key: cord-0941159-2tl5q8cu authors: Sinha, Saptarshi; Castillo, Vanessa; Espinoza, Celia R.; Tindle, Courtney; Fonseca, Ayden G.; Dan, Jennifer M.; Katkar, Gajanan D.; Das, Soumita; Sahoo, Debashis; Ghosh, Pradipta title: COVID-19 lung disease shares driver AT2 cytopathic features with Idiopathic pulmonary fibrosis date: 2022-02-28 journal: bioRxiv DOI: 10.1101/2021.11.28.470269 sha: 53995ec7386e85a55da0e991b8b8438885f1b5a1 doc_id: 941159 cord_uid: 2tl5q8cu Background In the aftermath of Covid-19, some patients develop a fibrotic lung disease, i.e., post-COVID-19 lung disease (PCLD), for which we currently lack insights into pathogenesis, disease models, or treatment options. Method Using an AI-guided approach, we analyzed > 1000 human lung transcriptomic datasets associated with various lung conditions using two viral pandemic signatures (ViP and sViP) and one covid lung-derived signature. Upon identifying similarities between COVID-19 and idiopathic pulmonary fibrosis (IPF), we subsequently dissected the basis for such similarity from molecular, cytopathic, and immunologic perspectives using a panel of IPF-specific gene signatures, alongside signatures of alveolar type II (AT2) cytopathies and of prognostic monocyte-driven processes that are known drivers of IPF. Transcriptome-derived findings were used to construct protein-protein interaction (PPI) network to identify the major triggers of AT2 dysfunction. Key findings were validated in hamster and human adult lung organoid (ALO) pre-clinical models of COVID-19 using immunohistochemistry and qPCR. Findings COVID-19 resembles IPF at a fundamental level; it recapitulates the gene expression patterns (ViP and IPF signatures), cytokine storm (IL15-centric) and the AT2 cytopathic changes, e.g., injury, DNA damage, arrest in a transient, damage-induced progenitor state, and senescence-associated secretory phenotype (SASP). These immunocytopathic features were induced in pre-clinical COVID models (ALO and hamster) and reversed with effective anti-CoV-2 therapeutics in hamsters. PPI-network analyses pinpointed ER stress as one of the shared early triggers of both diseases, and IHC studies validated the same in the lungs of deceased subjects with COVID-19 and SARS-CoV-2-challenged hamster lungs. Lungs from tg-mice, in which ER stress is induced specifically in the AT2 cells, faithfully recapitulate the host immune response and alveolar cytopathic changes that are induced by SARS-CoV-2. Interpretation Like IPF, COVID-19 may be driven by injury-induced ER stress that culminates into progenitor state arrest and SASP in AT2 cells. The ViP signatures in monocytes may be key determinants of prognosis. The insights, signatures, disease models identified here are likely to spur the development of therapies for patients with IPF and other fibrotic interstitial lung disease. Funding This work was supported by the National Institutes for Health grants R01-GM138385 and AI155696 and funding from the Tobacco-Related disease Research Program (R01RG3780). One Sentence Summary Severe COVID-19 triggers cellular processes seen in fibrosing Interstitial Lung Disease As the acute phase of the COVID-19 pandemic winds down, the chronic diseases in its aftermath have begun to 109 emerge. For example, many survivors are suffering from a mysterious long-haul form of the disease which 110 culminates in a fibrotic form of interstitial lung disease (post-COVID-19 ILD) (1) (2) (3) (4) (5) (6) (7) (8) (9) . The actual prevalence of post-111 COVID-19 ILD (henceforth, PCLD) is still emerging; early analysis indicates that more than a third of the 112 survivors develop fibrotic abnormalities. One of the important determinants for PCLD (10) is the duration of 113 disease; ~4% of patients with a disease duration of less than 1 week, ~24% of patients with a disease duration 114 between 1-3 weeks, and ~61% of patients with a disease duration > 3 weeks, developed fibrosis. Disability among 115 survivors of severe COVID-19 has been primarily attributed to reduced lung capacities(11). While COVID-19- (but not influenza). 126 As for how the fibrotic sequel of COVID-19 is managed at present, based on the shared pathological 127 features of overt fibrosis in both end-stage COVID-19 and ILDs, lung transplantation remains the mainstay option 128 for these patients (15) (16) (17) . Although corticosteroids appear to improve the risk of PCLD(2), beyond that, there is 129 no existing therapeutic option. 