OBJECTIVE:Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized by immune dysregulation and fibrosis, with myeloid-derived suppressor cells (MDSCs) emerging as important regulators of immune responses. However, the role of MDSCs in SSc-associated fibrosis and their interactions with other immune cell populations remain poorly understood. METHODS:Peripheral blood (SSc: N=37, healthy: N=27) and skin biopsies (SSc: N=9, healthy: N=5) were analyzed using flow cytometry and fluorescence microscopy, respectively. Bleomycin-induced mouse models of fibrosis were used to validate findings, including pharmacological disruption of granulocytic MDSC (G-MDSC) activity via Bruton's tyrosine kinase inhibition (ibrutinib). Single-cell RNA sequencing data from SSc and control skin and lung samples (skin: 19 SSc, 20 healthy; lung: 7 SSc, 15 healthy), complemented by spatial transcriptomics analysis of skin (12 SSc, 7 healthy), were integrated to define cellular interactions relevant to disease pathogenesis. Functional studies and bulk RNA sequencing were conducted to dissect the underlying G-MDSC interactions. RESULTS:G-MDSCs were significantly enriched in the circulation and skin of SSc patients, correlating with disease severity. Experimental fibrosis revealed enrichment of G-MDSCs, whereas monocytic MDSCs remained stable. Transcriptomic data revealed a strong interaction between MDSCs and type 2 innate lymphoid cells (ILC2s), promoting a pro-fibrotic environment via IL-13-driven TGFβ, Arginase1 and CCL24. Functional assays confirmed that G-MDSCs promote Tgfb1 expression in ILC2s, further driving tissue fibrosis. CONCLUSION:These findings explore a so far unknown G-MDSC-ILC2 axis as a driver of fibrosis in SSc. Targeting this interaction may represent a novel therapeutic strategy to mitigate fibrotic disease progression.

OBJECTIVES:Fibrosis progression in systemic sclerosis (SSc) has been attributed to matrix stiffness. Despite extensive research on fibroblast heterogeneity and subset imbalances in fibrotic disorders, the interplay between biomechanical cues and fibroblast dynamics remains largely unexplored. Here, we investigate how matrix stiffness alters fibroblast transcriptional state and influences lineage specification in fibrotic skin. METHODS:We employed a collagen I-based 3-dimensional culture system to expose fibroblasts to varying levels of matrix stiffness, followed by RNA sequencing to identify stiffness-responsive gene expression signature. We integrated single-cell RNA sequencing data from SSc and healthy skin samples to identify fibroblast subsets associated with this signature. Spatial transcriptomic analyses were performed to localise these fibroblasts and their associations with the fibrotic niche. RESULTS:Fibroblasts subjected to increased matrix stiffness exhibited a distinct transcriptional signature, amplified in SSc patients and enriched in PI16+ progenitor-like cells within the SFRP2+ fibrotic compartment. Further analysis indicated that PI16+ fibroblasts are predisposed to SFRP2+COMP+ PU.1+ myofibroblasts differentiation, whereas blocking mechanotransduction by focal adhesion kinase inhibition disrupts this process, suggesting that matrix stiffness is a key driver of this lineage transition. Spatial mapping revealed colocalisation of the PI16+ and COMP+ subsets in extracellular matrix-dense regions, highlighting the functional relevance of this relationship in fibrotic progression. CONCLUSIONS:Our findings suggest that increased matrix stiffness promotes fibroblast precursor differentiation into SFRP2+ COMP+ PU.1+ myofibroblasts, thereby sustaining the vicious cycle of persistent fibrosis in absence of inflammatory triggers. These insights reveal new aspects of fibrosis pathogenesis and highlight biomechanical signals as therapeutic targets in SSc.

