key: cord-0798305-bgjlbq0v authors: Musiu, C.; Caligola, S.; Fiore, A.; Lamolinara, A.; Frusteri, C.; Del Pizzo, F. D.; De Sanctis, F.; Cane', S.; Adamo, A.; Hofer, F.; Barouni, R. M.; Grilli, A.; Zilio, S.; Serafini, P.; Tacconelli, E.; Donadello, K.; Gottin, L.; Polati, E.; Girelli, D.; Polidoro, I.; Iezzi, P. A.; Angelucci, D.; Capece, A.; Chen, Y.; Shi, Z.-L.; Murray, P. J.; Chilosi, M.; Amit, I.; Bicciato, S.; Iezzi, M.; Bronte, V.; Ugel, S. title: Fatal cytokine release syndrome by an aberrant FLIP/STAT3 axis date: 2021-05-04 journal: nan DOI: 10.1101/2021.05.04.21256298 sha: 39c7d5bcec4753bc4572f50471d6ca61cb1b0be9 doc_id: 798305 cord_uid: bgjlbq0v Inflammatory responses rapidly detect pathogen invasion and mount a regulated reaction. However, dysregulated anti-pathogen immune responses can provoke life-threatening inflammatory pathologies collectively known as cytokine release syndrome (CRS), exemplified by key clinical phenotypes unearthed during the SARS-Cov-2 pandemic. The underlying pathophysiology of CRS remains elusive. We found that FLIP, a protein that controls caspase-8 death pathways, was highly expressed in myeloid cells of COVID-19 lungs. FLIP controlled CRS by fueling a STAT3-dependent inflammatory program. Indeed, constitutive expression of a viral FLIP homologue in myeloid cells triggered a STAT3-linked, progressive and fatal inflammatory syndrome in mice, characterized by elevated cytokine output, lymphopenia, lung injury and multiple organ dysfunctions that mimicked human CRS. As STAT3-targeting approaches relieved inflammation, immune disorders, and organ failures in these mice, targeted intervention towards this pathway could suppress the lethal CRS inflammatory state. We previously identified the anti-apoptotic cellular and viral FLICE converting enzyme)-like inhibitory proteins (hereafter c-FLIP and v-FLIP respectively) as a 123 factor that "reprogrammed" monocytes(35). FLIP isoforms are conventionally described to 124 control cell survival and proliferation as caspase-8 inhibitor and/or NF-κB activator (36) (37) (38) (39) (40) . 125 However, FLIPs regulate different biological processes based on their protein structure 126 (e.g. the presence of death effector domains in tandem)(41) or cellular localization (e.g. Table S1a ). In the 165 latter group, the respiratory tract was not altered by severe inflammation and showed a 166 tissue structure almost devoid of pathological aspects (Fig. 1a, infiltrating myeloid cells in SARS-CoV-2-infected mice expressed higher p-STAT3 levels 221 than the control group (Fig. 1f) . Moreover, we found an increased expression of p-STAT3 222 in c-FLIP-expressing cells, which morphologically resemble myeloid cells, in the lung of 223 SARS-CoV-2-infected mice (Fig. 1f ). Collectively, these data establish that myeloid subsets are converted into FLIP-and were obtained across WT and vFLIP mice. All the cells were visualized through t-284 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 4, 2021. Conversely, monocytes (~9% vFLIP; ~12% WT) and macrophages (~13% vFLIP; ~16% 292 WT) were comparable between the two groups. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 4, 2021. ; https://doi.org/10.1101/2021.05.04.21256298 doi: medRxiv preprint neutrophil markers including S100-family genes and Adam8 (cluster 0), inflammatory 327 chemokines Ccl3 and Ccl4 (cluster 1), high expression of Camp and Ngp genes (cluster 2) 328 and interferon response genes Isg15/Isg20 (cluster 3) ( Fig. 4a and Supplementary 329 information, Table S2 ). Similarly, in COVID-19 BAL, we outlined 5 different clusters 330 expressing marker genes similar to mouse neutrophil subsets. In particular, we observed 331 clusters expressing canonical neutrophil markers such as S100A8/S100A9 genes (cluster 332 a), CCL3/CCL4 chemokines as well as CTSB and CSTB genes (cluster b), interferon-333 response genes IFIT1, IFIT2 and ISG15/ISG20 (cluster c) and other 2 clusters expressing 334 ribosomal (cluster d) and heat shock protein (HSPs) genes (cluster e) ( Fig. 4b and 335 Supplementary information, Table S2 ). 