130 Here we seek to unravel the fundamental molecular mechanisms underlying PCLD, identifying key disease 131 drivers (cellular processes, immune pathways, and the signaling cascades that support those pathways). We use 132 artificial intelligence (AI) and machine learning derived gene signatures, the Viral Pandemic, ViP and severe(s) 133 ViP signatures that are induced in all respiratory viral pandemics(18). Besides the ViP signatures, we also use 134 gene signatures induced in the lungs of patients with severe COVID-19. These approaches, in conjunction with 135 experimental validation (in pre-clinical disease models and in human lung tissue) not only helped identify which 136 lung pathology shares fundamental molecular features with COVID-19 lung, but also revealed key mechanistic 137 insights into the pathogenesis of PCLD. The findings also provide clues into how to navigate, prognosticate and 138 recapitulate in pre-clinical models this emergent mysterious condition. The objectivity and precision of the AI-139 guided unbiased approaches enhance the translational potential of our findings. A study design that uses gene signatures as a computational framework to navigate COVID-19 lung disease 142 Because PCLD is still an emergent illness that lacks insights into disease pathophysiology, we resorted to a study 143 design (Figure 1 ) that is geared to achieve three goals: (i) maximize rigor by using diverse transcriptomic datasets 144 cohorts (a total of 2078 unique samples, human: 2019; mouse: 41, hamster: 18; see Supplemental Information 145 1), but (ii) minimize bias by using well characterized sets of gene signatures from independent publications (see 146 Supplemental Information 2), and (iii) retain objectivity and precision by using artificial intelligence/machine 147 learning (AI/ML) tools that are supported by precise mathematical algorithms and statistical tools (see Methods). 148 First, to ensure that the findings maintain relevance to respiratory viral pandemics and COVID-19, we 149 used a recently discovered 166-gene signature that was found to be conserved in all viral pandemics (ViP), 150 including COVID-19, and a subset of 20-genes within that signature that classifies disease severity(18); the latter 151 was found to be consistently and significantly induced in the most severe cases of COVID-19 and prognostic of 152 poor outcome, e.g., death or invasive respiratory support (see Supplemental Information 2). These ViP We began first by using the ViP signatures as a starting computational framework to navigate the 161 syndrome of COVID-19 lung disease, in conjunction with another independently reported(13) COVID-19-lung 162 derived signature to identify idiopathic pulmonary fibrosis (IPF)(21) as a computational match for COVID-19 163 lung disease (Figure 1, Step 1) . The subsequent steps in the study use the ViP signatures alongside IPF-specific 164 PBMC-derived prognostic signatures (Figure 1, Step 2) and signatures of alveolar cytopathic changes (Figure 1 , 165 Step 3) to systematically assess the degree of similarities between COVID-19 lung and IPF and the crossover of 166 pathophysiologic processes in the two conditions. In the final step (Figure 1, Step 4) , a set of gene signature-167 inspired protein-protein interaction (PPI) network analysis is used which pinpointed alveolar ER stress as a 168 potential early step in both IPF and PCLD, which is sufficient to recapitulate not just the ViP signatures, but also 169 the alveolar cytopathic features that are shared between the diseases. Key predictions are validated in human lung 170 tissues and plasma from COVID-19 subjects, hamster models of COVID-19 and in human pre-clinical lung 171 models (Figure 1, Steps 3-4) . COVID-19 lung disease and IPF induce a common set of gene expression signatures 173 We first asked which pathologic lung condition comes closest to COVID-19 lung disease regarding the host 174 immune response. To this end, we used the ViP/sViP signatures (found to be induced in all CoV samples tested 175 so far(18)) and a second set of CoV-lung disease gene signatures which were derived from a differential 176 expression analysis on lung samples from healthy controls vs. fatal COVID-19(13) (see Supplemental 177 Information 2). As for the lung diseases, we ensured that all four major pathologic conditions were represented: 178 neoplastic (e.g., carcinoids and adenocarcinoma), granulomatous (e.g., tuberculosis/TB and sarcoidosis), 179 allergic/infectious (e.g., severe asthma and community acquired pneumonia/CAP and tuberculosis) and 180 vasculopathic (e.g., pulmonary hypertension/PHT and chronic thromboembolic pulmonary 181 hypertension/CTEPH) (Figure 2A) . As expected, ViP/sViP signatures were induced in infectious diseases (CAP 182 and tuberculosis), and to our surprise, these signatures were upregulated also in sarcoidosis, a granulomatous 183 condition that progresses to fibrosis in ~20% of the patients(22, 23) (Figure 2A ). Only two diseases (tuberculosis 184 and IPF) showed significant induction of both ViP and CoV-lung signatures, of which only one condition (IPF) 185 manifests as a diffuse disease with patchy parenchymal involvement resembling COVID-19 lung disease ( Figure 186 2A). 187 We next analyzed a set of six signatures, each independently derived from diverse IPF-centric studies(24- classification and indicate that IPF is the closest computational match to COVID-19 lung disease ( Figure 2G ). 197 Consistent with the fact that PCLD occurs in survivors of severe disease that required hospitalization(32), we 198 found that sViP signature was induced significantly higher in hospitalized compared to non-hospitalized subjects 199 ( Figure 2H ). Finally, we asked what, if any relationship exists between another poorly understood feature of the PBMC-derived prognostic signatures for IPF also prognosticate outcome in CoV, and vice versa. 224 We found that alveolar macrophages (marked by CD-14(47)) in the lungs of deceased subjects contained the Figure 3D ; middle, right); among IPF subjects, the signature was prognostic exclusively in male subjects ( Figure 228 3D; middle). The 52-gene 'IPF-specific' prognostic signature(37) retained its ability to prognosticate outcome in 229 both male and female patients with IPF (as expected; Figure 3E ; middle, right), but also did so in the setting of IL15RA correlate with the ViP signature (as expected), IL15RA, but not IL15 correlate with TERC_UP and AT2-258 senescence signatures. Both ViP and sViP signatures correlate with AT2 senescence. Furthermore, when we 259 tested AT2-senescence and telomere dysfunction in other CoV datasets, we found that these features were 260 conserved in most CoV samples across diverse cohorts ( Figure 4D ). An independent sc-RNA seq IPF dataset 261 further confirmed that, much like CoV lung, the induction of ViP/sViP in alveolar epithelium (EpCAM+; Figure 262 4E) is associated with IL15/IL15RA induction, AT2-senescence and telomere dysfunction. These findings 263 support the existence of telomere dysfunction and DNA damage in both acute COVID-19 and IPF. These findings show that hamster lungs best recapitulate the host immune response, the nature of the 286 cytokine storm (IL15-centric), and the AT2 cytopathies that are shared between COVID-19 and IPF. It is also in 287 keeping with emerging data that Syrian hamsters best emulate COVID-19(58); they also recapitulate progressive 288 fibrosis in IPF(67). Validation of the predicted AT2 cytopathic features of COVID-19 in hamster and human lungs 291 We next sought to determine if the SARS-CoV-2 virus can induce the AT2 cytopathic changes, and whether 292 effective therapeutics can abrogate those changes. We analyzed by RNA seq and trichrome stain the lungs from 293 SARS-CoV-2-challenged golden Syrian hamsters who were pre-treated either with EIDD-2801 or anti-Spike 294 (CoV-2) mAb or untreated controls (see study protocol in Figure 5A ). Key genes associated with AT2 senescence 295 (Tp53 and Cdkn2a), ER stress (Hspa5/Grp78), DATP (Krt8 and Cldn4) and fibrosis (Col1a, Col3a and Serpine1) 296 were all induced in the infected lungs (compared to uninfected controls) but not in those that were treated ( Figure 297 5B). Trichrome staining confirmed that compared to uninfected controls, collagen deposition was induced in the 298 infected lungs, but not in those that were treated (Figure 5C-D) . Immunohistology studies confirmed that Cdkn2a, Krt8, Cldn4 and Tp53 proteins were expressed in the injured alveoli, but not in the treated lungs ( Figure 5E-F) . 