interaction.

Methods: The current study used site-directed mutagenesis, recombinant forms of IFNAR1-ECD and IFN-b, and a transient transfection system to express mutant forms of IFNAR1, to investigate the contribution of key residues to the formation of a stable, functional IFN-b/IFNAR1 complex. To observe the effect of mutation on IFN-b and/or IFNAR1 functionality, we used surface plasmon resonance to measure affinity of the protein-protein interaction, flow cytometry to measure down-regulation of cell surface IFNAR1, luciferase assays and western blots to observe activation of signaling molecules, Real-Time PCR to measure gene induction, and anti-viral and antiproliferative assays to observe the effect of mutation on biological outcomes.

We have identified a key interaction interface on the third subdomain (SD3) of IFNAR1 that stabilizes the ligand-receptor complex, and influences all aspects of IFN-b functionality investigated in this study. We demonstrated that the interface between two residues on IFNAR1-SD3, Y 240 and Y 274 , and the residues on IFN-b central to this interface (F 63 , L 64 , E 77 , T 78 , V 81 and R 82 ) were collectively crucial for the nano-molar affinity of IFN-b for IFNAR1, for the IFN-b-driven down-regulation of IFNAR1 from the cell surface, the efficient transmission of signals to activate STAT1 and induce ISG expression, and to drive the anti-viral and antiproliferative responses for which IFN-b is well characterized.

The results of our current study suggest that a key interaction interface, that influences IFN-b driven signals, is located on IFNAR1-SD3. This site may be a useful target for the development of therapeutics for treatment of IFNb-mediated diseases, including sepsis and autoinflammatory diseases. Introduction: Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the bloodbrain barrier [1] and blood-nerve barrier [2] , respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions.

Methods: To investigate the mechanism of antibody-mediated protection within the barrier-protected tissues, we used a mouse model of genital herpes infection.

Results: We examine the mechanism by which antibodies gain access to neuronal tissues to control infection. We demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-c and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus.

Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread. Introduction: Plasmacytoid dendritic cells (pDCs) function as the most potent producers of type-I and type-III interferon in viral infection [1, 2] . The expression of interferon by pDC is initiated by innate sensing of microbial or self DNA. Apart from the endosomal toll-like receptor 9 (TLR9)-mediated DNA-sensing pathway, a parallel pathway initiated by the cytosolic DNA-sensor, cyclic GMP-AMP synthase (cGAS) and mediated by stimulator of interferon genes (STING) was reported to induce immune activation in different human and murine cell types [3] [4] [5] . Here we report that a functional cGAS-STING pathway exists in human pDCs.

Methods: pDCs were isolated from PBMC of healthy donors by magnetic bead-based negative selection. The transcript and protein level of cGAS and STING were measured via qRT-PCR and intracellular flow cytometry, respectively. The expression of cGAS and STING were compared between two known subsets of human pDCs: CD2 high and CD2 low . PBMCs were treated with different viruses and cytokines to see how the expression of cGAS and STING are modified by different stimuli in pDCs through flow cytometry. After confirming the subcellular localization of cGAS in cytosolic compartment of pDCs, we transfected pDCs with non CpG doublestranded and single-stranded DNA to see their co-localization with cGAS by imaging flow. Purified pDCs were transfected with the STING agonist cyclic GMP-AMP (cGAMP), or treated with the dsDNA virus, herpes simplex 1 (HSV) and the synthetic TLR9-agonist, CpG-A. The production of type-I and type-III interferon was measured through intracellular flow cytometry and ELISA.

Results: cGAS and STING were found to be constitutively transcribed and translated in human pDCs. The expression of cGAS and STING were comparable in both CD2 high and CD2 low pDCs. cGAS expression was upregulated after treatment with type-I and type-III interferon and HSV, while STING expression remained unaltered. cGAS co-localized with non-CpG dsDNA in a time-dependent manner, but did not co-localize with ssDNA. Upon stimulation with cGAMP, pDCs produced IFN-a and IFN-k in a time-dependent and dosedependent manner.

Conclusion: A functional cGAS-STING pathway for cytosolic DNA sensing induces type-I and type-III interferon production in human pDCs, serving as a complement to the endosomal TLR9 pathway.

Disclosure of Interest: None declared. Methods: Mice are infected with F. novicida subcutaneously. Survival rates and bacterial burdens in the spleens and liver tissues are monitors following the infection.

Here, we showed that compared with infected wild-type mice, mice lacking the type I IFN receptor IFNAR2 exhibited increased resistance towards F. novicida infection, whereas mice lacking AIM2 were more susceptible. In addition, mice lacking IRF9, a core component of IFN-stimulated gene factor 3 that transduces type I IFN signaling, showed increased resistance following infection. These results suggested that while type I IFN signaling and AIM2 were essential for activation of the Francisella-induced inflammasome in macrophages, these components have strikingly contrasting effects in vivo. Importantly, we found that mice lacking both AIM2 and IFNAR2 were more resistant than infected wild-type mice and mice lacking AIM2. Methods: We used mice deficient for Irgm1, Irgm2, or Irgm3 expression to probe the involvement of these genes in the host response to LPS. These three paralogous genes are orthologs of the human gene IRGM which has been previously associated with the outcome of sepsis. To specifically examine the function of Irgm2 in LPS sensing pathways, Irgm2 -/mice were crossed with strains deficient for Tlr4 or caspase-11 expression. We performed cell culture studies using IFN-primed bone marrow derived macrophages (BMDMs) from mice of the various genetic backgrounds. To define the role of Irgm proteins in the pathogenesis of sepsis, mice were injected intraperitoneally with LPS and monitored for immune functions and morbidity. For experiments involving Tlr4-deficient mice the TLR3 agonists poly(I:C) was used to stimulate IFN production in vivo.

Results: Irgm1 -/and Irgm2 -/-BMDMs showed a marked increase in cell death, IL-1b, and IL-18 production following treatment with LPS. Consistent with these results, Irgm1 -/and Irgm2 -/mice exhibited decreased survival and increased serum IL-1b/IL-18 following LPS challenge. Mice and BMDMs deficient for both Tlr4 and Irgm2 showed increased sensitivity to LPS relative to mice singly lacking Tlr4 expression. On the other hand, BMDMs lacking Irgm2 and caspase-11 expression were protected against LPS-induced cytotoxicity.

Conclusion: These results show that Irgm1 and Irgm2 protect against LPS-induced inflammation on both a cellular and whole organismal level. Furthermore, the data implies Irgm1 and Irgm2 act as key regulators of caspase-11 activity. This supports a model wherein IFN signaling is not only required for a potent caspase-11 response but also helps to contract caspase-11-mediated inflammation.

Disclosure of Interest: None declared. Introduction: Interferons (IFNs) are classified into three different types, type I, type II and type III (or k) IFNs, based on their structure, receptor complexes and biological activities. In humans, type III IFNs (IFN-ks) are represented by IFN-k1, IFN-k2, IFN-k3 and a recently identified IFN-k4, which shares only a limited homology with other IFN-ks. There is a frequently occurring frame shift mutation in the Ifnl4 gene that can be used as a prediction marker for the responsiveness of patients chronically infected with HCV to IFN-abased therapies. Paradoxically, the presence of the intact Ifnl4 gene is associated with poor response to anti-HCV treatments, suggesting a unique function for IFN-k4. Studies of IFN-k4 have been tempered by difficulties in generating biologically active IFN-k4 protein, particularly from mammalian cells, due to its poor secretion from human cells.

We have generated a mammalian expression construct for human IFN-k4 with an N-terminal FLAG tag which resulted in the production of functional IFN-k4 protein when expressed in Chinese Hamster Ovary (CHO) cells. We used this mammalian cell-produced IFN-k4 protein to assess its receptor binding and biological activities. Introduction: Interferon lambdas (IFNLs) are potent antiviral cytokines that are widely implicated in many chronic inflammatory diseases [1] . While IFNLs are best known for their role in clearance of hepatitis C virus infection, there is substantial data supporting their role in acute viral infections such as influenza and viral gastroenteritis, as well as chronic autoimmune and liver disease [2, 3] . Numerous tissues have reported IFNL expression, but there remains controversy over which blood cell subsets are responsive to IFNL and thus contribute to inflammatory activity in chronic diseases.

Methods: Monocytes were isolated by CD14 selection, and differentiated into macrophages in vitro with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colonystimulating factor (GM-CSF) alone, or in the presence of IFNL3. Monocyte and macrophage IFNL responsiveness and IFNL receptor expression were assayed by quantitative PCR (qPCR) of interferon stimulated gene (ISG) expression, Western blot (phospho-STAT1) and flow cytometry. The pro-inflammatory role of IFNL3 during macrophage differentiation was examined using RNA sequencing and qPCR, mass spectrometry, and functional readouts including chemotaxis and phagocytosis assays. Fluorescent microscopy was used to identify IFNL receptor expressing macrophages in tissues affected by chronic inflammation.

Results: Upon examining IFNL sensitivity in vitro, we demonstrate macrophages but not monocytes are more responsive to IFNL3 than myeloid and plasmacytoid dendritic cells; the only other leukocyte populations with a significant IFNL response [4] . To investigate the role of IFNLs on macrophage maturation and function, human monocytes from healthy subjects were differentiated into macrophages using either GM-CSF (pro-inflammatory) or M-CSF (antiinflammatory), alone or in combination with IFNL3. Macrophages matured with GM-CSF expressed more IFNL receptor (IFNLR1) and were more responsive to IFNL3, supporting their role as proinflammatory cytokines. Moreover, IFNL3 increased the expression of numerous inflammatory/chemotactic cytokines and activation markers as measured by qPCR and flow cytometry respectively. Functionally, macrophages differentiated in the presence of IFNL3 were more phagocytic towards apoptotic cells and more chemoattractant towards CD3, NK and NKT cells.

Lastly, we demonstrated an accumulation of IFNLR1 expressing macrophages in numerous chronic inflammatory conditions, supporting their potential to drive a pro-inflammatory state.

Conclusion: Our data suggests that IFNLs may contribute to chronic inflammatory disease by stimulating a pro-inflammatory phenotype in macrophages, thus driving an adverse immune cell response.

It is unclear however, which components of the host immune response regulate this process. Using a mouse model of IAV, we sought to determine the relative roles of type I and II interferons (IFNs) in dictating the pulmonary inflammatory response to infection.

Methods: Pulmonary cellular responses in WT, Ifnar1 -/-, Ifngr1 -/and Stat1 -/mice were investigated by flow cytometry and gene expression analyzed qRT-PCR.

Results: Our recent work shows that in addition to its known antiviral activity, STAT1 signaling coordinates host inflammation during IAV infection in mice. This regulatory mechanism is dependent on both type I IFN and IFN-k receptor signaling and, importantly, requires functional interplay between the two pathways. Our work demonstrates that type I IFNs act to promote monocyte recruitment to the lungs of infected mice, as well as to prevent excessive activation of monocytes by IFNk. Interestingly, we show that type I IFNs preferentially regulate IFNk signalling in Ly6C lo compared to Ly6C hi mononuclear cell populations. In the absence of type I IFN signaling, Ly6C lo monocytes/macrophages become phenotypically and functionally more pro-inflammatory than Ly6C hi cells, revealing an unanticipated function of the Ly6C lo mononuclear cell subset in tissue inflammation. In addition to their effects on mononuclear cells, type I IFNs also suppress the recruitment of neutrophils into IAV-infected tissues, however the full neutrophil-suppressive effect requires the co-operation of both type I and II IFNs.

Conclusion: Our study demonstrates that type I and II IFN interplay links innate and adaptive anti-viral immunity to orchestrate tissue inflammation. Furthermore, this work reveals an additional level of complexity for IFN-dependent regulatory mechanisms that function to prevent excessive immunopathology while preserving antimicrobial functions.

Disclosure of Interest: None declared. Results: We first found both murine and human pDCs shared Lyn and Fyn expression (albeit at different relative levels) and that LYN activating residue was phosphorylated both before and after TLR stimulation. Next, we observed that FACS-purified pDCs from either Fyn or Lyn deficient mice exhibited impaired IFN-I and proinflammatory cytokine production after ex-vivo TLR7 and TLR9 stimulation, whereas no differences or enhanced cytokine production were observed in FACS-purified conventional DCs. Furthermore, a similar phenotype was recapitulated after in vivo infection with murine cytomegalovirus. Importantly, we could extend the aforementioned finding to human pDCs, showing that treatment with either a pan-SFKs inhibitor or bafetinib profoundly ablated TLRinduced IFN-I production in both CAL-1 cells and in primary pDCs from human peripheral blood. Furthermore, genetic ablation of LYN by CRISPR/Cas9 in CAL-1 cells also resulted in diminished TLR responses. Inhibition of SFK activity impaired key signaling events downstream TLR stimulation including activation of IKK-a/b, NF-jB, IRF7 and MTOR in human pDCs. Strikingly, BCAP phosphorylation, which is known to bridge PI3K and TLR pathways, was profoundly abolished by a pan-SFK inhibitor or bafetinib both before and after TLR engagement.

Conclusion: Taken together, our results contribute to understand the mechanisms tuning pDC responses, revealing a previously unappreciated positive role for SFKs, particularly LYN, in TLRinduced IFN-I and pro-inflammatory cytokine production that is conserved in both murine and human pDCs. In addition, our findings support a model in which pDCs receive constitutive SFK-mediated tonic signaling to enable an optimal response to subsequent pathogen encounter. Given the important roles of pDCs in multiple human illnesses, manipulation of SFK activity in pDCs, perhaps with small molecules such as bafetinib coupled to pDC specific antibodies, could bring about novel therapeutic strategies.

Disclosure of Interest: None declared. Methods: We used microarray, RNAi and molecular biology techniques to validate the role of IFIT1 in differentiated macrophage cells.

Results: Analysis of different steps in TLR activation in response to lipopolysaccharide (LPS) showed that knockdown of IFIT1 leads to enhanced upstream signaling activation such as p65 phosphorylation, p38 phosphorylation and p50 nuclear localization. Although no significant change was observed for IRF3 activation, a key compo-nent in the pathway for interferon expression, microarray analysis of IFIT1-depleted THP1 cells suggested a role for IFIT1 in positively regulating the interferon gene program induced by LPS. Unexpectedly, IFIT1 depletion led to a significant reduction in interferon-b (IFNb) expression and numerous interferon-stimulated genes. Consistent with these results, STAT1 activation was also significantly attenuated in IFIT1 depleted cells. We also observed reduced expression of IFNb1 in response to Influenza A virus, Sendai virus and Burkholderia cenocepacia bacterial infection in IFIT1 depleted cells, suggesting a broad role for IFIT1 in supporting . TBEV replication is inhibited by TRIM30D, a rodent-specific ISG that functions by binding and targeting the viral RNA polymerase (NS5) for lysosomal degradation. The closest human paralog of TRIM30D is TRIM5 which is a highly characterized restriction factor thought to be retrovirus-specific. Our study reveals that both human (h) and rhesus (rh) TRIM5 restrict replication of viruses belonging to the TBEV serogroup, but not WNV or DENV.

Methods: Replication of multiple flaviviruses was tested in HEK293 cells stably expressing h or rhTRIM5, or hTRIM22 as a control. Alternatively, TRIM5 expression was depleted using RNAi in A549 cells treated with IFNb.

Results: Ectopic expression of rhTRIM5 significantly reduced the production of TBEV by more than 99%, but did not affect WNV, DENV or ZIKV. hTRIM5 overexpression imposed a 90% reduction of TBEV titer, whereas knock-down of hTRIM5 partially rescued the antiviral effects of interferon treatment, demonstrating for the first time that hTRIM5 is functional against viral pathogens. TRIM5 reduced production of both viral RNA and protein, indicating that TRIM5 targets an early step in virus replication. However, unlike TRIM30D, TRIM5 did not interact with NS5 and instead interacts with and degrades NS3 (the viral protease critical to virus replication) by an unusual mechanism involving the proteasome and autophagy proteins LC3 and GABARAP. Moreover, TRIM5 colocalized with NS3 and dsRNA suggesting that TRIM5 disrupts the viral replication complex.

Conclusion: This work demonstrates that TRIM5 is an ISG that can target two very different viral proteins, the HIV capsid and the TBEV protease. Understanding the genetic trade-offs in TRIM5 that enable restriction of one virus versus another will illuminate how the evolution of antiviral interferon responses and host resistance is shaped by multiple pathogens.

Disclosure of Interest: None declared. Introduction: The outcomes of many diseases differ between women and men, with women experiencing a higher incidence and more severe pathogenesis of autoimmune and some infectious diseases. It has been suggested that this is partially due to toll-like receptor (TLR)-mediated activation of plasmacytoid dendritic cells (pDCs), the main producers of interferon (IFN)a, in response to TLR7 stimulation.

We investigated the induction of type I IFNs (IFN-I) upon TLR7 stimulation on isolated pDCs using qRT-PCR and intracellular cytokine staining. IFNa/b receptor (IFNAR) 1 and 2 surface expression was determined using flow cytometry.

Results: Our data revealed a sex-specific differential expression of IFN-I, with pDCs from females showing a significantly higher mRNA expression of all 13 IFNa-subtypes (P = 0.002). In addition, pDCs from females had higher levels of IFNb mRNA (P = 0.01) after stimulation, indicating that the sex differences seen in IFN-I production by pDCs were mediated by a signaling event upstream of the first loop of IFN-I mRNA transcription. Furthermore, the surface expression levels of the common IFNAR subunit 2 were significantly higher on pDCs from females in comparison to males.

Conclusion: These data indicate that higher IFNa production is already established at the mRNA level and propose a contribution of higher IFNAR2 expression on pDCs to the immunological differences seen between females and males. Introduction: Highly pathogenic avian influenza (HPAI) viruses continue to cause human infection with a reported mortality rate greater than 50%. However, the mechanism of enhanced disease severity following H5N1 virus infection is not fully understood.

Macrophages are important for protection against influenza virus infection but have been implicated in exacerbated disease following infection with H5N1 HPAI viruses via a dysregulation of macrophage antiviral responses. We have previously demonstrated that H5N1 influenza viruses are unique in their ability to replicate in macrophages, a feature that maps to the viral hemagglutinin (HA) gene. Further, disease severity in a mouse model of influenza virus infection correlated with the replicative capacity of the virus in macrophages. We hypothesize that H5N1 influenza viruses alter critical macrophage functions in a replication-dependent manner.

Methods: RAW264.7 macrophages were infected with the following influenza viruses: live/UV-inactivated H1N1 influenza virus A/ California/04/2009 (CA/09; does not replicate in macrophages) or live/UV-inactivated CA/09 expressing an H5 HA gene (CA/09-H5 HA; productively replicates in macrophages). Beginning at 5 h postinfection, the macrophages were treated with IFN-k and phagocytic capacity and nitric oxide production were measured. The IFN-k signaling response of macrophages infected as described above was measured by Western blotting for phosphorylated STAT1 and by RT-PCR for the expression of the IFN-k-regulated gene, IRF-1.

Results: Macrophages infected with the CA/09-H5 HA virus were less efficient at phagocytosis compared to cells infected with the CA/ 09 virus. Further, the CA/09-H5 HA virus inhibited IFN-k-dependent upregulation of phagocytosis and nitric oxide production. Importantly, these responses were inhibited in a replication-dependent manner as the macrophage responses could be rescued by UVinactivation of the CA/09-H5 HA virus. Moreover, the H5 HAexpressing virus blocks IFN-k signaling as the expression of IRF-1 was inhibited by CA/09-H5 HA virus in a replication-dependent manner. Phosphorylation of STAT1 was not inhibited by virus infection.