336 We used reference-based classification (see Methods section) to map mouse subsets into 337 human subsets. The results confirmed a conserved structure among neutrophil clusters 338 between the two species and pathologies (Fig. 4c) . In fact, about 89% of the human 339 cluster c was annotated as cluster 3 of mouse, about 81% of cluster d was annotated as 340 cluster 1 and, finally, clusters a, b and e were mainly annotated as cluster 1 of mouse (60-341 65%). Conversely, cluster 2 appeared to be a mouse-specific neutrophil subset with no 342 relevant correspondence in human subsets. These results recapitulate published reports 343 (69), in which 3 conserved modules between mouse and human were characterized by the 344 expression of Ccl3/CCL3, Cstb/CSTB (cluster 1 in vFLIP), type I interferon response genes 345 such as Ifit1/IFIT1, Irf7/IRF7 and Rsad2/RSAD2 (cluster 3 in vFLIP), and neutrophils 346 expressing canonical markers S100a8-a9/S100A8-A9 (cluster 0 in vFLIP). Table S2 ). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 4, 2021. ; https://doi.org/10.1101/2021.05.04.21256298 doi: medRxiv preprint and Cd300e (cluster 2), reproducing the immune landscape previously described in tumor-367 bearing mice(69). In monocytes/macrophages compartment from BAL patients, we 368 observed 5 clusters (Fig. 4f) Table S2 ), which was also the most 392 dysregulated in terms of cell proportion (~19% vFLIP; ~8% WT) (Fig. 4e) . Notably, the 393 up-regulation of JAK-STAT3 signaling pathway, in vFLIP cluster 4 compared to WT, was 394 paralleled by the expression of several genes in the pathway such as Jak2, Csf1, Il10rb 395 and Cd38 (Fig. 4h, bottom panels) . To investigate whether targeting STAT3 could be sufficient to dampen the uncontrolled 404 immune dysregulation in vFLIP mice, we evaluated therapeutic approaches involving either 405 pharmacological or RNA interference of STAT3. Initially, we tested in vivo the efficacy of 406 two STAT3 inhibitors: silibinin, a STAT3 inhibitor that blocks the Y705 phosphorylation-407 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 4, 2021. 11 related and STAT3 dimerization(70), or baricitinib, a clinically-approved inhibitor of JAK1 408 and JAK2 able to interfere with STAT3 signaling activation(71). Four weeks after the 409 establishment of BM chimerism in recipients (T0), mice were treated every two days by 410 intraperitoneal injection. Weight loss was significantly decreased in mice that had received 411 the drug treatments (Supplementary information, Fig. S5a ). Furthermore, STAT3 targeting 412 reduced the plasma concentration of several pro-inflammatory cytokines (Fig. 5a) , which 413 are produced at abnormal levels in untreated vFLIP mice and are also a feature of COVID- (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 4, 2021. ; 12 while Ifitm3, Stat1 and Isg15 genes, which are associated to interferon response 449 pathways, were up-regulated in baricitinib-treated vFLIP mice (Fig. 5f) To confirm the immunomodulatory capacity of STAT3 inhibitors, we tested the ability of showed also a reduction in systemic lymphopenia, with an increased frequency of both T 478 and B lymphocytes and a concomitant myeloid cell reduction in the spleen (Fig. 6c) and in 479 peripheral blood (Fig. 6d) . Finally, the 4PD-mediated, STAT3-specific shRNA delivery 480 normalized the plasma concentration of several pro-inflammatory cytokines (Fig. 6e) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 4, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 accepted by the scientific community. Our data establish FLIP-expressing myeloid cells as 490 a pivotal driver of CRS. Indeed, the vFLIP chimera mice show acute systemic inflammatory 491 symptoms ( Fig. 2 and Supplementary information, Fig. S2-3) , elevated cytokine levels 492 (Fig. 5a) , weight loss (Supplementary information, Fig. S5a) , lymphopenia (Supplementary 493 information, Fig. S2c ), lung injury (Fig. 2a) endothelial cell activation (Fig. 2d) and (Fig. 1d-e; Fig. 2i ). Monocytes from both COVID-19 patients (Fig. 1c) and vFLIP transgenic mice(35) display 526 immunosuppressive properties and are a source of pro-inflammatory cytokines (Fig. 1e 527 and Supplementary information, Fig. S1d ). We found that immunoregulatory functions of 528 monocytes