300 We next confirmed extensive deposition of eosin-positive collagenous materials in lung tissues obtained 301 at rapid autopsies on deceased subjects with COVID-19 compared to normal lung tissues (Figure 6A ; top). KRT8 pathogenic mechanisms in COVID-19(76, 77). It is noteworthy, that we found that levels of IL6 and platelets showed significant negative correlation (Supplementary Figure 3) . Because IL6 has been specifically implicated 337 in platelet hyper-reactivity and thrombosis(78), as well as in thrombus resolution(79), the strong negative 338 correlation may suggest the presence of thromboinflammation; the latter is one of the first and most distinctive 339 pathological feature of CoV-lung to be reported since the beginning of this pandemic(13). As for AT2-centric 340 processes, the PPI network analyses suggest that ER stress, which has been shown to serve as a causal role for 341 profibrogenic epithelial states (SASP) that is shared between aging-associated pulmonary fibrosis and IPF(80), 342 may also be a common underlying factor in fibrotic COVID-19 lung disease. 343 344 ER stress in AT2 cells is observed in COVID-19 and is sufficient to recapitulate the host immune response 345 in COVID-19 and IPF. We next sought to investigate if alveolar ER stress is a feature in CoV-lung. To this 346 end, we analyzed infected human ( Figure 8A ) and hamster ( Figure 8B ) lungs for the ER chaperone Grp78, a key 347 regulator of ER homeostasis expression by IHC. We found that compared to normal uninfected lungs, infected 348 lungs showed a significant increase in Grp78 staining and that the intensity of such staining was highest in the 349 epithelial cells lining the alveolar spaces in both species (Figure 8A-B) . 350 To determine if ER stress in AT2 cells may contribute to the epithelial dysfunction and fibrosis that is The major discovery we report here is that lung disease in severe COVID-19 resembles IPF, the most common KEY RESOURCE The lung specimens from the COVID-19 positive human subjects were collected as described in detail previously 507 (18, 70) using autopsy (study was IRB Exempt). All donations to this trial were obtained after telephone consent 508 followed by written email confirmation with next of kin/power of attorney per California state law (no in-person 509 visitation could be allowed into our COVID-19 ICU during the pandemic). The team member followed the CDC 510 guidelines for COVID-19 and the autopsy procedures 8, 9 . Lung specimens were collected in 10% Zinc-formalin 511 and stored for 72 h before processing for histology. Autopsy #2 was a standard autopsy performed by anatomical Source data are provided with this paper. All data is available in the main text or the supplementary materials. Publicly available datasets used are enlisted in Supplemental Information 1. Code availability 597 The software codes are publicly available at the following links: The authors declare no competing interests. Pulmonary fibrosis 621 secondary to COVID-19: a call to arms? An Observational Study of Corticosteroid Treatment Interstitial Lung Disease after COVID-19 Infection: A Catalog of 626 Uncertainties Lungs after COVID-19: Evolving Knowledge of Post Restrictive Lung Disease in Patients With Subclinical Coronavirus 630 Infection: Are We Bracing Ourselves for Devastating Sequelae? Association of COVID-19 and other viral 632 infections with interstitial lung diseases, pulmonary fibrosis, and pulmonary hypertension: A narrative review Post COVID-635 19 fibrosis, an emerging complicationof SARS-CoV-2 infection Patients Recovering From COVID-19 Critical Illness: A Prospective Longitudinal Point-of-Care Lung 638 Ultrasound Study Similarities Between Severe COVID-19 and Acute Exacerbation of 641 Connective Tissue Disease-Associated Interstitial Lung Disease (CTD-ILD) Functional 645 Outcomes and Their Association With Physical Performance in Mechanically Ventilated Coronavirus Disease 646 2019 Survivors at 3 Months Following Hospital Discharge: A Cohort Study Diffuse alveolar damage: a common phenomenon in progressive interstitial 648 lung disorders Thrombosis, and Angiogenesis in Covid-19 Immune signatures underlying post-652 acute COVID-19 lung sequelae Lung transplantation for 654 patients with severe COVID-19 Early outcomes 658 after lung transplantation for severe COVID-19: a series of the first consecutive cases from four countries AI-guided discovery 661 of the invariant host response to viral pandemics Childhood Multisystem Inflammatory Syndrome -A New Challenge in the Pandemic. N Engl 663 An AI-guided signature 21 Angiotensin-converting enzyme 2 protects from 668 severe acute lung failure From granuloma to fibrosis: sarcoidosis associated pulmonary 670 fibrosis Pulmonary fibrosis in sarcoidosis. Clinical features and outcomes Bayesian probit regression 674 model for the diagnosis of pulmonary fibrosis: proof-of-principle A novel 676 genomic