Background Persistently activated fibroblasts status leads to progressive extracellular matrix (ECM) deposition and tissue remodeling. The hallmark of systemic sclerosis (SSc) is collagen accumulation in organs, mainly in skin and lung. Despite intensive progress in understanding disease occurence, SSc remains an intriguing disease with unknown pathology and high mortality. Sorbin and SH3 domain-containing protein2, encoded by SORBS2 is a key member of the sorbin homology family of adapter and scaffold proteins. Recent studies suggest that SORBS2 plays a role in cardiac disease, however there is no available data about its role in fibrotic conditions. Objectives We aimed to investigate the role of SORBS2 in the pathogenesis of SSc. Methods To identify molecules specifically upregulated in persistently activated fibroblasts, human fibroblasts were chronically stimulated with TGF-β and analyzed by RNA-sequencing. To evaluate the functional implication of tissue elasticity on the transcriptome of fibroblasts, multiwell stiffness assays were performed. SORBS2 expression was further analyzed in skin samples of patients with SSc and murine models of fibrosis. Fibroblast specific SORBS2 knockout mice were challenged with bleomycin to induce skin and lung fibrosis. Further specific readouts like collagen content, skin thickness, myofibroblast count, CT scans were performed. Results Upon chronic TGF-β stimulation of human normal skin fibroblasts we identified SORBS2, as significantly upregulated molecule. SORBS2 is implicated in cytoskeletal organization, cell adhesion and different signaling pathways. Moreover, extracellular stiffness induced upregulation of SORBS2 mRNA level in fibroblasts. Deletion of SORBS2 in fibroblasts led to a change of the diameter of collagen fibers and modified the elastic index of the tissue. SORBS2 expression is not only elevated in different animal models of fibrosis, but also in fibrotic skin samples of SSc patients. SORBS2 knockout mice (KO) developed significantly less skin fibrosis upon bleomycin challenge in comparison to wild type mice (WT), as assessed by measurement of dermal thickness, myofibroblast counts and hydroxyproline content. Col1a1, Col1a2 and expression of αSMA were significantly lower in SORBS2 KO mice in comparison to SORBS2 WT mice. Similarly, fibroblast specific knockout of SORBS2 showed protective effects in bleomycin induced lung fibrosis. CT scans of the lungs showed statistically significant less fibrotic changes in SORBS2 KO mice in comparison to wild type mice. Conclusion SORBS2 is engaged in a vicious circle of fibrosis. Triggered by chronic TGF-β stimulation, SORBS2 is further upregulated by the increasing stiffness of the extracellular matrix. This leads to persistently high levels of SORBS2 resulting into further production of ECM products. Deleting SORBS2 has potent antifibrotic effects in animal models of skin and lung fibrosis. As most of the currently used therapeutic approaches are focussed on early stages of the disease, SORBS2 might be an interesting new therapeutic target in established stages of SSc. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests None Declared.

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 murine in vivo models. 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 (p<0,001). Linear regression analyses of the relationship between disease activity and circulating ILCs counts showed strongest correlation between ILC3s counts and DAPSA score. ILC3s counts also correlated with imaging signs of inflammation such as enthesitis, synovitis, erosions and/or ostoeproliferation as assessed by MRI and HR-pQCT. Musculoskeletal inflammation in mice was predominantly associated with p19 expression and IL-23R-signaling as assessed by RNA-seq. These effects were also accompanied by a strong upregulation of IL-17-producing lymphocytes within the inflamed joint niche with a dominant presence of ILC3s. Multi-channel immunofluorescence and confocal laser scanning microscopy revealed not only upregulation of ILC3 induced IL-17 production within the synovial membrane but also in peri-articular areas of the inflamed joints. Conclusion: 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: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by microangiopathy and fibrosis. In physiological wound healing, fibroblasts are transiently activated for tissue repair. In contrast, fibroblasts are persistently activated during fibrosis and thus resulted in progressive matrix deposition and tissue remodeling. However, the pathogenesis of the fibrotic process is not fully understood. Objectives: We aimed to identify molecules that play a role in chronically activated fibroblasts. Methods: To identify molecules specifically upregulated in human fibrotic fibroblasts, RNA-sequencing was performed. Identified adaptor proteins were further validated in skin biopsy samples of patients with limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc), and evaluated correlation between expression levels and clinical parameters. Respective overexpression and siRNA knockdown were further addressed in vitro . Functional effects were assessed by qPCR, hydroxyproline, proliferation and migration assays. Mouse models of systemic sclerosis were used to functionally validate adaptor proteins in vivo . Results: We identified adaptor proteins as significantly upregulated molecules in chronically active fibroblasts of skin biopsy samples from SSc patients compared to fibroblasts from healthy controls. Expression levels were correlated with the modified Rodnan skin score in the skin of SSc patients. We observed higher expression levels also in the mouse model of topoisomerase I induced skin fibrosis. This result was also observed in bleomycin induced lung fibrosis model suggesting important functions of adaptor proteins during fibrotic tissue remodeling across different organs. Fibroblast-specific knockout resulted into significantly attenuated bleomycin-induced fibrosis. Upon bleomycin challenge, hydroxyproline content was diminished in mice with genetic deficiency of adaptor proteins. In addition, COL1A1, COL1A2 and Lum mRNAs and also the number of myofibroblasts were significantly lower in knockout mice compared to wild type mice. In vitro , knockdown of adaptor proteins resulted into a significant alteration of the migratory potential of fibroblasts. Conclusion: Our results demonstrate that adaptor proteins play an essential role in the pathogenesis of systemic sclerosis. Understanding the molecular mechanism of adaptor proteins may lead to a novel therapeutic intervention in human SSc and related disorders. Disclosure of Interests: Yuko Ariza Employee of: Ono Pharmaceutical Co., Ltd., Stefanie Weber: None declared, Nils Neise: 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