isolated from COVID-19 patients correlated with the expression of PD-L1 529 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 4, 2021. Drug Administration to approve baricitinib in combination with remdesivir for the 564 treatment of severe COVID-19 patients. More important, we demonstrated that Jak1/Jak2 565 inhibitor did not affect negatively genes associated with type I IFN antiviral responses but, 566 on the contrary, pharmacological treatment was associated with a relative increase in 567 interferon-stimulated genes (Fig. 5e-f ), likely by re-programming the myeloid composition 568 in the lung environment ( Fig. 5d and Supplementary information, Fig. S5c ). This signaling 569 switch might be due to the activity of STAT1 occupying space on STAT3-activating 570 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 4, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 15 receptors, as suggested by the conversion of IL-6R signaling to a dominant STAT1 571 activation in 86) . Finally, the striking results on the normalization 572 of the immune landscape, organ pathology and cachexia following shSTAT3-based 573 treatment ( Fig. 6 and Supplementary information, Fig. S6 ) clearly finger at STAT3 as the 608 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This study was designed to define vFLIP + p-STAT3 + myeloid cells as a main driver for CRS 622 progression as well as to test STAT3-targeting approach as a possible strategy to 623 ameliorate CRS-undergoing hosts. All genetically transgenic mice and their respective 624 controls were gender and age-matched (typically 3-10 weeks) and both males and 625 females were used in this study. Mice were assigned randomly to experimental groups. Germ free C57BL/6 mice were originally purchased from Charles River Laboratories Inc., The in vivo effect of drugs treatment was investigated in the vFLIP-chimera mouse model, 642 four weeks after the bone marrow cells transplantation. Chimera mice that displayed at 643 least 20% of donor-derived cells were randomized before beginning treatment. Chimera 644 mice were treated using 8 intraperitoneal administrations of Baricitinib (10 mg/kg; Cayman 645 chemicals) or Silibinin (100mg/kg; Sigma-Aldrich) every two days, for a total of 9 646 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 4, 2021. ; https://doi.org/10.1101/2021.05.04.21256298 doi: medRxiv preprint 17 treatments. shRNA treatments were performed three times a week by injecting shRNA 647 (anti-STAT3 or scramble) loaded onto 4PD intravenously as previously described (72) Chemokines and chemokine receptors: positioning 676 cells for host defense and immunity Hematopoietic 678 cytokines can instruct lineage choice Fatal outcome of human influenza A (H5N1) is associated with high 681 viral load and hypercytokinemia Host-pathogen interactions in sepsis macrophages and abated by IL-1 blockade Cytokine release syndrome associated with chimeric-antigen receptor T-cell therapy: clinicopathological insights Cytokine-release syndrome in patients with B-cell chronic lymphocytic 691 leukemia and high lymphocyte counts after treatment with an anti-CD20 monoclonal 692 antibody (rituximab, IDEC-C2B8) Cytokine release syndrome after blinatumomab treatment related to 694 abnormal macrophage activation and ameliorated with cytokine-directed therapy Severe Cytokine-Release Syndrome after T Cell-Replete Peripheral Blood 697 Identifying and targeting pathogenic PI3K/AKT/mTOR signaling in 700 IL-6-blockade-refractory idiopathic multicentric Castleman disease Plasma proteomics identifies a 'chemokine storm' in idiopathic 703 multicentric Castleman disease The acute respiratory distress syndrome Clinical features of patients infected with 2019 novel coronavirus in Disease Control, Civil Liberties, and Mass Testing -Calibrating 709 Restrictions during the Covid-19 Pandemic A novel coronavirus outbreak of global 711 health concern A pneumonia outbreak associated with a new coronavirus of probable bat 713 origin Clinical Characteristics of Coronavirus Disease 2019 in China Pathological findings of COVID-19 associated with acute respiratory distress 717 syndrome Acute Lung injury evolution in Covid-19. medRxiv