Conclusion: Our data demonstrate that H5N1 influenza viruses decrease phagocytic capacity and IFN-k responsiveness of macro-phages and that these changes are dependent on influenza virus replication. This suggests that macrophage dysfunction during severe H5N1 disease is an effect of the unique ability of these viruses to productively replicate in macrophages. Introduction: Innate immune response to many microbial infections is initiated by intracellular pattern recognition receptors (PRR). Induction of Type I interferon (IFN), a major mediator of such a response, is often initiated by the recognition of microbial nucleic acids by the PRRs, TLR3 or cGAS/STING. TRIF is the sole adaptor for TLR3 signaling; here, we have investigated whether it is also required for STING signaling.

Methods: To test the requirement of TRIF in gene induction by STING, gene expression was measured by RT-qPCR, Western blot, ELISA and Microarray assays in TRIF -/cell lines and primary cells. To analyze TRIF-STING interaction and to identify their interaction regions, co-immunoprecipitation, pull-down assay and in vitro binding assay were used. STING activation was monitored by measuring its dimerization using biochemical assays and its intracellular membrane mobilization using fluorescent microscopy. The need of TRIF, for STING-mediated regulation of HSV1 replication, was studied by measuring viral and cellular gene expression and viral DNA synthesis.

Results: In mouse and human cells, IFN and other cytokine genes were not induced in TRIF -/cells after treatments with various STING ligands, such as cGAMP or c-di-GMP. Transcription profiling analysis showed that STING required TRIF for inducing all genes. The effect of TRIF was mediated by its direct interaction with STING via the carboxyl terminal domains of the two proteins. TRIF enhanced STING dimerization and membrane mobilization: dimeric STING was undetectable in TRIF -/cells after ligand treatment and distinct clustering of STING was observed upon ligand stimulation of Wt cells, but not TRIF -/cells. HSV1 infection triggers STING signaling to induce IFN-stimulated genes (ISGs) which inhibit virus replication. However, HSV1 could not induce ISGs expression in TRIF -/cells and the virus replicated more efficiently in the absence of TRIF. Others have observed association of specific TRIF mutations with Herpes Simplex Encephalitis in patients; we could attribute this association to defective STING signaling causing enhanced virus replication. For example, the mutant, TRIF P625L, is associated with Herpes Simplex Encephalitis; although it can mediate TLR3 signaling, we showed that it was defective in STING signaling because of its inability to bind STING. Consequently, HSV1 replicated more efficiently in TRIF P625L cells than in Wt cells.

We have uncovered an unexpected and obligatory need of TRIF for STING signaling, thus revealing a new connection between the two intracellular nucleic acid recognizing PRRs, TLR3 and STING. TRIF mediated STING signaling by promoting STING dimerization and membrane mobilization. Our results also indicate that defective STING signaling could be responsible for the observed genetic association between TRIF mutations and Herpes Simplex Encephalitis in patients. Introduction: The innate immune system represents a major barrier to viral infection and innate immunity is the first line of host defense. Myocarditis is clinically defined as inflammation and injury of the heart muscle, which can be brought on by an assortment of viruses and autoimmune processes then cause disease in target tissues. Coxsackie virus usually associated with virus infection mediated myocarditis.

Methods: To calculate lethal dose (LD50), 6-8 weeks old type1 interferon receptor knockout mice and wild-type mice were infected with different dose of CVB3 infected by intra-peritoneal injection (I.P.).

Elisa and qRT-PCR were used to measure cytokines and chemokine expression level. Tissues and blood were collected from infected wild-type mice. Plaque assay were used to titer the virus of different tissues from infected mice.

Results: We shown that the type1 interferon pathway determines the outcome of infection as type 1 interferon receptor knockout (IFNAR) mice are highly susceptible to CVB3 infection, animals infected with 10 5 Plaque-forming unit (PFU) die within 7 days, while the immuno-competent mice survived infection with when with 10 8 PFU. Next we examined the virus tissue distribution and demonstrated that IFN determines its ability to establish infection and replicate in specific tissues. Then we study induction of interferon beta and interleukin 1 beta from serum and tissues following CVB3 infection. We found that CVB3 induces tissue specific innate response such as type 1 interferon (IFN) response and inflammation response (IL1b, interleukin 1 beta) at the acute phase of virus infection until virus clearance. At late stages of infection, the wildtype mice developed robust neutralizing antibody response. We found that type 1 interferon regulates both inflammation response and adaptive immunity during CVB3 virus infection.

Conclusion: CVB3 induce tissue specific innate response such as type 1 interferon (IFN) response and inflammation response. Type 1 interferon is critical to control virus infection and regulate inflammation response, adaptive immunity during CVB3 virus infection in mice.

Disclosure of Interest: None declared. Introduction: The transcription factor ISGF3, comprised of IRF9 and tyrosine-phosphorylated STATs 1 and 2, transmits the signal from the type I interferon receptor to the genome. In ISGF3, STAT2 provides a potent transcriptional activation domain that recruits additional co-factors required to initiate gene expression. Compared to other STATs, relatively few post-translational modifications of STAT2 have been described. We now find that a major fraction of STAT2 is constitutively phosphorylated on threonine 387 (T387) in most untreated cell types, and that this phosphorylation inhibits signaling in response to type I IFN.

We used mass spectrometry to analyze STAT2 immunoprecipitated from 293T cells expressing the flag-tagged wild-type protein. After discovering STAT2 T387 phosphorylation, we mutated T387 to alanine to prevent its phosphorylation and then expressed either FLAG-tagged wild-type or T387A STAT2 in STAT2-deficient U6A cells and immortalized human mammary epithelial (HME) cells, followed by analysis of the IFN-b-induced expression of some typical ISGs by quantitative real-time PCR (qPCR) and Illumina Gene Expression Array. Besides ISG induction, we also tested the ability of IFN-b to inhibit virus replication and cell growth in these cells. To investigate the mechanism of T387 phosphorylation, we raised an antibody against T387 phosphorylated STAT2 and performed electrophoretic mobility shift assays (EMSAs) to test the DNA binding ability, and immunoprecipitation for ISGF3 integrity.

We have discovered a novel phosphorylation of STAT2 on T387 that negatively regulates the response to type I IFNs. In most untreated cell types, the majority of STAT2 (85% in 293T cells) is phosphorylated on T387 constitutively. In response to interferon-b, the T387A mutant of STAT2 is much more effective than wild-type STAT2 in mediating the expression of many interferon-stimulated genes, in protecting cells against virus infection, and in inhibiting cell growth. The level of STAT2 T387 phosphorylation varies in different cells and decreases after long-term treatment with IFN-b. T387 phosphorylation increases the level of STAT1/STAT2 heterodimers lack of tyrosine phosphorylation and decreases the amount of ISGF3 that is formed in response to IFN-b. Interferon-b-treated cells expressing wild-type STAT2 contain much less ISGF3 capable of binding to an interferon-stimulated response element than do cells expressing T387A STAT2. T387 lies in a cyclin-dependent kinase (CDK) consensus sequence, and CDK inhibitors decrease T387 phosphorylation.

The findings described in this study indicate that CDK inhibitors may accelerate and augment responses to type I IFNs by decreasing the phosphorylation of T387 of STAT2. Co-administration of a CDK inhibitor and type I IFN might be a novel strategy for treating cancers such as melanoma, which respond significantly to this therapy, and for treating multiple sclerosis and virus infections. Much more work remains to be done, including understanding how the phosphorylation of T387 modulates the interaction between U-STAT1 and U-STAT2, determining which CDKs catalyze T387 phosphorylation, and determining which phosphatases lead to T387 de-phosphorylation in response to IFN-b.

Disclosure of Interest: None declared. Methods: We analyzed flies that had mutations in Endoplasmic Reticulum (ER) stress machinery and found that ER stress was involved in the expression of immune response genes. From the gene expression profile, we identified the molecular mechanism of antiviral ER stress.

Results: We report that ER stress facilitates the expression of humoral proteins and activates the Jak-Stat pathway. Additionally, we show that ER stress regulates antiviral RNAi immunity, which cleaves viral RNA and suppresses viral replication in insects. Collectively, our study indicates that Drosophila antiviral immunity requires ER stress to activate an immune signaling pathway that leads to the expression of antiviral proteins.

Conclusion: ER stress is a highly conserved mechanism among eukaryotes, and mammalian studies have indicated that ER stress plays important roles in inflammation and antimicrobial immunity. Our research suggests that immunological ER stress is an evolutionarily conserved mechanism and would provide new insight into the evolution and adaptation mechanisms of arthropod-borne viruses such as Dengue virus and Zika virus. Introduction: IL-17 plays critical roles in host defense against extracellular bacterial and fungal pathogens through its heterodimeric receptor complex formed by IL-17RA and IL-17RC. IL-17R signaling has been shown to regulate both granulopoiesis as well as chemokine production leading to the optimal recruitment of neutrophils and the efficient eradication of various pathogens. Whereas IL-17 is predominantly produced by the lymphocytes, chemokines are thought to be made by IL-17 target cells.

Methods: IL-17 plays critical roles in host defense against extracellular bacterial and fungal pathogens through its heterodimeric receptor complex formed by IL-17RA and IL-17RC. IL-17R signaling has been shown to regulate both granulopoiesis as well as chemokine production leading to the optimal recruitment of neutrophils and the efficient eradication of various pathogens. Whereas IL-17 is predominantly produced by the lymphocytes, chemokines are thought to be made by IL-17 target cells.

Results: The conditional KO mice displayed an impaired ability to recruit neutrophils into the airway lumen and as well as a defect in controlling the bacterial burden upon infection with K. pneumoniae including the drug resistant isolate expressing New Delhi Metallobeta-lactamase-1. The impairment is associated with reduced epithelial expression of Cxcl5 and this defect could be restored by recombinant LIX, the gene product of murine Cxcl5.

Conclusion: Our data confirmed the requirement of epithelial IL-17R expression in the host defense against K. pneumoniae and suggested that agonizing epithelial IL-17R may serve as a novel therapeutic target in combating K. penumoniae including antibiotic resistant strains.

Disclosure of Interest: None declared. Introduction: BTK, a Tec kinase plays a critical role in B cell development. Mutations in the Btk gene result in B-cell immunodeficiency in humans. However, these patients are also known to be very susceptible to recurrent bacterial and viral infections, suggesting BTK's role also in innate immunity. We showed that absence of BTK impaired host interferon response. As a result, BTK-deficient mice are more resistant to p(I:C)-induced sepsis, suggesting that inhibiting BTK could be used to dampen the ''cytokine storm" resulting from exaggerated host immune response as observed in SARS coronavirus or Ebola virus crisis.

Methods: Computational modelling indicated that phospho-Tyr414 strengthens DDX41's interaction with STING.

Results: The innate immune system also senses foreign DNA and bacteria by initiating signaling via the adaptor STING to induce IFN response via DNA sensors like DDX41 helicases. BTK-deficient cells also have impaired IFN-b production when stimulated with STINGsignaling agonists or infected with infectious pathogens such as malaria parasites and Listeria monocytogenes. BTK phosphorylates DDX41 and is critical for STING-mediated IFN-b production.

We propose a biologics application in the generation of novel antibodies against Tyr414 of DDX41 as an antibody-based diagnostic biomarker of human viral diseases as well as screening and identifying novel BTK inhibiters and activators.

Disclosure of Interest: None declared. Introduction: Interferon Regulatory Factors (IRFs) are key transcription factor for innate immunity. While IRF3 and IRF7 are wellestablished factors for orchestrating an antiviral immune program, little is known about the specific role of IRF5 in response to virus infection. Our studies indicate that IRF5 plays a critical role in effective immune response to West Nile virus (WNV). Upon infection, bone marrow-derived dendritic cells (BMDCs) lacking IRF5 are defective in producing type I interferon (IFN-I), resulting in uncontrolled viral replication. Expression of other inflammatory cytokines and chemokines in response to WNV infection is also dependent on IRF5. These observations suggest that the effector function of IRF5 target genes is an essential component of anti-WNV immunity. However, few IRF5 transcriptional targets have been identified, its distinct or overlapping role with other IRFs has not been defined, and the overall actions of IRF5 in programming DC effector function are not known.

Methods: In this study, we addressed this question by a comprehensive genome-wide interrogation of BMDCs upon WNV infection. We undertook large-scale transcriptional profiling and high-resolution chromatin occupancy mapping to identify bona fide IRF5 primary target genes. First using an RNAseq approach, we compared the gene expression profiles of WNV-(and mock-) infected wildtype and Irf5-knockout BMDCs to identify WNV-responsive genes whose expression requires the presence of IRF5. We then performed chromatin-immunoprecipitation followed by sequencing (ChIPseq) in WNV-and mock-infected BMDCs to hone in on direct gene targets where IRF5 binding is induced upon WNV infection.

Results: The intersection of these two approaches allowed us to interrogate the global function of IRF5 in orchestrating antiviral immunity. Further gene ontology and network analyses revealed cellular pathways that constitute an IRF5-specific immune program, which likely plays a distinct and complementary role with other IRFs to coordinate an effective antiviral response.

Conclusion: Here, we present data that characterize the transcriptional antiviral program orchestrated by IRF5 in BMDCs for effective control of WNV infection.

Introduction: Human STING, encoded by the TMEM173 gene, is an innate immune sensor for cyclic dinucleotides (CDNs) critical in infection, inflammation, and cancer. We previously identified HAQ (R71H-G230A-R293Q) as the second most common human TMEM173 allele carried by billions of people worldwide. The in vivo significance of this HAQ allele remains unknown. Methods: In this study we used ''Il10 BAC-in transgene" (10BiT) IL-10 reporter mice to identify the cellular sources of IL-10 during infection with the hyper-virulent W-Beijing M. tuberculosis strain HN878.

Results: We observed that during the first 14-days of M. tuberculosis infection the predominant cells expressing IL-10 were innate Ly6C +CD11b+ monocytes. However, after day 21-post M. tuberculosis infection, IL-10 expression was also strongly detectable in T cells.

Mice deficient in T cell derived IL-10 (Il10 flox/flox CD4-Cre+ mice), but not mice deficient in monocyte derived IL-10 (Il10 flox/flox LysM-Cre+ mice), showed a significant reduction in lung bacterial loads during chronic M. tuberculosis infection when compared to fully IL-10 competent control mice. IL-10-expressing T cells from M. tuberculosis infected lungs were detected among both CD4+ and CD8+ T cells, expressed high levels of Tbet and CD44 and were able to co-produce IFN-k upon ex vivo stimulation. Additionally, Il10 expression in CD4+ T cells was partially regulated by IL-27 and type I IFN signaling.

Conclusion: In summary, our data reveal that, despite the multiple cellular sources of IL-10, it is mainly T cell-derived IL-10 that suppresses the protective immune response to M. tuberculosis infection.

Disclosure of Interest: None declared. Introduction: The RIG-I signaling pathway is essential for the recognition of viruses and the initiation of host IFN-mediated antiviral responses. Once activated, RIG-I interacts with polyubiquitin chains generated by TRIM25 and binds MAVS, leading to the production of type I IFN [1, 2] . We now show the specific interactions and the subcellular localization of key proteins in the pathway.

The interaction of key partners in the RLR pathway was examined by Bimolecular Fluorescence Complementation (BiFC) and Stimulated Emission Depletion (STED) Microscopy [3, 4] . This technique allowed us to isolate, visualize and analyze proteinprotein interactions in living cells.

Results: Dimers of RIG-I, TRIM25 and MAVS localize into different compartments in the cytoplasm of the cell. RIG-I/TRIM25 complexes accumulate in specific cytoplasmic dots whereas RIG-I/MAVS complexes are located similar to MAVS on the mitochondria. In addition, MAVS competes with TRIM25 for RIG-I binding, and this suggest that upon TRIM25 mediated activation of RIG-I, RIG-I moves away from TRIM25 to interact with MAVS at the mitochondria. For the first time, the distribution of the proteins was also analyzed in virus infected cells. We will show how different viral proteins modify specific complexes in the pathway.

The BiFC technique provided us with a tool that allowed us to define the spatial events and the different interactions among RIG-I, TRIM25 and MAVS in the RLR pathway. We were also able to show this distribution at a certain time point in the context of viral infection, showing again a striking formation of complexes displayed in different locations within the cell. Introduction: The human myxovirus resistance proteins MxA and MxB are expressed upon stimulation with type I and type III interferons (IFNs) induced by viral infection. While MxA is wellknown to elicit potent antiviral effects against a multitude of RNA and DNA viruses, MxB has long been unrecognized to serve any antiviral function. In 2013, three independent research groups provided first clear evidence for a potent antiviral effect of MxB against human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses. Subsequent evolutionary analyses however concluded that the amino acid motifs associated with lentiviral restriction were not subject to positive selection in primates. In addition, the residues that are associated with MxB GTPase function are highly conserved among mammals, but the capacity to bind and/ or hydrolyze GTP is not required to inhibit HIV-1 replication. Altogether, this indicates that MxB has additional antiviral (but non-lentiviral functions) that are yet to be uncovered.

Here, we argued that other potential viral targets of MxB would have a nuclear targeting process similar to HIV-1. Thus, we selected the large DNA virus herpes simplex virus type 1 (HSV-1) as promising candidate to be subjected to MxB susceptibility tests. First, we selected T98G cells as a cell culture model due to their high endogenous MxB expression and permissiveness to HSV-1 infection. Then, we tested MxB in absence of other interferon-induced genes by generating A549 cells stably overexpressing MxB (A549-MxB). In order to study HSV-1 replication as a function of MxB levels in the cells, we employed siRNAs targeting endogenous and/or exogenous MxB.

Results: We observed that the inhibition of HSV-1 replication in T98G cells after IFN treatment was reduced in presence of siRNAs targeting MxB, underlining the importance of MxB in the IFNmediated suppression of HSV-1. Furthermore, our results indicate that HSV-1 replication is strongly inhibited in A549-MxB cells as compared to control cells. In line with this, HSV-1 replication in A549-MxB cells recovered to a large extent after siRNA-mediated knock-down of MxB transgene expression. On the other hand, vesicular stomatitis virus (VSV), an RNA virus that is not dependent on nuclear entry to accomplish replication, was unaffected by the presence of MxB or MxB-directed siRNAs.

Conclusion: In summary, we established that MxB is a specific and effective HSV-1 restriction factor. Future experiments will help to elucidate the mechanisms that are responsible for the antiherpesviral functions of MxB.

Disclosure of Interest: None declared. Introduction: Toll-like receptors (TLRs), predominantly expressed in host immunocompetent cells, play a critical role in the early innate immune response to invading pathogens by sensing microorganisms. Although TLR2 is essential for the recognition of gram-positive bacteria, its function in infectious diseases is still debated. Rhodococcus aurantiacus is an intracellular gram-positive bacterium that induces granulomatous inflammation, resembling sarcoidosis, in wild-type (WT) mice. In the present study, we investigated the extent to which TLR2 contributes to the host innate response to this bacterium infection and to late granuloma formation using TLR2deficient mice.

Methods: After intravenous inoculation of 1 Â 10 8 CFUs of viable R. aurantiacus in PBS, the survival of WT and TLR2-deficient mice (7-8 week old females) was monitored and bacteria in their organs were enumerated; PBS alone was injected as a control. Local and systemic cytokine production and morphological changes in liver were assessed by ELISAs and H&E staining, respectively. Following stimulation with heat-killed R. aurantiacus (MOI of 2), the protein concentrations and gene expression levels of cytokines in mouse peritoneal macrophages were measured by ELISAs and real-time PCR, respectively.

