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Systemic sclerosis (SSc) is a rare systemic autoimmune disease associated with a high mortality. The first histopathological hallmarks are vasculopathy and inflammation, followed by fibrosis of the skin and internal organs. The molecular and cellular mechanisms are incompletely understood. Rodent models provide important insights into the pathogenesis of SSc and are a mainstay for the development of novel targeted therapies. Here we describe the mechanistic insights of inducible and genetic models, and also discuss in detail the limitations and pitfalls of the most frequently used SSc mouse models. We also describe protocols for running the established bleomycin-induced scleroderma skin fibrosis model.

Transforming growth factor-β (TGFβ) is a key mediator of fibroblast activation in fibrotic diseases, including systemic sclerosis. Here we show that Engrailed 1 (EN1) is reexpressed in multiple fibroblast subpopulations in the skin of SSc patients. We characterize EN1 as a molecular amplifier of TGFβ signaling in myofibroblast differentiation: TGFβ induces EN1 expression in a SMAD3-dependent manner, and in turn, EN1 mediates the profibrotic effects of TGFβ. RNA sequencing demonstrates that EN1 induces a profibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. EN1 regulates gene expression by modulating the activity of SP1 and other SP transcription factors, as confirmed by ChIP-seq experiments for EN1 and SP1. Functional experiments confirm the coordinating role of EN1 on ROCK activity and the reorganization of cytoskeleton during myofibroblast differentiation, in both standard fibroblast culture systems and in vitro skin models. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition and are partially protected from experimental skin fibrosis.

Background:Psoriasis (PsO) and psoriatic arthritis (PsA) are two types of chronic inflammatory diseases that share a similar cytokines profile. About 30% of PsO patients also develop a joint involvement, but the underlying mechanism is still unclear. Innate lymphoid cells (ILC) and specifically the type 3 ILCs (ILC3s) have raised increasing interest as possible player in the pathogenesis of both diseases, as they produce the pathological key cytokine IL-17A.Objectives:We addressed the contribution of ILC3s to the pathogenesis of PsO and PsA in patients as well as murinein vivomodels.Methods:130 patients satisfying the Classification Criteria for Psoriatic Arthritis (CASPAR), 40 patients with PsO and 35 healthy volunteers were enrolled in the study. Information regarding clinical features, laboratory parameters were collected and psoriasis area severity index (PASI), disease activity score 28 (DAS28), disease activity in psoriatic arthritis (DAPSA), minimal disease activity score (MDA) were calculated. Magnetic resonance imaging (MRI) and high-resolution peripheral CT (HR-pQCT) were taken and PsA MRI score (PsAMRIS) was assessed. Flow cytometric analysis was performed and IFNγ-producing ILC1s, IL-4/IL-5-producing ILC2s and IL-17/IL-22-producing ILC3s were identified among ILCs. Multivariate linear regression and Receiver-Operating Characteristic (ROC) Curve analysis was performed using the IBM SPSS Statistics software. Different in vivo models were used to assess functional implications of ILCs at different time points of the disease. Joint inflammation was assessed through MRI and H&E staining of ankle areas. Peripheral blood was obtained from mice of each group and flow cytometry analysis was performed. High dimensional analyses including RNA-seq was performed to identify phenotypic characteristics of ILCs implemented into the pathogenesis of the disease.Results:Total number of circulating ILCs were increased in PsA patients compared to PsO and healthy controls (pConclusion:ILC3s not only correlate with various facets of PsA manifestations but also functionally contribute to synovitis and enthesitis suggesting them as interesting target for upcoming treatment strategies in the near future.Disclosure of Interests:Maria Gabriella Raimondo Grant/research support from: Celgene, Partner Fellowship, Simon Rauber: None declared, Markus Luber: None declared, Aleix Rius Rigau: None declared, Stefanie Weber: None declared, Charles Gwellem Anchang: None declared, Rahul Agarwal: None declared, Alina Soare: None declared, Michael Sticherling Grant/research support from: Novartis, Consultant of: Advisory boards Abbvie, Celgene, Janssen Cilag, Lilly, Pfizer, MSD, Novartis, Amgen, Leo, Sanofi, UCB, Speakers bureau: Abbvie, Celgene, Janssen Cilag, Leo, MSD, Novartis, Pfizer, Jürgen Rech Consultant of: BMS, Celgene, Novartis, Roche, Chugai, Speakers bureau: AbbVie, Biogen, BMS, Celgene, MSD, Novartis, Roche, Chugai, Pfizer, Lilly, Arnd Kleyer Consultant of: Lilly, Gilead, Novartis,Abbvie, Speakers bureau: Novartis, Lilly, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Andreas Ramming Grant/research support from: Pfizer, Novartis, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Speakers bureau: Boehringer Ingelheim, Roche, Janssen