signature with translational significance for human idiopathic pulmonary fibrosis Detecting the Molecular 679 A robust data-driven genomic signature for idiopathic 682 pulmonary fibrosis with applications for translational model selection Idiopathic Pulmonary Fibrosis Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis Single-cell RNA-seq reveals 688 ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis Persistent viral activity, cytokine storm, and lung 691 fibrosis in a case of severe COVID-19 3-month, 6-month, 9-month, and 12-month respiratory 693 outcomes in patients following COVID-19-related hospitalisation: a prospective study The lasting misery of coronavirus long-haulers Fatigue in idiopathic pulmonary 697 fibrosis measured by the Fatigue Assessment Scale during antifibrotic treatment Highly Prevalent in Patients with IPF or Sarcoidosis Perceptions of fatigue in 702 patients with idiopathic pulmonary fibrosis or sarcoidosis Validation of a 52-gene 704 risk profile for outcome prediction in patients with idiopathic pulmonary fibrosis: an international, multicentre, 705 cohort study Host adaptive 709 immunity deficiency in severe pandemic influenza Isolation of a novel 718 coronavirus from a man with pneumonia in Saudi Arabia Host Transcriptional Response 720 to Influenza and Other Acute Respiratory Viral Infections--A Prospective Cohort Study Single-cell landscape of bronchoalveolar immune 723 cells in patients with COVID-19 TREM-2 promotes macrophage 725 survival and lung disease after respiratory viral infection Distinct developmental 727 pathways from blood monocytes generate human lung macrophage diversity Interleukin-15 is associated 729 with disease severity in viral bronchiolitis Interleukin (IL) 15 is a 731 novel cytokine that activates human natural killer cells via components of the IL-2 receptor SCINA: A Semi-Supervised Subtyping 734 Algorithm of Single Cells and Bulk Samples Alveolar Epithelial Type II Cells as Drivers of Lung 736 Fibrosis in Idiopathic Pulmonary Fibrosis Insights from human genetic studies of lung and organ fibrosis Molecular mechanisms of pulmonary fibrosis Cell-Derived Damage-Associated Transient Progenitors that Mediate Alveolar Regeneration Alveolar regeneration 744 through a Krt8+ transitional stem cell state that persists in human lung fibrosis Persistence of a regeneration-746 associated, transitional alveolar epithelial cell state in pulmonary fibrosis Drives Progressive Pulmonary Fibrosis Molnupiravir and Favipiravir results in a potentiation of antiviral efficacy in a SARS-CoV-2 hamster infection 751 model Therapeutically administered ribonucleoside analogue MK-753 EIDD-2801 blocks SARS-CoV-2 transmission in ferrets Characterization of orally 755 efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia Quantitative efficacy paradigms of 758 the influenza clinical drug candidate EIDD-2801 in the ferret model An orally bioavailable 760 broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple 761 coronaviruses in mice Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral 764 EIDD-766 2801) inhibits SARS-CoV-2 replication and enhances the efficacy of favipiravir in a Syrian hamster infection 767 model Molnupiravir, an Oral Antiviral 769 Treatment for COVID-19. medRxiv Isolation of potent SARS-CoV-2 771 neutralizing antibodies and protection from disease in a small animal model Angiotensin-converting enzyme 2 (ACE2) 773 mediates influenza H7N9 virus-induced acute lung injury Pulmonary fibrosis in the 775 aftermath of the COVID-19 era (Review) Efficacy and tolerability of bevacizumab in 777 patients with severe Covid-19 Adult stem cell-derived 779 complete lung organoid models emulate lung disease in COVID-19 An organoid-781 derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells The STRING database in 784 2021: customizable protein-protein networks CLUGO: a clustering algorithm for automated 787 functional annotations based on gene ontology Platelets amplify 791 endotheliopathy in COVID-19 Endothelial 793 dysfunction and immunothrombosis as key pathogenic mechanisms in COVID-19 Pathophysiological Association of Endothelial Dysfunction with Fatal 796 Outcome in COVID-19 Interleukin-6 798 mediates the platelet abnormalities and thrombogenesis associated with experimental colitis Crucial Involvement of IL-6 in 801 Thrombus Resolution in Mice via Macrophage Recruitment and the Induction of Proteolytic Enzymes Loss in Epithelial Progenitors Reveals an 83 Signaling that Regulates Lung Alveologenesis by Controlling Epithelial Self-Renewal and Differentiation Endoplasmic reticulum stress activates 815 telomerase Shorter telomere 817 lengths in patients with severe COVID-19 disease Short telomeres and severe COVID-19: The connection conundrum A transitional stem cell state in the lung Cell senescence and fibrotic lung diseases Cellular Senescence: Pathogenic Mechanisms in Lung Fibrosis An AI-guided signature 825 reveals the nature of the shared proximal pathways of host immune response in MIS-C and Kawasaki disease. 