Results: TLR2-deficient mice were hypersusceptible to R. aurantiacus infection and displayed high mortality rates compared to their WT counterparts. These mice also showed decreased production of proand anti-inflammatory cytokines including TNF-a, IFN-k, IL-6, and IL-10 in the liver during the early phase of infection, and a high bacterial load in the organs. Moreover, histological studies revealed a reduction in the number and size of hepatic granulomas at day 14 post-infection in TLR2-deficient mice. Upon stimulation with heatkilled bacteria, macrophages from TLR2-deficient mice showed lower expression of TNF-a, IL-6, and IL-10 at both the gene and protein level compared to that of WT mouse macrophages.

Conclusion: TLR2 deficiency attenuates not only the inflammatory cytokine response but also the anti-inflammatory cytokine response to R. aurantiacus infection. Decreased production of pro-inflamma-tory cytokines contributes to a reduction in bacterial elimination from organs in the early phase of infection and inhibition of hepatic granuloma formation during the late phase. However, the decreased level of anti-inflammatory cytokine IL-10 exacerbates mouse survival, as we previously showed that IL-10 is a key element in protecting mice from mortality. These findings thus indicate the essential role of TLR2 in regulating the activation and function of immunocompetent cells, especially macrophages, during the initial phase of systemic R. aurantiacus infection.

Disclosure of Interest: None declared. Introduction: Tyrosine kinase 2 (TYK2) is a Janus kinase with a pivotal role in immunity to infections, tumour surveillance and cytokine signalling, most prominently type I interferons (IFN) and interleukin 12 (IL-12). Previously it was shown that TYK2-deficient (Tyk2 -/-) mice are highly susceptible to Listeria monocytogenes infection due to impaired IFNk production by T cells. The role of other cell types and the molecular mechanisms responsible for the decreased survival in the absence of TYK2, however, are ill-defined.

Methods: To investigate kinase-independent and cell type-specific functions of TYK2 we challenged kinase-inactive mutant TYK2 (Tyk2 K923E ) and conditional TYK2 knockout mice (Tyk2 fl/fl ) intraperitoneally with L. monocytogenes. Survival rates and bacterial burden in spleen and liver were determined. Furthermore, IFNk serum levels were evaluated using enzyme-linked immunosorbent assay (ELISA). In addition, splenocytes of mice lacking TYK2 in the NK cell compartment (Tyk2 ÄNcr1 ) and of Tyk2 fl/fl were tested for intracellular expression of IFNk by flow cytometry.

Results: In contrast to Tyk2 -/mice, infected Tyk2 K923E mice produce IFNk, although significantly delayed compared to wild-type (WT) mice. Nevertheless, upon high dose of infection Tyk2 K923E mice exhibit a similar survival as Tyk2 -/mice. Surprisingly, upon challenge with a low dose of L. monocytogenes Tyk2 K923E mice display higher bacterial burden than WT mice and succumb to infection. Thus, our data reveal that despite late IFNk production, expression of kinase-inactive TYK2 is not sufficient to protect mice against low-dose systemic L. monocytogenes infection and worsens disease compared to complete loss of TYK2. Regarding cell typespecific functions of TYK2, our data unveil a critical role of TYK2 in NK cells in the anti-Listeria immunity. In contrast, mice with a macrophage/neutrophil-specific deletion of TYK2 were more resistant to infection. Deletion of TYK2 in dendritic cells did not grossly impact on survival.

Conclusion: We thus hypothesize that due to its employment in different cytokine pathways and cell types, TYK2 exhibits both protective and detrimental functions in the immune response against bacterial infections. Introduction: Interferon stimulated genes (ISGs) are a diverse set of genes upregulated in response to interferon and are major components of cellular immunity to intracellular pathogens such as viruses. Despite the importance of these ISGs in cellular immunity, the functions of most ISGs are unknown. In recent screening efforts, we found multiple ISGs to be inhibitory to the infection of a panel of viruses. Interestingly, a small number of ISGs were found to enhance infection of multiple viruses. One of the more robustly enhancing ISGs was mucolipin-2 (MCOLN2), which promoted infection by a diverse set of viruses. The aim of this study is to determine the mechanism of action of MCOLN2 in viral infection to in turn better understand the purpose of seemingly enhancing viral infection from the host standpoint.

Methods: Lentiviral ectopic expression and CRISPR/Cas9-based gene editing were used to modulate MCOLN2 expression. Viral infectivity was quantitated using a flow cytometry based system. Viral replication was quantified by plaque assays or using subgenomic replicons. Experiments analyzing viral entry used RT-qPCR based, cell fractionation based, and confocal microscopy based approaches.

Results: We found that ectopic expression of MCOLN2 enhanced infection of multiple, unrelated, enveloped viruses. These included influenza A virus, yellow fever virus, and equine arterivirus. A role for MCOLN2 in viral infection was confirmed with CRISPR/Cas9 mediated knockout experiments. Mechanistic studies showed that MCOLN2 specifically enhances viral entry after receptor mediated endocytosis of the virus but prior to viral replication.

We have shown that MCOLN2 is important for the optimal infection of multiple, clinically significant and unrelated viruses in vitro. Mechanistic studies have found that MCOLN2 affects a specific stage in the viral life cycle prior to replication yet after endocytosis of the virus. Further studies will help to clarify the role of enhancing ISGs such as MCOLN2 in cellular immunity. Introduction: Host genetic background plays a key role in determining the outcome of pathogenic processes, such as infections, but how and at what extent remains to be established. The individual genetic profile can influence the cytokines network during the early phases of respiratory infections, orchestrating innate immunity. Here, we aim to define the host genetic profiles associated to cytokines networks and their relevance on disease severity during P. aeruginosa respiratory infection.

Methods: To achieve our aim we exploited a system biology approach and in vivo model of acute respiratory infection using a highly genetically-diverse mouse resource population, named the Collaborative Cross (CC) mice. To model acute pneumonia, 23 CC inbred lines were challenged by P. aeruginosa, monitoring disease severity including mortality and morbidity up to seven days. Next, we quantified the specific markers of disease phenotypes (3 mice/ each inbred line), including bacterial load and a panel of 41 cytokines and chemokines (by Luminex technology) in the murine lung at early stage (20 h) of infection.

Results: The CC murine population exhibited a broad range of pathological phenotypes ranging from lethal disease to complete recovery from the infection. We defined two distinct groups of CC inbred lines, ''resistant" (11 CC inbred lines) and ''susceptible" (12 CC inbred lines), performing cluster analysis based on mean survival time, bacterial burden and the levels of KC, MIP-2 and TNF-a. Next, when ''resistant" and ''susceptible" CC inbred lines were directly compared, we found that 15 cytokines out of 41 were differently secreted. In particular, we observed that disease severity is associated with a reduced bioavailability of IL-12p40 and increased levels of IL-12p70 and IL-17A, while the levels of IFN-k and IL-23 were similar between the two groups. Of note, the disease dynamic associated to the cytokine network was generally conserved among CC inbred lines, although single cytokine profiles may have specific regulation, probably due to the individual genetic background.

In conclusion, the diversity of host genetic profile determines the outcome of the respiratory infections through the regulation of highly conserved immunological mechanisms. In particular, we suggest that balance of the IFN-k/IL-17 mediated response may be critical in determining disease severity during P. aeruginosa respiratory infections in genetically-diverse murine population.

Disclosure of Interest: None declared.

Introduction: Certain bat species are asymptomatic carriers of highly pathogenic viruses, but the mechanisms underlying viral resistance remain unclear. Characterization of the antiviral mechanisms in bats may provide insights that could be used to develop targeted antiviral therapies. A key defense mechanism against viral disease is the rapid activation of the interferon (IFN) response, which results in the expression of a pool of antiviral effectors known as IFNstimulated genes (ISGs). We are studying the IFN/ISG system in Pteropus alecto bat cells to determine whether IFN signaling or ISG effector functions have unique features in this species.

Methods: We used RNA-seq to define the temporal regulation of ISGs by type I IFN in bat cells. nCounter Nanostring technology was used to validate RNA-seq data as well as compare ISG induction between bat and human cell lines. To study functional differences between bat and human ISGs, we used a high-throughput, flow cytometry-based screening assay to compare the antiviral activity of 80 bat ISGs relative to their human orthologs.

Results: We observed that treating bat cells with IFN induces the expression of genes that have been identified as ISGs in humans as early as 2 h after treatment. Bat cells seem to have a faster and more potent response to IFN than human cells as measured by ISG induction. Using our functional screens, we identified bat IRF7 as a potent antiviral effector across multiple virus families. Bat IRF7, unlike human IRF7, is capable of inducing an IFN-like antiviral state in cells in the absence of viral challenge, even in cells lacking an intact IFN pathway. Results: We assessed the relative in vivo contributions of the different DC subsets involved in priming of HSV-specific CD8 + T cells.

Assessing the endogenous, polyclonal HSV-specific CD8 + T cell response, we found that effective in vivo T cell priming was dependent on the presence of DC subsets specialized in crosspresentation, while Langerhans cells and plasmacytoid DCs were dispensable. Crucially, we demonstrate in vivo that this requirement for cross-presenting DCs was not related to their infection, but instead reflected their true capacity to cross-present HSV-derived antigen in vivo.

Conclusion: These findings are consistent with the view that skinresident DCs, either directly infected or containing virally infected material, migrate from the skin to the draining lymph nodes and hand over their cargo to LN-resident CD8a + DC for priming of CD8 + T cells. This work demonstrates a division of labor between distinct DC subsets, highlighting that in vivo HSV-specific CD8 + T cell priming depends heavily, if not entirely, on cross-priming by DCs.

Disclosure of Interest: None declared. demonstrating an initial lack of pro-inflammatory cytokine response, followed by an exponential increase (termed Cytokine Storm) 1-2 days prior to death. Both of these aspects contribute to the pathogenesis of the disease and therefore represent potential targets for the generation of post-exposure therapeutics.

We have developed a suboptimal antibiotic mouse model of infection to determine the full impact of cytokine storm during infection.

Results: Over the last few years our laboratory has focused on the use of anti-inflammatory compounds (either alone or in combination with antimicrobials) to potentially increase survival and/or extend time to death. We have demonstrated proof of principle that the use of a host-targeted therapy (i.e. anti-HMGB1 antibodies), in combination with levofloxacin during a F. tularensis Schu S4 infection can increase survival and widen the window of opportunity for traditional antibiotic treatments. Further, using immuno-histochemistry, the potential role of macrophages and regulatory T cells in the pathogenesis of the disease was also identified. Recently we have explored the role of regulatory T cells, which appear to be absent during the early stages of infection with F. tularensis, and the consequences of this in the context of immune dysregulation. Using DEREG mice, which specifically deplete FOXP3 regulatory T cells through the administration of diphtheria toxin, we have shown that bacterial burden in the liver and spleen is significantly reduced compared to wild type controls during an infection with F. tularensis live vaccine strain.

Conclusion: Our ongoing research on the host immune response looks to establish the key immune cells/molecules during infection in order to improve the likelihood of identifying a novel (hosttargeting) post-exposure therapeutic that can treat tularemia and/or increase the efficacy of existing antimicrobials. Introduction: Cytosolic sensing of nucleic acids alerts cells of invading pathogens and leads to initiation of programs aimed at limiting infection. These programs are mediated by cytosolic receptors and cofactors for DNA (c-GAS/STING) and RNA (RIG-I/ IPS-1) sensing and include initiation of pro-inflammatory responses -tight regulation is paramount so as to limit collateral damage. Cell cycle and fertilization represent two scenarios when an inappropriate response to non-pathogen-derived nucleic acids would have dramatic consequences.

We report on an unbiased approach to identify negative regulators of nucleic acid sensing and the discovery of a putative rheostat during fertilization.

Results: We demonstrate that this regulator interacts physically with components of nucleic acid sensing pathway, targets TBK1 (a kinase critical for signaling downstream of both DNA and RNA sensing) for ubiquitination and degradation, and delineate domains that mediate the inhibitory functions. Furthermore, we identify a naturally occurring human nonsense mutation associated with male sterility that results in loss of protein function and hyper responses to nucleic acids.

Conclusion: This discovery points to a fundamental requirement to control nucleic acid sensing machinery and suggest that its regulation may be critical in germline.

Disclosure of Interest: None declared. Results: Human pDCs picked up FLARE signal in a dose-dependent manner, which was inhibited by cold conditions. A very low percentage of cells that contained AF633 signal also contained DSRed signal, indicating decreased spore viability after uptake by pDCs. Dectin-1 expression on the surface of human pDCs was comparable to that of human monocytes, which are known to express Dectin-1. Dectin-1 was upregulated after stimulation with all viral stimuli. Curdlan induced the upregulation of costimulatory markers CD40, CD83, CD80, and CD86, as well as the production of IL-6 (but not IFN-a) and the upregulation of Dectin-1 itself. Finally, pDCs from HIV patients expressed modestly higher baseline levels of Dectin-1 on their cell surface, which remains consistent after curdlan and viral stimulation.

We have shown that human pDCs phagocytose and kill Aspergillus spores directly. Dectin-1 expression, regulation, and IL-6 responsiveness of pDCs after curdlan stimulation indicates Dectin-1 is present and functional on human pDCs, suggesting Dectin-1 may be used by human pDCs to recognize the b-glucan component of fungal spores. Finally, the higher baseline Dectin-1 levels on HIV patient pDCs may be explained by low-level chronic immune activation experienced by HIV patients, which has been described to negatively affect pDC functionality.

Disclosure of Interest: None declared. Methods: IL-10 and IL-12 cytokine detection was performed by ELISA method in supernatant of splenic OX-62+CD4+ and CD4dendritic cells culture of Plamodium berghei infected rat.

And then production of cytokine was correlated to either age anaimal or to the survival.

Results: While 100% of young rats were succumbed to infection, all adults survived and cleared blood parasites. CD4 phenotypic analysis of spleen dendritic cells revealed an age-related distribution of two subsets of cells. While OX62+CD4-dendritic cells were associated with susceptibility and induced Th2 cytokines profile through high amounts of IL-10, in young rats and OX62+CD4+ dendritic cells exhibited Th1 cytotoxicity profile with high amounts of IL-12 and increased CD8 lymphocytes, NK cells in resistant adults rats.

Conclusion: Our investigation focused on malaria protective mechanisms that may link to parasites cytotoxicity induced by dendritic cells that maturity might be related to the expression of CD4 phenotype in non susceptible adults.

Disclosure of Interest: None declared. Introduction: Toll like receptor signaling (TLRs) is known to be initiated in multiple cell types by exposure to a variety of DAMPs following tissue injury. We have studied the role of TLR signaling pathways, particularly TLRs3/4 in regulating the acute response to injury using the acetaminophen (APAP) induced acute liver injury model in mice.

Methods: Age matched C57Bl/6 and gene knock out male mice were fasted overnight for about 16 h prior to intraperitoneal injection of 300 mg APAP/kg body weight.Serum ALT/AST were measured from whole blood and hepatic damage was assessed from formalin fixed paraffin sections stained with H&E.Sections were stained with fluorescentley labelled K167 to detect cell proliferation and cyclin D1 was measured from whole liver total protein extract by western blotting.

Results: Mice with deficiency in TRIF, one of 2 adaptor proteins that determine signaling events downstream of TLRs3/4, exhibit exacerbated response to intraperitoneal injection of APAP. This is reflected in elevated serum AST/ALT levels and increased areas of necrosis visualized by H&E staining of liver sections. Comparison of age matched wild type and TRIF-deficient mice showed markedly enhanced hepatocyte proliferation evidenced by increased Ki67 staining and cyclin D1 expression up to 72 h after APAP challenge. Comparable outcomes were observed in mice deficient in the transcription factor IRF3 and the interferon inducible genes IFITs1-3 implicating a significant role for interferon signaling in reducing injury.

Conclusion: APAP overdose results in rapid hepatocyte cell death due to the generation of toxic metabolites while recovery from injury depends upon the infiltration of inflammatory monocytes. Further experiments are underway to evaluate the roles for IFN signaling in distinct populations to test the hypothesis that the response to injury involves interplay between multiple cell types.

Disclosure of Interest: None declared. Introduction: Ras GTPase-activating Protein-binding protein 1 (G3BP1) is ubiquitously expressed in mammalian cells and plays diverse biological roles. Notably, it is a key component of stress granules, which are formed within the cell cytoplasm upon various stress signals. Recent studies have shown that stress granule formation impairs viral replication, and some viruses express proteins that cleave G3BP1 and inhibit stress granule formation, suggesting a potential role for G3BP1 and stress granules in anti-viral response.

Methods: siRNA-knockdown of G3BP1 results in impaired poly:(IC)mediated IFN production.

Results: Here, we showed that G3BP1 plays a critical role in driving IFNb production following Poly:(IC) stimulation of RAW264.7 macrophages. Mechanistically, G3BP1 binds TBK1 and is required for Poly:(IC) induced IFN production. G3BP1 also interacts and acts synergistically with Downstream of Kinase (DOK)-3, which has been found earlier to act as a critical mediator in TLR-3 dependent TRIF signaling. We also showed that in addition to conventional stress signals (Heat shock, Arsenite treatment), stress granule formation can be induced by Poly:(IC) stimulation on RAW264.7 cells, indicating a link between stress granule formation and IFN signaling.

Conclusion: G3BP1 participates in host innate stress-granules formation and IFN production upon virus infection. Methods: Methods for propagation of mouse norovirus, infection of mice, and analysis of viral growth and the immune response have been previously described [1] [2] [3] [4] .

Results: We previously found that IFNk is critical for control and clearance of CR6 from the intestine and functions independent of B and T cells. However, IFNk receptor deficiency does not allow intestinal CW3 replication, emphasizing the importance of viral genes in determining tropism. In the present study we find that selective IFNab receptor deficiency is sufficient for systemic CW3 persistence in spite of functional B and T cell responses. CW3 has a cellular tropism for macrophages and dendritic cells; we find that deletion of the IFNab receptor on CD11c-or LysM-expressing myeloid cells but not on Villin-expressing intestinal epithelial cells results in increased early viral titers and systemic persistence without persistent fecal shedding. Persistence in this setting is correlated with increased cellular and humoral adaptive immune responses that control but do not clear CW3 infection. Furthermore, increased early replication and persistence are also observed in mixed bone marrow chimeras where 50% of the CD11c-positive DCs are unable to respond to IFNab demonstrating that a cell-intrinsic IFNab response is required for control and clearance of systemic norovirus infection.

Conclusion: Taken together with prior studies, these findings indicate that (1) norovirus tropism determines the relative importance of IFNab compared to IFNk and (2) that the IFN response regulates norovirus persistence independent of adaptive immune function.

Disclosure of Interest: None declared. Introduction: Convergent evolution dictates that diverse groups of viruses will target the same host factors in order to manipulate the immune response and improve infection. Yet, immunity to infection is not a linear response; complex interactions govern disease outcome and successful viruses must employ a combination of approaches to combat host immunity. Even among closely related viruses, diverse strategies may produce similar infection results achieved through distinct mechanisms. Therefore, susceptibility to a particular host factor may not be uniform across a viral family or apply to all viruses infecting the same tissue. In this project, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome.

Methods: Building upon an established systems biology platform, we compared the host immune response across a variety of phenotypic outputs following infection with highly pathogenic coronaviruses (SARS & MERS-CoV) as well as pathogenic influenza A viruses (H5N1-VN1203 & H1N1-2009). Our results identified differential regulation of a subset of interferon stimulated genes (ISGs) following infection. Comparing across RNA expression, proteomics, and epigenetics data, we have developed a consensus list of host ISGs differentially regulated between and within viral families. We predict that modulation of an ISG by one or more viruses indicates a possible role in host immunity to virus infection. From this list, we utilized both in vitro siRNA knockdown as well as Crisper generated knockout mice to identify ISGs with efficacy against viruses that did not manipulate those targets.

Results: Our initial screen identified several ISG targets with differential activity following infection including Ace2, Serping1, Lamp3, and others. The absence of Serping1 or Lamp3, down regulated by MERS-CoV in vitro, produced increased pathogenesis relative to control mice following SARS-CoV challenge. Importantly, this increased susceptibility was not governed by increases in viral replication, but rather altered aspects of pathogenesis including cytokine stimulation, immune infiltration, and overall immune activation. Further studies seek to identify the mechanisms of action for multiple target ISGs.

The data highlights the utility of leveraging viral cross comparisons as a means to identify novel host factors impacting disease outcomes. The results identify multiple targets that have an impact on pathogenesis, but were unlikely to be explored using traditional in vitro screens based on viral replication. Together, the studies define a new approach to define critical host factors impacting viral infection and pathogenesis. Introduction: Obesity is characterized as a public health concern worldwide mainly in childhood and adolescence period. The origin of obesity-related complications is the result of chronic adipose tissue and systemic inflammation. Studies have shown that the release of inflammatory cytokines is related with increased production of inflammatory cytokines.

Objective: Identify and quantify serum levels of IL-1, 6, 8, 10, 12 and TNF-alpha of adolescents with three levels of weight excess.

Methods: A cross sectional study including 97 adolescents from both gender with weight excess and cared for in the Adolescent Outpatient Clinic. The adolescents were allocated into three groups: Overweight (OW), Obese (OB) and Extremely Obese (EO). The study included anthropometric evaluations, IL-6 dosages and biochemical tests to characterize the Metabolic Syndrome. The OW, OB and EO definitions are according to the American Academy of Pediatrics (AAP). Metabolic Syndrome was characterized according to IDF [1] , and central obesity was identified when more than three parameters were altered. Quantification of IL-6 was carried out using the Cytometric Bead Array (CBA) BD TM kit and monoclonal antibodies were marked with PE. Fluorescence was measured by a BDFACS-Canto II flow cytometer, and the number of counted events was 300 particles/events. Values were expressed as pg/mL.

Results: Levels of IL-6 were higher in the EO adolescent group (median; p25-p75: 3.650; 0.37-5.90 pg/mL), followed by the OB and OW groups (median = 0.8 pg/mL; median = 0.09 pg/mL). Levels of IL-6 were also higher in Metabolic Syndrome individuals than in adolescents without the Syndrome (3.17 vs 0.09 pg/mL, p = 0.001). Similar data were observed for IL-1 beta and IL10 levels; Extremely Obese individuals had higher levels of these parameters than the OB and OW individuals.

Conclusion: Levels of IL-1 beta, IL-6 and IL-10 are associated with excess body weight in adolescents, and these parameters have potential such as diagnostic markers for the Metabolic Syndrome in this age group. Introduction: In obesity and metabolic syndrome, manifestation of a latent adipose tissue inflammation and, consequently, systemic inflammation is closely related to disturbance of insulin signaling resulting in the development of insulin resistance and type 2 diabetes. Insulin-dependent glucose uptake via Glut-4 activation is mediated by PI3K/Akt pathway, with phosphorylation of Akt kinase on Thr-308 which is governed by insulin directly and phosphorylation of Ser-473 which mostly depends on mTORC2 activity and also on insulin action. The distortion of this signal in inflammatory conditions could be reduced or abolished by the agents skewing inflammatory M1 macrophages (Mph) to acquire alternative, antiinflammatory M2 phenotype. In our work we studied the influence of activation of SIRT1 in M1 Mph on insulin-dependent Akt phosphorylation in adipocytes.

We used in vitro model of ''chemokine" co-culture of polarized RAW 264.7 (Mph) and differentiated 3T3L1 adipocytes. As a SIRT1 activator, we used novel small-molecule compound DCHC (3-(2,4-Dichlorophenyl)-7-hydroxy-4H-chromen-4-one). Mph M1/ M2 polarization was characterized by real time PCR. Also, we modeled paracrine effect of M0, M1, M2 and M1-DCHC Mphs on differentiated 3T3L1 adipocytes by using macrophage conditioned media (CM). Then, after short-term loading by insulin, 3T3L1 cells were analyzed on Akt phosphorylation by Western-blotting.

Results: We have found that M1-Mphs express predominantly inflammatory markers (TNF-a, CXCL9) while M2-type cells produce mostly anti-inflammatory factors (ALOX-15, IGF-1). M1 Mphs treated with DCHC (M1-DCHC Mphs) were characterised by intermediate expression of inflammatory markers and anti-inflammatory markers which was closer to M2 phenotype. We have found that M1-cell CM exert a decrease of pAkt-Thr308 while increasing pAkt-Ser473 in 3T3L1 adipocytes. These effects were not observed if M2 and M1-DCHC cells' media was used for adipocyte treatment. On the contrary, M2 Mph and M1-DCHC Mph CM induced the increase of pAkt-Thr308 and decrease in pAkt-Ser473.

The decline of Thr-308 phosphorylation in inflammatory conditions may be due to regulatory inhibitory Ser/Thr phosphorylation of IRS-1. This regulatory mechanism can be operated via a variety of protein kinases, including proinflammatory kinase IKK. The proposed mechanism of pAkt-Ser473 increase in inflammatory conditions may be associated with IKK-dependent activation of mTORC2, the possibility of which was shown in some studies. Thus, possible mechanism mediating the influence of polarized Mphs on insulin-dependent Akt-phosphorylation may be associated with divergent regulation of IKK-kinase in target cells. Introduction: Inflammation is a biological response that protects organisms from harmful stimuli. During the occurrence of inflammation, activated immune cells secrete many inflammatory mediators, including cytokines and chemokine, which promote inflammatory responses. Lung and gut share many similarities in physiological and inflammatory pathologies. Cigarette smoke (CS) is harmful mixture, which can cause acute or chronic inflammation. Thus, we investigated CS exposure-induced lung and gut crosstalk inflammatory response.

Methods: C57BL/6 mice were exposed to CS for 2 h per day for 4 weeks. The mice were placed in the smoking chamber, which was filled with smoke from Kentucky reference cigarettes (3R4F). Mice were exposed to CS for 30 min, after which they were rested in a fresh air environment for 30 min. The total duration of exposure to CS was 2 h per day. Individual body weights were measured twice a week, and the mice were sacrificed on day 28. Hematoxylin and eosin (H&E) staining was performed in lung and colon tissue to determine histological changes. Tumor necrosis factor (TNF)-a, interleukin (IL)-10 and monocyte chemoattractant protein (MCP)-1 synthesis in the bronchoalveolar lavage fluid (BALF) and colon tissue were analyzed using enzyme-linked immunosorbent assay (ELISA).

Results: The CS group mice body weights were 5% decreased, while the CON group mice body weights were 13% increased. And, the CS group showed histological changes with infiltration of immune cells surrounding the perivascular and peribronchial regions in the lung. Similarly, CS exposure induced the cell infiltration in the intestinal mucosa. In addition, our data showed the increase of proinflammatory cytokines (TNF-a and MCP-1), and decrease of IL-10 as anti-inflammatory cytokine in the BALF and colon tissue.

Conclusion: Our study confirmed the accumulation of immune cells into the lung and colon tissue, and the excessive secretion of inflammatory cytokines in the both inflamed tissue. These results indicated that CS exposure induces inflammation in the lung as well as bowel.

Disclosure of Interest: None declared. Introduction: Protein arginine methylation is a novel posttranslational modification, which is mediated by protein arginine methyltransferases (PRMTs) [1] . PRMT1, the major isoform of PRMT family, is responsible for diverse biological functions including cellular differentiation [2, 3] . Although the important function of PRMT1 in various tissues is increasingly recognized, its role in RANKL-induced osteoclastogenesis and osteoporosis has not yet been described.

Here we showed that PRMT1 is a key molecule involved in osteoclast differentiation.

Methods: Osteoclastogenesis was determined by TRAP staining, F actin ring formation, and bone resorption assay. siRNA for PRMT1 and scrambled siRNA were used for silencing the endogenous PRMT1 expression. NF-kB and MAPKs activation were analyzed by transfection assay and Western blot, respectively. Real-time PCR was performed to examine the expression of osteoclastogenesis-related genes. Histological changes, increase of TRAP-positive cells, and cathepsin K expression were examined in the metaphysis of OVX mice. Density of bone marrow was evaluated by micro-CT.

Results: RANKL treatment increased PRMT1 and ADMA expressions in BMDMs in a JNK dependent manner. Silencing of PRMT1 by siRNA attenuated RANKL-induced osteoclastogenesis. Results also revealed that PRMT1 regulates NF-jB transcription activity and interacts with NF-jB in RANKL-treated BMDMs. In vivo study showed that PRMT1 deficiency has a protective effect against bone loss in OVX mice.

Conclusion: Taken together, our results suggest that PRMT1 plays important roles in the progression of osteoporosis through JNK-PRMT1-NF-jB signaling cascades, and may also be a good therapeutic target for postmenopausal osteoporosis.

Methods: We studied 485 subjects (95 subjects with T2D, 96 with atherosclerosis, 94 subjects with both T2D and atherosclerosis and 200 healthy controls). SNP alleles and serum levels of IL-6, sIL-6R and sgp130 were analyzed.

Results: We confirm that sIL-6R and sgp130 are able to neutralize IL-6 in vitro and show that the capacity of this buffer is controlled by the SNP rs2228145 within the IL6R. When we compared T2D patients with healthy controls, we found sIL-6R and sgp130 levels to be significantly lower (p < 0.001) and IL-6 levels inversely correlated to sIL-6R levels (r = -0.57, p < 0.001). Interestingly, the difference in sIL-6R levels between genotypes was lost in type-2 diabetes patients.

Conclusion: As the sIL-6R/sgp130 buffer is able to neutralize IL-6, these data suggest that this protective buffer system is disturbed in T2D patients and that, due to the chronic overload of the buffer by increased IL-6 levels, loss of sIL-6R and sgp130 in the serum might be closely associated to T2D pathophysiology. Introduction: Post-traumatic stress disorder (PTSD) is a psychiatric condition, defined by an array of affective and attentional disturbances, that can result from the experience or witnessing of traumatic or life-threatening events, such as combat exposure. Emerging evidence suggests PTSD and its psychobiological manifestations are linked to a systemic low-grade inflammatory state characterized by increased circulating pro-inflammatory mediators, as well as decreased anti-inflammatory mediators. Few studies, however, have investigated potential alterations in inflammatory biomarker indices in military PTSD and have yielded mixed results. The aim of this study was to compare levels of key pro-and antiinflammatory markers in combat veterans with PTSD vs. healthy trauma-exposed and non-deployed soldiers.

Methods: Eligible participants were 18-60 years, male treatmentseeking veterans of any era who had a diagnosis of current PTSD related to their combat experiences and fulfilled DSM-IV criteria for PTSD, with a Clinician Administered PTSD Scale (CAPS) score =50. Trauma-exposed (TE) soldiers had experienced at least one traumatic event (CAPS =15); TE and non-deployed (ND) controls had no history of PTSD or DSM-IV Axis-I psychopathology. The study was approved by Human Research Ethics Boards of Western University, CAMH and DRDC, and informed consent was obtained from participants. Circulating levels of selected pro-/anti-inflammatory mediators, including [interleukin (IL)-1b, -2, -4, -5, -8, -10, -12, tumor necrosis factor (TNF)-a, interferon (IFN)-c], serum amyloid protein A (SAA), C-reactive protein (CRP), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, matrix metalloproteinases (MMP)-1,3,9, and myeloperoxidase (MPO)] were analyzed by Meso-Scale Discovery (MSD Ò ) MULTI-ARRAY TM platform using MULTI-SPOT Ò 96-well V-plex Human Biomarker Immunoassay kits and SECTOR TM instrumentation. Statistical significance (p < 0.05) evaluated by unpaired Student's ttest and Kruskal-Wallis One-way ANOVA.

Results: Relative to both TE and ND controls, combat veterans with PTSD exhibited significant alterations in basal levels of multiple inflammatory markers. In particular, markedly higher circulating concentrations of IL-6, IL-8, TNF-a, CRP, sICAM-1, MMP9, and MPO were detected in the PTSD group relative to both ND and TE groups. By contrast, IL-1b was undetectable in over 90% of samples and no significant differences were observed between PTSD and controls for IL-2, IL-4, SAA, sVCAM-1. Importantly, IL-10 levels were found to be significantly higher in TE controls relative to both PTSD and ND.

Conclusion: Our findings, showing elevated pro-inflammatory cytokines, vascular adhesion and injury molecules, and lower antiinflammatory IL-10 in combat veterans diagnosed with PTSD compared with healthy controls, support the hypothesis that PTSD is associated with chronic, low-grade systemic inflammation among symptomatic individuals. This dysregulation may be a central link between PTSD and poor physical health, foremost an increased risk of atherothrombotic disease.

Disclosure of Interest: None declared. Introduction: The zinc-finger transcription factor GATA-3 is having important functions in T cells. It maintains hematopoietic stem cell renewal in bone marrow, leads T cell maturation in thymus, regulates Th2 cell differentiation in class II immune organs and transcripts IL-4, IL-5, IL-13 expression [1, 2] . EHMT1 (Euchromatichistone-lysine N-methyltransferase [1] has enzymatic activity on the zinc finger domain of PRDM16 gene, directly [3] . GATAs responses to VEGF-A expression levels. We have previously found that VEGF-A repression induced brown adipocytes differentiation in the white adipose tissue with overexpression of GATA-1 and brown adipocyte specific factor PRDM16 [4] .

Methods: A tetracycline-regulated reversible VEGF-A repression transgenic mouse model (VEGFtetO/tetO:bAKtg/tg) was developed in the study. H&E staining and blood smear followed Wright's staining were carried out for the hematological analysis. Quantitative RT-PCR, western blotting, immunofluorescence were used for measuring gene or protein expression levels in the experiments.

Results: First, we have found that the circulation red blood cell number was reduced for 20%(P < 0.01) and hemoglobin was reduced for 13%(P < 0.01), and lymphocyte number was increased for 28% (P < 0.05). Megakaryocyte number was increased in both bone marrow and the spleen. GATA-1 and GATA-3 gene expression was increased in both bone marrow and circulation. VEGF-A repression induced GATA-1, GATA-3, PU.1, Runx1, ALAS-2, Elf-1 gene expression increase when GATA-2 was decreased. The IL-7R+ subgroup of hematopoietic stem cell was increased in the bone marrow. EHMT1 can be regulated by VEGF-A in our result, which may directly regulate GATA-1 and GATA-3 expression by the methylation activity. Introduction: Interleukin-13 (IL-13)-mediated hyperactive fibroblasts of breast cancer stroma are associated with the malignant development of breast cancer, but IL-13-targetd factors are unclear. In order to explore the mechanism of the effects of IL-13 on breast cancer, we studied the effect of IL-13 on epidermal growth factor (EGF) expression of fibroblasts co-cultured with breast cancer cells and breast cancer growth.

Methods: Human breast cancer cell line MDA-MB-231 and human fibroblast line ESF were co-cultured by Transwell inserts method. The effects of IL-13 on EGF expression in fibroblasts co-cultured with breast cancer cells were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western-blotting in vitro. The effects of IL-13 on EGF expression of fibroblasts of breast cancer tissue in tumor-burdened nude mice in vivo were analyzed using immunofluorescence and laser confocal microscope, and the breast cancer volumes of tumor-burdened nude mice were examined. Data were presented as mean ± SD and analyzed using SPSS statistics software17.0. Different analysis between two groups was performed by Student's t-test. P values of <0.05 were considered to be a statistically significant difference.

Results: EGF expression of ESF cells in MDA-MB-231+ESF+IL-13 group was higher than the other groups (P < 0.05). It indicated that IL-13 could promote EGF expression of fibroblasts under the condition that breast cancer cells and fibroblasts were co-cultured in vitro. It also showed that tumor microenvironment played a synergistic role in function of IL-13. EGF expression in breast cancer tissue of tumor-burdened nude mice was marked by fluorescent antibody and observed using laser confocal microscopy. The fluorescent intensity in MDA-MB-231+ESF+IL-13 group of breast tumor-burdened nude mice was much stronger than the other groups (P < 0.05). The fluorescence intensity was no significantly different between MDA-MB-231+IL-13 and MDA-MB-231 groups of breast tumor-burdened nude mice. It indicated that IL-13 targeted EGF of fibroblasts not breast cancer cells of breast cancer tissues in tumor-burdened nude mice. EGF was expressed in MDA-MB-231 group of tumor-burdened nude mice which suggested that there was the basal expression of EGF in breast cancer cells, but IL-13 had no significant effect on EGF expression of breast cancer cells. From the 7th to 21th day after injection of IL-13, the tumor volumes in MDA-MB-231+ESF+IL-13 group of tumor-burdened nude mice were significantly larger than MDA-MB-231+ESF group (P < 0.05). IL-13 promoted the growth of transplanted tumor in nude mice. The results of IL-13 and EGF immunohistochemistry showed that the density value of IL-13 and EGF in human breast cancer tissues was higher than breast hyperplasia (P < 0.05). Methods: Colostrum-deprived germ-free piglets (GF) were colonized with EcN or LA 4 h after hysterectomy or bred as GF piglets. One week later they were infected with ST for 24 h. Bacterial colonization of the intesine (CFU counting), secretion of IL-8, IL-12/23 p40 and TNF alpha (xMAP technology) and transcriptions of claudin-1 and occludin (RT-qPCR) and their protein expression (IF) were monitored.

Results: The infection with ST caused bacteremia, somnolence, anorexia, and diarrhea in the piglets and high levels of IL-8, IL-12/23 p40 and TNF alpha. Their levels in the piglets preliminary associated with EcN were almost completely suppressed but not in the case of preliminary associated with LA. The transcription of claudin-1 in GF, EcN and LA piglet groups in colon were comparable but in ST group highly increased. In contrast, the transcription of occludin was depressed in ST group. Introduction: Both type I and type III interferons (IFNs) are now well recognized for their non-redundant importance in limiting viral infection; however their roles as inflammatory mediators are less well characterized. Both IFN types signal through the common Jak/ STAT pathway, each type through their distinct receptor complex. While there is functional redundancy between type I and type III IFNs, the restriction of the type III IFN receptor expression primarily to epithelial cells suggests an important role for the type III IFNs at mucosal surfaces. Inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn's disease, involves perturbation of the complex interactions between the mucosal immune system and the commensal bacteria of the gut, with cytokines acting as important cross-regulators of these multi-layered interactions.

Methods: To characterize the contributions of type I and type III IFNs to the formation, progression, and resolution of UC, we used mice deficient in type I IFN, type III IFN, and type I/III IFN signaling and a murine model of acute UC, which follows exposure of the mice to dextran sodium sulfate (DSS)-containing water.

Results: The importance of IFN signaling in the acute phase of inflammation was demonstrated by the onset of enhanced pathology and weight loss concurrent with the peak of IFN-induced Mx2 promoter-driven luciferase expression in DSS-treated transgenic Mx2-Luciferase reporter mice. Accordingly, experiments revealed enhanced sensitivity of double type I and type III IFN receptordeficient mice to DSS-induced disease as measured by decreased body weight, colon length, diffuse destruction of the colonic epithelium and increased mortality. Furthermore, distinct expression profiles of several pro-inflammatory and antiinflammatory cytokines in both colon homogenates and serum, as well as differences in innate immune cell infiltration, were observed in IFN receptor-deficient mice in comparison with wild type mice.

Conclusion: These data suggest that both type I and type III IFNs play important roles in modulating the inflammatory response, tissue homeostasis and repair during DSS-induced colitis, where loss of IFN signaling may exacerbate the inflammatory response and retard mucosal wound healing.

Disclosure of Interest: None declared. Introduction: Psoriasis is a chronic inflammatory skin disease characterized by hyper-proliferation and impaired differentiation of keratinocytes, coupled with an influx of inflammatory cells in the epidermis and especially the dermis. The mechanisms underlying psoriasis in humans and in mouse models are still incompletely understood, although the IL-17 cytokine has been clearly shown to make crucial contributions and anti-IL-17 antibodies have proven to be highly efficacious in treating the disease. IL-17 cytokines signal via the obligatory adaptor CIKS/Act1.

We have identified critical roles of CIKS/Act1-mediated signaling in imiquimod-induced psoriatic inflammation, a mouse model that shares features with the human disease.

Results: IL-17 cytokine/CIKS-mediated signaling into keratinocytes was essential for neutrophilic micro-abscess formation and contributed to hyper-proliferation and markedly attenuated differentiation of keratinocytes, in part via direct effects of this cytokine on keratinocytes. Interestingly, IL-17 signaling into keratinocytes also exerted a negative feedback, delimiting the accumulation of IL-17producing cdT cells in skin; this led to reduced IL-17 production over time, as dermal cdT are the primary source of this cytokine in this model. We now demonstrate that the negative feedback is mediated in part via IL-19, a member of the IL-10 family of cytokines. IL-19 is strongly induced by IL-17-stimulated keratinocytes and appears to function at least in part via an autocrine/paracrine mechanism to suppress recruitment of IL-17-producing cdT cells, thereby dampening IL-17 production. This stands in contrast to IL-17 cytokines/CIKS-mediated signaling into non-keratinocytes, particularly dermal fibroblasts during the early phase of the inflammation, which promoted cellular infiltration and enhanced accumulation of IL-17-producing cdT cells in skin, comprising an initial positive feedforward mechanism.

Conclusion: Our data show that CIKS-mediated signaling is dynamic and central to the development of both dermal and epidermal hallmarks of psoriasis, inducing distinct pathologies via target cellspecific effects, but also initiating mechanisms that ultimately mitigate further damage. The latter mechanisms may offer additional avenues for therapeutic intervention. is G-enteric pathogen that causes self-limiting gastroenteritis in the human and the pig. The aim of this work was to modify LPS of Salmonella Typhimurium to modify TLR4 signaling and local production of inflammatory cytokines.

Methods: One-week-old hysterectomy-derived germ-free piglets (GF) were infected with S. Typhimurium (ST) or its isogenic rfaG mutant for 24 h. Salmonella CFU counting, TLR4, MyD88 and TRIF transcriptions in the ileum were evaluated by RT-qPCR, IL-8, TNFalpha and HMGB1 proteins by ELISA.

Results: ST and its rfaG mutant comparably colonized the ileum of the gnotobiotic piglets. While TLR4 transcription was increased in ST group, the transcriptions in rfaG group were similar with GF piglets. MyD88 was decreased but TRIF increased in ST group while rfaG group was comparable with GF piglets. IL-8, TNF-alpha and HMGB1 were induced in ST piglets only.

Conclusion: TLR4 is the receptor for LPS. While ST induced high levels of IL-8, TNF-alfa and HMGB1 in ileum that may be harmful, the rough LPS mutant did not induce them at all. Future incorporation of other molecules related to LPS and TLR4 as MD-2, CD-14 and LBP would contribute to clarify relations of LPS type and TLR4-signaling. Introduction: Human inflammatory bowel disease (IBD) is a complex and heterogeneous. Interaction of various genetic and environmental factors leads to a loss of immunological tolerance against the gut residing microbial flora and subsequent chronic inflammation in the gastrointestinal tract. No single IBD animal model accurately mimics the complexity and heterogeneity of human disease. However, mechanistic animal models are useful for hypothesis testing and to interrogate biological pathways for drug discovery.

Methods: We aimed to characterize, with greater temporal and biological resolution, a model of innate acute colitis induced by stimulation of the proinflammatory receptor CD40 with an agonistic antibody in lymphopenic RAG2-/-mice in order to gain better insights into the pathobiology.

Results: Our comprehensive analyses of the time course of inflammation show the dynamic nature of the colonic versus systemic immune response, changes in cytokine network and histopathological changes in the gut, spleen and liver. Unexpectedly, we found that the inflammatory response causes time-related dysbiosis in the gut flora. Colitis induction in this model is dependent upon the presence of innate lymphocytes. Treatment with antibodies against the proinflammatory cytokines IL-12, IL-23 or TNF-alpha attenuated the inflammatory cascade differently, indicating unique functional roles for pathways activated by these cytokines.

Conclusion: Our data reveal novel disease mechanisms for this model leading to a better understanding of the pathobiology in this colitis model. Introduction: Intestinal epithelial cells (IECs) constitute the primary barrier that separate us from the outside environment. Their apical side faces the lumen of the gut while their basolateral side faces the lamina propia. While being fully functional to combat pathogen intruders, IECs have developed specific mechanisms to regulate their intrinsic immune response to tolerate the presence of the commensal flora in the lumen of the gut. This tailored response is key to maintain gut homeostasis but little is known on the molecular mechanisms that lead to this perfectly balanced immune tolerance.

Methods: Here, we use human IECs grown on transwells to recapitulate the epithelium architecture in order to address how immune homeostasis is achieved in the gut.

Results: We addressed whether the polarized nature of IECs influences their innate immune response upon viral infection. We found that when infection of IECs initiates from the basolateral side a stronger innate immune response is triggered. This response is characterized by a prolonged production of type III interferon (IFN) compared to infection emanating from the apical side. Using CRISPR/ Cas9 gene editing and molecular trans-complementation approaches, we demonstrated that this asymmetric response is mediated by the RIG-I like receptor pathway and is fully dependent on the polarized nature of IECs. Additionally, we found, unlike in mouse, human IECs are fully responsive to both type I and III IFNs. Importantly, we define, for the first time, functional differences between these two IFNs in epithelial cells.

Conclusion: Our results demonstrate that human IECs have developed unique mechanisms to regulate their immune response as function of infection side and by specifically functionalizing type III IFN. This polarized response would represent a strategy to maintain gut immune homeostasis by avoiding excessive response against viruses located in the luminal side while maintaining full responsiveness against viruses that have passed the epithelium barrier.

Disclosure of Interest: None declared. Introduction: IFN-k4, a recently discovered type-III interferon (IFN), is genetically regulated, and has clinical significance because of its association with impaired clearance of hepatitis C virus (HCV) infection 1 . Type-III IFNs play a critical role in the innate immune response to viral infections 2 . Although IFN-k4 induces a similar antiviral profile as other type-III IFNs, its specific contribution to the innate immune response is unclear. Delineating the functional properties of IFN-k4 that distinguish it from other type-III IFNs may give insights into its unique role during HCV, and other viral infections.

Methods: We compared antiviral responses induced by recombinant IFN-k4 versus IFN-k3; the proteins were expressed in stable inducible cell lines or used exogenously in the HepG2 human hepatoma cell line. We measured several IFN-induced responses, including activation of a luciferase reporter under control of an IFNstimulated response element (ISRE), phosphorylation of STAT1 and STAT2, induction of IFN-stimulated genes (ISGs), and the ability to induce antiviral activity against an infectious challenge with Sendai virus (SeV).

Results: IFN-k4 showed significantly higher antiviral activity than IFN-k3 within the first 4 h of treatment, antiviral activity of both IFNs was comparable by 8 h, but antiviral activity of IFN-k4 declined sharply after 24 h unlike IFN-k3 which maintained its activity. This data was supported by a stronger but more transient induction of STAT phosphorylation by IFN-k4 compared to IFN-k3. We also show that despite its poor secretion, IFN-k4 was significantly more potent than IFN-k3 in HepG2 cells engineered to express GFP-tagged IFN-k3 or IFN-k4 and IFN-k4 was more effective at inhibiting Sendai virus infection at early time points compared to IFN-k3.

Introduction: Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants, although the specific molecular mechanisms responsible for NEC development remain unclear. NEC is characterized by intestinal epithelial barrier disruption, enterocyte apoptosis and impaired mucosal healing. Recent evidence suggests a critical role for IL-22 in the regulation of intestinal epithelial barrier integrity and promotion of epithelial healing. We hypothesized that IL-22 signaling is important in protecting against NEC, and subsequently, IL-22 deficient mice would be susceptible to NEC.

Methods: Experimental NEC was induced in neonatal (postnatal day 7) wild-type (WT), IL-22 knockout, and intestinal deficient IL-22Ra1 (villin-cre X IL22ra1 fl/fl ) mice using gavage formula feeds (5 times/ day, 4 d) and intermittent hypoxia (5%O 2 , 95%N 2 , 10 min, twice daily). Recombinant IL-22 was administered intraperitoneally (1 mg/ gram body weight) daily in WT mice. NEC severity was assessed using histology and small intestinal mRNA expression of proinflammatory markers, iNOS and IL-6. Immunohistochemistry was performed for enterocyte proliferation and apoptosis.

Results: In WT mice, experimental NEC resulted in disruption of the intestinal mucosal architecture, decreased enterocyte proliferation and increased expression of pro-inflammatory cytokines IL-6 and iNOS within the intestine. Importantly, IL-22 knockout and intestinal deficient IL-22Ra1 mice were very susceptible to experimental NEC development and demonstrated accelerated NEC-related mortality, derangement of the villi architecture on histology, and upregulation of pro-inflammatory markers IL-6 and iNOS. Immunohistochemistry revealed decreased enterocyte proliferation and increased enterocyte apoptosis in IL-22 knockout and intestinal deficient IL-22Ra1mice compared to controls. Strikingly, we further demonstrate that recombinant IL-22 attenuates experimental NEC severity in WT mice. Introduction: Propolis is a resinous product made by honeybees from various plant parts and has attracted the attention of researchers because of its biological properties, highlighting its immunomodulatory effects. Monocytes are circulating mononuclear phagocytes that can adopt different roles dependent on the activating stimuli in their microenvironment. LPS is one of the most potent activators of monocytes driving CD4 + T cells polarization. This work aimed to investigate propolis immunomodulatory action on cytokine production by LPS-activated human monocytes, assessing cell viability and TNF-a, IL-6, and IL-10 production.

Methods: Human monocytes were obtained from healthy donors and stimulated with LPS (1 ìg/mL), propolis (P -5 and 25 ìg/mL) and their combination (LPS + P) for 18 h. A possible cytotoxic effect was assessed by MTT assay, and LPS-induced cytokines production were determined by ELISA. Significant differences were determined by analysis of variance (ANOVA), followed by Dunnett's test (P < 0.05).

Results: Data showed that cell viability was not affected after monocytes incubation with propolis alone or in combination with LPS. Propolis inhibited TNF-a and IL-6 production by LPS-activated human monocytes, whereas the combination (LPS + P) increased IL-10 release.

Conclusion: Propolis displayed an anti-inflammatory activity by decreasing LPS action in TNF-a and IL-6 production by human monocytes; moreover, propolis induced IL-10 production even in the presence of LPS. These findings may provide a novel therapeutic approach modulating monocytes activity by propolis.

Disclosure of Interest: None declared. Introduction: Even highly biocompatible materials, such as titanium-based devices, cause some degree of host response, thought to play a significant role in subsequent regenerative responses. However, the mechanisms involved in triggering host response, as well the role of host response in the ensuing regeneration, remains unknown. The aim of this study was to evaluate the role of MyD88 signaling molecule, part of CD14/TLR/MyD88 signaling complex, in the genesis and modulation of inflammatory immune response to a titanium (Ti) implantation and its impact in the following healing/ regeneration.

The experimental groups were comprised of eight-weekold, male wild-type (WT) C57BL/6 mice and mice with targeted disruption of the MyD88 (MyD88KO) (CEEPA-FOB/USP, process #012/2014). Ti-discs were implanted in the subcutaneous tissue of C57BL/6(WT) and MyD88KO mice, comparatively evaluated using microscopic and molecular (RealTimePCRarray/ELISA/LAL Assay) parameter assessments at 1, 3, 7 and 21 days time points. Values of p < 0.05 were considered statistically significant.

Results: Ti-disc implantation in WT mice results in the local release of HMGB1 (measured by ELISA), the major endogenous ligand (and the better characterized DAMP) of CD14/TLR/MyD88 complex; while no exogenous ligands, such as LPS (measured by LAL Assay) and LTA (measured by ELISA) were detected in the implantation site. HMGB1 release was followed by a moderate and transitory leukocyte influx, and the inflammatory process resolution in parallel with fibrous connective tissue formation and discs encapsulation. MyD88KO mice presented a significant decrease in inflammatory response and delayed connective tissue capsule formation and maturation as evidenced by histomorphometric and birefringence analysis. RealTi-mePCRarray showed that the impaired healing response in the absence of MyD88 was associated with decreased levels of growth factors, cytokines and healing markers, such as FGF2, TGFb1, VEGFa, Col1a2, MMP2, MMP9, IL1beta, IL6, TNFalpha, CCL2, CCL5 and CXCL12; being this data confirmed by ELISA. Of note, several MSC markers (CD106, COT4, NANOG, CD146, NES and CD105) were reduced in MyD88 KO mice, suggesting MyD88-mediated host response also contributes to MSCs migration and/or activation after Ti-discs implantation.

Conclusion: In summary, MyD88 (possibly activated by HMGB1) is a critical mediator of host inflammatory and healing responses to classic titanium-based biomaterials in subcutaneous tissue compartment, as evidenced by the significant decrease in inflammatory response and delayed connective tissue formation, as well as the decreased levels of molecular markers in MyD88KO mice compared to WT mice. These evidences contribute to the overall understanding of the role of host immune response in the healing process after biomaterial grafting.

Disclosure of Interest: None declared. Introduction: BATF3 has been shown to be a critical transcription factor for the development of a distinct subset of dendritic cells (DCs) characterized by the expression of CD24 and CD103 in the periphery and CD8a in lymphoid tissues. Using mice deficient for BATF3, these DCs have been shown in multiple models to be the required antigen presenting cells (APCs) for priming of CD8+ T cells, and are a critical source of the IL-12 needed to generate a protective T cell response during infection with the protozoan parasite Toxoplasma gondii. Protection from infection by T. gondii is dependent on a T cell response that can be generated by immunization the non-replicating CPS strain of T. gondii. Despite this low dose of parasite antigen, CPS immunization results in a T cell response with the same magnitude as that during active infection. It is not well understood what cells and cytokine signals are required for CPS to generate this T cell response. Surprisingly, it is shown here that BATF3-/-mice generate neither a CD8+ nor CD4+ T cell response to CPS immunization despite a previously undescribed role for BATF3 dependent DCs (BATF3 DCs) in priming CD4+ T cells. The aim of these studies is to determine what role BATF3 DCs play in priming CD4+ T cells during CPS immunization.

Methods: Mice were immunized with the CPS, CPS-OVA, or CPS-Cre-mCherry parasites by intraperitoneal injection of 10^5 parasites. Peritoneal exudate cells (PECs) were obtained by peritoneal lavage using 8 mL of ice cold PBS. The omentum, LN, and spleen were then processed through 40 mm filters to generate single cell suspensions. Single cells suspensions were then stained for flow cytometry analysis on a LSR Fortessa (BD Biosciences). Statistical analysis was performed using PRISM software (Graphpad Software).

Results: Intraperitoneal immunization with a low dose of CPS is capable of generating a CD4+ and CD8+ T cell response that is primed within two days in the omentum as shown by CD69 expression on T cells in the peritoneum, omentum, draining lymph nodes, and spleen from 0 to 3 days post-immunization (dpi). To track the interactions of CPS with host immune cells in these tissues, Ai6 reporter mice were immunized with CPS-Cre-mCherry parasites and analyzed on 1 and 2 dpi. It is shown that resident peritoneal macrophages are the cells that primarily interact with the parasite, while BATF3 DCs do not directly interact with CPS. The role of IFN-g dependent migration of infected or injected cells from the peritoneum to the omentum in T cell priming was analyzed by blocking IFN-g on -1, 0, and 1 dpi of Ai6 mice with CPS-Cre-mCherry. Indeed, IFN-g was required for parasite antigen transport to the omentum as no cells that interacted with the parasite were observed in the omentum and T cells did not increase expression of CD69 at 2 dpi compared to mice injected with control IgG antibody.

Conclusion: We hypothesize that CPS immunization induces IL-12 that promotes NK cells to produce this early IFN-g in the peritoneum. Future work aims to determine whether this DC subset is required for the processing and presentation of parasite antigen to CD4+ T cells during CPS immunization, or if these DCs serve as a required source of the IL-12 that induces NK cell IFN-g production and subsequent delivery of CPS antigen to APCs in the omentum. Introduction: The immune system generates innate and adaptive responses to ensure clearance of pathogens. Both are essential for host protection, but to develop sustained immunity, antigen-specific memory responses are needed. Dendritic cells (DCs) are key players as they bridge innate and adaptive immune responses. They activate and polarise T cells into effector cells which are crucial for adaptive immunity. The induction of immune responses is crucial to fight infections, but it can be also detrimental to the host and tight regulation is needed. Ubiquitination is an important mechanism of regulating immune responses. There are multiple E3 ubiquitin ligases, which have been studied in context of immunity, but the role of Pellino proteins is just emerging. Significant progress has been made on delineating the role of Pellino1 and Pellino3 but the physiological roles of Pellino2 are poorly understood. The current study aimed to use Pellino2-deficient mice to explore Pellino2 function in immune responses.

Methods: Spleens and lymph nodes of Pellino2-deficient mice and matching wild-type littermates were analysed by flow cytometry to assess normal development of the immune system. To study cellular responses, immune cells were purified and their cytokine production and proliferation was examined following agonist antibodies or mitogen activation. To study dendritic cell biology, bone marrowderived DCs (BMDCs) were generated and their activation was assessed following stimulation with various pathogen associated molecular patterns.

Results: The immune system of young adult Pellino2-deficient mice develops normally. There is no significant difference in various cell types, including T and B cell, frequency or number in spleens or lymph nodes between wild-type and Pellino2-deficient mice. Purified T cells proliferate and produce similar levels of proinflammatory cytokines when stimulated in vitro. Furthermore, Pellino2-deficient BMDC stimulated with various TLR ligands express similar levels of CD40 and CD86 and secrete comparable levels of pro-inflammatory cytokines when compared to wild-type cells indicating that Pellino2 is dispensable for DC activation. However, deletion of Pellino2 inhibited the production of IL-12 and IL-23 by dendritic cells, which are key cytokines in driving cellular responses in T cells.

Conclusion: Our findings highlight a novel mechanism in regulating immune responses and increase our understanding of Pellino protein functions. While Pellino1 and Pellino3 were described to regulate various immune signalling pathways, they were never implicated in regulating dendritic cell functions. Overall, this study shows that Pellino2 specifically regulates IL-12 and IL-23 secretion by DC which implicates its important role in cellular responses. Introduction: Propolis is produced by honeybees from several parts of plants. This product has been used since ancient times due its many biological and pharmacological properties. Retinoic acid (RA) is a metabolite of vitamin A and is a potent regulator of immune cells, playing an important role in T cell differentiation. The goal of this study was to verify propolis influence on RA-stimulated human monocytes regarding cytokine production (TNF-a, IL-6 and IL-10).

Methods: Human monocytes were obtained from healthy donors (n = 5) and incubated with propolis (5 ìg/mL), retinoic acid (10 -8 M) and the combination propolis + RA in the same concentrations for 18 h. Cell viability was assessed by the MTT assay and cytokine production was determined by ELISA. Significant differences were determined by analysis of variance (ANOVA), followed by Dunnett's test (P < 0.05).

Results: Propolis, RA and the combination did not affected cell viability. No alterations were seen in TNF-a and IL-10 levels after incubation with propolis, RA and the combination propolis + RA, and their production were similar to control. On the other hand, RA increased IL-6 production whereas propolis counteracted RA action and led to IL-6 levels similar to control.

Conclusion: Propolis exerted a modulatory action by preventing RAinduced IL-6 production. Since this cytokine is involved in plenty of biological activities and in the differentiation of Th17 cells as well, our results may provide a new approach for the development of immunomodulatory drugs containing propolis to treat inflammatory/autoimmune diseases.

Disclosure of Interest: None declared. Introduction: Herpesviruses including the human pathogen human cytomegalovirus (HCMV) replicate within and disseminate from mucosal organs. Experiments using the murine cytomegalovirus (MCMV) model of beta-herpesvirus infection have revealed the importance of CD4 + T cells in controlling persistence in an important site of virus replication, the salivary glands. We have demonstrate that virus-specific CD4 + T cells within MCMV-infected salivary glands are reactive to multiple viral epitopes and express the immune regulatory cytokine interleukin-10.

We utilized CD4 cre IL-10 flox mice to examine the impact that IL-10 production by T cells has on MCMV persistence, and studied IL-10 (10-Bit) reporter mice in protein phenotyping and RNA sequencing to characterize mucosal IL-10 + CD4 + T cells.

Results: Mice lacking T cell derived-IL-10 exhibit heightened virusspecific T cell responses and improved control of virus replication. These data suggest that targeting mechanisms that regulate the generation of mucosal IL-10 + CD4 + T cells will improve antiviral control. Development of salivary gland IL-10 + T cells was independent of the previously identified IL-10-inducing cytokines IL-27, IL-12 and TGF-beta. Transcriptomic analyses and protein phenotyping of IL-10 + CD4 + T cells revealed that these cells exhibit a distinct transcription factor and surface receptor expression profile as compared to IL-10 -CD4 + T cells that implicates multiple pathways, currently being examined in the laboratory, in the development of this important regulatory T cell population.

The development of IL-10 + CD4 + T cells is a critical determinant of herpesvirus persistence in mucosal tissue. IL-10 + CD4 + T cells exhibit a distinct transcriptional signature that may inform strategies that aim to enhance protective antiviral immunity in mucosal surfaces. Results: IL-39 is produced by macrophages in response to various stimulating agents including TNFa, IL-17a, IL-4 and IL-12 whereas TGFb and IFN? inhibit IL-39 production. Moreover, levels of IL-39 in circulation rise in response to inflammation induced by thioglycollate injection into the peritoneal cavity. In order to elucidate the function of IL-39, we have produced an IL-39 -/mouse. Although IL-39 deficient mice have comparable numbers of major immune cell populations in their immune organs, the effector functions of some of these cells are altered. For example, activated splenocytes from IL-39 -/mice produce abnormal levels of cytokines when compared to splenocytes isolated from their WT littermates.To elucidate the role of IL-39 in innate and/or adaptive immune responses, we tested the IL-39 -/mice in a model of acute T. gondii infection. We found that levels of IL-39 in serum and peritoneal cavity significantly increase in response to infection. Surprisingly, IL-39 deficient mice were more resistant to the T. gondii infection as indicated by reduced pathogen load and increased survival. Analysis of various cytokines and chemokines in serum from infected WT and IL-39 deficient mice revealed that IL-39 -/mice had lower levels of several proinflammatory mediators including CXCL1 and CCL2. High levels of these chemokines have strong correlation with increased mortality suggesting that with sublethal doses of T. gondii infection, mice die from sepsis rather than from the infection itself.

Conclusion: Therefore, our results indicate that reduced inflammation levels in IL-39 -/mice protect them from immune-mediated tissue damage during T. gondii infection. Together, our studies indicate that IL-39 is a pro-inflammatory cytokine that plays important roles in both innate and/or adaptive immune responses.

[4] Roth RB, Hevezi P, Lee Methods: Cell lines lacking inflammasome machinery were infected with a inducible and dimerizable caspase-1 contruct. Cell death and IL-1b activation and secretion were measured by western blot, ELISA assay, flow cytometry, fluorsecence microscopy, MTTS viability assays and ELISpot assays following both caspase-1 induction and dimerization. To study MLKL-induced inflammasome activation bone marrow derived macrophages derived from a variety of necroptotic gene deficient mice were stimulated with either inflammasome or necroptotic stimuli and inflammasome activity, cell death and DAMP released measured by ELISA, Western blot and flow cytometry. Monocytic THP1 cells harbouring inducible wildtype and mutant MLKL constructs were created to directly assay the domains, and role, of MLKL in inducing inflammasome activity.

Results: Caspase-1 cleaves and activates the pro-inflammatory cytokine interleukin-1 beta (IL-1b), yet the mechanism of IL-1b release and its dependence on cell death remains controversial. To address this issue, we generated a novel inflammasome independent system in which we directly activate caspase-1 by dimerization. In this system caspase-1 dimerization induced the cleavage and secretion of IL-1b, which did not require processing of caspase-1 into its p20 and p10 subunits. Moreover, direct caspase-1 dimerization allowed caspase-1 activation of IL-1b to be separated from cell death. Introduction: We have shown that recurrent activation of acute peritoneal inflammation drives tissue damage and fibrosis. Here, transition to a more chronic disease setting is controlled by IL-6, which promotes the expansion of pro-fibrotic interferon (IFN)-c secreting CD4 Th1 cells as a response to repeat inflammatory activation. To investigate potential changes in the provision of competent host defence versus inflammation-induced tissue injury, we have now used RNA-seq to profile gene signatures in 'resolving' and 'pro-fibrotic' peritoneal lining tissues.

Methods: Acute peritoneal inflammation was established in wild type (WT) and IL-6-deficient (Il6 -/-) or IL-6R-deficient (Cd126 -/-) mice -stimulated using a cell-free supernatant of Staphylococcus epidermidis (SES), or a fluorescently labelled Staphylococcus epidermidis isolate. At optimal time points, peritoneal lining tissue (for RNA extraction or western blot determination), inflammatory leukocytes (both resident and infiltrating cells) and peritoneal lavage samples were collected for analysis using RNA-seq, multiparameter flow cytometry and immuno-detection assays.

Results: Profiling of IL-6-mediated (occurring via IL-6 trans-signalling) STAT1 and STAT3 signalling in peritoneal lining tissues following SES challenge identified 3 and 6 h as optimal time points for RNA-seq analysis. Bioinformatic analysis of RNA-seq data from WT and Il6 -/mice identified gene signatures relevant to the control of anti-microbial immunity, leukocyte recruitment and adhesion, leukocyte effector function, vascular permeability and the homeostatic turnover of extracellular matrix. Consistent with these changes, Il6 -/mice showed altered neutrophil recruitment and impaired anti-microbial innate responses (phagocytosis and respiratory burst) following infection with Staphylococcus epidermidis. To mimic the tissue microenvironment associated with the onset of peritoneal fibrosis, mice were adoptively transferred (i.p.) with either naïve CD4 T-cells, or 'pro-fibrotic' Th1 cells expanded ex vivo with conditioned media from SES challenged resident peritoneal monocytic cells. Here, adoptive transfer of Th1 cells together with SES triggered substantive alterations in the capacity of the stromal peritoneal tissues to respond to acute inflammation -as defined using RNA-seq. In the context of IL-6 signalling, the presence of Th1 cells led to an increase in peritoneal IL-6, sIL-6R and sgp130 levelswith RNA-seq analysis identifying changes in Il6st (gp130) splicing.

Conclusion: These data provide insight into the dynamic relationship between IL-6 and IFN-k in determining the balance between competent host defence and inflammation-induced tissue damage. It is proposed that the initial IL-6 expansion of Th1 cells leads to a retrograde amplification IL-6 bioactivity and acute inflammatory responses controlled via the stromal tissue following recurrent infection.

Disclosure of Interest: None declared. Most of the researches on propolis and immunity were performed using animals and there are few studies regarding propolis immunomodulatory action in human cells. Monocytes were incubated simultaneously with propolis and a human tumor antigen (MAGE-1), which is known to induce the production of proinflammatory cytokines and Th1 profile. This work aimed to investigate the modulatory action of propolis on MAGE-1-induced cytokine production (TNF-a, IL-6 and IL-10) by human monocytes.

Methods: Human monocytes were obtained from healthy donors (n = 10) and incubated with propolis (P-5 and 25 ìg/mL), MAGE-1 (M-10 ìg/mL) and the combination MAGE-1/propolis (M + P -10/5 and 10/25 ìg/mL) for 18 h. Cell viability was assessed by the MTT assay, the cytokine production was determined by ELISA. Significant differences were determined by analysis of variance (ANOVA), followed by Dunnett's test (P < 0.05).

Results: Propolis, MAGE-1 and combination M + P did not affect monocytes viability. MAGE-1 stimulated TNF-a and IL-6 production, with no effect on IL-10 production. On the other hand, the combination M10 + P5 decreased TNF-a and IL-6 production and increased IL-10 production. Results: Previous studies in our lab and others have revealed that HCV acts to regulate the innate immune response in infected cells.

The virus replicates in hepatocytes where it acutely activates RIG-Ilike receptor signaling through viral detection by RIG-I. This response is quickly blocked by HCV-directed processes that prevent activation of the downstream interferon regulatory factor 3 (IRF3) transcription factor, supporting viral persistence in hepatocytes. However, despite this regulation of innate immune defense in the hepatocyte, the HCV-infected liver is most typically in a state of persistent innate immune activation. This outcome suggests a role for immune crosstalk in which signaling-competent liver cells direct the hepatic response to infection. Importantly, we have found that HCV produced during chronic infection is phagocytosed by liver macrophages, called Kupffer cells, wherein the internalized virion triggers the NLRP3 inflammasome. HCV does not productively infect Kupffer cells but induces potent IL-1b production and release to drive hepatic inflammation that underlies liver disease. Investigations of the macrophage as a hub of inflammatory signaling have revealed a hereto unappreciated role for IL-1 signaling in the regulation of both inflammatory as well as interferon/antiviral response genes. We therefore hypothesize that the pro-inflammatory signaling molecule IL-1b acts to drive innate immune activation from the Kupffer cell to the hepatic compartment in response to viral insult in the liver, and that the resulting maladaptive, non-resolving response to chronic infection underlies lasting hepatic injury.

Conclusion: By investigating the cellular response to HCV in the hepatocyte/macrophage co-culture setting, we have identified an IL-1b/IFN crosstalk signaling network and cytokine mediators that underscore liver disease outcome. Furthermore, we are able to link these profiles with degree of liver injury to predict which pathways are immunoprotective versus immunopathogenic during chronic HCV infection.

Disclosure of Interest: None declared. Introduction: Accurate and precise measurement of cytokine expression is essential for meaningful assessment of both innate and adaptive immune processes. While mice are a ubiquitous model for many infectious diseases, questions about the relevance and translatability of the results from these studies have led to increased usage of non-human primate (NHP) models of infection and disease. As part of the development work for a rhesus macaque periodontal disease model, 2 different cytokine measurement methodologies were assessed in spike and recovery assays.

Methods: The 2 kits assessed were a Luminex technology kit (MILLIPLEX MAP Non-Human Primate Cytokine Magnetic Bead Panel) and an electrochemiluminescence kit [Meso Scale Diagnostics V-PLEX Proinflammatory Panel 1 (NHP) Kit]. While both kits consist of human specific antibodies and controls, these reagents are reported to be cross-reactive with cytokines produced by rhesus macaques and other NHP species. The 11 recombinant macaque cytokines assessed were acquired from two separate venders (MyBioscience and R&D Systems).

Results: These experiments revealed great variability in assessed cytokine concentrations. There was accurate detection of some cytokines (e.g. IL-13), but over detection (e.g. IL-4), under detection (e.g. IFN-g) and no detection (e.g. IL-2) of other cytokines. Furthermore, the detection of cytokines across a range of concentrations was not always linear. Finally, variability was observed between the different kits, suggesting different sensitivities and degrees of cross-reactivity in the kit reagents. The limitations of human reagent cross-reactivity with NHP cytokines was further illustrated by experimentation with another Luminex technology kit (Bio-Plex Pro TM Human Cytokine 27-plex Assay) as only 4 of the 11 recombinant macaque cytokines assessed were detected by this kit.

Conclusion: Taken together these data suggest that caution should be used when drawing conclusions about immune polarization based on NHP cytokine data that have not been corroborated through alternative methods that do not rely on detection with human specific antibody (e.g. qPCR). These data also highlight the need to move beyond the use of cross-reactive human reagents and develop NHP-specific reagents as these animal models continue to increase in both usage and importance. Introduction: The cytokine interleukin-6 (IL-6) is known to be a causal factor in neuroinflammatory disease in humans. However, an understanding of the mechanisms via which IL-6 mediates central nervous system (CNS) inflammation and injury is unclear. A transgenic mouse (termed GFAP-IL6 mice) model with CNS-restricted production of IL-6 replicates many of the key neuropathological and behavioural changes found in human neuroinflammatory disease [1] . Microglia, the tissue-resident macrophages of the CNS, may be a major target of IL-6 and effector cells in IL-6-mediated neuroinflammation. Therefore the objective of this study was to gain an understanding of the nature of the microglial response to IL-6 in the CNS.

Methods: WT and GFAP-IL6 mice with EGFP-labelled macrophages (MacGreen -MG) were generated. Combinations of in situ (immunohistochemistry and confocal microscopy) and ex vivo (flow cytometry) approaches were employed to determine the morphology, distribution, number and molecular signature of microglia [2] in the brain of age-matched MG/WT compared with MG/GFAP-IL6 mice.

Results: Compared with MG/WT, MG/GFAP-IL6 mice displayed significantly increased numbers of EGFP+ cells throughout the brain, which were negative for the peripheral monocyte-specific marker 4C12. This increase in microglia number was due to significantly increased proliferation, particularly in younger GFAP-IL6 mice, as shown by BrdU incorporation. Three-dimensional reconstructions and morphometric analysis revealed MG/WT microglia exhibited a typical highly ramified morphology with long, fine primary and secondary processes. In contrast, MG/GFAP-IL6 microglia exhibited a dramatically altered morphology, with stunted, thickened primary processes and reduced secondary branching. Finally, compared with MG/WT, microglia in MG/GFAP-IL6 mice had an altered molecular signature with significantly decreased P2RY12 levels, while FCRLS levels were increased and 4D4 levels were unchanged.

These studies indicate that microglia are a major responder cell to IL-6 in the CNS that undergo dramatic morphological transformation and increased proliferation. In addition, exposure to IL-6 in the CNS can mediate a marked alteration in the unique molecular signature that defines these tissue-resident myeloid cells. Introduction: The Th17 response has been associated with host defense as well as with autoimmune diseases in patients and in experimental animal models. Th17 cells cause tissue damage by producing a stereotypic profile of pro-inflammatory cytokines, including Interleukin (IL)-17A, IL-17F, Granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-22. IL-17A is recognized as the Th17 signature cytokine and IL-17-producing T cells are pathogenic effectors in models of autoimmunity.

Methods: Using a recently developed model of spontaneous uveitis in R161H mice, which express a transgenic T cell receptor specific for the retinal autoantigen IRBP, we investigated susceptibility to disease of mice on an IL-17A deficient background. Additionally, IRBP-specific T cells from IL17A sufficient and deficient R161H mice were polarized under Th17 conditions and were adoptively transferred to WT hosts to investigate their cytokine profile and their ability to induce uveitis.

Results: We found that IL-17A deficiency did not reduce the severity of uveitis in R161H mice. However there are only a dozen of mucosal vaccines approved for human use. 3', 5'-Cyclic di-GMP (CDG) is an attractive mucosal vaccine adjuvant candidate that activates STING (stimulator of interferon genes) in pulmonary dendritic cells when administered intranasally. Pulmonary dendritic cells consist of two functional distinct subsets: CD103 + DC and CD11B + DC. How CDG targets these pulmonary DC subsets to promote its mucosal adjuvant activity is not known.

Methods: Intranasal vaccination of Batf3 -/-, IRF4 fl/fl CD11c cre and TNFR -/mice with pneumococcal protein antigen PspA and the mucosal adjuvant CDG. Two weeks after the last immunization, PspA-specific antibody responses are determined in Bronchoalveolar lavage (BAL) and blood. PspA-specific Th1/Th2/Th17 cellular responses are determined in the lung and spleen cells by ELISA. The maturation of pulmonary DC subsets is determined by flow cytometry analysis.

Results: Here, we investigated the role of different pulmonary DCs subsets in mediating the mucosal adjuvant activity of CDG. Using Batf3 -/mice, we found that CD103 + DCs do not mediate the CDGinduced antibody production. The population of pulmonary CD11b + DCs is still intact in the Batf3 -/mice, suggesting that CD11b + DCs are sufficient for CDG-induced humoral immunity. Furthermore, deleting CD11b + DCs in the IRF4 fl/fl CD11c cre mice demonstrated that CD11b + DCs are needed for CDG-induced antibody production and Th1/Th2/Th17 responses in the lung. In addition, CDG adjuvant activity completely depends on TNFa signaling and not type I interferon. We found that CDG-induced TNFa signaling is required for the activation of pulmonary CD11b + DCs.

Conclusion: Our results demonstrate that CDG targets and activates CD11b + pulmonary DCs to promote its mucosal adjuvant activity. Introduction: Macrophage migration inhibitory factor (MIF) is a pluripotent pro-inflammatory factor that acts as a mediator of innate immunity and is implicated in the pathogenesis of numerous autoinflammatory disorders, including Gout. Levels of serum MIF correlate with disease outcomes and clinical studies have shown correlations between elevated levels of IL-1b and MIF in serum of patients with autoimmune disorders. To date, it is unclear whether MIF specifically regulates the expression and secretion of IL-1 family cytokines.

The biological activity of MIF in murine bone marrow derived macrophages (BMDM) was inhibited using a small molecule inhibitor, COR123625. Concurrently, BMDM derived from Mif -/mice were employed to evaluate the effects of MIF depletion on regulation of IL-1 family cytokines. mRNA expression was analyzed by qRT PCR. Protein expression and secretion of both pro-IL-1b and secreted IL-1b was assessed by western blot. Levels of secreted IL-1a, IL-1b and IL-18 were determined by ELISA.

Results: We show that depletion of MIF in macrophages significantly reduced IL-1 cytokine family release in response to NLRP3-activating stimuli, but has no effect on the secretion of TNF-a, IL-6 and MCP-1 (CCL2). Moreover, diminished IL-1 responses were independent of NF-kB function and production of pro-IL-1b. Instead, MIF depletion specifically inhibits NLRP3-mediated responses; IL-1 cytokine secretion was unaffected following activation of the AIM2 or NLRC4 inflammasomes.

Conclusion: Our findings reveal a novel role for MIF in the modulation of IL-1-dependent inflammatory responses, linking MIF directly to NLRP3 inflammasome activation. This study for the first time implicates a specific role for MIF in the release of IL-1 family cytokines and highlights the potential of targeting MIF in IL-1dependent pathologies. Introduction: Pathogenesis of allergic diseases including asthma is strongly associated with robust responses of Th2 cells, which produce IL-4, IL-5, and IL-13. Thymic stromal lymphopoietin (TSLP) is a cytokine that is expressed upon epithelial infection or dysfunction and is implicated in the pathogenesis of allergic disorders. Although the functions of TSLP in stimulating dendritic cells and promoting Th2 differentiation are well appreciated, direct mechanisms of action of TSLP on the Th2 effector state remain to be elucidated. Previously, TSLP was shown to increase responsiveness of human CD4 T cells to IL-2 and enhance survival of murine CD4 cells by inducing JAK1/JAK2/STAT5 signaling pathway [1, 2] . Now we sought to determine the role of TSLP in Th2 cell differentiation and gain insight into its molecular mechanism of action during TCR activation.