Background: Activation of the immune system is a characteristic feature of SSc. Numerous studies have suggested that type 2 are key drivers of progressive fibrosis. Recently, innate lymphoid cells (ILC) are emerging as an important cellular source of type 2 cytokines triggering fibrotic tissue remodeling independently of the adaptive immune system. Increased levels of ILC2 were found in patients with SSc. However, the contributive role of ILC2 in pathogenesis of SSc is not completely understood. Objectives: We aim in our 3 years observational study to evaluate the predictive role of ILC2 in SSc patients. Methods: We conducted an observational retrospective study on 52 patients with SSc fulfilling the 2013 ACR/EULAR classification criteria. Yearly clinical, laboratory and investigational data according to EUSTAR recommendations were collected. Blood samples collected between 15.09.2014 and 15.01.2015 were analyzed by flow cytometry and ILC2 counts were measured. The predictive value of ILC2 during a 3-year follow-up was analyzed using SPSS 21.0. Results: 52 patients were included in the study, 78% female, 63% limited cutaneous SSc with a mean follow-up time of 2.85 ± 1.28 years. At baseline we have shown that circulating ILC2s are significantly increased compared to gender and age-matched healthy controls. Increased numbers of ILC2s significantly correlated with worsening of mRSS calculated by five point increase in mRSS or 25% increase from baseline (p < 0.001; 95% CI 1.39 – 3.26). ILC2 counts also correlated with 5% decrease of diffusion capacity of carbon monoxide (DLCO) during the follow-up time (p < 0.0001; 95% CI 1.83 – 3.49). Worsening of forced vital capacity (FVC) assessed as 5% decrease over 2 years was also significantly correlated with an increased number of ILC2s (p < 0.0001; 95% CI 1.27 – 3.04). In contrast, we did not find any correlation regarding increase in pulmonary arterial pressure assessed by echocardiography. Although new appearance of digital ulcers could not be predicted by ILC2 counts, increased numbers of ILC2s were correlated with digital ulcers at follow-up. Conclusion: Here, we provide first evidence for a role of ILC2s as potential prognostic marker of disease progression in SSc. Disclosure of Interests: Alina Soare: None declared, Stefanie Weber: 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

Background: Since vascular manifestations such as Raynaud’s phenomenon often precede the onset of other clinical manifestations of systemic sclerosis (SSc), the identification of pathways linking vasculopathy to organ fibrosis might thus provide important insights into early disease mechanisms and allow early targeted intervention for both fibrotic and vascular events. Objectives: In this study we performed high dimensional (HD) analyses to identify mediators that link vasculopathy to organ fibrosis. Methods: HD techniques including RNA-seq, ChIP-seq, ATAC-seq and FISH-seq have been performed to identify mediators in vessels and fibrotic lesions of human skin samples of SSc patients and healthy volunteers. In addition, murine skin and lung tissue samples were analyzed by multi-channel immunofluorescence (IF) and confocal laser scanning microscopy. Microvascular endothelial cells, smooth muscle cells and fibroblasts have been further processed to address their functional attributes with regard to their proliferative, migratory and chemotactic capacity. In vivo models and e x vivo mouse fetal metatarsal assays were performed to study fibrotic and angiogenic processes. Results: Bioinformatic HD analyses revealed the ETS transcription factor PU.1 as molecular checkpoint of a network of factors that drive matrix production and fibrotic imprinting in SSc. Within this network ATF3 was significantly upregulated in fibroblasts of skin biopsies of SSc patients and of various organs of fibrosis models. ATF3 deficiency ameliorated fibrosis in various mouse models. Notably, ATF3 was significantly upregulated in vascular cells of fibrotic tissues of SSc patients. Multi-channel IF and confocal laser scanning microscopy of skin and lung biopsies of SSc patients revealed an increased expression of ATF3 especially in microvascular endothelial cells and smooth muscle cells. ATF3 overexpression in smooth muscle cells led to an extensively enhanced proliferation and increased migratory capacity whereas endothelial cells showed a SSc-like phenotype with reduced proliferation and migration. After ATF3 overexpression, tube formation capacity was completely altered as assessed by cumulative tube length, tube numbers and capillary sprouting. To investigate vessel outgrowth from a different perspective, we used the ex vivo fetal mouse metatarsal assay. ATF3 knockout mice showed a completely altered angiogenic response as assessed by tube length, number of branches and number junctions compared to wildtype controls. Conclusion: We identified PU.1 and ATF3 as key factors in disturbed vasculature and endogenous activated fibroblasts suggesting this axis as a potential therapeutic target intervening both fibrotic and vascular manifestations. Disclosure of Interests: Charles Gwellem Anchang: None declared, Bettina Matalobos Lawaree: None declared, Stefanie Weber: None declared, Simon Rauber: None declared, Thomas Wohlfahrt: None declared, Markus Luber: 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

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 vitro culture 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

Background Systemic sclerosis (SSc) is a chronic autoimmune disease with a high morbidity and mortality. Activation of the immune system is a characteristic feature of SSc. Numerous studies have suggested that type 2 and type 3 cytokines are key drivers of progressive fibrosis. Recently, innate lymphoid cells (ILC) are emerging as an important cellular source of type 2 and type 3 cytokines triggering fibrotic tissue remodeling independently of the adaptive immune system. ILCs are characterized by the absence of conventional lineage markers. Similar to T cells, they are categorized into three groups (ILC1, ILC2, ILC3), according to distinct pattern of cytokine production and the requirement of specific transcription factors guiding their development and function. Increased levels of ILC2 were found in patients with SSc. However, the contributive role of ILC2 and ILC3 in pathogenesis of SSc is not completely understood. Objectives We aim in our study to evaluate the predictive role of ILC2 and ILC3 in SSc patients. Methods We conducted an observational retrospective study on 52 patients with SSc fulfilling the 2013 ACR/EULAR classification criteria. Yearly clinical, laboratory and investigational data according to EUSTAR recommendations were collected. Blood samples collected between 15.09.2014 and 15.01.2015 were analyzed by flow cytometry and ILC2 and ILC3 counts were measured. The predictive value of ILC2 and ILC3 during a 2-year follow-up was analyzed using SPSS 21.0. ILC3 counts were also analyzed in skin sections (10 patients with SSc and 10 healthy controls) by immunofluorescence (IF) staining using two complementary panels of markers. Cytokine production of skin resident ILC3s was additionally analyzed by IF staining. Results 52 patients were included in the study, 78% female, 63% limited cutaneous SSc with a mean follow-up time of 2.85 ± 1.28 years. At baseline we have shown that circulating ILC2s are significantly increased compared to gender and age-matched healthy controls. Increased numbers of ILC2s significantly correlated with worsening of mRSS calculated by five point increase in mRSS or 25% increase from baseline (p < 0.001; 95% CI 1.39 – 3.26). ILC2 counts also correlated with 5% decrease of diffusion capacity of carbon monoxide (DLCO) during the follow-up time (p < 0.0001; 95% CI 1.83 – 3.49). Worsening of forced vital capacity (FVC) assessed as 5% decrease over 2 years was also significantly correlated with an increased number of ILC2s (p < 0.0001; 95% CI 1.27 – 3.04). In contrast, we did not find any correlation regarding increase in pulmonary arterial pressure assessed by echocardiography. Although new appearance of digital ulcers could not be predicted by ILC2 counts, increased numbers of ILC2s were correlated with digital ulcers at follow-up. ILC2s did not have a predictive value for death during the follow-up time. In contrast to ILC2, circulating ILC3s were not found to be correlated with worsening of disease activity. However, ILC3s were prominent in fibrotic skin of SSc patients compared to healthy controls, and showed strong production of IL-17. Conclusion Here, we provide first evidence for a role of ILC2s as potential prognostic marker of disease progression in SSc. Circulating ILC3 counts were not elevated in SSc. However, ILC3s showed strong cytokine production in the fibrotic skin of SSc patients. The functional impact has to be further evaluated. Disclosure of Interests: Alina Soare: None declared, Stefanie Weber: None declared, Markus Luber: None declared, Simon Rauber: None declared, Thomas Wohlfahrt: None declared, Georg Schett: None declared, Jörg Distler: None declared, Andreas Ramming Grant/research support from: Novartis

Background Persistent activation of fibroblast with excessive release of extracellular matrix is a hallmark of systemic sclerosis (SSc). Fibroblasts can either acquire a “pro-fibrotic” phenotype with excessive matrix production or a “pro-inflammatory” phenotype with releasing of matrix-degrading enzymes and subsequent tissue destruction. Despite these well-characterized phenotypic differences, the molecular mechanisms that drive polarization of fibroblasts into these two functionally opposing phenotypes remain enigmatic. Objectives We aimed to evaluate the transcriptional network that promotes the extracellular matrix-producing fibrotic fibroblast fate. Methods We investigated the transcriptional network that induces the profibrotic phenotype of fibroblasts by in silico and immunofluorescence analyses of human fibrotic skin, lung, liver and kidney, and performed functional assays to address the fibrogenic potential of fibroblasts in vitro and in several mouse models of systemic sclerosis. The heterocyclic diamidine DB1976 was used as new therapeutic compound to induce regression of fibrosis. Results We identified the ETS transcription factor PU.1 as molecular checkpoint for acquisition of a “pro-fibrotic” phenotype of fibroblasts. Our data demonstrate that expression of PU.1 is effectively silenced in fibroblasts during tissue homeostasis. When the epigenetic control of PU.1 is lost and PU.1 expression is induced, fibroblasts differentiate into a fibrotic phenotype that includes the transcription of numerous pro-fibrotic mediators. PU.1 polarized resting fibroblasts and even repolarized extracellular matrix-degrading inflammatory fibroblasts to an extracellular matrix-producing fibrotic phenotype. PU.1 is associated with a network of pro-fibrotic factors including members of the TEAD–HIPPO, canonical TGF-β–SMAD and AP1 signaling pathways. Other transcription factors with fibrotic abilities, such as SNAI2 and myocyte enhancer factor (MEF) 2, bind in close vicinity to PU.1-binding sites within the genome and contribute to the recruitment of the transcription machinery that drives the switch towards the fibrotic phenotype. PU.1 has a major coordinating role within this complex network of transcription factors in fibroblasts, as the inactivation of PU.1 alone is sufficient to prevent fibrotic polarization in vitro and in vivo. Finally, we investigated pharmacological targeting of PU.1 as a potential strategy to prevent uncontrolled fibrotic tissue remodelling. DB1976 showed anti-fibrotic effects in vivo in various fibrosis models and across several organs. Treatment with DB1976 not only prevented bleomycin-mediated skin fibrosis, but also induced regression of pre-established fibrosis, and was well tolerated. Conclusion These findings suggest that PU.1 inhibition may represent a novel and effective therapeutic approach to treat a wide range of fibrotic diseases. Inactivation of PU.1 effectively reverted the fibrotic phenotype of fibroblasts to a resting state and induced the regression of tissue fibrosis: Disclosure of Interests: Thomas Wohlfahrt: None declared, Simon Rauber: None declared, Markus Luber: None declared, Alina Soare: None declared, Stefanie Weber: None declared, Alexandru-Emil Matei: None declared, Chih-Wei Chen: None declared, Emmanuel Karouzakis: None declared, Hans Kiener: None declared, Elena Pachera: None declared, Clara Dees: None declared, Alexander Kreuter: None declared, Astrid Juengel: None declared, Steffen Gay: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Georg Schett: None declared, Jörg Distler: None declared, Andreas Ramming Grant/research support from: Novartis

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs.

Innate lymphoid cells (ILC) have a high potency for cytokine production independent of specific Ag stimulation. Imbalance of ILC subsets may influence cytokine production in humans and hence be associated with the development of inflammatory disease. Evidence for an imbalance of ILC homeostasis in human disease, however, is very limited to date. In this study we show that psoriatic arthritis (PsA), a severe disease of the joints depending on the activation of the IL-23/IL-17 pathway, is characterized by a skewed ILC homeostasis. Circulating ILC3s as potent source of IL-17/IL-22 were elevated in active PsA, whereas ILC2s, which produce proresolving cytokines, were decreased. The ILC2/ ILC3 ratio was significantly correlated with clinical disease activity scores and the presence of imaging signs of joint inflammation and bone damage. Multivariable analysis showed that a high ILC2/ILC3 ratio is associated with remission in PsA, suggesting that specific alterations of ILC homeostasis control disease activity in PsA.

Background Evaluation of actual immunopathology in psoriatic arthritis (PsA) is challenging. Current composite measures approved for PsA are very useful tools to assess disease activity in clinical routine. Nonetheless, because of subjective patients’ estimations that widely affect the scoring, in particular the distinction between remission and low disease activity is a common question of debate. Innate lymphoid cells (ILC) subsets may couple different aspects of PsA disease activity. Objectives To address whether PsA is associated with an altered composition of innate lymphoid cells and whether such changes are associated with disease activity and structural damage in PsA. Methods 124 patients satisfying the Classification Criteria for Psoriatic Arthritis (CASPAR) and 26 healthy volunteers were enrolled in the study. Information regarding clinical features, laboratory parameters were collected and disease activity score 28 (DAS28), disease activity in psoriatic arthritis (DAPSA), minimal disease activity score (MDA) were calculated. MRI and high-resolution peripheral CT were taken and PsA MRI score (PsAMRIS) was assessed. Flow cytometric analysis was performed and IFNg-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. Results Total number of circulating ILCs were increased in PsA patients compared to healthy controls (p<0,001). Linear regression analyses of the relationship between disease activity and circulating ILC counts showed that ILC2 negatively and ILC1 and ILC3 positively correlated with DAPSA score. The strongest correlation was observed when the ratio of ILC2 to ILC3 was analysed (R=-0.5709; p<0.0001). ILC2/3 ratio was also reduced in patients with active psoriatic skin disease, presence of enthesitis or a history of concomitant uveitis. Extend of synovitis or tenosynovitis or presence of bone erosions or osteophytes on MRI was inversely correlated with the ILC2/3 ratio (R=-0.6753; p<0.0001, R=-0.5828; p=0.0011 and p<0.001 respectively). Consistently, presence of erosions and/or osteoproliferation assessed by HR-pQCT was correlated with a significant lower ILC2/3 ratio. Furthermore, ROC Curve was used to test the performance of the ILC2/3 ratio as marker in differentiating between remission and disease activity of PsA. Indeed, a cut-off 0.57 exhibited highest sensitivity (92.9%) and a 84.7% specificity in identifying remission. Conclusions The ILC2/3 ratio correlates with various facets of PsA manifestations and might be a useful tool to evaluate disease activity in PsA patients. Disclosure of Interest None declared

Inflammatory diseases such as arthritis are chronic conditions that fail to resolve spontaneously. While the cytokine and cellular pathways triggering arthritis are well defined, those responsible for the resolution of inflammation are incompletely characterized. Here we identified interleukin (IL)-9-producing type 2 innate lymphoid cells (ILC2s) as the mediators of a molecular and cellular pathway that orchestrates the resolution of chronic inflammation. In mice, the absence of IL-9 impaired ILC2 proliferation and activation of regulatory T (Treg) cells, and resulted in chronic arthritis with excessive cartilage destruction and bone loss. In contrast, treatment with IL-9 promoted ILC2-dependent Treg activation and effectively induced resolution of inflammation and protection of bone. Patients with rheumatoid arthritis in remission exhibited high numbers of IL-9+ ILC2s in joints and the circulation. Hence, fostering IL-9-mediated ILC2 activation may offer a novel therapeutic approach inducing resolution of inflammation rather than suppression of inflammatory responses.

OBJECTIVE:Type 2 innate lymphoid cells (ILC2s), a recently identified population of lymphoid cells lacking lineage-specific receptors, promote type 2 immunity and tissue remodelling. However, the contributive role of ILC2s in the pathogenesis of systemic sclerosis (SSc) is unknown. We aimed to evaluate the levels and correlations with fibrotic manifestations in SSc. METHODS:69 patients with SSc and 47 healthy controls were included. Blood samples and skin sections were analysed by flow cytometry and immunohistochemically by staining two complementary panels of markers. RESULTS:Dermal and circulating ILC2s were significantly elevated in patients with SSc compared with controls. Dermal, but not circulating ILC2s were activated. Stratification of the SSc population in patients with limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc) demonstrated increased levels of ILC2s in both subgroups with significantly higher frequencies in dcSSc compared with lcSSc. Moreover, dermal and circulating ILC2 counts correlated closely with the modified Rodnan skin score and with the presence of pulmonary fibrosis. CONCLUSIONS:ILC2 counts are elevated in patients with SSc and correlate with the extent of skin fibrosis and the presence of interstitial lung disease providing compelling evidence for profibrotic effect of ILC2s in SSc.

Background Type 2 innate lymphoid cells (ILC2s), are recently identified as population of cells with lymphoid morphology lacking re-arranged antigen-specific receptors. Objectives We aimed to evaluate the contributive role of ILC2s in the pathogenesis of systemic sclerosis (SSc), in particular their levels and correlations with fibrotic manifestations in SSc and various fibrotic mouse models. Methods Human blood samples and skin sections (69 patients with SSc and 47 healthy controls) as well as skin and lung tissue of various fibrotic mouse models were analyzed by flow cytometry and immunohistochemistry using two complementary panels of markers each. ILC2s were further phenotyped for activation and homing parameters. ILC2 counts were correlated with clinical manifestations of SSc Results Significantly elevated numbers of ILC2s were detected in the skin (10-fold increase) and blood (4-fold increase) of SSc patients by two independent established sets of ILC2 markers compared to healthy controls. In contrast to circulating ILC2s, skin-resident ILC2s express various activation markers and stained positive for skin homing markers. Furthermore, our data suggest that ILC2s might be involved in the pathogenesis of fibrosis in SSc by showing multiple associations of ILC2 counts with fibrotic manifestations in SSc patients. Significantly higher frequencies were observed in diffuse cutaneous (dc)SSc patients compared to limited cutaneous SSc (lcSSc). The modified Rodnan Skin Score (mRSS) positively correlated with ILC2 counts and pulmonary involvement. In parallel, we detected significantly elevated numbers of ILC2s in the fibrotic skin of bleomycin-induced and DNA topoisomerase I-induced mice compared to control mice. Moreover, in the tight-skin (Tsk)-1model of fibrosis resembling less inflammatory stages of SSc significantly increased ILC2 counts were detected compared to control mice. Kinetic analyses revealed an early upregulation of ILC2s in experimental models of fibrosis. In contrast to lineage positive lymphocytes that peak at early inflammatory stages of fibrosis, however disappear over time, ILC2s persist also in later stages of established fibrosis. Conclusions Here, we provide first evidence for a role of ILC2s in the pathogenesis of SSc by demonstrating increased ILC2 counts in the skin and blood of human SSc patients. These findings were indirect supported by elevated numbers of ILC2s in the fibrotic skin and lung sections of various mouse models for SSc, reflecting different pathophysiological aspects of the disease. Proliferation of ILC2s in the fibrotic skin, the activated state of dermal ILC2s and correlations of ILC2 counts with dermal and pulmonary fibrosis together with supporting findings of these cells in fibrotic areas of murine skin and lung samples suggest a central role of ILC2s in the pathogenesis of fibrosis. Disclosure of Interest None declared