826 bioRxiv ACE and ACE2 in inflammation: a tale of two enzymes. Inflamm 828 Allergy Drug Targets Ly6d marks the earliest 830 stage of B-cell specification and identifies the branchpoint between B-cell and T-cell development A Dynamic Variation of Pulmonary ACE2 Is Required to Modulate Neutrophilic Inflammation in Response to Pulmonary Angiotensin-Converting Enzyme 2 (ACE2) and Inflammatory Lung Disease Multiple functions of angiotensin-converting enzyme 2 and its 838 relevance in cardiovascular diseases Increased 840 Odds of Death for Patients with Interstitial Lung Disease and COVID-19: A Case-Control Study Hospitalization for COVID-19 in Patients with Interstitial Lung Disease. An International Multicenter Study COVID-19 and Interstitial Lung Disease: Keep Them Separate Risk Factors for Mortality 848 after COVID-19 in Patients with Preexisting Interstitial Lung Disease Pirfenidone in Two Patients of COVID 19 Covid Lung: A Case Series Post-inflammatory pulmonary fibrosis in a discharged COVID-19 patient: 855 Effectively treated with Pirfenidone. Archives of Pulmonology and Respiratory Care The Role of Cytokines including Interleukin-6 in Expression of 861 IL-15 in inflammatory pulmonary diseases Role of IL-15 in interstitial 863 lung diseases in amyopathic dermatomyositis with anti-MDA-5 antibody Serum IL-15 in patients with 865 early systemic sclerosis: a potential novel marker of lung disease Critical role of 869 natural killer cells in lung immunopathology during influenza infection in mice Interferon-stimulated genes: a complex web of host 872 defenses Short telomeres increase the 874 risk of severe COVID-19 Pulmonary 876 fibrosis 4 months after COVID-19 is associated with severity of illness and blood leucocyte telomere length Age Acceleration and Telomere Shortening in COVID-19 Survivors Contributions of alveolar epithelial cell quality control to pulmonary fibrosis Reveals an Age-linked Role for Endoplasmic Reticulum Stress in Pulmonary Fibrosis Hyaluronan and TLR4 promote surfactant-886 protein-C-positive alveolar progenitor cell renewal and prevent severe pulmonary fibrosis in mice Single-cell RNA sequencing identifies 889 diverse roles of epithelial cells in idiopathic pulmonary fibrosis Premature lung aging and cellular senescence in the 891 pathogenesis of idiopathic pulmonary fibrosis and COPD/emphysema TGF-β1/IL-11/MEK/ERK signaling mediates 893 senescence-associated pulmonary fibrosis in a stress-induced premature senescence model of Bmi-1 deficiency PAI-1 Regulation of TGF-β1-896 induced Alveolar Type II Cell Senescence, SASP Secretion, and SASP-mediated Activation of Alveolar 897 SARS-CoV-2 899 infects lung epithelial cells and induces senescence and an inflammatory response in patients with severe 900 COVID-19 Virus-induced 902 senescence is a driver and therapeutic target in COVID-19 Inactivated trivalent 910 influenza vaccination is associated with lower mortality among patients with COVID-19 in Brazil Influenza Vaccination 913 Primes Human Myeloid Cell Cytokine Secretion and NK Cell Function A cell-based systems biology 915 assessment of human blood to monitor immune responses after influenza vaccination Endogenous production 918 of interleukin 15 by activated human monocytes is critical for optimal production of interferon-gamma by 919 natural killer cells in vitro Boolean implication networks derived from 921 large scale, whole genome microarray datasets MiDReG: a method of 923 mining developmentally regulated genes using Boolean implications IHC Profiler: an open source plugin for the quantitative 926 evaluation and automated scoring of immunohistochemistry images of human tissue samples Unbiased Boolean analysis of public gene expression data for cell 929 cycle gene identification Extracting binary signals from microarray time-course 931 data Telomere dysfunction induces 933 environmental alterations limiting hematopoietic stem cell function and engraftment