Background:Engrailed 1 (EN1) is a homeodomain-containing transcription factor with essential roles in embryonic development. In most cell types, the expression of EN1 is restricted to embryonic development. However, under pathological conditions, EN1 can be re-expressed to promote phenotypical adaptation. En1 is transiently expressed in the developing dermis of murine embryos in a distinct fibroblast lineage and silenced before birth (1). Former EN1-expressing cells give rise to a subpopulation of fibroblasts that has a high capacity for extracellular matrix production in adult murine skin. The role of EN1 in systemic sclerosis (SSc) was previously not explored.Objectives:To study the role of EN1 in the pathological activation of fibroblasts in tissue fibrosis.Methods:Bulk RNA-Seq and EN1 or SP1 ChIP-Seq were performed from cultured human dermal fibroblasts. The expression of EN1 was inhibited by siRNA. Cytoskeletal drugs paclitaxel, vinblastin and ROCK inhibitor (Y27632) were used to modulate the cytoskeleton in EN1 knockdown or overexpressing dermal fibroblasts. The role of EN1 in fibroblast activation was evaluated by functional experiments with EN1 knockdown or overexpression in standard 2D culture systems as well as in 3D skin equivalent models. The role of EN1 in skin fibrosis was further studied in En1fl/fl X Col6Cre mice, with fibroblast-specific knockout of En1 in three complementary mouse models: overexpression of a constitutively active TGFß-receptor I (TBRICA), bleomycin-induced skin fibrosis and TSK1 mice.Results:Pathologically activated dermal fibroblasts from SSc patients express higher levels of EN1 compared with age and sex matched healthy individuals in the skin and in vitro. TGFβ induces EN1 expression in fibroblasts in a SMAD3-dependent manner both in cultured fibroblasts and in murine skin. Knockdown of EN1 prevents TGFβ-induced fibroblast activation, whereas overexpression of EN1 fosters the pro-fibrotic effects of TGFβ with increased expression of αSMA, stress fibers and collagen. RNA sequencing demonstrates that EN1 induces a pro-fibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. In silico analyses of the promoters of En1 target genes coupled with siRNA-mediated knockdown demonstrated that EN1 regulates these pro-fibrotic target genes by modulating the activity of regulatory modules that contain transcription factors of the specificity protein (SP) family. Functional experiments with selective modulators of ROCK and of microtubule polymerization confirm the coordinating role of EN1 on ROCK activity and the re-organization of cytoskeleton during myofibroblast differentiation in both conventional culture systems and 3D skin equivalents. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition, reduced dermal thickening and impaired collagen deposition in the TBRICA, bleomycin-induced and TSK1 models.Conclusion:We characterize the homeodomain transcription factor EN1 as a molecular amplifier of TGFβ signaling in myofibroblast differentiation that coordinates cytoskeletal organization in a SP-dependent manner. EN1 might thus be a novel candidate for molecular targeted therapies to interfere with myofibroblast differentiation in fibrotic diseases.References:[1]Rinkevich Y, Walmsley GG, Hu MS, Maan ZN, Newman AM, Drukker M, et al. Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science. 2015;348(6232):aaa2151.Disclosure of Interests:Andrea-Hermina Györfi: None declared, Alexandru-Emil Matei: None declared, Maximilian Fuchs: None declared, Aleix Rius Rigau: None declared, Xuezhi Hong: None declared, ZHU Honglin: None declared, Markus Luber: None declared, Christina Bergmann: None declared, Clara Dees: None declared, Ingo Ludolph: None declared, Raymund Horch: None declared, Oliver Distler Consultant of: Actellion, AbbVie, Acceleron Pharma, Anamar, Amgen, Blade Therapeutics, CSL Behring, ChemomAb, Ergonex, Glenmark Pharma, GSK, Inventiva, Italfarmaco, iQvia, Medac, Medscape, Lilly, Sanofi, Target BioScience, UCB, Bayer, Boehringer Ingelheim, Catenion, iQone, Menarini, Mepha, Novartis, Mitsubishi, MSD, Roche, Pfizer, Georg Schett: None declared, Meik Kunz: None declared, Jörg H.W. Distler Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB., Grant/research support from: Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB

Background:Fibrotic diseases are characterized by excessive extracellular matrix production as a result of immune-mediated permanent fibroblast activation. Innate lymphoid cells type II (ILC2) are an only recently discovered cell type involved in barrier integrity and tissue homeostasis. There is upcoming evidence that ILC2s play a central role in mediating fibrotic diseases.Objectives:The aim of the study was to further elucidate the role of ILC2s in fibrotic tissue remodeling and fibroblast activation.Methods:Skin biopsies of patients with systemic sclerosis (SSc) or sclerodermatous chronic graft versus host disease (scGvHD) as well as lung biopsies of patients with idiopathic pulmonary fibrosis (IPF) were analyzed by immunofluorescence (IF) staining. Single cell RNA-sequencing (scRNA-seq) was performed on ILCs from fibrotic skin and lung of bleomycin-challenged mice. Further characterization of ILC2 phenotypes in fibrosis models was done by flow cytometry.In vitroculture of fibroblasts and ILC2s was used to study cellular interaction and fibrotic activation. Quantitative realtime-PCR, western blot, IF staining and ELISA were used as readouts.Results:Two different subtypes of ILC2s were found in skin of SSc and scGvHD patients as well as in lungs of IPF patients with one subpopulation being particularly increased in fibrotic tissue. Single cell RNA-sequencing confirmed the existence of two major populations of ILC2s in experimental fibrosis. One subtype showed features of immature ILC2 progenitors and was actively recruited from the bone marrow during fibrotic tissue remodeling. The other ILC2 subset was highly activated and expressed pro-fibrotic cytokines. These profibrotic ILC2s directly interacted with fibroblasts in a cell contact dependent manner. Semaphorin 4A (SEMA4A) expressed by ILC2s bound to Plexin D1 (PLXND1) on fibroblasts. This interaction resulted into fibrotic imprinting with high expression levels of the transcription factor PU.1 which was recently described as central regulator of the pro-fibrotic gene expression program (Wohlfahrt et al. 2019). Signaling through Jagged 1 (JAG1) and Notch receptor 2 (NOTCH2) was identified as a second mechanism of interaction between fibroblasts and ILC2s. JAG1 expressed by fibroblasts activated NOTCH2 signaling in ILC2s which emphazised the secretion of pro-fibrotic cytokines.Conclusion:We identified a bidirectional interaction between ILCs and fibroblasts incorporating a vicious circle of fibrotic tissue remodelling. As ILCs are still not accessible as therapeutic targets these results might contribute to the development of new strategies for anti-fibrotic therapies.References:[1]Wohlfahrt, Thomas, Simon Rauber, Steffen Uebe, Markus Luber, Alina Soare, Arif Ekici, Stefanie Weber, Alexandru-Emil Matei, Chih-Wei Chen, Christiane Maier, Emmanuel Karouzakis, Hans P. Kiener, Elena Pachera, Clara Dees, Christian Beyer, Christoph Daniel, Kolja Gelse, Andreas E. Kremer, Elisabeth Naschberger, Michael Stürzl, Falk Butter, Michael Sticherling, Susetta Finotto, Alexander Kreuter, Mark H. Kaplan, Astrid Jüngel, Steffen Gay, Stephen L. Nutt, David W. Boykin, Gregory M. K. Poon, Oliver Distler, Georg Schett, Jörg H. W. Distler, and Andreas Ramming. 2019. ‘PU.1 controls fibroblast polarization and tissue fibrosis’,Nature, 566: 344-49.Disclosure of Interests:Stefanie Weber: None declared, Charles Gwellem Anchang: None declared, Simon Rauber: None declared, Markus Luber: None declared, Maria Gabriella Raimondo Grant/research support from: Celgene, Partner Fellowship, Yuko Ariza Employee of: Ono Pharmaceutical Co., Ltd., Aleix Rius Rigau: None declared, Alexander Kreuter: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim, Andreas Ramming Grant/research support from: Pfizer, Novartis, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Speakers bureau: Boehringer Ingelheim, Roche, Janssen