Thomas Wohlfahrt

Resolution of inflammation by interleukin-9-producing type 2 innate lymphoid cells

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

Type 2 innate lymphoid cell counts are increased in patients with systemic sclerosis and correlate with the extent of fibrosis

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

Composition of TWIST1 dimers regulates fibroblast activation and tissue fibrosis

Objectives TWIST1 is a member of the class B of basic helix-loop-helix transcription factors that regulates cell lineage determination and differentiation and has been implicated in epithelial-to-mesenchymal transition. Here, we aimed to investigate the role of TWIST1 for the activation of resident fibroblasts in systemic sclerosis (SSc). Methods The expression of Twist1 in fibroblasts was modulated by forced overexpression or siRNA-mediated knockdown. Interaction of Twist1, E12 and inhibitor Of differentiation (Id) was analysed by co-immunoprecipitation. The role of Twist1 in vivo was evaluated using inducible, conditional knockout mice with either ubiquitous or fibroblast-specific depletion of Twist1. Mice were either challenged with bleomycin or overexpressing a constitutively active transforming growth factor (TGF)β receptor I. Result The expression of TWIST1 was increased in fibroblasts in fibrotic human and murine skin in a TGFβ/SMAD3-dependent manner. TWIST1 in turn enhanced TGFβ-induced fibroblast activation in a p38-dependent manner. The stimulatory effects of TWIST1 on resident fibroblasts were mediated by TWIST1 homodimers. TGFβ promotes the formation of TWIST1 homodimers by upregulation of TWIST1 and by induction of inhibitor of DNA-binding proteins, which have high affinity for E12/E47 and compete against TWIST1 for E12/E47 binding. Mice with selective depletion of Twist1 in fibroblasts are protected from experimental skin fibrosis in different murine models to a comparable degree as mice with ubiquitous depletion of Twist1. Conclusions Our data identify TWIST1 as a central pro-fibrotic factor in SSc, which facilitates fibroblast activation by amplifying TGFβ signalling. Targeting of TWIST1 may thus be a novel approach to normalise aberrant TGFβ signalling in SSc.

The transcription factor GLI2 as a downstream mediator of transforming growth factor-β-induced fibroblast activation in SSc

Objectives Hedgehog 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. Methods The 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. Results TGF-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. Conclusions Our 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.

The histone demethylase Jumonji domain-containing protein 3 (JMJD3) regulates fibroblast activation in systemic sclerosis

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 (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. 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.

OP0051 Dipeptidyl-Peptidase-4 (DPP4) Characterizes A Subpopulation of Fibrosis-Promoting Fibroblasts and Is A Molecular Target for The Treatment of Fibrosis