Pulmonary post-mortem findings in a series of COVID-19 cases from 721 northern Italy: a two-centre descriptive study Immune mechanisms of 723 pulmonary intravascular coagulopathy in COVID-19 pneumonia Extrapulmonary manifestations of COVID-19 Endothelial cell infection and endotheliitis in COVID-19 COVID-19: consider cytokine storm syndromes and immunosuppression Targeting potential drivers of COVID-19: Neutrophil extracellular traps Association of COVID-19 inflammation with activation of the C5a-C5aR1 736 axis COVID-19 and the human innate immune system Deciphering the state of immune silence in fatal COVID-19 patients An inflammatory cytokine signature predicts COVID-19 severity and 744 survival Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell 746 Compartment Induction of immunosuppressive functions and NF-κB by FLIP in monocytes Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death 750 receptors Inhibition of death receptor signals by cellular FLIP Modulation of virus-induced NF-κB signaling by NEMO coiled coil mimics The c-FLIP-NH2 terminus NF-kappaB activation Safa, c-FLIP, a master anti-apoptotic regulator Viral and cellular FLICE-inhibitory proteins: a comparison of their roles in 759 regulating intrinsic immune responses Modulation of Wnt signaling by the nuclear localization of cellular FLIP Nuclear localization of c-FLIP-L and its regulation of AP-1 activity Viruses and apoptosis Hepatitis C virus core protein inhibits tumor necrosis factor alpha-mediated 767 apoptosis by a protective effect involving cellular FLICE inhibitory protein Multiple Functions of Cellular FLIP Are Essential for Replication of Hepatitis 770 B Virus HTLV-1 Tax protects against CD95-mediated apoptosis by induction of 772 the cellular FLICE-inhibitory protein (c-FLIP) HIV-1 Tat protein concomitantly down-regulates apical caspase-10 and 774 up-regulates c-FLIP in lymphoid T cells: a potential molecular mechanism to escape TRAIL 775 cytotoxicity Modulation of caspase-8 and FLICE-inhibitory protein expression 777 as a potential mechanism of Epstein-Barr virus tumorigenesis in Burkitt's lymphoma Cellular inhibitor of apoptosis protein cIAP2 protects against 780 pulmonary tissue necrosis during influenza virus infection to promote host survival Two distinct immunopathological profiles in autopsy lungs of COVID-19 The role of macrophages in interstitial lung diseases: Number 3 in the Series 785 "Pathology for the clinician The contributions of lung macrophage and monocyte 788 heterogeneity to influenza pathogenesis Immunosuppression by monocytic myeloid-derived suppressor cells in 790 patients with pancreatic ductal carcinoma is orchestrated by STAT3 Monocytes in the Tumor Microenvironment STAT3 regulates arginase-I in myeloid-derived suppressor cells 795 from cancer patients STATs in cancer inflammation and immunity: a leading role for 797 STAT3 Pathological inflammation in patients with COVID-19: a key role for 799 monocytes and macrophages Baricitinib restrains the immune dysregulation in patients with severe 801 COVID-19 Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human 803 Angiotensin-Converting Enzyme 2 SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 805 pathology X-ray-induced P-selectin 807 localization to the lumen of tumor blood vessels Neutrophil extracellular traps contribute to immunothrombosis in 809 COVID-19 acute respiratory distress syndrome Neutrophil extracellular traps kill bacteria Cytokines induced neutrophil extracellular traps formation: implication 813 for the inflammatory disease condition Regulation of lymphocyte proliferation and death by FLIP SARS-CoV-2 infection of human ACE2-transgenic mice causes severe 817 lung inflammation and impaired function Baricitinib treatment resolves lower-airway macrophage inflammation 819 and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals 821 Silibinin is a direct inhibitor of STAT3 JAKs and STATs in immunity, immunodeficiency, 826 and cancer 4PD Functionalized Dendrimers: A Flexible Tool for In Vivo Gene Silencing of Tumor-Educated Myeloid Cells Chronic intestinal inflammation in mice expressing viral Flip in epithelial 830 cells Kaposi sarcoma