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Chronic graft-versus-host disease (cGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGVHD remain poorly understood, and targeted therapies for clinical use are not well established. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGVHD (sclGVHD). WNT signaling was activated in human sclGVHD with increased nuclear accumulation of the transcription factor β-catenin and a WNT-biased gene expression signature in lesional skin. Treatment with the highly selective tankryase inhibitor G007-LK, the CK1α agonist pyrvinium, or the LRP6 inhibitor salinomycin abrogated the activation of WNT signaling and protected against experimental cGVHD, without a significant impact on graft-versus-leukemia effect (GVL). Treatment with G007-LK, pyrvinium, or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d) → BALB/c (H-2d) and LP/J (H-2b) → C57BL/6 (H-2b) models of sclGVHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect inflammation-dependent effects in sclGVHD. Our findings may have direct translational potential, because pyrvinium is in clinical use, and tankyrase inhibitors are in clinical trials for other indications.

Summary Background Interstitial lung disease (ILD) is the most common cause of death in systemic sclerosis. To date, the progression of systemic sclerosis-associated ILD is judged by the accrual of lung damage on CT and pulmonary function tests. However, diagnostic tools to assess disease activity are not available. Here, we tested the hypothesis that quantification of fibroblast activation by PET-CT using a 68Ga-labelled selective inhibitor of prolyl endopeptidase FAP (68Ga-FAPI-04) would correlate with ILD activity and disease progression in patients with systemic sclerosis-associated ILD. Methods Between Sept 10, 2018, and April 8, 2020, 21 patients with systemic sclerosis-associated ILD confirmed by high-resolution CT (HRCT) within 12 months of inclusion and with onset of systemic sclerosis-associated ILD within 5 years or signs of progressive ILD and 21 controls without ILD were consecutively enrolled. All participants underwent 68Ga-FAPI-04 PET-CT imaging and standard-of-care procedures, including HRCT and pulmonary function tests at baseline. Patients with systemic sclerosis-associated ILD were followed for 6 months with HRCT and pulmonary function tests. We compared baseline 68Ga-FAPI-04 PET-CT uptake with standard diagnostic tools and predictors of ILD progression. The association of 68Ga-FAPI-04 uptake with changes in forced vital capacity was analysed using mixed-effects models. Follow-up 68Ga-FAPI-04 PET-CT scans were obtained in a subset of patients treated with nintedanib (follow-up between 6–10 months) to assess change over time. Findings 68Ga-FAPI-04 accumulated in fibrotic areas of the lungs in patients with systemic sclerosis-associated ILD compared with controls, with a median standardised uptake value (SUV) mean over the whole lung of 0·80 (IQR 0·60–2·10) in the systemic sclerosis-ILD group and 0·50 (0·40–0·50) in the control group (p Interpretation Our study presents the first in-human evidence that fibroblast activation correlates with fibrotic activity and disease progression in the lungs of patients with systemic sclerosis-associated ILD and that 68Ga-FAPI-04 PET-CT might improve risk assessment of systemic sclerosis-associated ILD. Funding German Research Foundation, Erlangen Anschubs-und Nachwuchsfinanzierung, Interdisziplinares Zentrum fur Klinische Forschung Erlangen, Bundesministerium fur Bildung und Forschung, Deutsche Stiftung Systemische Sklerose, Wilhelm-Sander-Foundation, Else-Kroner-Fresenius-Foundation, European Research Council, Ernst-Jung-Foundation, and Clinician Scientist Program Erlangen.

The activation of fibroblasts is required for physiological tissue remodelling such as wound healing. However, when the regulatory mechanisms are disrupted and fibroblasts remain persistently activated, the progressive deposition of extracellular matrix proteins leads to tissue fibrosis, which results in dysfunction or even loss of function of the affected organ. Although fibrosis has been recognized as a major cause of morbidity and mortality in modern societies, there are only few treatment options available that directly disrupt the release of extracellular matrix from fibroblasts. Intensive research in recent years, however, identified several pathways as core fibrotic mechanisms that are shared across different fibrotic diseases and organs. We discuss herein selection of those core pathways, especially downstream of the profibrotic TGF-β pathway, which are druggable and which may be transferable from bench to bedside.

Background and purpose Genetic deletion and pharmacological studies suggest a role for lysophosphatidic acid (LPA1 ) receptor in fibrosis. We investigated the therapeutic potential in systemic sclerosis (SSc) of a new orally active selective LPA1 receptor antagonist using dermal fibroblasts from patients and an animal model of skin fibrosis. Experimental approach Dermal fibroblast and skin biopsies from systemic sclerosis patients were used. Myofibroblast differentiation, gene expression and cytokine secretion were measured following LPA and/or SAR100842 treatment. Pharmacolgical effect of SAR100842 was assessed in the tight skin 1 (Tsk1) mouse model. Key results SAR100842 is equipotent against various LPA isoforms. Dermal fibroblasts and skin biopsies from patients with systemic sclerosis expressed high levels of LPA1 receptor. The LPA functional response (Ca2+ ) in systemic sclerosis dermal fibroblasts was fully antagonized with SAR100842. LPA induced myofibroblast differentiation in systemic sclerosis dermal and idiopathic pulmonary fibrosis lung fibroblasts and the secretion of inflammatory markers and activated Wnt markers. Results from systemic sclerosis dermal fibroblasts mirror those obtained in a mouse Tsk1 model of skin fibrosis. Using a therapeutic protocol, SAR100842 consistently reversed dermal thickening, inhibited myofibroblast differentiation and reduced skin collagen content. Inflammatory and Wnt pathway markers were also inhibited by SAR100842 in the skin of Tsk1 mice. Conclusion and implications The effects of SAR100842 on LPA-induced inflammation and on mechanisms linked to fibrosis like myofibroblast differentiation and Wnt pathway activation indicate that LPA1 receptor activation plays a key role in skin fibrosis. Our results support the therapeutic potential of LPA1 receptor antagonists in systemic sclerosis.

OBJECTIVE Expression of dipeptidylpeptidase 4 (DPP-4) identifies a dermal fibroblast lineage involved in scarring during wound healing. The role of DDP-4 in tissue fibrosis is, however, unknown. The aim of the present study was to evaluate DPP-4 as a potential target for the treatment of fibrosis in patients with systemic sclerosis (SSc). METHODS Expression of DPP-4 in skin biopsy samples and dermal fibroblasts was analyzed by real-time polymerase chain reaction, immunofluorescence, and Western blot analyses. The activity of DPP-4 was modulated by overexpression, knockdown, and pharmacologic inhibition of DPP4 using sitagliptin and vildagliptin. The effects of DPP4 inhibition were analyzed in human dermal fibroblasts and in different mouse models of SSc (each n = 6). RESULTS The expression of DPP-4 and the number of DPP-4-positive fibroblasts were increased in the fibrotic skin of SSc patients, in a transforming growth factor β (TGFβ)-dependent manner. DPP-4-positive fibroblasts expressed higher levels of myofibroblast markers and collagen (each P < 0.001 versus healthy controls). Overexpression of DPP4 promoted fibroblast activation, whereas pharmacologic inhibition or genetic inactivation of DPP4 reduced the proliferation, migration, and expression of contractile proteins and release of collagen (each P < 0.001 versus control mice) by interfering with TGFβ-induced ERK signaling. DPP4-knockout mice were less sensitive to bleomycin-induced dermal and pulmonary fibrosis (P < 0.0001 versus wild-type controls). Treatment with DPP4 inhibitors promoted regression of fibrosis in mice that had received bleomycin challenge and mice with chronic graft-versus-host disease, and ameliorated fibrosis in TSK1 mice (each P < 0.001 versus untreated controls). These antifibrotic effects were associated with a reduction in inflammation. CONCLUSION DPP-4 characterizes a population of activated fibroblasts and shows that DPP-4 regulates TGFβ-induced fibroblast activation in the fibrotic skin of SSc patients. Inhibition of DPP4 exerts potent antifibrotic effects when administered in well-tolerated doses. As DPP4 inhibitors are already in clinical use for diabetes, these results may have direct translational implications for the treatment of fibrosis in patients with SSc.

ObjectiveX-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein with important functions in apoptosis, cellular differentiation and cytoskeletal organisation and is emerging as potential target for the treatment of various cancers. The aim of the current study was to investigate the role of XIAP in the pathogenesis of systemic sclerosis (SSc).MethodsThe expression of XIAP in human skin samples of patients with SSc and chronic graft versus host disease (cGvHD) and healthy individuals was analysed by quantitative PCR, immunofluorescence (IF) and western blot. XIAP was inactivated by siRNA-mediated knockdown and pharmacological inhibition. The effects of XIAP inactivation were analysed in cultured fibroblasts and in the fibrosis models bleomycin-induced and topoisomerase-I-(topoI)-induced fibrosis and in Wnt10b-transgenic mice.ResultsThe expression of XIAP, but not of other inhibitor of apoptosis protein family members, was increased in fibroblasts in SSc and sclerodermatous cGvHD. Transforming growth factor beta (TGF-β) induced the expression of XIAP in a SMAD3-dependent manner. Inactivation of XIAP reduced WNT-induced fibroblast activation and collagen release. Inhibition of XIAP also ameliorated fibrosis induced by bleomycin, topoI and overexpression of Wnt10b in well-tolerated doses. The profibrotic effects of XIAP were mediated via WNT/β-catenin signalling. Inactivation of XIAP reduces binding of β-catenin to TCF to in a TLE-dependent manner to block WNT/β-catenin-dependent transcription.ConclusionsOur data characterise XIAP as a novel link between two core pathways of fibrosis. XIAP is overexpressed in SSc and cGvHD in a TGF-β/SMAD3-dependent manner and in turn amplifies the profibrotic effects of WNT/β-catenin signalling on fibroblasts via transducin-like enhancer of split 3. Targeted inactivation of XIAP inhibits the aberrant activation of fibroblasts in murine models of SSc.

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.

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.

BackgroundNintedanib is an inhibitor targeting platelet-derived growth factor receptor, fibroblast growth factor receptor and vascular endothelial growth factor receptor tyrosine kinases that has recently been approved for the treatment of idiopathic pulmonary fibrosis. The aim of this study was to analyse the effects of nintedanib in the fos-related antigen-2 (Fra2) mouse model of systemic sclerosis (SSc).MethodsThe effects of nintedanib on pulmonary arterial hypertension with proliferation of pulmonary vascular smooth muscle cells (PVSMCs) and luminal occlusion, on microvascular disease with apoptosis of microvascular endothelial cells (MVECs) and on fibroblast activation with myofibroblast differentiation and accumulation of extracellular matrix were analysed. We also studied the effects of nintedanib on the levels of key mediators involved in the pathogenesis of SSc and on macrophage polarisation.ResultsNintedanib inhibited proliferation of PVSMCs and prevented thickening of the vessel walls and luminal occlusion of pulmonary arteries. Treatment with nintedanib also inhibited apoptosis of MVECs and blunted the capillary rarefaction in Fra2-transgenic mice. These effects were associated with a normalisation of the serum levels of vascular endothelial growth factor in Fra2 mice on treatment with nintedanib. Nintedanib also effectively blocked myofibroblast differentiation and reduced pulmonary, dermal and myocardial fibrosis in Fra2-transgenic mice. The antifibrotic effects of nintedanib were associated with impaired M2 polarisation of monocytes and reduced numbers of M2 macrophages.ConclusionNintedanib targets core features of SSc in Fra2-transgenic mice and ameliorates histological features of pulmonary arterial hypertension, destructive microangiopathy and pulmonary and dermal fibrosis. These data might have direct implications for the ongoing phase III clinical trial with nintedanib in SSc-associated interstitial lung disease.