Methods: Adoptive transfer of wild-type (WT) and TSLP receptordeficient CD4 cells into Tcra KO mice followed by house dust mite (HDM) administration was used to define CD4 T cell specific effects of TSLP in an asthma model. Naïve CD4 cells were activated by TCR in the presence or absence of TSLP or IL-4 to assess cytokine production of differentiated cells in primary and then secondary responses after adoptive transfer into WT mice. Flow cytometry, RNA-seq, and ChIP-seq analyses were performed to explore cellular and molecular profiles of TSLP-and IL-4-primed Th2 cells.

Results: We found that TSLP signaling in CD4 T cells during allergen exposure of the lung enhanced allergic airway inflammation. In vitro analysis revealed that TSLP-primed CD4 cells produced increased amounts of pro-allergic cytokines, notably IL-5 and IL-13, in primary as well as secondary responses, and this was accompanied by distinctive epigenetic changes in the Th2 cytokine locus. Importantly, TSLP induced Th2 cytokine expression independently of IL-4 after TCR activation, but both cytokines are required for a sustained Th2 differentiation with enhanced production of pro-allergic cytokines. Introduction: Interleukin-15 (IL-15) signaling is essential for the development and maintenance of natural killer (NK) cells, with STAT5A and STAT5B proteins being key transcription factors that mediate the actions of this cytokine. We previously reported that splenic NK cells are decreased in Stat5a/Stat5b double knockin (DKI) tetramer-deficient mice, but how STAT5 tetramers act to maintain normal numbers of NK cells remains unknown.

Methods: Using flow cytometric analysis, we analyzed NK cells from bone marrow, thymus, liver, and spleen in wild type (WT) and Stat5 DKI mice. We also compared proliferative responses by CFSE labeling, determined NK cell viability using 7AAD and Annexin V staining, and measured active caspases using FLICA staining. Moreover, we used RNA-Seq and ChIP-Seq analyses to identify STAT5 tetramer regulated mRNAs and STAT5 tetramer target genes in mouse NK cells.

Results: We found markedly decreased CD11b + CD27 low mature NK cells, especially in the bone marrow of Stat5 DKI mice, indicating an essential role for STAT5 tetramers in NK maturation. Despite this developmental defect, Stat5 DKI NK cells from bone marrow and spleen exhibited similar proliferation to that observed with WT NK cells. Interestingly, tissue resident NK cells in Stat5 DKI liver developed normally. We also found decreased expression of Prdm1 in bone marrow NK cells and of Prdm1 and Tbx21 in CD11b -CD27 + splenic NK cells in Stat5 DKI mice, which potentially explains the partial block in the late-stage maturation of NK cells. Moreover, Stat5 DKI NK cells were more susceptible than WT NK cells to cytokine withdrawal-induced cell death, with direct regulation of Bcl2 expression by STAT5 tetramers in response to IL-15 stimulation and increased active caspases in Stat5 DKI NK cells.

Conclusion: These findings reveal critical roles of STAT5 dimers in the early development of NK cells and indispensable roles of STAT5 tetramers in the late-stage maturation and survival of conventional NK cells in vivo.

Disclosure of Interest: None declared. Methods: To dissect the role of miRNAs in ILC2 cell homeostasis, we used mice with conditional deletion of Dgcr8. We further looked for specific miRNA role in ILC2 cell regulation by using mice with conditionally deleted miR 17~92 cluster. We isolated ILC2s from the lungs and cultured them with IL-33/TSLP/IL-7 and examined the cytokine production and growth of these cells. For the in vivo study of ILC2 function, mice were challenged with the natural allergen papain.

Results: ILC2 cells were severely reduced in Dgcr8 knockout mice. miR 17~92 cluster knockout ILC2 cells proliferation and IL-5 and IL-13 expression was significantly reduced compare to wild type ILC2 cells in response to IL-33/TSLP/IL-7 in vitro. Knockout ILC2 cells transfected with mature miR 19a mimic showed increased IL-13 production compared to control mimic transfected cells. Knockout ILC2 cells exhibited increased expression of the miR 17~92 cluster direct targets Tnfaip3, Socs1, Rora and Bmpr2. Knockout ILC2 cells showed increased IL-13 expression by inhibition of Tnfaip3 and Socs1 via siRNA approach. ILC2 cells expansion and functions were impaired in knockout mice in the Papain allergic model.

Conclusion: miRNAs regulate diverse aspects of ILC2 cell biology including homeostasis and function. miR 17~92 cluster is required to support Th2 cytokine production in ILC2 cells and repress the expression of its direct targets, the Th2 signaling inhibitors SOCS1 and Tnfaip3. miR 17~92 amplified similar signals to support the production of Th2 cytokines in both Th2 and ILC2 cells, which suggest that miR 17~92 might be a useful drug target of allergic airway inflammation.

Disclosure of Interest: None declared. are intracellular negative regulators often induced to limit cytokine signaling in a classic negative feedback loop. They are characterized by a central SH2 domain and a C-terminal SOCS box sequence motif which recruits an E3 ubiquitin ligase complex.

We have used a multidisciplinary approach to show that CIS (encoded by Cish) is the critical negative regulator of IL-15 signaling in NK cells.

Results: Cish was rapidly induced in response to IL-15 and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-k production and cytotoxicity towards tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, specifically inhibiting its enzymatic activity and targeting JAK1 for proteasomal degradation.

Cish -/mice were resistant to experimental melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Remarkably, Cish -/mice treated with antibodies against PD-1 and CTLA-4 (currently one of the most effective treatments against advanced melanoma) developed even fewer metastases than control Cish -/mice, highlighting the potential therapeutic benefit that could be achieved if anti-CTLA-4 and anti-PD-1 therapy was combined with loss of CIS function.

Conclusion: This study has uncovered a potent intracellular checkpoint in NK cell-mediated tumor immunity and holds promise for novel cancer immunotherapies directed at blocking CIS function.

CRP and suPAR were both predictors of 30-day mortality with HRs of 3.40 (95% CI 3.07-3.77) for log10(CRP) and 3.08 (2.86-3.32) for log2 (suPAR) when adjusting for age and sex. When the model was adjusted for both CRP and suPAR, the HRs were 2.41 (2.16-2.68) for log10(CRP) and 2.38 (2.18-2.59) for log2(suPAR). There was no interaction between suPAR and CRP (P 0.39).

The AUC for ROC curve analysis for predicting 30-day mortality was 0.79 (95% CI 0.78-0.80) for sex and age. Adding CRP improved the AUC to 0.84 (95% CI 0.83-0.85, P < 0.0001). Adding suPAR was better than adding CRP, AUC 0.86 (95% CI 0.85-0.87, P 0.005). Combining sex, age, CRP, and suPAR improved the AUC to 0.88 (95% CI 0.87-0.89, P < 0.0001).

Conclusion: Like CRP, suPAR is positively correlated with components of innate immunity, including CRP, total leukocyte count, monocytes, and neutrophils. CRP and suPAR are strong predictors of mortality in acute medical patients after adjusting for other risk factors, and suPAR adds significant prognostic information to the established inflammation marker CRP. Both biomarkers may therefore aid in the risk stratification in the acute care setting, as patients with increased CRP and suPAR are at higher risk of dying. Introduction: Th17 cells drive autoimmune inflammation through production of pro-inflammatory cytokines including IL-17, GM-CSF and IFNk. Beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) has physiological roles in central nervous system (CNS) development including myelination, but is best known for its role in promoting amyloid plaque formation in Alzheimer's disease. BACE1 is also thought to contribute to neurodegenerative processes triggered by CNS injury, and is upregulated in response to inflammatory stimuli. On the other hand, BACE1 deficiency causes impaired myelination of the CNS and has been reported to reduce repair following peripheral nerve injury. We therefore sought to investigate the role of BACE1 in Th17-induced neuroinflammation in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE).

Methods: Active EAE was induced by immunizing with MOG(35-55) in CFA. Passive transfer of EAE was achieved by culturing cells from immunized donors with MOG(35-55) and IL-23, or by activating 2D2 cells under Th17 polarizing conditions, followed by transfer into naive recipients. Clinical scores were monitored, and T cells analyzed by flow cytometry and ELISA. Naive T cells were also differentiated under Th0, Th17, Th1 conditions and analyzed by flow cytometry, ELISA, qPCR and RNA-Seq.

Results: BACE1 -/mice were more susceptible to EAE, as expected from myelin defects, but the severity of EAE was not increased. Unexpectedly, BACE1 deficiency resulted in defective IL-17 but not IFNk production by MOG-reactive T cells. When BACE1 deficiency was restricted only to T cells, this resulted in protection from disease in both active EAE and in passive transfer of myelin-reactive Th17 cells. BACE1 promotion of IL-17 was T cell intrinsic, as demonstrated by in vitro and in vivo co-cultures, and acted downstream of RORct. Expression of other Th17 factors IL-17F, GMCSF and RORct were not affected by BACE1, as confirmed by RNA-Seq of BACE1 -/and WT Th17 cells.

We have uncovered BACE1 as a novel regulator of Th17 cell function in autoimmune inflammation, through cell-intrinsic promotion of IL-17 production, and are now investigating mechanisms through which this fine-tuning of Th17 function is achieved.

Disclosure of Interest: None declared. Introduction: Systemic lupus erythematosus (SLE) is a complex autoimmune disease that affects multiple organs including kidneys, skin, nervous system, heart and the vascular system. It is characterized by abnormalities of both adaptive and innate immune cells and inflammatory cytokines synthesized by these cells play a pivotal role in the pathogenesis of the disease. Several studies have shown that blockade of inflammatory cytokines or their cytokine receptors is a feasible approach for the treatment of SLE.

Tofacitinib is an inhibitor of a Janus kinases (JAKs) that blocks signaling downstream of multiple cytokines. Several ongoing and completed clinical trials have shown that tofacitinib exhibits significant clinical efficacy in diseases ranging from rheumatoid arthritis to psoriasis, inflammatory bowel disease.

Methods: To evaluate the impact of tofacitinib on the pathology of SLE, we utilized the MRL/lpr lupus-prone mice model.

: Tofacitinib-treated mice displayed decreased serum cytokines levels including IL-17, IFN-k, and TNF-a and splenocytes showed reduced expression of type I/II IFN signature genes (IP-10, MX1, and STAT1). Reduced renal and skin inflammation, lower levels of ANA, diminished deposition of immunoglobulin complex and decreased albumin/creatinine ratio in the urine were also observed. Furthermore, neutrophils derived form the treated mice showed reduced production of neutrophil extracellular traps and the drug induced significant improvements in endothelium-dependent vasorelaxation and endothelial differentiation.

Conclusion: Taken together, these data suggest that, by modulating both the adaptive and innate arms of the immune system and interfering with cytokine signaling and the interferon-driven genetic program, tofacitinib ameliorates the SLE phenotype and vascular dysfunction in lupus-prone mice. This supports the concept that tofacitinib and other JAK inhibitors may be beneficial in SLE. Despite their high metabolism, bats have very long lifespans 3.5 times those of comparably sized non-flying mammals 3 . However, the molecular mechanisms for these observations are totally unknown. As aberrant or excessive inflammasome activation has recently been linked to viral pathogenesis and age-related disease, one can hypothesize that differential regulation of inflammasome signaling may play a role in some of the biological features of bats.

Methods: Here we investigated the inflammasome activation in bat primary immune cells and cell lines.

Results: Our data show that activation is significantly reduced in bat cells compared to human or mouse counterparts. There is low induction of ASC speck formation and caspase-1 activation in response to various natural and synthetic stimuli.

Conclusion: Our result so far suggests the involvement of multiple molecular mechanisms at different steps of the signaling pathway. These include unique loss of a sensor gene family at the genomic level and altered expression and function of key inflammasome proteins.

inflammation. The non-chemotactic function of CCR6 in the CD4 T cells during inflammation and autoimmunity is not known. Being a GPCR, CCR6 has the ability to activate battery of downstream signaling pathways in the cell. Thus we hypothesized that intrinsic signaling from CCR6 along with other co-stimulatory signals can influence the differentiation, plasticity and function of Th17 and regulatory CD4 T cells (Tregs) during autoimmunity.

Methods: Acute colitis was induced in the C57BL/6 or CCR6 -/mice by giving 2% dextran sodium sulphate (DSS, w/v) in the drinking water. Development and progression of colitis was monitored daily for weight loss, loose stool and bloody diarrhea. Spleen, mesenteric lymph node, Payer's patches and lamina propria CD4+ effector T cells and CD4+CD25+Foxp3+ T cells (Treg) were analyzed by flow cytometry and immunohistological staining. Splenic CD4+CD25-T cells from naïve C57BL/6 or CCR6 -/mice were cultured in presence of anti-mouse CD3e mAb, IL-2, and irradiated T cell depleted spleenocytes together with CD4+ T cell polarizing reagents (for Th17, IL-6, TGF-b and IL-23; for Treg, TGF-b). CCR6-CCL20 signaling was studied in CCR6gfp-Jurkat cells and in primary CD4 cells sorted from CCR6gfp +/mice by stimulating them with recombinant CCL20.

Results: CCL20 inhibits TGF-b1 induced Treg differentiation and directing them towards pathogenic Th17 lineage. CCL20 increased the expression of Th17 associated molecules such as Aryl hydrocarbon receptor (Ahr), IL-21, IL-22, IL-23 and IL-23R on the cultured Tregs, and inhibited the surface expression of suppressor molecules such as CD39, CD73 and FasL on cultured Tregs. Under Th17 differentiating condition, CCR6 signaling further increased the RORct expression and Th17 associated molecules. Furthermore, CCR6 induces phosphorylation of Akt, mTOR, and STAT3. CCR6 activation promotes binding of RORct on conserved non-coding region 5 (CNS5) of IL-17A promoter. CCR6-/-CD4 T cells showed reduced phosphorylation of STAT3 and differentiation of Th17 cells and prevented the development of autoimmune colitis. Analysis of PBMCs of human inflammatory bowel disease (IBD) patients showed increased RORct+ expression on CCR6+ CD4 T cells compared to healthy volunteers.

In conclusion, CCR6 activation modulates the critical balance between Treg and Th17 cells required for maintenance of gut tolerance. Targeting the CCR6 will not only inhibit the migration of pathogenic Th17 cells in the inflamed tissues but also help in maintaining the increased Treg/Th17 ratio which in turn may help in controlling the inflammation and autoimmunity.

Disclosure of Interest: None declared. Introduction: Parasitic worms (helminths) are successful pathogens that infect approximately 25% of the world's population. They cause chronic infections that are associated with type-2 and regulatory immune responses. Helminths not only suppress the anti-parasite immune response but can also combat pathological inflammation, including autoimmunity. Indeed, regions with a high prevalence of helminth infections have a low incidence of autoimmune disease. Consequently, helminths are being trialled as potential therapies for autoimmune diseases. However, the use of live helminths as a treatment raises many regulatory and practical concerns. This has led for the search to identify specific helminth immunoregulatory molecules. Our lab has demonstrated that Fasciola hepatica excretory-secretory products (FHES) can attenuate the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, by suppressing the infiltration of diseasemediating Th1 and Th17 cells into the CNS. Protection was mediated by the type-2 cytokines IL-5 and IL-33.

Methods: As FHES is a mixture of many proteins and small molecules, it provides the basis for identifying and characterising individual immunomodulatory components. To address this, we fractionated FHES by size-exclusion chromatography. Fractions were assayed in vivo for the ability to promote type-2 responses. Positive fractions were screened by mass spectrometry. Several candidate proteins were identified, cloned, transformed into yeast, expressed, and purified. Macrophages were treated in vitro with these proteins to assay their ability to promote alternative activation. To assay the effects of candidate proteins on autoimmunity, we injected mice with EAE with the candidate proteins, beginning on day -1 and continuing throughout the disease course. Parallel to this, we isolated small extracellular vesicles (exosomes) from FHES by various centrifugation steps and tested their efficacy to promote type-2 responses in vivo.

The results demonstrated that the FHES proteins and the exosomes have anti-inflammatory properties. The candidate proteins promote the production of the anti-inflammatory cytokine IL-10 and the alternative activation of bone marrow-derived and peritoneal macrophages. Functional studies demonstrated that the proteins had regulatory activity in vivo, attenuating the clinical severity of EAE. In vivo, the exosomes increased eosinophil recruitment and reduced neutrophil numbers in the peritoneum. This effect correlated with an increase in alternative activation markers on macrophages and production of the anti-inflammatory cytokine IL-1RA.

Conclusion: Our findings demonstrated that exosome or specific proteins from the helminth F. hepatica can promote regulatory immune responses that could be exploited for the treatment of human inflammatory or autoimmune diseases.

Disclosure of Interest: None declared. Introduction: Epidemiology studies have shown that there is a lower incidence of allergy and autoimmune diseases in developing countries with a high prevalence of parasitic infections and this has been attributed to the induction of Th2 or regulatory T (Treg) cells.

We have previously demonstrated that infection of mice with the helminth parasite Fasciola hepatica, or treatment with the excretorysecretory products from F. hepatica attenuates experimental autoimmune encephalomyelitis (EAE); a mouse model for multiple sclerosis (MS), through suppression of pathogenic myelin antigen (MOG)-specific Th1 and Th17 cells. The aim of this study was to examine whether we could exploit the immunomodulatory properties of a helminth parasite to prevent the development of autoimmunity either by inducing autoantigen-specific Treg cells or by training the innate immune system to be more regulatory.

Methods: C57BL/6 mice were subcutaneous injected with total extract (TE) from F. hepatica or TE and the myelin peptide MOG, MOG only or PBS 21 and 7 days before the induction of EAE. Mice were monitored daily for clinical signs of the disease. On day 16 after EAE induction, mice were sacrificed and mononuclear cells were isolated from the brain and analysed by flow cytometry.

Results: Prophylactic treatment of mice with MOG in the presence of F. hepatica TE suppressed MOG-specific effector T cells responses and prevented the onset of EAE. Interestingly, prophylactic administration of F. hepatica TE alone (without antigen) also protected against the development of EAE. This was associated with a reduction of CNS infiltrating pathogenic IL-17 and IFNk-producing T cells. Furthermore, we found that TE suppresses LPS-induced IL-1b and IL-23 production by bone marrow derived macrophages and dendritic cells, in a manner that is independent of IL-10. Moreover, TE was found to suppress IL-17A, IL-17F and IFNy production and gene expression by cd T cells stimulated with IL-1b and IL-23. Interestingly, TE also suppressed IL-1b and IL-23 induced expression of IL-1R1 and IL-23R on cd T cells. Finally, pretreatment with TE primed macrophages to be more anti-inflammatory, leading to increased IL-10 and decreased TNF-a production upon re-stimulation with LPS after a period of rest.

Conclusion: Our results demonstrate that pre-treatment of mice with parasite products can prevent the development of T cell mediated autoimmune disease by antigen-independent innate immune regulation. Our findings suggest that in addition to innate immune training or memory leading to heighten inflammatory responses, the innate immune system can also be trained to become more anti-inflammatory.

Disclosure of Interest: None declared. Introduction: NCX 2121, a nitric oxide (NO)-releasing derivative of indomethacin, has been reported to possess potentially beneficial properties including chemopreventive and anti-inflammatory effects. However, the effect of NCX 2121 against inflammatory periodontal disease has not yet been reported. In the present study, the influences and possible mechanisms of NCX 2121 on the production of proinflammatory mediators were examined using murine macrophages activated by LPS isolated from Prevotella intermedia, a pathogen that has been implicated in periodontal diseases.

Methods: LPS from P. intermedia ATCC 25611 was isolated employing the standard hot phenol-water protocol. Conditioned culture media were assayed for NO and IL-1b. Real-time PCR was carried out to measure murine iNOS, IL-1b and SOCS1 mRNA levels. iNOS protein expression and levels of signaling proteins were assessed by immunoblot analysis. NF-jB-dependent SEAP reporter assay was performed using a reporter cell line. DNA-binding activities of NF-jB subunits were analysed by using the ELISA-based NF-jB transcription factor assay kit.

Results: NCX 2121 significantly down-regulated P. intermedia LPSinduced production of iNOS-derived NO and IL-1b as well as their mRNA expression in RAW264.7 cells. Of note, NCX 2121 was much more effective than the parental compound indomethacin in reducing LPS-induced production of NO and IL-1b. NCX 2121 exerted suppressive effects on the production of NO and IL-1b in P. intermedia LPS-activated murine macrophages through the inhibition of NF-jB, STAT1 and STAT3 activation and SOCS1 induction.

Conclusion: NCX 2121 could potentially be useful in the host modulation of inflammatory periodontal disease, although further studies are required to verify this.

Disclosure of Interest: None declared. Introduction: Strawberry notch homolog 2 (Sbno2) is a putative transcription factor the expression of which we have identified previously 1 to be both constitutive and strongly upregulated by various cytokines (including IL-6) and by lipopolysaccharide (LPS) in astrocytes in the central nervous system (CNS). Although these observations suggest Sbno2 may have roles in astrocyte homeostasis and in the regulation of the inflammatory response little is known concerning its function in the healthy or inflamed CNS. Therefore, the aim of this study was to further delineate the function of Sbno2 by generating a mouse model for the cell-specific deletion of Sbno2 in astrocytes.

Methods: Mice (Sbno2 fl/fl ) were generated in which the introns flanking exons 8-10 of the murine Sbno2 gene were floxed. The Sbno2 fl/fl mice were then crossed with GFAP-Cre mice to conditionally disrupt the Sbno2 gene in astrocytes (Sbno2-AstKO mice). Histological examination was performed on the brain from Sbno2-AstKO and control mice at different ages. Primary cultures of astrocytes were obtained from Sbno2-AstKO and control mice and the physical and molecular phenotypes of these cells were compared. Mice were given dual, staggered intra-peritoneal injections of purified E. coli LPS and various markers for neuroinflammation were analysed.

Results: Sbno2-AstKO offspring developed normally and displayed no outward phenotype up to at least 12 months of age. In cultured astrocytes from Sbno2-AstKO mice neither un-recombined Sbno2 mRNA nor SBNO2 protein were detectable, confirming the Cremediated disruption of the Sbno2 gene. The morphology of astrocytes from Sbno2-AstKO mice was indistinguishable from control mice despite having increased levels of STAT3 protein and the astrocyte markers glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). The level of GFAP mRNA was higher in the cerebrum of adult Sbno2-AstKO mice compared with age-matched control littermates. Following LPS injection, the expression of various inflammatory and glial markers was found to be similarly affected between Sbno2-AstKO and control mice.

Conclusion: A new model allowing the conditional deletion of the Sbno2 gene in specific cell types was successfully generated. Our initial findings reveal that loss of Sbno2 in astrocytes, (1) does not compromise the development or viability of these cells and is associated with only subtle phenotypic changes and (2) does not overtly alter the neuroinflammatory response following systemic LPS challenge. It is expected that further insight to the function of Sbno2 in astrocytes will be gained from ongoing studies profiling the transcriptome of Sbno2-AstKO cells.

10 or greater), interferon signature, and medication use, indicating that each of these factors exerted a different impact on monocyte gene expression that could be separately identified. A subset of antiinflammatory gene set expressing NCLs was inversely correlated with anti-dsDNA titers (rho = -0.77, p = 0.0051) and positively correlated with C3 complement (rho = 0.68, p = 0.030) in the SLE patient group.

Conclusion: Using single cell gene expression, we have identified a unique gene expression patterns that reflect the major clinical and immunologic characteristics of the SLE patients which are not evident in bulk cell data, supporting the critical importance of the single cell technique.

Disclosure of Interest: None declared.

Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy

A High-Throughput-Compatible 3D Microtissue Co-Culture System for Phenotypic RNAi Screening Applications

COPD, a multicomponent disease: implications for management

Chronic obstructive pulmonary disease: effects beyond the lungs

Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary

Commensal-innate immune miscommunication in IBD pathogenesis

Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients

A novel method in the induction ofreliable experimental acute and chronic ulcerative colitis in mice. Gastroenterol

Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin

Disclosure of Interest: None declared

Intrinsic antiviral immunity

Immunologic messenger molecules: cytokines, interferons, and chemokines

Type I interferons and the virus-host relationship: a lesson in détente

Pathogen recognition with Toll-like receptors

Interferon-stimulated genes: a complex web of host defenses

Concomitant TLR/RLH Signaling of Radioresistant and Radiosensitive Cells Is Essential for Protection against Vesicular Stomatitis Virus Infection

Disclosure of Interest: None declared

THERAPEUTIC ELIMINATION OF THE TYPE 1 INTERFERON RECEP-TOR FOR TREATING PSORIATIC SKIN INFLAMMATION

Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells

The nature of the principal type 1 interferonproducing cells in human blood

Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects

Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA

cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING

Interferon lambdas: the next cytokine storm

IL28B is associated with response to chronic hepatitis C interferon-alpha andribavirin therapy

Interferon-lambda rs12979860 genotype and liver fibrosis in viral and non-viral chronic liver disease

The biology of type III interferons

IRF-3, IRF-5, and IRF-7 coordinately regulate the type I IFN response in myeloid dendritic cells downstream of MAVS signaling

Disclosure of Interest: None declared

HUMAN HAQ STING DOES NOT RESPOND TO CYCLIC DINUCLEO-TIDE IN VIVO AND IN VITRO

United States; 2 Department of Immunology and Microbia Disease

Critical Role for Interferon Regulatory Factor 3 (IRF-3) and IRF-7 in Type I Interferon-Mediated Control of Murine Norovirus Replication

Persistent Enteric Murine Norovirus Infection Is Associated with Functionally Suboptimal Virus-Specific CD8 T Cell Responses

A Single-Amino-Acid Change in Murine Norovirus NS1/2 Is Sufficient for Colonic Tropism and Persistence

Interferon-k cures persistent murine norovirus infection in the absence of adaptive immunity

Disclosure of Interest: C. Kurokawa Grant/research support from: Fapesp

Chemokine expression in IBD. ucosal chemokine expression is unselectively increased in both ulcerative colitis and Crohn's disease

Inflammatory bowel disease: cause and immunobiology

Cytokines in chronic obstructive pulmonary disease. The European respiratory journal

Arginine methylation of the histone H3 tail impedes effector binding

The clinical significance of asymmetric dimethylarginine

The PRMT1 gene expression pattern in colon cancer

GATA-3 function in innate and adaptive immunity

GATA-3 regulates the selfrenewal of long-term hematopoietic stem cells

Resistance to obesity by repression of VEGF gene expression through induction of brown-like adipocyte differentiation

EHMT1 controls brown adipose cell fate and thermogenesis through the PRDM16 complex

A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus

Guarding the frontiers: the biology of type III interferons

Disclosure of Interest: None declared

Olivia Parks 2 , C. Ma 2 , A. Bolock 2

United States; 2 Department of Pediatrics, Children's Hospital of Pittsburgh of UPMC

Immune responses to implants -a review of the implications for the design of immunomodulatory biomaterials

Damage associated molecular patterns within xenogeneic biologic scaffolds and their effects on host remodeling

Cytokines: past, present, and future

Search of Magic Bullets: The Golden Age of Immunotherapeutics

Effects of RNA degradation on gene expression analysis of human postmortem tissues

At 12, 24, and 48 later, serum was collected and levels of sIL-15 complexes were mesured using an ELISA specific for IL-15/IL-15Ra complexes. To assess the requirements for type I IFN signaling, IFNAR-/-mice were examined. To determine the cellular source of sIL-15 complexes, IL-15Rafloxed X CD11-Cre, IL-15Rafloxed X LysM-Crem, and IL-15Rafloxed control mice were treated with STING agonists

Neurologic disease induced in transgenic mice by cerebral overexpression of interleukin-6

A progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin 6 in the brain

Identification of a unique TGF-beta-dependent molecular and functional signature in microglia

Cutting edge: direct action of thymic stromal lymphopoietin on activated human CD4+ T cells

Thymic stromal lymphopoietin-mediated STAT5 phosphorylation via kinases JAK1 and JAK2 reveals a key difference from IL-7-induced signaling

Disclosure of Interest: None declared. P214 FATTY ACID METABOLISM SUSTAINS ILC2 MEDIATED BARRIER IMMUNITY DURING INFECTION AND MALNUTRITION

Disclosure of Interest: None declared

Laboratory for Innate Immune Systems

A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special?

Bats and viruses: friend or foe?

Life history, ecology and longevity in bats

Can eradication therapy for Helicobacter pylori really improve the thrombocytopenia in idiopathic thrombocytopenic purpura. Our experence and a literature

Clinical characteristics and treatment outcomes in patients with Helicobacter pylori-positive chronic immune thrombocytopenic purpura

Helicobacter pylori, Eradication in Patients with Immune Thrombocytopenic Purpura: A Review and the Role of Biogeography

Improvement of thrombocytopenia after treatment for Helicobacter pylori, in a patient with immunologic thrombocytopenic purpura

Helicobacter pylori eradication in patients with chronic immune thrombocytopenic purpura

Disclosure of Interest: None declared

Mills School of Biochemistry and Immunology

Strawberry notch homolog 2 is a novel inflammatory response factor predominantly but not exclusively expressed by astrocytes in the central nervous system

Acknowledgements: The help rendered by the faculty from Neurology department is kindly acknowledged.

Our results show that IFN-k4 is more active during the early stages of viral infection compared to other type-III IFNs which may be a clinically important window for restricting viral infection in the host that may affect disease outcome. These results demonstrate the potential importance of IFN-k4 in the innate immune response to viral infection. 1 Microbiology and Immunology, UNESP, Botucatu, Brazil; 2 Center for Neurosciences and Cellular Biology, University of Coimbra, Coimbra, Portugal Conclusion: We propose that TSLP acts in coordination with IL-4 to generate a stable pathogenic Th2 state that could underlie various allergic disorders.

The protein chip showed a simple, fast and accurate method for H. pylori detection, by analyzing the H. pylori-associated antibodies CagA, VacA, Ure provied an accurate classification for ITP, and had a high clinical value in diagnosis and treatment of H. pylori infected ITP patients.

Introduction: IL-15 is a stimulatory cytokine for CD8 T cells and NK cells, which can mediate responses by being transpresented via the IL-15Ra protein or in a paracrine manner through soluble IL-15/IL-15Ra complexes (sIL-15 complexes). Our previous studies have shown that sIL-15 complexes are induced by various inflammatory stimuli, such as type I IFNs. Since activation of the Stimulator of Interferon Genes (STING) pathway is important for regulating type I Interferons (IFN), we set out to determine if sIL-15 complexes are regulated by STING signaling. Since production of sIL-15 complexes is also induced upon total body irradiation and is partially dependent on type I interferons (IFN), we also investigated the role of STING in inducing sIL-15 complexes after lymphodepletion. 1 University of Bonn, Bonn, Germany; 2 NIH, Bethesda, United States; 3 Université Laval, Quebec City, Canada; 4 USDA, Beltsville, United States Introduction: Survival of the host relies on the establishment of a functional barrier immune defense that must be maintained in fluctuating states of food availability. Innate lymphoid cells (ILC) are an important component of tissue immunity involved in the maintenance and repair of tissue barriers such as the lung, skin and intestine but how their function is metabolically controlled is unknown.Methods: To test the metabolic regulation of ILC2 we used fluorescently labeled fatty acids or glucose together with pharmacological inhibition of fatty acid uptake, fatty acid metabolism and glycolysis in models of helminth infection and malnutrition (vitamin A deficiency). In addition we analyzed the cytokine levels and cellular source by ELISA and flow cytometry and performed extracellular flux analysis and quantitative PCR to reveal the metabolic regulation on a cellular level.Results: We found that ILC2 take up high amounts of fatty acid from the environment and predominantly depend on fatty acid metabolism during helminth infection. Ablation of fatty acid metabolism or dietary fat adsorption impairs the ILC2 mediated immune response and expulsion of tissue dwelling parasites. Furthermore, in situations where an essential nutrient, such as vitamin A, is limited, ILC2 increase in numbers, sustain their function and selectively maintain IL-13 production via increased acquisition and utilization of fatty acid. This metabolic adaptation of ILC2 is regulated on a cellular level by an increase in genes involved in fatty acid metabolism and a corresponding decrease in genes regulating glycolysis.Conclusion: Together, these results reveal that ILC2 depend on fatty acid metabolism to mediate barrier immunity in the context of helminth infection and malnutrition. Thus we identified an important mechanism that allows the host through metabolic adaptation to maintain barrier immunity and to promote host survival in the face of limited nutritional availability.

Introduction: Recent studies have identified novel lymphocytes that do not express Rag-dependent antigen specific receptors but are able to produce large amounts of cytokines, collectively called innate lymphoid cells (ILCs). ILCs are classified into three groups based of their cytokine expression patterns. Among ILCs, group 2 ILC (ILC2) including natural helper (NH) cells, nuocytes and innate helper type 2 (Ih2) cells produce type 2 cytokines such as IL-5, IL-6 and IL-13 in response to IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) that are produced by epithelial cells. We have focused on the regulatory mechanisms of ILC2 functions in helminth infection and allergic inflammation.Methods: We employed a parasitic infection model with Nippostrongylus brasiliensis and an allergic infllamation model with Alternaria alternata to examine the activation mechanisms of NH cells or ILC2.Results: We found that the corticosteroid-resistance observed as a result of airway inflammation triggered by sensitization and exposure to allergen is induced via the IL-33/ILC2 axis. Thymic stromal lymphopoietin (TSLP) synthesized during airway inflammation plays a pivotal role in the induction of ILC2 corticosteroid resistance in vitro and in vivo, by controlling phosphorylation of Stat5. Blockade of TSLP/Stat5 signaling pathway restores corticosteroid sensitivity. We also examined the regulatory mechanisms of ILC2 functions and demonstrate here that tissue-resident ILC2 proliferate in situ without migration during inflammatory responses. Both type I and type II IFNs, and interleukin-27 (IL-27) strongly suppressed proliferation and function of ILC2 in a Stat1-dependent manner. ILC2-mediated lung inflammation was enhanced in the absence of IFN-k receptor on ILC2 in vivo, demonstrating the importance of endogenous IFN-k in the termination of ILC2-mediated inflammation. IFN-k effectively suppressed the function of ILC2 but not inflammatory ILC2 induced by IL-25, and IL-27 suppressed ILC2 but not T H 2 in Alternaria-induced lung inflammation.Conclusion: Our results demonstrate that TSLP is a Stat5-dependent activator for ILC2 and Stat1-dependent suppression by IFN and IL-27-is a negative feedback mechanism for ILC2 function in vivo.Disclosure of Interest: None declared. Introduction: Identifying patients at risk of adverse outcomes is crucial in acute medicine. C-reactive protein (CRP) is a wellestablished biomarker widely used in clinical routine as a marker of infection and inflammation, while soluble urokinase plasminogen activator receptor (suPAR) is a novel inflammatory biomarker associated with immune activation, disease severity, and mortality. Here, we aimed to compare suPAR and CRP and their association to mortality in acute medical patients.Methods: This study included 17,312 unselected patients who were admitted to the acute medical department between November 18th 2013 and September 30th 2015 at Copenhagen University Hospital Hvidovre, Denmark. CRP and suPAR were measured as a part of the standard admission blood test panel. Follow-up information, including diagnoses and vital status, was extracted from national Danish registries after 30 days. Statistical analysis was performed using correlation analysis, receiver operating characteristic (ROC) curve analysis, and Cox regression analysis. Results are presented as medians with interquartile range (IQR), Kendall's tau b correlation coefficients, and hazard ratios (HRs) or area under the curves (AUCs) with 95% confidence intervals (CIs).Results: Median CRP was 5.0 mg/l (IQR 1.0-31.0, n = 16,817) and median suPAR was 2.8 ng/ml (IQR 1.9-4.3, n = 17,312). CRP and suPAR were positively correlated (tau b 0.36, P < 0.0001), but 1155 patients (6.9%) had low CRP (<10 mg/l) and high suPAR (=4.5 ng/ml).CRP and suPAR were both negatively correlated with lymphocyte count (tau b -0.22 and -0.20, respectively) and eosinophils (-0.17 and -0.11), and positively correlated with total leukocyte count (0.27 and 0.14), monocytes (0.28 and 0.16), and neutrophils (0.32 and 0.18) (All P < 0.0001).Disclosure of Interest: None declared. Introduction: Many immunosuppressive therapeutics for autoimmune diseases can have the side effect of inhibiting immune responses that control infection and tumors. Induction of autoantigen-specific regulatory T (Treg) cells has the capacity to specifically suppress immune responses to self-antigens without affecting response to foreign antigens and has been dubbed the 'holy grail' of autoimmune disease therapy. However the induction of autoantigen-specific Treg cells is not straightforward.

We immunized C57Bl/6 mice with the CNS self-antigen myelin oligodendrocyte glycoprotein (MOG) together with RA as an adjuvant prior to the induction of experimental autoimmune encephalomyelitis (EAE) or prior to transfer of CD4 + T cells to recipient mice with EAE. Lymph nodes, spleens and brains cells were isolated and their phenotype and cytokine production were analysed by flow cytometry, RT-PCR and ELISA.Results: Here we demonstrated that immunization of mice with a peptide from the CNS self-antigen myelin oligodendrocyte glycoprotein (MOG), in combination with the vitamin A derivative retinoic acid (RA) as an adjuvant, promotes the induction of MOG-specific Treg cells. These Treg cells had a Tr1-type phonotype, secreting high concentrations of IL-10 and TGF-b, but not IFN-k, IL-17 or IL-4. Prophylactic immunization with MOG and RA prevented the development of experimental autoimmune encephalomyelitis (EAE), and this was associated with a significant reduction of CNSinfiltrating Th1 and Th17 cells. Furthermore, transfer of CD4 + T cells from mice immunized with MOG and RA prevented the development of EAE in recipient mice and reduced the migration of IL-17A and IFN-k producing T cells in their CNS, demonstrating that the protective effect was transferable and dependent on antigen-specific CD4 + T cells.

Our study provides an effective vaccination approach against CNS autoimmunity, which has application across a range of T cell-mediated autoimmune diseases.Disclosure of Interest: None declared. Methods: CD14 + + CD16classical monocytes (CLs) and CD14 dim CD16 + non-classical monocytes (NCLs) from SLE patients were purified by magnetic separation. The Fluidigm single cell capture and Rt-PCR system was used to quantify expression of 87 monocyte-related genes.Results: NCL monocytes had higher IFN scores than CL monocytes. Unsupervised hierarchical clustering of the entire data set demonstrated two unique clusters found only in SLE patients, one related to high disease activity and one related to prednisone use. Independent clusters in the SLE patients were related to disease activity (SLEDAI