herpesvirus vFLIP oncoprotein induces B cell transdifferentiation and tumorigenesis in mice Down-regulation of FoxO-dependent c-FLIP 838 expression mediates TRAIL-induced apoptosis in activated hepatic stellate cells Comparative Transcriptome Analysis Reveals the Intensive Early Stage 841 Responses of Host Cells to SARS-CoV-2 Infection Roles of c-FLIP in Apoptosis, Necroptosis, and Autophagy Moonlighting Proteins Are Important Players in Cancer Immunology Disease-association analysis of an inflammation-related feedback loop COVID-19: A New Virus, but a Familiar Receptor Multi-organ proteomic landscape of COVID-19 autopsies JAK inhibition reduces SARS-CoV-2 liver infectivity and modulates 853 inflammatory responses to reduce morbidity and mortality Baricitinib plus Remdesivir for Hospitalized Adults with Covid-19 Alternative activation of STAT1 and STAT3 in response to interferon-857 gamma The JAK-STAT signaling pathway: input and output integration An experimental model of idiopathic pneumonia syndrome after bone 861 marrow transplantation: I. The roles of minor H antigens and endotoxin Integrating single-cell 864 transcriptomic data across different conditions, technologies, and species Normalization and variance stabilization of single-cell RNA-seq 867 data using regularized negative binomial regression Scrublet: Computational Identification of Cell Doublets 871 in Single-Cell Transcriptomic Data Reference-based analysis of lung single-cell sequencing reveals a transitional 873 profibrotic macrophage Immunological Genome Project, The Immunological Genome 875 Project: networks of gene expression in immune cells Remodeling of epigenome and transcriptome landscapes with aging 877 in mice reveals widespread induction of inflammatory responses Mapping the Mouse Cell Atlas by Microwell-Seq BLUEPRINT: mapping human blood cell epigenomes The ENCODE project An expression atlas of 885 human primary cells: inference of gene function from coexpression networks Deconvolution of Human Immune Cell Types Fast gene set enrichment analysis. bioRxiv Gene set enrichment analysis: a knowledge-based approach for 891 interpreting genome-wide expression profiles GSVA: gene set variation analysis for microarray 894 and RNA-seq data Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 The Molecular Signatures Database (MSigDB) hallmark gene set 898 collection CLIP 2020) and M.I.; 941 Computational analysis and data interpretation: Si.C., A.G. and S.B.; 942 Methodology for the in vivo RNA delivery experiments and data interpretation Methodology of human ACE2 mouse model, pathological analysis and data interpretation: 947 Y.C Writing and data interpretation hold proprietary rights on the patent applications about engineered 955 cells for inducing tolerance by BioNTech Representative IF staining of lung-infiltrating 983 neutrophils and macrophages in WT or vFLIP mice. Cells were stained for DAPI (blue), 984 neutrophil elastase (NE) (red, middle panel) and F4/80 (green, bottom panel). Scale bar, 985 200 μm. (C) Representative IF staining of lung-infiltrating neutrophils and macrophages in 986 WT or vFLIP mice. Scale bar, 20 μm. Cells were stained for DAPI (blue), NE (red, middle 987 panel) or F4/80 (green, bottom panel) and pSTAT3 Tyr705 (grey). (D) CD62P presence in 988 lung of WT Representative confocal analysis of NET in WT (left panel; 50 μm) or vFLIP mice Cells were stained for DAPI (white), NE (red) and H3cit (blue). (F) Dot plots of IL-6 CD45.2 + Ly6G + cells) cells isolated from vFLIP mice (n=17). (G) Representative IF 993 staining of spleen-infiltrating neutrophils (NE + cells) and monocytes/macrophages (F4/80 + 994 cells) in WT or vFLIP mice Scale bar, 200 μm. (H) Representative IF staining of spleen-996 infiltrating neutrophils and macrophages in WT or vFLIP mice. Cells were stained for DAPI 997 (blue), NE (red, middle panel) or F4/80 (green, bottom panel) Dot plots of IL-6 and TNF-α in myeloid cells in spleen of WT (n=8) or vFLIP mice 999 (n=9) No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. syndrome. 