ObjectivesJanus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-β. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts.MethodsThe antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance.ResultsThe antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis.ConclusionFibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.

ObjectivesCoactivators are a heterogeneous family of transcriptional regulators that are essential for modulation of transcriptional outcomes and fine-tune numerous cellular processes. The aim of the present study was to evaluate the role of the coactivator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in the pathogenesis of systemic sclerosis (SSc).MethodsExpression of PGC-1α was analysed by real-time PCR, western blot and immunofluorescence. Modulation of autophagy was analysed by reporter studies by expression of autophagy-related genes. The effects of PGC-1α knockdown on collagen production and myofibroblast differentiation were analysed in cultured human fibroblasts and in two mouse models with fibroblast-specific knockout of PGC-1α.ResultsThe expression of PGC-1α was induced in dermal fibroblasts of patients with SSc and experimental murine fibrosis. Transforming growth factor beta (TGFβ), hypoxia and epigenetic mechanisms regulate the expression of PGC-1α in fibroblasts. Knockdown of PGC-1α prevented the activation of autophagy by TGFβ and this translated into reduced fibroblast-to-myofibroblast differentiation and collagen release. Knockout of PGC-1α in fibroblasts prevented skin fibrosis induced by bleomycin and by overexpression of a constitutively active TGFβ receptor type I. Moreover, pharmacological inhibition of PGC-1α by SR18292 induced regression of pre-established, bleomycin-induced skin fibrosis.ConclusionPGC-1α is upregulated in SSc and promotes autophagy to foster TGFβ-induced fibroblast activation. Targeting of PGC-1α prevents aberrant autophagy, inhibits fibroblast activation and tissue fibrosis and may over therapeutic potential.

Background: Interstitial lung disease (ILD) is the most common cause of death in systemic sclerosis (SSc). However, the course of ILD in this population is highly variable. Effective diagnostic tools to assess activity and to predict the individual course of the disease are lacking. Here, we tested the hypothesis that in vivo quantification of fibroblast activation may correlate with ILD activity and improve risk stratification in SSc patients. Methods: Fibroblast activation was visualized by positron emission tomography/computed tomography (PET/CT) using the 68Gallium-labeled selective inhibitor of fibroblast activation protein (68Ga-FAPI-04). We correlated 68Ga-FAPI-04 uptake with changes in lung function parameters, high resolution CT scan (HRCT) and patient reported outcomes. 68Ga-FAPI-04 PET/CTs, HRCTs and lung function tests were evaluated in a blinded manner. Findings: In this single center study, we enrolled 21 patients with SSc-ILD, five patients with dermatomyositis-associated-ILD, ten non-SSc/non-ILD control patients and ten non-diseased control individuals. 68Ga-FAPI-04 accumulated in fibrotic areas of the lungs in SSc-ILD with significantly higher wlSUVmean (p 1.0 or a wlTL-FAPI > 500 cm3 demonstrated radiologic progression and decline of FVC after 6 months, but none of the patients with less 68Ga-FAPI-04 accumulation. In consecutive 68Ga-FAPI-04-PET/CTs, changes in 68Ga-FAPI-04 uptake correlated with response to the fibroblast-targeting antifibrotic agent nintedanib. Interpretation: Our study presents first in human evidence that fibroblast activation correlates with fibrotic activity and disease progression in the lungs of SSc-ILD patients and that 68Ga-FAPI-04-PET/CT may improve risk stratification. Funding: German Research Foundation, ELAN-Foundation-Erlangen and Bundesministerium fur Bildung und Forschung. Conflict of Interest: O.D. has consulted for, or has received research funding from, 4D Science, Actelion, Active Biotech, Bayer-Schering, Biogen, Biovitrium, BMS, Boehringer, EpiPharm, Ergonex, GSK, Inventiva, Medac, Novartis, Pfizer, Roche/Genentech, Sanofi/Genzyme, Serodapharm, Sinoxa and United BioSource Corporation; JHWD has consultancy relationships and/or has received research funding from Actelion, BMS, Celgene, Bayer Pharma, Boehringer Ingelheim, JBTherapeutics, Sanofi-Aventis, Novartis, UCB, GSK, Array Biopharma, Galapagos, Inventiva and Active Biotech in the area of potential treatments of SSc and is stock owner of 4D Science. Ethical Approval: File number 30_19B, Ethics Committee Friedrich-Alexander-University Erlangen.

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, Jorg Distler: None declared, Andreas Ramming Grant/research support from: Novartis

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:Interstitial lung disease (ILD) is the most common cause of death in systemic sclerosis (SSc). To date, the progression of SSc-ILD is judged by the accrual of lung damage on computed tomography (CT) and functional decline (forced vital capacity). However, this approach does not directly assess the activity of tissue remodeling. Moreover, prediction of the course of ILD in individual SSc patients remains challenging. Fibroblast Activation Protein (FAP) is a specific, ex vivo validated marker for activated fibroblasts.Objectives:The aims of this study were: 1. To assess differences in the uptake of 68GA-FAPI 04 in SSc-ILD patients compared to controls, to analyze 2. whether 68GA-FAPI 04 uptake at baseline correlates with other risk factors of disease progression and 3. Whether 68GA-FAPI 04 uptake is associated with the course of SSc-ILD.Methods:Between September 2018 and April 2020, 21 patients with SSc-ILD confirmed by HRCT and onset of SSc-ILD within ≤ 5 years or signs of progressive ILD and 21 controls without ILD were consecutively enrolled. All participants underwent 68Ga-FAPI-04 PET/CT imaging and standard-of-care procedures including HRCT and lung function testing (PFT) at baseline. Patients with SSc-ILD patients were followed-up for 6 months with HRCT and PFT. Follow-up 68Ga-FAPI-04 PET/CT scans were obtained in a subset of patients treated with nintedanib. We compared baseline 68Ga-FAPI-04 PET/CT uptake to standard diagnostic tools and currently used predictors of ILD progression. The association of 68Ga-FAPI-04 uptake with changes in FVC was analyzed using mixed-effects models.Results:68Ga-FAPI-04 accumulated in fibrotic areas of the lungs in SSc-ILD compared to controls with a median (q1-q3 interval) wlSUVmean of 0.8 (0.6 to 2.1) in the SSc-ILD group and 0.5 (0.4 to 0.5) in the control group (p3 (163.4 to 442.3) and the median wlTL-FAPI was 183.6 cm3 (98.04 to 960.7). 68Ga-FAPI-04 uptake was higher in patients with extensive disease, with previous ILD progression or high EUSTAR activity scores. Increased 68Ga-FAPI-04 uptake at baseline was associated with progression of ILD independently of extent of involvement on HRCT scan and the forced vital capacity at baseline. In consecutive 68Ga-FAPI-04-PET/CTs, changes in 68Ga-FAPI-04 uptake was concordant with the observed response to the fibroblast-targeting antifibrotic agent nintedanib.Conclusion:Our study presents first in human evidence that 68Ga-FAPI-04-fibroblast uptake correlates with fibrotic activity and disease progression in the lungs of SSc-ILD patients and that 68Ga-FAPI-04-PET/CT may be of potential to improve risk assessment of SSc-ILD.Figure 1.A and B:68Ga-FAPI-04 PET/CT scan from a patient with SSc-ILD with selective 68Ga-FAPI-04 uptake in fibrotic areas of the left- and right lower lung lobes (red arrows), but not in non-fibrotic areas such as the middle lobe (green arrow). B Corresponding CT component.Acknowledgements:We gratefully acknowledge Prof. Uwe Haberkorn (University Hospital Heidelberg and DKFZ, Heidelberg, Germany) and iTheranostics Inc. (Dulles, VA, USA) for providing the precursor FAPI-04.Disclosure of Interests:Christina Bergmann: None declared, Jörg H.W. Distler Speakers bureau: Actelion, Anamar, ARXX, Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, and UCB, Grant/research support from: Anamar, Active Biotech, Array Biopharma, ARXX, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB, Christoph Treutlein: None declared, Koray Tascilar Speakers bureau: Gilead sciences GmbH, Pfizer Turkey, UCB Turkey, Anna-Theresa Mueller: None declared, Armin Atzinger: None declared, Alexandru-Emil Matei: None declared, Johannes Knitza: None declared, Andrea-Hermina Györfi: None declared, Anja Lueck: None declared, Clara Dees: None declared, Alina Soare: None declared, Andreas Ramming: None declared, Verena Schönau: None declared, Oliver Distler Speakers bureau: Arxx Therapeutics, Baecon Discovery, Blade Therapeutics,Bayer, Böhringer Ingelheim, Catenion,Competitive Drug Development International Ltd, Corbuspharma, CSL Behring, ChemomAb, Horizon Pharmaceuticals, Ergonex, Galaapagos NV, Glenmark Pharmaceuticals,GSK, Inventiva, Italfarmaco, IQvia, Kymera, Lilly, Medac, Medscape, MSD, Novartis, Pfizer, Roche, Sanofi, Taget Bio Sciencec, UCB, Grant/research support from: Bayer,Böhringer Ingelheim, Mitsubishi Tanabe Pharma, Olaf Prante: None declared, Philipp Ritt: None declared, Theresa Ida Goetz: None declared, Markus Koehner: None declared, Michael Cordes: None declared, Tobias Baeuerle: None declared, Torsten Kuwert Speakers bureau: Honoraria for occasional lectures by Siemens Healthineers, Grant/research support from: Research grant to the Clinic of Nuclear Medicine by this entity covering projects in the field of SPECT/CT, Georg Schett: None declared, Christian Schmidkonz: None declared

Background:Excessive activation of fibroblasts with a TGFβ-biased gene signature and deposition of extracellular matrix are key features of fibrotic diseases. The mechanisms underlying these transcriptional changes remain poorly understood. Deregulation, mutations and malfunctions of transcriptional co-regulators, which can interact with multiple transcription factors and enable a broad-spectrum regulation of transcriptional networks, have been implicated as driving factors in a large number of diseases and pathologies.Objectives:In the present study, we aimed to analyze the role of the co-regulator Nuclear Receptor Co-Activator 3 (NCOA3) in fibroblast activation and tissue fibrosis, and to evaluate a potential interaction of NCOA3 with fibrosis-relevant transcription factors.Methods:NCOA3 was inhibited genetically by siRNA transfection and pharmacologically by the SRC3 inhibitor-2 (SI-2). We performed bulk RNASeq of human dermal fibroblasts and in silico transcription factor binding site screening of differentially expressed genes (DEGs). The interaction of NCOA3 and TGFβ-SMAD signaling was analyzed by reporter and CoIP assays.Results:The expression of NCOA3 in skin biopsies of SSc patients compared to normal controls demonstrated that SSc fibroblasts express modestly, but significantly reduced levels of NCOA3, which persisted in cultured SSc fibroblasts. Stimulation of normal fibroblasts with chronically high levels of TGFβ as they also occur in fibrotic tissue remodeling strongly decreased NCOA3 expression to a similar extent as in SSc fibroblasts. Furthermore, NCOA3 expression is also deregulated in different murine models of skin fibrosis. To investigate the functional effects of decreased NCOA3 levels, we targeted the expression of NCOA3 in normal fibroblasts. SiRNA-mediated knockdown of NCOA3 ameliorated TGFβ-induced gene expression, collagen release, myofibroblast differentiation and cell proliferation. In contrast, knockdown of NCOA3 had no effects on collagen release, expression of contractile proteins or gene expression in unstimulated fibroblasts, suggesting that NCOA3 is not required for cellular homeostasis. To characterize the molecular mechanisms, we performed RNASeq upon NCOA3 knockdown. We identified 343 significant differentially expressed genes (220 downregulated and 123 upregulated with a Benjamini-Hochberg false discovery rate FDR < 0.25 and fold change > 1.5) between TGFβ-stimulated fibroblasts with and without NCOA3 knockdown (NCOA3-DEGs) including the fibrosis-relevant genes EDNRB, COL5A3, HES1, IL11 or IL33. Functional analysis of the NCOA3-DEGs showed enrichment of pathway terms such as collagen binding and extracellular matrix organization. In silico screening of the promoters of the NCOA3-DEGs for potential transcription factor binding motifs revealed binding motifs of core transcription factors of fibroblast activation and tissue fibrosis such as SMAD2/3/4, RBPJ, ZEB1, TCF4, REL, and SNAIL2 amongst the downregulated NCOA3-DEGs. Experimental validation of our biostatistical results using SMAD3 as example demonstrated a higher percentage of NCOA3-pSMAD3 double-positive fibroblasts in skin sections of SSc patients compared to healthy controls. In addition, knockdown of NCOA3 reduced TGFβ-induced SMAD-reporter activity. Furthermore, stimulation with TGFβ increased the interaction of NCOA3 with SMAD3 as analyzed by co-immunoprecipitation. Simultaneous knockdown of NCOA3 and SMAD3 showed no additional reductions compared to the single knockdowns, suggesting that NCOA3 controls SMAD3-dependent gene transcription under fibrotic conditions. Finally, inhibition of NCOA3 showed anti-fibrotic effects in different murine models of experimental skin and lung fibrosis.Conclusion:Our findings characterize NCOA3 as regulator of multiple pro-fibrotic transcription programs. Pharmaceutical inhibition of NCOA3 might be a strategy to interfere simultaneously with several core pro-fibrotic mediators in fibrotic diseases such as SSc.Acknowledgements:We thank Lena Summa, Vladyslav Fedorchenko, Wolfgang Espach and Regina Kleinlein for excellent technical assistance.The study was funded by grants DI 1537/7-1, DI 1537/8-1, DI 1537/9-1 and -2, DI 1537/11-1, DI 1537/12-1, DI 1537/13-1, DI 1537/14-1, DI 1537/17-1, DE 2414/2-1, DE 2414/4-1, and RA 2506/3-1 of the German Research Foundation, SFB CRC1181 (project C01) and SFB TR221/ project number 324392634 (B04) of the German Research Foundation, grants J39, J40 and A64 of the IZKF in Erlangen, grant 2013.056.1 of the Wilhelm-Sander-Foundation, grants 2014_A47, 2014_A248 and 2014_A184 of the Else-Kröner-Fresenius-Foundation, grant 14-12-17-1-Bergmann of the ELAN-Foundation Erlangen, BMBF (Era-Net grant 01KT1801), MASCARA program, TP 2 and a Career Support Award of Medicine of the Ernst Jung Foundation.Disclosure of Interests:Clara Dees: None declared, Sebastian Poetter: None declared, Maximilian Fuchs: None declared, Christina Bergmann: None declared, Alexandru-Emil Matei: None declared, Andrea-Hermina Györfi: None declared, Alina Soare: None declared, Andreas Ramming: None declared, Paolo Ceppi: None declared, 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, ARXX, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB

ObjectivesHedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways.MethodsThe GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-β (TGF-β) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-β receptor I.ResultsTGF-β upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBRact-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis.ConclusionsOur data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.

Background:Fibroblast growth factor receptor 3 (FGFR3) is a member of the family of different fibroblast growth factor receptors with several ligands called fibroblast growth factors (FGFs) in humans. Each FGFR has different isoforms resulting from natural alternative splice variants. Upon binding FGF ligands, fibroblast growth factor receptors (FGFRs) trigger various intracellular signaling pathways to regulate important biological processes. Systematic evaluation of FGF/FGFR signaling in the context of SSc has not been performed so far.Objectives:The aim of this study was to characterize FGFR3/FGF9 signaling in the context of fibroblast activation and to evaluate FGFR3 as a potential molecular target for antifibrotic treatment in SSc.Methods:Differential expression profiling of dermal cells from SSc patients and healthy volunteers were performed employing GEArray cDNA microarray. Real-time PCR, Western Blot, immunohistochemistry and immunofluorescence were done in skin tissues and fibroblasts from SSc patients. Selective inhibitors in conjunction with genetic knockdown and knockout strategies were used to target FGFR3 signalingin vitroand in mouse models of SSc: skin fibrosis induced by bleomycin and by overexpression of a constitutively active transforming growth factor receptor 1 (TBR) and tight skin-1 (TSK) mice. Affymetrix gene arrays in dermal fibroblasts from mice with constitutive FGFR3 signaling and mice lacking FGFR3.Results:Expression of FGFR3, specifically the isoform FGFR3IIIb and its ligand FGF9, was significantly upregulated in the dermis and dermal fibroblasts of SSc patients as compared to healthy volunteers. Furthermore, an increase of FGFR3 IIIb/FGF9 expression comparable to that in SSc fibroblasts could also be obtained by stimulating normal healthy dermal fibroblasts with transforming growth factor (TGFβ)in vitroand in mice constitutively overexpressing active TGFβ receptor type I.Transcriptome profiling,in silicoanalysis and functional experiments revealed that FGFR3 synergistigically induces multiple profibrotic pathways including Endothelin-, Interleukin-4- and CTGF-signaling in a CREB-dependent manner. FGFR3 exerts profibrotic effects by modulating phosphorylation of CREB by ERK-, AKT-, CAMK2- and p38-kinases. Activation of FGFR3 in healthy or SSc dermal fibroblasts by stimulation with recombinant FGF9 was sufficient to induce resting fibroblast-to-myofibroblast differentiation along with increased collagen secretion and alpha-SMA production.Genetic knockout of Fgfr3 abrogates myofibroblast differentiationin vitroand ameliorates skin fibrosis in TSK and TBR mice and in bleomycin-induced fibrosis. Further confirming the translational potential of these findings in the preclinical models of SSc, we demonstrate that pharmacological inactivation of FGFR3 by PD173074 could induce the regression of experimental fibrosis invitroand in bleomycin-challenged, TSK and TBR mice.Conclusion:Our findings characterize FGFR3 as an upstream regulator of a network of profibrotic mediators in SSc and thus, we could demonstrate successfully that the targeted inhibition of FGFR3 could inhibit multiple signaling pathwaysin vitroand ameliorated fibrosis in different preclinical models of SSc. These findings may have direct translational implications as FGFR3 inhibitors are currently in development.Disclosure of Interests:Debomita Chakraborty: None declared, Honglin Zhu: None declared, Astrid Juengel: None declared, Lena Summa: None declared, Yi-Nan Li: None declared, Christina Bergmann: None declared, Alexandru-Emil Matei: None declared, Thuong Trinh-Minh: None declared, Chih-Wei Chen: None declared, Clara Dees: None declared, Andreas Ramming: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim

Background:Tissue fibrosis caused by a pathological activation of fibroblasts is a major hallmark of systemic sclerosis (SSc). Epigenetic gene silencing of anti-fibrotic genes is thought to play a central role to establish the persistently activated phenotype of fibroblasts independent of external stimuli such as TGFβ, which has been identified as key-mediator of fibroblast activation.Objectives:The aims of the present study were to investigate whether the aberrant activation of JAK2-STAT3 signaling in fibrosis might be caused by epigenetic silencing of SOCS expression and whether re-establishment of the endogenous, SOCS-dependent control of JAK / STAT signaling may prevent aberrant fibroblast activation and ameliorate tissue fibrosis.Methods:The methylation status of SOCS3 in fibroblasts was evaluated by methylation-specific PCR and MeDIP assays. 5-aza-2-deoxycytidine (5-aza) and siRNA was used to inhibit DNA methyltransferases (DNMTs)in vitroandin vivo. Knockdown and overexpression experiments served to analyze the mechanism of action in cultured fibroblasts. Fibroblast-specific knockout mice were additionally used to analyze the role of SOCS3 and DNMTsin vivo.Results:Chronically increased levels of TGFβ reduced the expression of SOCS3 in normal fibroblasts to a level also found in SSc fibroblasts. Consistently, the expression of SOCS3 was severely downregulated in skin of SSc patients compared to healthy individuals with only minor differences between limited and diffuse cutaneous SSc. Methylation analyses demonstrated a prominent promoter hypermethylation of SOCS3 in SSc fibroblasts and in normal fibroblasts exposed to persistently high levels of TGFβ. Increased DNMT activity and a time-dependent induction of DNMT3A and DNMT1 expression upon chronic exposure to TGFβ resulted in promoter hypermethylation of SOCS3. Knockdown of SOCS3 induced an SSc-like phenotype in normal dermal fibroblasts with increased activation of JAK2-STAT3 signaling, enhanced expression of myofibroblast markers, increased collagen release, and aggravated experimental tissue fibrosis with increased activation of JAK2-STAT3 signaling. This effect was mimicked by overexpression of mutant JAK2 with mutations in the SOCS3 binding motif. Vice versa, forced overexpression of SOCS3 reduced TGFβ-mediated fibroblast activation and ameliorated the endogenous activation of SSc fibroblasts. Pharmacological inhibition or selective knockdown of DNMTs restored the normal expression of SOCS3, reduced fibroblast activation and collagen release, blocked STAT3-responsive transcription, and exerted potent antifibrotic effects in bleomycin- and TBRIact-induced dermal fibrosis. In addition, treatment with 5-aza or knockdown of either DNMT1 or DNMT3A induced regression of established fibrosis.Conclusion:These findings identify a novel pathway of epigenetic imprinting of fibroblasts in fibrotic disease with translational implications for the development of new targeted therapies in fibrotic diseases. We demonstrate that the chronic activation of TGFβ signaling in fibrotic diseases perturbs the epigenetic control of STAT signaling by DNMT-induced silencing of SOCS3 expression. Our data might thus strengthen the scientific rational for targeting DNA methylation in fibrotic diseases.Disclosure of Interests:Clara Dees: None declared, Sebastian Poetter: None declared, Yun Zhang: None declared, Christina Bergmann: None declared, xiang zhou: None declared, Markus Luber: None declared, Emmanuel Karouzakis: None declared, Andreas Ramming Grant/research support from: Pfizer, Novartis, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Speakers bureau: Boehringer Ingelheim, Roche, Janssen, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, 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

Background:Pathologic activation of fibroblasts is a central feature of fibrotic tissue disease in Systemic Sclerosis (SSc). Although individual key signaling pathways of fibroblast activation such as transforming growth factor β (TGFβ) and WNT/β-catenin signaling have been identified, the consequences of the concomitant upregulation of these pathways and their crosstalk are incompletely characterized. Given the high medical need, the identification of mutual activation and amplification loops of profibrotic signals is essential to identify novel candidates for antifibrotic therapies. XIAP (X-linked inhibitor of apoptosis protein) is a ubiquitously expressed member of the IAP protein family which are implicated in the regulation of various cellular functions and tissue turnover. XIAP was recently described to be implicated in WNT/β-catenin signaling and TGFβ signaling.Objectives:The aim of this study is to characterize the role of XIAP in fibrotic disease.Methods:XIAP-expression was analyzed by qPCR, IF and Western blot. XIAP was targeted pharmacologically and with siRNA. The activation of WNT/β-catenin signaling was assessed by analyses of WNT target genes, by TOPflash/FOPflash luciferase reporter assay and in reporter mice.In vivo,XIAP inhibition was analysed in two different models of fibrosis.Results:The expression of XIAP is increased in the skin of SSc patients compared to matched healthy individuals with a particular prominent expression in fibroblasts. The overexpression of XIAP is more pronounced in SSc patients with diffuse and active skin fibrosis compared to SSc patients with limited and inactive disease. The overexpression of XIAP is also reflected in several experimental fibrosis models: the model of sclerodermatous graft versus host disease, the model of bleomycin induced skin fibrosis and Topoisomerase I induced fibrosis (TopoI) mice. TGFβ induces the expression of XIAP in vitro and in vivo and treatment with the TGFβ1 receptor antagonist SD208 reverses the TGFβ induced expression of XIAP. Inhibition of XIAP with embelin or siRNA reduces the TGFβ induced activation of fibroblasts with reduced collagen release and reduced expression of myofibroblast markers. In addition, XIAP inhibition reverted the activated fibroblast phenotype in SSc fibroblasts with reduced expression of stress fibers and αSMA. The antifibrotic effects of XIAP inhibition occurred in non-toxic doses as demonstrated by MTT and by TUNEL staining. In vivo, inhibition of XIAP reduced skin fibrosis in the models of bleomycin induced skin fibrosis and in TopoI-induced skin and lung fibrosis as demonstrated by analysis of dermal thickening, dermal hydroxyproline content and by analysis of myofibroblast differentiation. Mechanistically, XIAP inhibition reduced the activation of WNT/β-catenin signaling as demonstrated by TOPflash reporter assays and by the analysis of WNT target genes.Conclusion:XIAP is upregulated in SSc fibroblasts and murine SSc models in a TGFβ-dependent manner and promotes fibroblast activation by fostering canonical WNT signaling. Our data suggest that XIAP mediates an amplification loop between TGFβ and WNT/β-catenin signaling. Inhibition of XIAP may thus be a novel approach to target aberrant WNT/β-catenin signaling in fibrotic diseases.Disclosure of Interests:Christina Bergmann: None declared, Ludwig Hallenberger: None declared, Benita Merlevede: None declared, Clara Dees: None declared, Chih-Wei Chen: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, 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

Background SHP2, encoded by the PTPN11 gene, is a ubiquitously expressed non-receptor tyrosine phosphatase (PTP). Altered expression and activity levels of SHP2 have also been implicated into the pathogenesis of autoimmune diseases. Objectives In our study, we aim to characterise the role of SHP2 as a possible novel molecular checkpoint of transforming growth factor-β (TGFβ)-induced JAK2/STAT3 signalling in fibrotic diseases. Methods Mice carrying Shp2fl/flxCol1a2-CreER were generated to specifically knockout Shp2 in fibroblasts. SHP2 was inhibited using NSC-87877 inhibitor. The role of Shp2 was investigated in three different mouse models: Bleomycin- and TBRICA induced dermal fibrosis as well as TSK1 genetic model of skin fibrosis. The construct pCMV3-Flag-JAK2 was used for overexpression and for in vitro mutagenesis of human JAK2. Co-immunoprecipitation was performed to confirm the interaction between SHP2 and JAK2. STAT3 activation was analysed by reporter assay. Protein and mRNA expression levels were analysed by Western Blot and qPCR. Results Our data characterise SHP2 as an important regulator of TGFβ signalling in fibroblasts. Although SHP2 expression is decreased in systemic sclerosis (SSc), the moderate downregulation of SHP2 expression is not sufficient to counterbalance the hyper-activation of TGFβ signalling in SSc. However, Shp2-deficient murine fibroblasts are less responsive to TGFβ. An impaired response to the profibrotic effects of TGFβ was also observed in human SSc fibroblasts with siRNA-mediated knockdown of SHP2 and upon pharmacological inhibition of SHP2. We show that SHP2 can dephosphorylate JAK2 at the inhibitory Y570 site to promote TGFβ-dependent activation of JAK2 and its downstream mediator STAT3. In contrast, pharmacologic inhibition of SHP2, overexpression of a catalytically inactive SHP2 mutant or knockout of SHP2 inhibits dephosphorylation of pJAK2Y570 preventing phosphorylation of JAK2 at the activation site Y1007/Y1008 and subsequent activation of STAT3 in TGFβ-stimulated fibroblasts and in experimental fibrosis. Fibroblasts overexpressing SHP2 are more susceptible to the profibrotic effects of TGFβ, whereas the stimulatory effects of TGFβ on myofibroblast differentiation and collagen release are reduced in Shp2 knockout fibroblasts. In addition, fibroblast-specific inactivation of Shp2 in vivo protected from experimental fibrosis induced by overexpression of a constitutively active TGFβ receptor type I, by bleomycin injection and ameliorated fibrosis in TSK1 mice. Conclusions The present study describes for the first time a role of SHP2 in the pathogenesis of SSc. SHP2 is required for the activation of JAK2 and STAT3 by TGFβ. Inactivation of SHP2 prevents fibroblast activation and tissue fibrosis, providing evidence that SHP2 may be a potential target for the treatment of fibrosis. Disclosure of Interest A. Zehender: None declared, J. Huang: None declared, A.-H. Gyorfi: None declared, A.-E. Matei: None declared, C. Dees: None declared, C. Beyer: None declared, K. Gelse: None declared, A. Ramming: None declared, C. Bergmann: None declared, W. Birchmeier: None declared, O. Distler Grant/research support from: Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, Roche/Genentech, Medac, Biovitrium, Boehringer Ingelheim, Novartis, 4D Science, Active Biotech, Bayer, Sinoxa, Serodapharm, EpiPharm, GSK, Pharmacyclics and Biogen, Consultant for: Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, Roche/Genentech, Medac, Biovitrium, Boehringer Ingelheim, Novartis, 4D Science, Active Biotech, Bayer, Sinoxa, Serodapharm, EpiPharm, GSK, Pharmacyclics and Biogen, G. Schett: None declared, J. Distler Shareholder of: 4D Science GmbH, Grant/research support from: Anamar, Active Biotech, Array Biopharma, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, GSK, Novartis, Sanofi-Aventis and UCB, Consultant for: Actelion, Active Biotech, Anamar, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB

Background The pathologic activation of fibroblasts is a key feature of fibrotic disorders such as Systemic Sclerosis (SSc). Deregulation of TGFβ- and Hedgehog signalling has been shown to be critical for the persistent, uncontrolled activation of fibroblasts in SSc.1–3 However, the consequences of the concomitant upregulation of multiple profibrotic pathways are unknown and cross-talk between individual pathways in fibrotic diseases is currently poorly characterised. Mutual activation and amplification of profibrotic signals might be central for the persistent activation of fibroblasts. Objectives The aim of this study is to characterise the crosstalk between AP1- and hedgehog signalling in fibrotic tissue disease in SSc. Methods Co-localization of cJUN and GLI2 in fibrotic skin was analysed by confocal microscopy and interaction was shown by Co-IP. cJUN/AP1 signalling and GLI2 signalling were inhibited in vitro and in vivo using the pharmacological inhibitors T5224 and GANT61. Hedgehog signalling was activated in mice by fibroblast-specific overexpression of constitutively active Smoothend (SmoACT mice). Results Expression profiling of all AP1 family members revealed most pronounced differences for cJUN in SmoACT mice. The expression of cJUN colocalized with the hedgehog transcription factor GLI2. Overexpression and colocalization of cJUN and GLI2 were also observed in fibroblasts in the skin of SSc patients. The number of GLI2- and cJUN double-positive fibroblasts was strongly increased in involved SSc skin compared to healthy controls and was particularly high in samples of SSc patients with diffuse and progressive disease. Based on the upregulation of both cJun and GLI2 and their colocalization, we hypothesised that cJUN and GLI2 might interact with each other to amplify fibroblast activation and tissue fibrosis. Stimulation of resting fibroblasts with TGFβ induces both cJUN and GLI2 mRNA and protein in a time-dependent manner. cJUN and GLI2 are also induced upon stimulation with Sonic hedgehog (SHH), demonstrating that TGFβ and SHH are both capable to activate cJUN and GLI2-dependent transcription. This crosstalk occurs by direct interaction of cJUN and GLI2, which amplifies the transactivation potential of both transcription factors. Co-immunoprecipitation demonstrated that stimulation of fibroblasts with TGFβ or SHH induces direct interaction of cJUN and GLI2. Overexpression of cJun and Gli2 resulted in activation in both AP1 and Hedgheog target genes. The central role of the crosstalk between cJUN and GLI2 for tissue fibrosis was further highlighted by the finding that hedgehog-induced fibrosis was strongly reduced by AP1 inhibition. SmoACT mice developed extensive skin fibrosis. Treatment with a cJUN/AP1 inhibitor T5224, however, strongly ameliorated hedgehog-induced fibrosis in SmoACT mice. Conclusions We demonstrate in the present study that the concomitant activation of AP1- and hedgehog signalling amplifies signalling through both cascades to promote excessive fibroblast activation and tissue fibrosis. This finding may open venues for combined inhibition of AP1- and hedgehog signalling for the treatment of fibrosis. Disclosure of Interest None declared

Background Aberrant activation of profibrotic pathways is a key feature of systemic sclerosis (SSc). Extensive evidence characterises TGFβ- and canonical WNT-signalling as key drivers of fibroblast activation. The crosstalk between those pathways, however, remains largely unknown. A better understanding of the interplay of different profibrotic pathways may be the key to the development of effective targeted therapies. XIAP (X-linked inhibitor of apoptosis protein) is an ubiquitously expressed member of the IAP protein family with important functions in tissue turnover. XIAP was recently described to be implicated in canonical Wnt signalling and TGFβ signalling. Objectives The aim of this study is to characterise the role of XIAP in fibrotic disease. Methods XIAP-expression was analysed by qPCR, IF and Western blot. XIAP was targeted pharmacologically with Embelin. The activation of the canonical Wnt pathway was assessed by analyses of Wnt target genes and by TOPflash/FOPflash luciferase reporter assay. In vivo, XIAP inhibition was analysed in two different models of fibrosis. Results The expression of XIAP is increased in the skin of SSc patients compared to matched healthy individuals with a particularly prominent expression in fibroblasts. The overexpression of XIAP is more pronounced in SSc patients with diffuse and active skin fibrosis compared to SSc patients with limited and inactive disease. The overexpression of XIAP is also reflected in several experimental fibrosis models: the model of sclerodermatous graft versus host disease, the model of bleomycin induced skin fibrosis and in Wnt10b transgenic mice. Stimulation with either recombinant Wnt1 or TGFβ cytokine induces the expression of XIAP in cultured fibroblasts. Inhibition of XIAP reduced the Wnt1- and TGFβ induced activation of fibroblasts with reduced collagen release and expression of myofibroblast markers. In addition, XIAP inhibition reverted the activated fibroblast phenotype in SSc fibroblasts with reduced expression of stress fibres and αSMA. The antifibrotic effects of XIAP inhibition occurred in non-toxic doses. Mechanistically, XIAP inhibition reduced the activation of canonical Wnt signalling as demonstrated by TOPflash reporter assays and by the analysis of canonical Wnt target genes. XIAP inhibition also reduced the expression of canonical Wnt target genes in Wnt10b transgenic mice. To analyse the effects of XIAP inhibition in vivo, two mouse models were used: the model of bleomycin-induced dermal fibrosis and Wnt10b-transgenic mice. In both models, the pharmacological inhibition of XIAP exerted anti-fibrotic effects with reduced dermal thickening, reduced myofibroblast counts and reduced hydroxyproline contents. Conclusions XIAP is upregulated in SSc fibroblasts in a TGFβ-dependent manner and promotes fibroblast activation by fostering canonical WNT signalling. Our data suggest that XIAP mediates an amplification loop between TGF-β and canonical Wnt signalling. Inhibition of XIAP may thus be a novel approach to target aberrant canonical WNT signalling in fibrotic diseases. Disclosure of Interest None declared

Background Dipeptidyl-peptidase-4 (DPP4) plays a role in tissue scaring and its inhibition leads to reduced scar formation. Its function in tissue fibrosis, however, is unknown. Objectives The aim of the study was to investigate the expression of DPP4 in fibrotic tissue of systemic sclerosis (SSc) patients, to characterise DPP4 positive cells, to study the mechanism of action of DPP4 in fibroblasts and to evaluate the antifibrotic effect of pharmacological and genetically inhibition of DPP4 in different preclinical models of SSc. Methods Expression of DPP4 in human and murine skin was analysed. Mouse fibroblasts were isolated and DPP4 positive cells properties were assessed. Pulmonary fibrosis was induced by bleomycin in DPP4 knockout (KO) mice and wildtype littermates. Fibrosis of the lungs was additionally evaluated by computer tomography scans (CT). Two oral DPP4 inhibitors were tested in two concentrations in bleomycin-induced skin fibrosis and in sclerodermatous chronic graft-versus-host disease (scl-cGvHD) model. Antiinflammatory effects of DPP4 inhibition were assessed by CD45 staining of fibrotic and non-fibrotic mouse tissue upon DPP4 inhibition. Moreover, chimeric mice were generated by transplanting bone marrow from DPP4-KO mice in WT-littermates (DPP4→WT) and vice versa (WT→DPP4) and fibrosis was by intratracheal injections of bleomycin. Results DPP4 positive fibroblasts were increased in fibrotic skin of SSc patients and also in murine models of fibrosis. DPP4 expression is induced by TGF-β in an Erk-dependent manner. DPP4-positive fibroblasts strongly express stress fibres after TGF-β stimulation and released increased amounts of collagen. Mechanistically, inhibition of DPP4 selectively interferes with the TGF-β induced activation of ERK signalling, but does not inhibit TGF-β induced SMAD signalling, or other non-canonical TGF-β pathways involving Fra2, c-Jun, p38, Akt or STAT3. Furthermore, pharmacological inhibition of DPP4 reduced the release of collagen and the expression of myofibroblast markers. DPP4-KO mice are less sensitive to bleomycin-induced pulmonary fibrosis as shown by milder changes on CT, reduced Ashcroft scores and reduced hydroxyproline content. DPP4-KO mice also show reduced skin fibrosis upon bleomycin challenge. Moreover, treatment with DPP4 inhibitors demonstrated potent antifibrotic effects in bleomycin-induced skin fibrosis and experimental scl-cGvHD mouse model. Treatment with DDP4 inhibitors also reduced leukocyte infiltrations into the skin. The extent of pulmonary fibrosis of DPP4→WT was comparable to that of WT→WT control mice. Fibrosis was strongly ameliorated in WT→DPP4 mice and results were comparable to that of DPP4→DPP4 mice, characterising resident cells such as fibroblasts as major target cells for the antifibrotic effects of DPP4 inhibitors. Conclusions DPP4 characterises an activated subpopulation of fibroblasts in SSc. Moreover, inhibitors of DPP4 show a significant anti-fibrotic effect in several mouse models of SSc in well tolerated doses. These results may have direct translational implications as DPP4 inhibitors are already in clinical use for diabetes. Acknowledgements AS received a scientific training bursary from the European League Against Rheumatism. Disclosure of Interest None declared

Fibroblasts are key effector cells in tissue remodeling. They remain persistently activated in fibrotic diseases, resulting in progressive deposition of extracellular matrix. Although fibroblast activation may be initiated by external factors, prolonged activation can induce an "autonomous," self-maintaining profibrotic phenotype in fibroblasts. Accumulating evidence suggests that epigenetic alterations play a central role in establishing this persistently activated pathologic phenotype of fibroblasts. We demonstrated that in fibrotic skin of patients with systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease, TGF-β induced the expression of DNA methyltransferase 3A (DNMT3A) and DNMT1 in fibroblasts in a SMAD-dependent manner to silence the expression of suppressor of cytokine signaling 3 (SOCS3) by promoter hypermethylation. Downregulation of SOCS3 facilitated activation of STAT3 to promote fibroblast-to-myofibroblast transition, collagen release, and fibrosis in vitro and in vivo. Reestablishment of the epigenetic control of STAT3 signaling by genetic or pharmacological inactivation of DNMT3A reversed the activated phenotype of SSc fibroblasts in tissue culture, inhibited TGF-β-dependent fibroblast activation, and ameliorated experimental fibrosis in murine models. These findings identify a pathway of epigenetic imprinting of fibroblasts in fibrotic disease with translational implications for the development of targeted therapies in fibrotic diseases.

Although fibrosis is becoming increasingly recognized as a major cause of morbidity and mortality in modern societies, there are very few treatment strategies available that specifically target the pathogenesis of fibrosis. Early in disease, inflammation and vascular changes and an increase in reactive oxygen species play pivotal roles. After inflammation has subsided, fibrosis and scarring are predominant in later phases. Fibrosis is driven by a complex, not-yet fully understood interplay between inflammatory cells on one hand and endothelium and fibroblasts on the other hand. The latter are regarded as the key players due to their extensive synthesis of extracellular matrix components which results in skin and organ fibrosis. Various cytokines orchestrate altered functions of the mentioned cell types. There are promising targets with therapeutic potential that have been extensively characterized in recent years connected with the hope to translate these preclinical results into clinical practice.

ObjectivesThe enzyme poly(ADP-ribose) polymerase-1 (PARP-1) transfers negatively charged ADP-ribose units to target proteins. This modification can have pronounced regulatory effects on target proteins. Recent studies showed that PARP-1 can poly(ADP-ribosyl)ate (PARylate) Smad proteins. However, the role of PARP-1 in the pathogenesis of systemic sclerosis (SSc) has not been investigated.MethodsThe expression of PARP-1 was determined by quantitative PCR and immunohistochemistry. DNA methylation was analysed by methylated DNA immunoprecipitation assays. Transforming growth factor-β (TGFβ) signalling was assessed using reporter assays, chromatin immunoprecipitation assays and target gene analysis. The effect of PARP-1 inactivation was investigated in bleomycin-induced and topoisomerase-induced fibrosis as well as in tight-skin-1 (Tsk-1) mice.ResultsThe expression of PARP-1 was decreased in patients with SSc, particularly in fibroblasts. The promoter of PARP-1 was hypermethylated in SSc fibroblasts and in TGFβ-stimulated normal fibroblasts. Inhibition of DNA methyltransferases (DNMTs) reduced the promoter methylation and reactivated the expression of PARP-1. Inactivation of PARP-1 promoted accumulation of phosphorylated Smad3, enhanced Smad-dependent transcription and upregulated the expression of TGFβ/Smad target genes. Inhibition of PARP-1 enhanced the effect of TGFβ on collagen release and myofibroblast differentiation in vitro and exacerbated experimental fibrosis in vivo. PARP-1 deficiency induced a more severe fibrotic response to bleomycin with increased dermal thickening, hydroxyproline content and myofibroblast counts. Inhibition of PARylation also exacerbated fibrosis in Tsk-1 mice and in mice with topoisomerase-induced fibrosis.ConclusionPARP-1 negatively regulates canonical TGFβ signalling in experimental skin fibrosis. The downregulation of PARP-1 in SSc fibroblasts may thus directly contribute to hyperactive TGFβ signalling and to persistent fibroblast activation in SSc.

ObjectivesSystemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis.MethodsJMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP.ResultsThe expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFβ)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFβ. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2.ConclusionWe present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.

BackgroundActivating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of transcription factors, regulates cellular response to stress including oxidative stress. The aim of this study was to analyse the role of ATF3 in fibroblast activation in systemic sclerosis (SSc).MethodsATF3 was analysed by reverse transcription quantitative PCR, western blot and immunohistochemistry. ATF3 knockout fibroblasts and mice were used to study the functional role of ATF3. Knockdown experiments, reporter assays and coimmunoprecipitation were performed to study the effects of ATF3 on Smad and activation protein 1 (AP-1) signalling. The role of c-Jun was analysed by costaining, specific inactivation and coimmunoprecipitation.ResultsTransforming growth factor-β (TGFβ) upregulates the expression of ATF3 in SSc fibroblasts. ATF3-deficient fibroblasts were less sensitive to TGFβ, whereas ectopic expression of ATF3 enhanced the profibrotic effects of TGFβ. Mechanistically, ATF3 interacts with Smad3 directly on stimulation with TGFβ and regulates Smad activity in a c-Jun-dependent manner. Knockout of ATF3 protected mice from bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active TGFβ receptor I. Reporter assays and analyses of the expression of Smad target genes demonstrated that binding of ATF3 regulates the transcriptional activity of Smad3.ConclusionsWe demonstrate for the first time a key role for ATF3 in fibrosis. Knockout of the ATF3 gene reduced the stimulatory effect of TGFβ on fibroblasts by interfering with canonical Smad signalling and protected the mice from experimental fibrosis in two different models. ATF3 might thus be a candidate for molecular targeted therapies for SSc.

Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-β (TGF-β) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-β target genes, thereby limiting pro-fibrotic TGF-β effects. Even though temporary upregulation of TGF-β in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-β signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-β signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.

ObjectivesStimulators of soluble guanylate cyclase (sGC) are currently investigated in clinical trials for the treatment of fibrosis in systemic sclerosis (SSc). In this study, we aim to investigate the role of protein kinases G (PKG) as downstream mediators of sGC–cyclic guanosine monophosphate (cGMP) in SSc.MethodsMice with combined knockout of PKG1 and 2 were challenged with bleomycin and treated with the sGC stimulator BAY 41-2272. Fibroblasts were treated with BAY 41-2272 and with the PKG inhibitor KT 5823.ResultsPKG1 and 2 are upregulated in SSc in a transforming growth factor-β1 (TGFβ1)-dependent manner, as an attempt to compensate for the decreased signalling through the sGC–cGMP–PKG pathway. Inhibition or knockout of PKG1 and 2 abrogates the inhibitory effects of sGC stimulation on fibroblast activation in a SMAD-independent, but extracellular signal-regulated kinase (ERK)-dependent manner. In vivo, sGC stimulation fails to prevent bleomycin-induced fibrosis in PKG1 and 2 knockout mice.ConclusionsOur data provide evidence that PKGs are essential mediators of the antifibrotic effects of sGC stimulators through interfering with non-canonical TGFβ signalling. TGFβ1 promotes its profibrotic effects through inhibition of sGC–cGMP–PKG signalling, sGC stimulation exerts its antifibrotic effects by inhibition of TGFβ1-induced ERK phosphorylation.

Background Chronic and exaggerated fibroblast activation is a central hallmark of Systemic Sclerosis (SSc) fibrotic disease and results in a high morbidity and mortality. Epigenetic changes might play important roles in mediating chronic fibroblast activation. Trimethylation of H3 at lysine residue K27 (H3K27me3) is a repressive epigenetic mark that was recently identified as an important negative regulator of fibroblast activation [1]. Jumonji domain containing protein 3 (JMJD3) mediates H3K27me3–demethylation. JMJD3 inhibitors are being tested as therapeutic strategies in malignant diseases. Objectives The aim of this study was to characterize the role of JMJD3 in fibrotic disease and to explore JMJD3 as a potential drug target in SSc. Methods Expression analyses of JMJD3 were performed using qPCR, IF and Western blot. siRNA mediated knockdown and the pharmacologic H3K27me3-demethylase inhibitor GSKJ4 were used to target JMJD3. In vivo, we analyzed the effects of GSKJ4 in bleomycin-induced dermal fibrosis and in Topoisomerase-I-induced (TopoI) fibrosis. H3K27me3 levels at the Fra2 promotor were analyzed by CHIP. Results We observed increased expression of JMJD3 in SSc skin compared to healthy controls. Fibroblast-specific overexpression of JMJD3 was also reflected in experimental fibrosis models. TGFβ upregulated JMJD3. Inhibition of JMJD3 increased H3K27me3 in vitro and in vivo . Inhibition of JMJD3 reverted the activated fibroblast phenotype in SSc fibroblasts and decreased the expression of contractile fibers and of α-smooth muscle actin. In addition, JMJD3 inhibition reduced the basal and TGFβ induced collagen secretion of SSc fibroblasts. JMJD3 regulated the TGFβ induced expression of Fra2. GSKJ4 reverted the TGFβ induced reduction of H3K27me3 at the Fra2 promotor. Moreover, the anti-fibrotic effects of JMJD3 inhibition were evened in Fra2 knockout fibroblasts. Overexpression of Fra2 in JMJD3-knockdown fibroblasts restored the profibrotic effect of JMJD3. In vivo , inhibition of JMJD3 ameliorated fibrosis in bleomycin- and TopoI- induced experimental fibrosis and reduced dermal thickening, hydroxyproline content and myofibroblast differentiation. Conclusions We present first evidence that JMJD3 contributes to the activated phenotype of SSc fibroblasts. TGFβ upregulated JMJD3. Inhibition of JMJD3 prevented the aberrant activation of fibroblasts in vitro and ameliorated dermal fibrosis in several mouse models in vivo . The profibrotic effects of JMJD3 might be mediated by reducing the H3K27me3 at the Fra2 promotor and consecutive overexpression of Fra2. References Kramer, M., et al., Inhibition of H3K27 histone trimethylation activates fibroblasts and induces fibrosis. Ann Rheum Dis, 2013. 72(4): p. 614–20. Disclosure of Interest None declared

Background Fos-related antigen 2 (Fra2) is a transcription factor belonging to the Fos family proteins which is part of the AP-1 transcription complex. We recently described a Fra2 transgenic (tg) mouse model which develops a multi-organ inflammatory phenotype affecting skin, lungs, thymus, liver and salivary glands. We have observed abnormalities in the T cell compartment, particularly in regulatory T (Treg) cells, which led us to hypothesize that Fra2 tg mice develop a T cell driven autoimmune phenotype. Objectives To demonstrate the autoimmune phenotype of Fra2 tg mice and to characterize the mechanisms leading to Treg cell abnormality. Methods We used previously generated Fra2 tg overexpressing mice. T lymphocyte populations were analyzed by flow cytometry for expression of activation markers and secretion of cytokines. We transferred purified CD4+ T cells into Rag2-/- mice lacking T and B cells, and we generated Rag2-/-Fra2 tg mice. Bone marrow cells were transferred into lethally irradiated recipients to create Fra2-WT bone marrow chimeric mice. Results Fra2 tg mice backcrossed onto a Rag2-/- background did not develop inflammatory manifestations (n=6), demonstrating the dependence on T and/or B cells of the autoimmune phenotype. In line with this, the transfer of purified CD4+ cells from 16 week-old Fra2 tg mice into Rag2-/- recipients was sufficient to transfer the disease phenotype (n=3). Analysis of T cell populations from Fra2 tg mice showed the presence of activated CD4+ and CD8+ cells in the spleen and lungs. After in vitro stimulation, we found that CD4+ T cells from Fra2 tg mice produced the Th2 cytokines IL-4, IL-5 and IL-13. Thus, these data strongly suggest a T cell-driven autoimmune disease in these mice. We previously reported a striking decrease of Treg cells in Fra2 tg mice, which might explain the autoimmune phenotype observed. Supporting this idea, we found that 3 week-old mice were devoid of organ manifestations and of T cell activation, but presented the same defect in the Treg cell population (n=6, p Conclusions Our data suggest that Fra2 controls tTreg cell development, possibly by modulating IL-2 signaling in T cells, which leads to autoimmunity in this mouse model. This new pathway could be targeted in a translational approach to modulate the capacity of T cells to differentiate in Tregs during autoimmune disease. Disclosure of Interest F. Renoux Grant/research support from: Swisslife, M. Stellato: None declared, D. Impellizzieri: None declared, A. Vogetseder: None declared, R. Huang Employee of: Sanofi-Genzyme, A. Subramaniam Employee of: Sanofi-Genzyme, C. Dees: None declared, J. Distler Shareholder of: 4D Science, Grant/research support from: Anamar, Active Biotech, Array Biopharma, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, GSK, Novartis, Sanofi-Aventis, UCB, Consultant for: Actelion, Active Biotech, Anamar, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, G. Kania: None declared, O. Boyman: None declared, O. Distler Grant/research support from: Actelion, Bayer, Boehringer Ingelheim, Pfizer, Sanofi, Consultant for: 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, ChemomAb, EpiPharm, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, Mepha, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Pfizer, Sanofi, Serodapharm, Sinoxa, Speakers bureau: AbbVie, iQone Healthcare, Mepha

Objective Nuclear receptors regulate cell growth, differentiation, and homeostasis. Selective nuclear receptors promote fibroblast activation, which leads to tissue fibrosis, the hallmark of systemic sclerosis (SSc). This study was undertaken to investigate the effects of constitutive androstane receptor (CAR)/NR1I3, an orphan nuclear receptor, on fibroblast activation and experimental dermal fibrosis. Methods CAR expression was quantified by quantitative polymerase chain reaction, Western blotting, immunohistochemistry, and immunofluorescence. CAR expression was modulated by small molecules, small interfering RNA, forced overexpression, and site-directed mutagenesis. The effects of CAR activation were analyzed in cultured fibroblasts, in bleomycin-induced dermal fibrosis, and in mice overexpressing a constitutively active transforming growth factor β (TGFβ) receptor type I (TβRI-CA). Results Up-regulation of CAR was detected in the skin and in dermal fibroblasts in SSc patients. Stimulation of healthy fibroblasts with TGFβ induced the expression of CAR messenger RNA and protein in a Smad-dependent manner. Pharmacologic activation or overexpression of CAR in healthy fibroblasts significantly increased the stimulatory effects of TGFβ on collagen synthesis and myofibroblast differentiation, and amplified the stimulatory effects of TGFβ on COL1A2 transcription activity. Treatment with CAR agonist increased the activation of canonical TGFβ signaling in murine models of SSc and exacerbated bleomycin-induced and TβRI-CA–induced fibrosis with increased dermal thickening, myofibroblast counts, and collagen accumulation. Conclusion Our findings indicate that CAR is up-regulated in SSc and regulates TGFβ signaling. Activation of CAR increases the profibrotic effects of TGFβ in cultured fibroblasts and in different preclinical models of SSc. Thus, inactivation of CAR might be a novel approach to target aberrant TGFβ signaling in SSc and in other fibrotic diseases.

Objectives S100A4 is a calcium binding protein with regulatory functions in cell homeostasis, proliferation and differentiation that has been shown to promote cancer progression and metastasis. In the present study, we evaluated the role of S100A4 in fibroblast activation in systemic sclerosis (SSc). Methods The expression of S100A4 was analysed in human samples, murine models of SSc and in cultured fibroblasts by real-time PCR, immunohistochemistry and western blot. The functional role of S100A4 was evaluated using siRNA, overexpression, recombinant protein and S100A4 knockout (S100A4 −/− ) mice. Transforming growth factor β (TGF-β) signalling was assessed by reporter assays, staining for phosphorylated Smad2/3 and analyses of target genes. Results The expression of S100A4 was increased in SSc skin and in experimental fibrosis in a TGF-β/Smad-dependent manner. Overexpression of S100A4 or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, knockdown of S100A4 reduced the pro-fibrotic effects of TGF-β and decreased the release of collagen. S100A4 −/− mice were protected from bleomycin-induced skin fibrosis with reduced dermal thickening, decreased hydroxyproline content and lower myofibroblast counts. Deficiency of S100A4 also ameliorated fibrosis in the tight-skin-1 (Tsk-1) mouse model. Conclusions We characterised S100A4 as a downstream mediator of the stimulatory effects of TGF-β on fibroblasts in SSc. TGF-β induces the expression of S100A4 to stimulate the release of collagen in SSc fibroblasts and induce fibrosis. Since S100A4 is essentially required for the pro-fibrotic effects of TGF-β and neutralising antibodies against S100A4 are currently evaluated, S100A4 might be a candidate for novel antifibrotic therapies.

Objectives: Casein kinase II (CK2) is a constitutively active serine/threonine protein kinase that plays a key role in cellular transformation and tumorigenesis. The purpose of the study was to characterise whether CK2 contributes to the pathologic activation of fibroblasts in patients with SSc and to evaluate the antifibrotic potential of CK2 inhibition. Methods: Activation of CK2, JAK2 and STAT3 in human skin and in experimental fibrosis was analysed by immunohistochemistry. CK2 signalling was inhibited by the selective CK2 inhibitor 4, 5, 6, 7-Tetrabromobenzotriazole (TBB). The mouse models of bleomycin-induced and TGFβ receptor I (TBR)-induced dermal fibrosis were used to evaluate the antifibrotic potential of specific CK2 inhibition in vivo. Result: Increased expression of CK2 was detected in skin fibroblasts of SSc patients. Inhibition of CK2 by TBB abrogated the TGFβ-induced activation of JAK2/STAT3 signalling and prevented the stimulatory effects of TGFβ on collagen release and myofibroblasts differentiation in cultured fibroblasts. Inhibition of CK2 prevented bleomycin-induced and TBR-induced skin fibrosis with decreased dermal thickening, lower myofibroblast counts and reduced accumulation of collagen. Treatment with TBB also induced regression of pre-established fibrosis. The antifibrotic effects of TBB were accompanied by reduced activation of JAK2/STAT3 signalling in vivo. Conclusions: We provide evidence that CK2 is activated in SSc and contributes to fibroblast activation by regulating JAK2/STAT3 signalling. Inhibition of CK2 reduced the pro-fibrotic effects of TGFβ and inhibited experimental fibrosis. Targeting of CK2 may thus be a novel therapeutic approach for SSc and other fibrotic diseases.

Canonical Wnt signalling belongs to the so-called morphogen pathways and plays essential roles in development and tissue homeostasis. Being such a crucial regulatory pathway, Wnt signalling is tightly controlled at different levels. However, uncontrolled activation of canonical Wnt signalling has been implicated into the pathogenesis of various human disorders. In the last years, aberrant Wnt signalling has been demonstrated in fibrotic diseases including systemic sclerosis (SSc). In this review, we will discuss the current state of research on canonical Wnt signalling in SSc. Activation of canonical Wnt signalling induces fibroblast activation with subsequent myofibroblast differentiation and excessive collagen release resulting in tissue fibrosis. Genetic or pharmacological blockade of Wnt activation ameliorates experimental fibrosis in different preclinical models. These findings have direct translational implications because several small molecule inhibitors of Wnt signalling are currently evaluated in clinical trials and some already showed first promising results.

ObjectivesTo assess whether the discrepancy between the strong antifibrotic effects of tyrosine kinase inhibitors (TKIs) in animal models and the inconsistent results in clinical studies might be related to the activation levels of drug targets.MethodsSkin sections of bleomycin, TSK1, Fra-2 transgenic mice, SSc patients and controls were analysed by histology and immunohistochemistry. Subgroups of mice were treated with the TKIs nilotinib or imatinib. Differences in the activation levels of the TKI targets p-PDGFRβ (platelet derived growth factor β) and p-c-abl were assessed.ResultsIn bleomycin and TSK1 mice, expression of activated p-PDGFRβ (platelet derived growth factor receptor β) and p-c-abl was ubiquitous with strong upregulation compared with controls. Treatment with TKIs resulted in successful target inhibition and consequently reduced dermal fibrosis. In the Fra-2 model, the activation levels of p-PDGFRβ and p-c-abl were much lower than in the bleomycin and the TSK1 models. Accordingly, nilotinib did not prevent dermal fibrosis and target inhibition was unsuccessful. Notably, in skin biopsies of SSc patients, the mean activation levels of TKI targets were only moderate and in the majority of patients resembled those of the non-responsive Fra-2 model.ConclusionsAnimal models for proof-of-concept studies should be selected based on a similar activation level and expression pattern of drug targets as in human SSc.

Chronic graft-versus-host disease (cGVHD) is a prognosis limiting complication of allogeneic stem cell transplantation. The molecular mechanisms underlying cGVHD are incompletely understood, and targeted therapies are not yet established for clinical use. Here we examined the role of the hedgehog pathway in sclerodermatous cGVHD. Hedgehog signaling was activated in human and murine cGVHD with increased expression of sonic hedgehog and accumulation of the transcription factors Gli-1 and Gli-2. Treatment with LDE223, a highly selective small-molecule antagonist of the hedgehog coreceptor Smoothened (Smo), abrogated the activation of hedgehog signaling and protected against experimental cGVHD. Preventive therapy with LDE223 almost completely impeded the development of clinical and histologic features of sclerodermatous cGVHD. Treatment with LDE223 was also effective, when initiated after the onset of clinical manifestations of cGVHD. Hedgehog signaling stimulated the release of collagen from cultured fibroblasts but did not affect leukocyte influx in murine cGVHD, suggesting direct, leukocyte-independent stimulatory effects on fibroblasts as the pathomechanism of hedgehog signaling in cGVHD. Considering the high morbidity of cGVHD, the current lack of efficient molecular therapies for clinical use, and the availability of well-tolerated inhibitors of Smo, targeting hedgehog signaling might be a novel strategy for clinical trials in cGVHD.

ObjectivesFibrosis is a predominant cause of death in systemic sclerosis (SSc). First epigenetic modifications have recently been shown to contribute to activation of SSc fibroblasts. Here, we investigated inhibition of sumoylation as a novel antifibrotic approach.MethodsSumoylation was inhibited by siRNA-mediated knockdown of the Small Ubiquitin-like MOdifiers (SUMO) E2-conjugating enzyme Ubc9, which is essential for sumoylation. The effects of knockdown of Ubc9 were analysed in bleomycin-induced dermal fibrosis, and in the model of fibrosis induced by overexpression of a constitutively active TGF-beta receptor type I (TBR). SUMO-1 and phosphorylated Smad3 were detected by immunohistochemistry.ResultsIncreased staining for SUMO-1 was detected in patients with SSc and in experimental fibrosis. Inhibition of sumoylation exerted potent antifibrotic effects and prevented dermal thickening, myofibroblast differentiation and accumulation of collagen induced by bleomycin, or by overexpression of constitutively active TBR. Moreover, knockdown of Ubc9 reduced the accumulation of phosphorylated Smad3 in experimental fibrosis indicating that inhibition of sumoylation may normalise canonical TGF-β signalling in vivo.ConclusionsWe demonstrate that inhibition of sumoylation reduces canonical TGF-β signalling and prevents experimental fibrosis in different preclinical models. These data provide first evidence that targeting of aberrant sumoylation may be a novel therapeutic approach for fibrotic diseases.

Objective To investigate whether c-Jun and c-Fos contribute to the pathologic activation of fibroblasts in systemic sclerosis (SSc) and to evaluate the antifibrotic potential of selective activator protein 1 (AP-1) inhibition. Methods Expression of c-Jun and c-Fos was determined by real-time polymerase chain reaction (PCR) and immunohistochemical analysis. Fibroblasts were stimulated with transforming growth factor β (TGFβ) and incubated with T-5224, a small-molecule inhibitor of AP-1, or were transfected with small interfering RNA (siRNA) duplexes against c-Jun and c-Fos. Collagen synthesis was quantified by real-time PCR and hydroxyproline assay. Differentiation of resting fibroblasts into myofibroblasts was assessed by staining for α-smooth muscle actin and stress fibers. The antifibrotic potential of T-5224 was evaluated in mouse models of dermal fibrosis induced by bleomycin or by adenoviral overexpression of a constitutively active TGFβ receptor type I. Results Up-regulation of c-Jun and c-Fos was detected in mouse models of SSc and in the skin and dermal fibroblasts of patients with SSc. Stimulation of healthy fibroblasts with TGFβ induced the expression of c-Jun and c-Fos. Treatment with T-5224 or nucleofection with siRNA directed against c-Jun and c-Fos abrogated the profibrotic effects of TGFβ. T-5224 decreased the release of collagen selectively in SSc fibroblasts. T-5224 was well tolerated and prevented dermal fibrosis induced by bleomycin or by adenoviral activation of TGFβ signaling. Conclusion AP-1 is up-regulated in a TGFβ-dependent manner in SSc. The selective AP-1 inhibitor T-5224 reduced collagen synthesis selectively in SSc fibroblasts and efficiently prevented the development of experimental dermal fibrosis. Thus, AP-1 might be a promising new molecular target for the treatment of SSc.

ObjectivesTissue fibrosis is a leading cause of death in patients with systemic sclerosis (SSc). Effective antifibrotic treatments are not available. Here, the authors investigated inhibition of hedgehog signalling by targeting Smoothened (Smo) as a novel antifibrotic approach.MethodsThe activation status of the hedgehog pathway was assessed by immunohistochemistry for Gli transcription factors and by quantification of hedgehog target genes. Hedgehog signalling was inhibited by the selective inhibitor LDE223 and by small interfering RNA against Smo in the models of bleomycin-induced dermal fibrosis and in tight-skin-1 mice.ResultsHedgehog signalling is activated in SSc and in murine models of SSc. Inhibition of Smo either by LDE223 or by small interfering RNA prevented dermal thickening, myofibroblast differentiation and accumulation of collagen upon challenge with bleomycin. Targeting Smo also exerted potent antifibrotic effects in tight-skin-1 mice and did prevent progression of fibrosis and induced regression of pre-established fibrosis.ConclusionsInhibition of hedgehog signalling exerted potent antifibrotic effects in preclinical models of SSc in both preventive and therapeutic settings. These findings might have direct translational implications because inhibitors of Smo are already available and yielded promising results in initial clinical trials.

OBJECTIVE: Tissue fibrosis caused by pathologic activation of fibroblasts with increased synthesis of extracellular matrix components is a major hallmark of systemic sclerosis (SSc). Notch signaling regulates tissue differentiation, and abnormal activation of Notch signaling has been implicated in the pathogenesis of various malignancies. The present study was undertaken to investigate the role of Notch signaling in SSc and to evaluate the therapeutic potential of Notch inhibition for the treatment of fibrosis. METHODS: Activation of the Notch pathways was analyzed by staining for the Notch intracellular domain (NICD) and quantification of levels of HES-1 messenger RNA. In the mouse model of bleomycin-induced dermal fibrosis and in tight skin 1 mice, Notch signaling was inhibited by the γ-secretase inhibitor DAPT and by overexpression of a Notch-1 antisense construct. RESULTS: Notch signaling was activated in SSc in vivo, with accumulation of the NICD and increased transcription of the target gene HES-1. Overexpression of a Notch antisense construct prevented bleomycin-induced fibrosis and hypodermal thickening in tight skin 1 mice. Potent antifibrotic effects were also obtained with DAPT treatment. In addition to prevention of fibrosis, targeting of Notch signaling resulted in almost complete regression of established experimental fibrosis. CONCLUSION: The present results demonstrate that pharmacologic as well as genetic inhibition of Notch signaling exerts potent antifibrotic effects in different murine models of SSc. These findings might have direct translational implications because different inhibitors of the γ-secretase complex are available and have yielded promising results in cancer trials. Copyright © 2011 by the American College of Rheumatology.

Objectives Transforming growth factor β (TGFβ) has been identified as a key player in fibrotic diseases. However, the molecular mechanisms by which TGFβ activates fibroblasts are incompletely understood. Here, the role of JunD, a member of the activator protein 1 (AP-1) family of transcription factors, as a downstream mediator of TGFβ signalling in systemic sclerosis (SSc), was investigated. Methods The expression of JunD was analysed by real-time PCR, immunofluorescence, western blotting and immunohistochemistry. The canonical Smad pathway was specifically targeted by small interfering (si)RNA. The expression of extracellular matrix proteins in JunD deficient (JunD −/− ) fibroblasts was analysed by real-time PCR and hydroxyproline assays. The mouse model of bleomycin-induced dermal fibrosis was used to assess the role of JunD in experimental fibrosis. Results JunD was overexpressed in SSc skin and in cultured fibroblasts in a TGFβ dependent manner. The expression of JunD colocalised with pSmad 3 in fibrotic skin and silencing of Smad 3 or Smad 4 by siRNA prevented the induction of JunD by TGFβ. JunD −/− fibroblasts were less responsive to TGFβ and released less collagen upon stimulation with TGFβ. Moreover, JunD −/− mice were protected from bleomycin-induced fibrosis with reduced dermal thickening, decreased myofibroblast counts and lower collagen content of lesional skin. Conclusions These data demonstrate that JunD is overexpressed in SSc and that JunD is a mediator of the profibrotic effects of TGFβ. Considering that inhibitors of AP-1 signalling have recently been developed and are available for clinical trials in SSc, these findings may have translational implications.

OBJECTIVE: The transcription factor STAT-4 has recently been identified as a genetic susceptibility factor in systemic sclerosis (SSc) and other autoimmune diseases. The aim of this study was to investigate the contribution of STAT-4 in the development of a fibrotic phenotype in 2 different mouse models of experimental dermal fibrosis. METHODS: STAT-4-deficient (stat4(-/-) ) mice and their wild-type littermates (stat4(+/+) ) were injected with bleomycin or NaCl. Infiltrating leukocytes, T cells, B cells, and monocytes were quantified in the lesional skin of stat4(-/-) and stat4(+/+) mice. Inflammatory and profibrotic cytokines were measured in sera and lesional skin samples from stat4(-/-) and stat4(+/+) mice. The outcome of mice lacking STAT-4 was also investigated in the tight skin 1 (TSK-1) mouse model. RESULTS: Stat4(-/-) mice were protected against bleomycin-induced dermal fibrosis, with a reduction in dermal thickening (mean ± SEM 65 ± 3% decrease; P = 0.03), hydroxyproline content (68 ± 5% decrease; P = 0.02), and myofibroblast counts (71 ± 6% decrease; P = 0.005). Moreover, the number of infiltrating leukocytes, especially T cells, was significantly decreased in the lesional skin of stat4(-/-) mice (mean ± SEM 63 ± 5% reduction in T cell count; P = 0.02). Stat4(-/-) mice also displayed decreased levels of inflammatory cytokines such as tumor necrosis factor α, interleukin-6 (IL-6), IL-2, and interferon-γ in lesional skin. Consistent with a primary role of STAT-4 in inflammation, STAT-4 deficiency did not ameliorate fibrosis in TSK-1 mice. CONCLUSION: The results of this study demonstrate that the transcription factor STAT-4 exerts potent profibrotic effects by controlling T cell activation and proliferation and cytokine release. These findings confirm the results of genetics studies on the role of STAT-4 in the development of SSc. Copyright © 2011 by the American College of Rheumatology.

Objective. Cannabinoids are derivates of the marijuana component 9 -tetrahydrocannabinol that exert their effects on mesenchymal cells and immune cells via CB1 and CB2 receptors. The aim of the present study was to evaluate the role of CB1 in systemic sclerosis. Methods. CB1-deficient (CB1 / ) mice and wildtype littermates (CB1 / mice) were injected with bleomycin. CB1 signaling was activated in vivo with the selective agonist N-(2-chloroethyl)-5Z,8Z,11Z,14Zeicosatetraenamide (ACEA). Bone marrow transplantation experiments were performed to investigate whether the phenotype of CB1 / mice was mediated by leukocytes or mesenchymal cells. The role of CB1 was also investigated in the TSK-1 mouse model. Results. CB1 / mice were protected from bleomycin-induced dermal fibrosis, with reduced dermal thickening, hydroxyproline content, and myofibroblast counts. Inactivation of CB1 decreased the number of infiltrating T cells and macrophages in lesional skin. In contrast, activation of CB1 with ACEA increased leukocyte infiltration and enhanced the fibrotic response to bleomycin. The phenotype of CB1 / mice was mimicked by transplantation of CB1 / mouse bone marrow into CB1 / mice, demonstrating that CB1 exerts its profibrotic effects indirectly by regulating leukocyte infiltration. Consistently, knockdown of CB1 did not prevent fibrosis in the inflammationindependent TSK-1 mouse model. Conclusion. We demonstrate that the cannabinoid receptor CB1 is crucial for leukocyte infiltration and secondary fibroblast activation and that inactivation of CB1 exerts potent antifibrotic effects in inflammationdriven models of fibrosis.

Objective Systemic sclerosis (SSc) is a connective tissue disease that is characterized by microvascular disease and tissue fibrosis. Progressive loss and irregular architecture of the small blood vessels are well characterized, but the potential involvement of the lymphatic vessel system has not been analyzed directly in SSc. This study was undertaken to assess whether the lymphatic vascular system is affected in SSc, and whether changes to the lymphatic vessels are associated with dystrophic changes and tissue damage in patients with SSc. Methods Lymphatic endothelial cells in skin biopsy samples from patients with SSc and age- and sex-matched healthy volunteers were identified by staining for podoplanin and prox-1, both of which are specifically expressed in lymphatic endothelial cells but not in blood vascular endothelial cells. CD31 was used as a pan–endothelial cell marker. Statistical analyses were performed using Kruskal-Wallis, Mann-Whitney U, and Spearman's rank correlation tests. Results The numbers of podoplanin- and prox-1–positive lymphatic vessels were significantly reduced in patients with SSc as compared with healthy individuals. The number of podoplanin-positive lymphatic precollector vessels was significantly lower in SSc patients with fingertip ulcers than in SSc patients without ulcers. Moreover, the number of lymphatic vessels correlated inversely with the number of fingertip ulcers at the time of biopsy and with the number of fingertip ulcers per year. The inverse correlation between lymphatic precollector vessel counts and fingertip ulcers remained significant after statistical adjustment for the blood vessel count, age, and modified Rodnan skin thickness score. Conclusion These results demonstrate a severe reduction in the number of lymphatic capillaries and lymphatic precollector vessels in patients with SSc. Patients with decreased lymphatic vessel counts may be at particularly high risk of developing fingertip ulcers.

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc.

ObjectivesNotch ligands and receptors have recently been shown to be differentially expressed in osteoarthritis (OA). We aim to further elucidate the functional role of Notch signalling in OA using Notch1 antisense transgenic (Notch1 AS) mice.MethodsNotch and hedgehog signalling were analysed by real-time PCR and immunohistochemistry. Notch-1 AS mice were employed as a model of impaired Notch signalling in vivo. Experimental OA was induced by destabilisation of the medial meniscus (DMM). The extent of cartilage destruction and osteophyte formation was analysed by safranin-O staining with subsequent assessment of the Osteoarthritis Research Society International (OARSI) and Mankin scores and µCT scanning. Collagen X staining was used as a marker of chondrocyte hypertrophy. The role of hairy/enhancer of split 1 (Hes-1) was investigated with knockdown and overexpression experiments.ResultsNotch signalling was activated in human and murine OA with increased expression of Jagged1, Notch-1, accumulation of the Notch intracellular domain 1 and increased transcription of Hes-1. Notch1 AS mice showed exacerbated OA with increases in OARSI scores, osteophyte formation, increased subchondral bone plate density, collagen X and osteocalcin expression and elevated levels of Epas1 and ADAM-TS5 mRNA. Inhibition of the Notch pathway induced activation of hedgehog signalling with induction of Gli-1 and Gli-2 and increased transcription of hedgehog target genes. The regulatory effects of Notch signalling on Gli-expression were mimicked by Hes-1.ConclusionsInhibition of Notch signalling activates hedgehog signalling, enhances chondrocyte hypertrophy and exacerbates experimental OA including osteophyte formation. These data suggest that the activation of the Notch pathway may limit aberrant hedgehog signalling in OA.