Background Dipeptidyl-peptidase-4 (DPP4/CD26) has been recently identified as a marker for a special fibroblast lineage responsible for the tissue remodeling during physiological wound healing. Fibrotic disease may be considered as a consequence of persistent, exaggerated and uncontrolled tissue repair processes. Systemic sclerosis (SSc) is associated with the highest mortality among the connective tissue disorders and effective antifibrotic therapies are still lacking. DPP4 inhibitors are already used in treatment of diabetes. Objectives The aim of the study was to characterize the DPP4 positive cells, investigate the expression of DPP4 in SSc skin and to evaluate the antifibrotic effect of DPP4 inhibitors in preclinical models of systemic sclerosis. Methods Mouse fibroblasts were isolated and DPP4 positive cells characterized by fluorescence activating cell sorting. Expression of DPP4/CD26 in human and murine skin was analyzed by immunofluorescence. DPP4 inhibitors were tested in two different concentrations administered orally (Sitagliptin 3mg/kg/d and 10mg/kg/d, Vildagliptin 1,5mg/kg/d and 15mg/kg/d) in bleomycin induced skin fibrosis and in sclerodermatous chronic graft-versus-host disease mouse model (cGvHD). The antifibrotic effect on skin was assessed by hydroxyproline assay, alpha smooth muscle cells quantification and measuring the dermal thickness. Inflammatory infiltrate was assessed by CD45 immunofluorescence staining. Results We have demonstrated that DPP4/CD26 positive cells are a unique population of cells implicated in fibrosis. DPP4/CD26 positive cells were increased not only in experimental fibrosis, but also in skin biopsies from SSc patients as compared to healthy volunteers. Treatment with DPP4 inhibitor reduced dermal thickness in both mouse models (p<0.05). The differentiation of resting fibroblasts into myofibroblasts was also significantly decreased (p<0.05) in all treatment groups. Hydroxyproline content of the skin diminished by 40% in comparison with NaCl injected mice or syngeneic transplanted mice. Moreover, DDP4 inhibitors reduced the inflammatory infiltrate in a dose dependent manner in the bleomycin injected skin. Conclusions DPP4/CD26 identifies a subpopulation of fibrosis-promoting fibroblasts that plays a key role in the pathogenesis of fibrosis in SSc. Moreover, inhibitors of DPP4 show a significant antifibrotic effect in several mouse models of SSc in well tolerated doses. These results may have direct clinical implications as DPP4 inhibitors are already in clinical use for diabetes. Acknowledgement AS received an EULAR Scientific Training Bursary (2014) and was an Articulum Fellow (2015). Disclosure of Interest None declared

FRI0410 TgfΒ promotes fibrosis by myst1-dependent epigenetic regulation of autophagy

Background Autophagy is catabolic process allowing cells to degrade unnecessary or dysfunctional cellular organelles. Aberrant activation of autophagy has been also implicated into the pathogenesis of fibrotic diseases. Several stimuli present in fibrosis such as pro-fibrotic cytokines are known to activate autophagy. Objectives The objective of this work is characterise the regulation of autophagy by TGFβ and analyse whether targeting of autophagy in fibroblasts may prevent their aberrant activation in fibrotic diseases. Methods To selectively disable autophagy in fibroblasts we generate Atg7fl/flx Col1a2;CreER mice. The role of the autophagy was investigated in the model of bleomycin- and TβRIact-induced dermal and pulmonary fibrosis. Overexpression of Myst1 was achieved by adenovirus encoding for Myst1. Collagen release and protein expression were measure by Western blot. Target genes were analysed by RT-PCR. Co-immunoprecipitation and reporter assay were performed to study physical and functional interactions between MYST1 and SMAD3. To monitor the autophagic flux in vitro and in vivo we generate an adenovirus encoding for tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3), defined as reliable autophagy maker. Results We provide evidence that transforming growth factor-β (TGFβ) activates autophagy by an epigenetic mechanism to amplify its profibrotic effects. TGFβ induces autophagy in fibrotic diseases by SMAD3-dependent downregulation of the H4K16-histoneacetlytransferase MYST1, which controls the expression of core components of the autophagy machinery such as ATG7 and BECLIN1. Activation of autophagy in fibroblasts promotes collagen release and is both, sufficient and required, to induce tissue fibrosis. Forced expression of MYST1 abrogates the stimulatory effects of TGFβ on autophagy and re-establishes the epigenetic control of autophagy in fibrotic conditions. Interference with the aberrant activation of autophagy inhibits TGFβ-induced fibroblast activation and ameliorates experimental dermal and pulmonary fibrosis. These findings link uncontrolled TGFβ signalling to aberrant autophagy and altered epigenetics in fibrotic diseases and may open new avenues for therapeutic intervention in fibrotic diseases. Conclusions We demonstrate that the epigenetic control of autophagy is disturbed by a TGFβ-dependent downregulation of MYST1. The increased activation of autophagy induces fibroblast-to-myofibroblast transition and promotes fibrotic tissue remodelling. Re-expression of MYST1 prevents aberrant autophagy, limits the profibrotic effects of TGFβ and ameliorates experimental fibrosis. Restoration of the epigenetic control of autophagy might thus be a novel approach to ameliorate fibrotic tissue remodelling. Disclosure of Interest A. Zehender: None declared, N.-Y. Lin: None declared, A. Stefanica: None declared, C.-W. Chen: None declared, A. Soare: None declared, T. Wohlfahrt: None declared, S. Rauber: None declared, C. Bergmann: None declared, A. Ramming: 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, 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, Ga-lapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB

SAT0021 Type 2 Innate Lymphoid Cells Are Rapidly and Persistently Recruited in Experimental Fibrosis and Their Counts Correlate with The Severity of Skin and Lung Fibrosis in Systemic Sclerosis

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

SAT0186 The Homeoprotein Engrailed-1 Regulates Canonical TGF-β Signaling via Fibroblast Differentiation and Tissue Fibrosis

Background Systemic sclerosis (SSc) is a chronic fibrotic connective disease of unknown etiology that affects the skin and internal organs. Transforming growth factor-β (TGFβ) has been characterized as a key-mediator of fibroblast activation in SSc. However, the intracellular signaling cascades that control TGFβ signaling and the TGFβ-induced activation of fibroblasts are still incompletely understood. Homeobox containing transcription factor, Engrailed-1 plays a key role in embryonic development and has also been linked to disease, including cancer. EN-1 is induced by proinflammary cytokines and oxidative stress which play an important role in Systemic Sclerosis (SSc). Objectives The aim of this study was to determine the role of the embryonic homeoprotein EN-1 in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc). Methods The expression of EN-1 in skin tissue and in human dermal fibroblasts was determined by real-time PCR, Western blot and immunofluorescence. Knock-down and overexpression strategies were used to evaluate the effect of EN-1 on fibroblast activation. The outcome of mice with fibroblast-specific knockout of EN-1 (EN1 fibKO) was evaluated in bleomycin-induced skin fibrosis; fibrosis induced by overexpression of a constitutively active TGF-β receptor I (TBRIact) and in the Tsk model which resembled the later stages of SSc. Co-IP and CAGA Reporter assay were performed to study the physical and functional interaction between EN-1 and Smad3. Results A five-fold increased expression of EN-1 was detected in the upper layer of the dermis of SSc patients on fibroblasts double stained for EN-1 and anti-prolyl-4-hydroxylase. EN-1 expression was induced by TGF-β in cultured fibroblasts and treatment with the TBR inhibitor SD-208 prevented the induction of EN-1 by two-fold decrease in experimental fibrosis. Fibroblasts lacking EN-1 were less sensitive to the pro-fibrotic effects of TGF-β with impaired induction of target genes mRNA and protein. Additionally, overexpression of EN-1 enhanced the profibrotic effect of TGF-β with myofibroblast differentiation and increased collagene release as well as mRNA and protein levels of target genes. Function studies demonstrated that EN-1 interacts with Smad3 to regulate the pro-fibrotic effects of TGF-β. Co-IP demonstrated that TGF-β induces binding of EN-1 to Smad3. Reporter study and analyses of the expression of classical Smad target genes such as PAI-1 demonstrated that the binding of EN-1 to Smad3 stimulates the transcriptional activity of Smad3. In the model of bleomycin-induced fibrosis dermal thickening, hydroxyproline content and myofibroblast counts were significantly decreased in EN1 fibKO mice as compared to wildtype littermates. EN1 fibKO also protected from TBRIact-induced fibrosis and ameliorated fibrosis in the Tsk1 model. Conclusions We demonstrate for the first time a role of EN-1 in fibroblast activation and tissue fibrosis. Deficiency in EN-1 reduced the stimulatory effect of TGF-β on fibroblasts by interfering with canonical Smad and protected from experimental fibrosis in different mouse models. Considering the potent anti-fibrotic effects observed in this study, EN-1 might be a candidate for molecular targeted therapies of SSc. Disclosure of Interest None declared