1004 (A) tSNE representation of scRNA-seq from 2 WT and 2 vFLIP mice samples (WT: 9,174; 1005 vFLIP: 9,847) colored according to cell type. Stacked bar plots representing cell type 1006 proportions across WT and vFLIP conditions. (B) Bar plot representing the up-regulated 1007 (NES > 0, adjusted p-value < 0.05) hallmark gene sets in the analysis of vFLIP vs WT cells 1008 obtained through GSEA analysis. (C) Violin plots showing the expression of key genes that 1009 drive the up-regulation of inflammatory response, TNF-α signaling via NF-κB and JAK-1010 STAT3 signaling pathway in the lung of vFLIP mice. The asterisks denote statistically 1011 significant up D) Bar plot representing the up-regulated (NES > 0, 1013 adjusted p-value < 0.05) hallmark gene sets in bulk RNA-seq data obtained through GSEA 1014 analysis of ACE2-transgenic mice infected with Sars-CoV-2 (Winkler et al., 2020) 1015 comparing day 7 post infection (dpi) with mock-infected (0 dpi No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity BAL-derived immune cells obtained from COVID-19 patients. 1024 (A) tSNE representation and stacked bar plot showing cluster analysis of neutrophils 1025 coming from scRNA-seq analysis of lung tissue of mice (n=7) and (B) COVID-19 patients 366) are visualized through 1027 tSNE projection and colored according to cluster analysis. (C) Reference-based 1028 classification of BAL neutrophil clusters using average expression of mice neutrophil 1029 clusters (see Methods). (D) Violin plots showing the expression of key inflammatory and 1030 interferon response genes among mouse neutrophil clusters. The asterisks denote 1031 statistically significant up-regulation in the comparison between vFLIP and WT conditions 1032 (*p < 0.05, **p < 0.01, ***p < 0.001). (E) Subset analysis of monocytes/macrophages 1033 of mouse (n = 7) and (F) COVID-19 BALs (n = 7). Monocytes/macrophages of mouse 1034 (6,115) and human (3,445) are visualized through tSNE projection and colored according 1035 to cluster analysis. (G) GSVA scores for inflammatory response Violin plots showing the expression level of key 1038 inflammatory response and JAK-STAT3 signaling pathway genes across mouse 1039 monocytes/macrophages subsets. The asterisks denote statistically significant up-1040 regulation in the comparison between vFLIP and WT conditions Analysis of cytokines levels in serum of vFLIP mice before treatment (T0) or at the 1046 end of treatment (untreated, n=9; silibinin, n=9; baricitinib, n=9). (B) Lymphocytes and 1047 macrophages quantification in spleens of untreated (n=14), silibinin (n=8) and baricitinib 1048 (n=16) vFLIP mice by H&E staining. Scale bar, 200 μm. (C) Pathological score of lungs of 1049 untreated (n=14), silibinin (n=8) and baricitinib (n=16) vFLIP mice by H&E staining. Scale 1050 bar, 200 μm. (D) tSNE representation of scRNA-seq from untreated (4,662) mice and 1051 treated with silibinin (3,414) and baricitinib (4,177) colored according to cell type. Stacked 1052 bar plots representing cell type proportions across conditions. (E) Bar plot representing 1053 the up-and down-regulated (adjusted p-value < 0.05) hallmark gene sets obtained in the 1054 bulk-like analysis of treated compared to untreated vFLIP chimera cells obtained through 1055 GSEA analysis. (F) Violin plots showing the expression of genes involved in inflammatory 1056 response, JAK-STAT3 signaling pathway and interferon response in the lung of vFLIP 1057 chimera mice 001 by Student's t-test, Mann-Whitney test. 1059 1060 1061 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. immunopathological disorders in vFLIP mice. 1064 (A) Pathological score of lungs of vFLIP mice Scale bar, 200 μm. (B) Flow cytometry analysis of CD45 + 1066 leukocytes isolated from lungs of vFLIP mice (untreated, n=6 Lymphocytes and macrophages quantification in 1068 spleens of vFLIP mice (untreated, n=6; sh-scramble, n=5; sh-STAT3, n=5) by H&E 1069 staining. Scale bar, 400 μm. (D) Flow cytometry analysis in peripheral blood in vFLIP mice 1070 (untreated, n=6; sh-scramble, n=5; sh-STAT3, n=5) or WT mice (n=5). e Analysis of 1071 cytokines levels in serum of WT (n=5) Data are reported as mean ± S.E.M. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 by Mann-1073 Whitney test No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity