OBJECTIVE:Members of the superfamily of nuclear receptors have been implicated in inflammatory processes and pathologic tissue remodeling and have emerged as attractive targets for pharmaceutical intervention. However, the role of testicular receptor 4 (TR4; or Nr2c2) in fibroblast activation and rheumatologic diseases has not yet been investigated. METHODS:TR4 expression in human skin and experimental fibrosis were determined by immunofluorescence staining and Western blot. RNA sequencing (RNAseq) was performed in transforming growth factor β (TGFβ)-stimulated fibroblasts with or without TR4 knockdown by small interfering RNA. Myofibroblast differentiation was assessed by α-smooth muscle actin and extracellular matrix staining, and Rho-associated protein kinase (ROCK) activity was assessed by enzyme-linked immunosorbent assay. Fibroblast-specific Tr4 knockout mice were subjected to constitutively active TGFβ receptor type I-, bleomycin-, and chronic graft-versus-host disease-induced dermal fibrosis and bleomycin-induced pulmonary fibrosis. Precision-cut skin slices (PCSSs) from patients with systemic sclerosis (SSc) were used for TR4 knockdown studies. RESULTS:TR4 was up-regulated in fibroblasts in the skin of patients with SSc and in murine models of SSc. TGFβ induced TR4 in fibroblasts in an SMAD3-dependent manner. Knockdown of TR4 prevented fibroblast-to-myofibroblast transition, and fibroblast-specific knockout of Tr4 ameliorated experimental murine skin and pulmonary fibrosis. RNAseq and functional experiments identified the profibrotic effects of TR4 were dependent on G protein subunit alpha 12- and ROCK-associated cytoskeletal remodeling. PCSSs confirmed that TR4 regulates ROCK signaling and the expression of profibrotic genes in SSc skin. CONCLUSION:TR4 is up-regulated in SSc in a TGFβ-dependent manner and drives fibroblast activation. Inhibition of TR4 prevents ROCK activation and fibroblast-to-myofibroblast transition and ameliorates dermal and pulmonary fibrosis, highlighting TR4 as a potential therapeutic target in SSc and related diseases.

Key Points1.The porcupine inhibitor Wnt-C59 ameliorated clinical signs of sclGvHD, reduced inflammation and fibrotic tissue remodeling without significant toxicity both as monotherapy and in combination with ruxolitinib, belumosudil or ibrutinib in experimental sclGvHD.2. Porcupine inhibition showed synergistic effects with belumosudil in preventing fibrotic remodeling of the skin in sclGvHD.

Transforming growth factor β (TGF-β) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-β remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-β in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-β. The activation of latent TGF-β requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-β, rebalanced TGF-β signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-β in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Background Activation of melanocortin 1 receptor (MC1R) is known to exert broad anti-inflammatory and anti-fibrotic effects. The purpose of this study is to investigate the potential of dersimelagon, a novel oral MC1R agonist, as a therapeutic agent for systemic sclerosis (SSc). Methods The effects of dersimelagon phosphoric acid (MT-7117) on skin fibrosis and lung inflammation were evaluated in bleomycin (BLM)-induced SSc murine models that were optimized for prophylactic and therapeutic evaluation. Microarray-based gene expression analysis and serum protein profiling were performed in the BLM-induced SSc models. The effect of MT-7117 on transforming growth factor-β (TGF-β)-induced activation of human dermal fibroblasts was evaluated in vitro. Immunohistochemical analyses of MC1R expression in the skin of SSc patients were performed. Results Prophylactic treatment with MT-7117 (≥ 0.3 mg/kg/day p.o.) significantly inhibited skin fibrosis and lung inflammation, and therapeutic treatment with MT-7117 (≥ 3 mg/kg/day p.o.) significantly suppressed the development of skin fibrosis in the BLM-induced SSc models. Gene array analysis demonstrated that MT-7117 exerts an anti-inflammatory effect via suppression of the activation of inflammatory cells and inflammation-related signals; additionally, vascular dysfunction was extracted as the pathology targeted by MT-7117. Serum protein profiling revealed that multiple SSc-related biomarkers including P-selectin, osteoprotegerin, cystatin C, growth and differentiation factor-15, and S100A9 were suppressed by MT-7117. MT-7117 inhibited the activation of human dermal fibroblasts by suppressing TGF-β-induced ACTA2 (encoding α-smooth muscle actin) mRNA elevation. MC1R was expressed by monocytes/macrophages, neutrophils, blood vessels (endothelial cells), fibroblasts, and epidermis (keratinocytes) in the skin of SSc patients, suggesting that these MC1R-positive cells could be targets for MT-7117. Conclusions MT-7117 demonstrates disease-modifying effects in preclinical models of SSc. Investigations of its mechanism of action and target expression analyses indicate that MT-7117 exerts its positive effect by affecting inflammation, vascular dysfunction, and fibrosis, which are all key pathologies of SSc. The results of the present study suggest that MT-7117 is a potential therapeutic agent for SSc. A phase 2 clinical trial investigating the efficacy and tolerability of MT-7117 in patients with early, progressive diffuse cutaneous SSc is currently in progress.

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.

OBJECTIVES Interstitial lung disease (ILD) is a key driver of mortality in patients with systemic sclerosis (SSc). A lack of approved treatments encompasses a high unmet medical need. Nintedanib has recently been approved for treatment in SSc-associated ILD (SSc-ILD) following SENSCIS®, a Phase III clinical trial showing that nintedanib slows the loss of pulmonary function in patients with SSc-ILD relative to placebo, as measured by annual rate of decline in forced vital capacity over 52 weeks. The aim of this study was to compare the activity of nintedanib and mycophenolate mofetil (MMF) in a transgenic Fra2 mouse model of SSc-ILD. METHODS Fra2 transgenic mice were treated with MMF or nintedanib. Haematoxylin and Eosin and Sirius Red staining were used to identify pulmonary fibrosis and vascular remodelling in whole lung sections. Fibrosis was quantified by Ashcroft scoring, fold change in fibrotic area, and hydroxyproline. Ki67, SM22a, CD31, and caspase-3 staining was used to quantify proliferating vascular smooth muscle cells and apoptotic endothelial cells. RESULTS Nintedanib effectively ameliorated pulmonary vascular remodelling and fibrosis in Fra2 transgenic mice. Pulmonary fibrotic and vascular remodelling parameter scores and the apoptosis of dermal endothelial cells were significantly reduced compared with vehicle-treated Fra2 transgenic mice. Treatment with MMF had only mild antifibrotic effects and no effect on pulmonary vascular remodelling. CONCLUSIONS In this model of SSc-ILD, nintedanib ameliorated pulmonary fibrosis, remodelling of pulmonary vasculature, and the apoptosis of endothelial cells. In contrast, MMF had minor effects on pulmonary fibrosis and no effects on vascular manifestations.

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-beta 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-beta-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.

Objective Systemic sclerosis ( SS c) is characterized by fibrosis, vascular disease, and inflammation. Adenosine signaling plays a central role in fibroblast activation. We undertook this study to evaluate the therapeutic effects of adenosine depletion with PEG ylated adenosine deaminase ( PEG ‐ ADA ) in preclinical models of SS c. Methods The effects of PEG ‐ ADA on inflammation, vascular remodeling, and tissue fibrosis were analyzed in Fra‐2 mice and in a B10.D2→ BALB /c (H‐2 d ) model of sclerodermatous chronic graft‐versus‐host disease ( GVHD ). The effects of PEG ‐ ADA were confirmed in vitro in a human full‐thickness skin model. Results PEG ‐ ADA effectively inhibited myofibroblast differentiation and reduced pulmonary fibrosis by 34.3% (with decreased collagen expression) ( P = 0.0079; n = 6), dermal fibrosis by 51.8% ( P = 0.0006; n = 6), and intestinal fibrosis by 17.7% ( P = 0.0228; n = 6) in Fra‐2 mice. Antifibrotic effects of PEG ‐ ADA were also demonstrated in sclerodermatous chronic GVHD (reduced by 38.4%) ( P = 0.0063; n = 8), and in a human full‐thickness skin model. PEG ‐ ADA treatment decreased inflammation and corrected the M2/Th2/group 2 innate lymphoid cell 2 bias. Moreover, PEG ‐ ADA inhibited proliferation of pulmonary vascular smooth muscle cells (reduced by 40.5%) ( P < 0.0001; n = 6), and prevented thickening of the vessel walls (reduced by 39.6%) ( P = 0.0028; n = 6) and occlusions of pulmonary arteries (reduced by 63.9%) ( P = 0.0147; n = 6). Treatment with PEG ‐ ADA inhibited apoptosis of microvascular endothelial cells (reduced by 65.4%) ( P = 0.0001; n = 6) and blunted the capillary rarefication (reduced by 32.5%) ( P = 0.0199; n = 6). RNA sequencing demonstrated that treatment with PEG ‐ ADA normalized multiple pathways related to fibrosis, vasculopathy, and inflammation in Fra‐2 mice. Conclusion Treatment with PEG ‐ ADA ameliorates the 3 cardinal features of SS c in pharmacologically relevant and well‐tolerated doses. These findings may have direct translational implications, as PEG ‐ ADA has already been approved by the Food and Drug Administration for the treatment of patients with ADA ‐deficient severe combined immunodeficiency disease.

Systemic sclerosis is a fibrosing chronic connective tissue disease of unknown etiology. A major hallmark of systemic sclerosis is the uncontrolled and persistent activation of fibroblasts, which release excessive amounts of extracellular matrix, lead to organ dysfunction, and cause high mobility and motility of patients. Systemic sclerosis-associated interstitial lung disease is one of the most common fibrotic organ manifestations in systemic sclerosis and a major cause of death. Treatment options for systemic sclerosis-associated interstitial lung disease and other fibrotic manifestations, however, remain very limited. Thus, there is a huge medical need for effective therapies that target tissue fibrosis, vascular alterations, inflammation, and autoimmune disease in systemic sclerosis-associated interstitial lung disease. In this review, we discuss data suggesting therapeutic ways to target different genes in distinct tissues/organs that contribute to the development of SSc.

Background: Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is the best studied member of the family of coactivators. PGC-1α was initially identified through its interaction with PPARγ in brown adipose tissue. Recent evidence further indicates that PGC-1α may also modulate the transcription of autophagy-related genes, which has recently been shown to be required for fibroblast-to-myofibroblast differentiation under fibrotic conditions. However, the role of PGC-1α in the pathogenesis of SSc has not been investigated. Objectives: The aim of the present study was to evaluate the role of the coactivator PGC-1α on autophagy and to evaluate its role in the pathologic activation of fibroblasts in SSc. Methods: Expression of PGC-1α was analyzed by RT-PCR, Western blot and immunofluorescence. Modulation of autophagy was analyzed by reporter studies by expression of autophagy related genes. The effects of PGC-1α knockdown on collagen production and myofibroblast differentiation were analyzed in cultured human fibroblasts and in two mouse models with fibroblast-specific knockout of PGC-1α. Results: PGC-1α overexpression was detected by immunohistochemistry in skin sections of SSc patients and in experimental fibrotic murine skin, particularly in fibroblasts. Knockdown of PGC-1α inhibited the stimulatory effects of TGFβ on fibroblast activation with impaired induction of collagen as compared to control fibroblasts. Fibroblasts specific knockout of PGC-1α ameliorates experimental fibrosis in bleomycin-induced and adTBR-induced murine dermal fibrosis with decreased dermal thickness, hydroxyproline and myofibroblast counts compared to wild-type fibrotic mice. Incubation of dermal fibroblasts with TGFβ activated autophagy in control fibroblasts with increased expression of the autophagy-related genes ATG7 and BECLIN-1, enhanced conversion of LC3 I to LC3 II and decreased ratios of ILC3 I EGFP to LC3 II RFP in LC3 reporter assays. The expression levels of ATG7, BECLIN-1 and ILC3 II of TGFβ-stimulated PGC-1α knockout fibroblasts decreased compare to TGFβ stimulated wild-type fibroblasts. The ratio of ILC3 I EGFP to LC3 II RFP of TGFβ-stimulated PGC-1α knockout fibroblasts in reporter assays were comparable to unstimulated fibroblasts. Conclusion: PGC-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. References: [1]Finck BN, Kelly DP. PGC-1 coactivators: inducible regulators of energy metabolism in health and disease. The Journal of clinical investigation. 2006 Mar; 116(3):615-622 [2]Lindholm D, Eriksson O, Makela J, Belluardo N, Korhonen L. PGC-1alpha: a master gene that is hard to master. Cellular and molecular life sciences: CMLS. 2012 Aug; 69(15):2465-2468. [3]Li SY, Susztak K. The Role of Peroxisome Proliferator-Activated Receptor gamma Coactivator 1alpha (PGC-1alpha) in Kidney Disease. Semin Nephrol. 2018 Mar; 38(2):121-126. [4]Vainshtein A, Tryon LD, Pauly M, Hood DA. Role of PGC-1alpha during acute exercise-induced autophagy and mitophagy in skeletal muscle. American journal of physiology Cell physiology. 2015 May 1; 308(9):C710-719. [5]Zehender A LN, Stefanica A, Chen CW, Soare A, Wohlfahrt T, Rauber S, Bergmann C, Ramming A, Distler O, Schett G, Distler J. TGFβ Promotes Fibrosis By MYST1-Dependent Epigenetic Regulation of Autophagy [abstract]. Arthritis Rheumatol 2017; 69 (suppl 10). Disclosure of Interests: Yun Zhang: None declared, Katja Dreißigacker: None declared, Diana Distler: None declared, Andrea-Hermina Györfi: None declared, Christina Bergmann: None declared, xiang zhou: None declared, Lichong Shen: None declared, Ingo Ludolph: None declared, Raymund Horch: None declared, Andreas Ramming Grant/research support from: Pfizer, Novartis, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Speakers bureau: Boehringer Ingelheim, Roche, Janssen, 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

Objectives Coactivators 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). Methods Expression 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. Results The 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. Conclusion PGC-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: 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 vitro and in 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 DNMTs in 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 TBRI act -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 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

Objectives The 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. Methods The 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. Results The 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. Conclusion PARP-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.

Objectives Systemic 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. Methods JMJD3 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. Results The expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGF beta)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGF beta. 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. Conclusion We 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.