Search

Objective Systemic sclerosis (SSc) is characterized by dysregulation of type I interferon (IFN) signaling. CD52 is known for its immunosuppressive functions in T cells. This study was undertaken to investigate the role of CD52 in monocyte adhesion and type I IFN signaling in patients with SSc. Methods Transcriptome profiles of circulating CD14+ monocytes from patients with limited cutaneous SSc (lcSSc), patients with diffuse cutaneous SSc (dcSSs), and healthy controls were analyzed by RNA sequencing. Levels of CD52, CD11b/integrin αΜ, and CD18/integrin β2 in whole blood were assessed by flow cytometry. CD52 expression was analyzed in relation to disease phenotype (early, lcSSc, dcSSc) and autoantibody profiles. The impact of overexpression, knockdown, and antibody blocking of CD52 was analyzed by gene and protein expression assays and functional assays. Results Pathway enrichment analysis indicated an increase in adhesion- and type I IFN-related genes in monocytes from SSc patients. These cells displayed up-regulated expression of CD11b/CD18, reduced expression of CD52, and enhanced adhesion to intercellular adhesion molecule 1 and endothelial cells. Changes in CD52 expression were consistent with the SSc subtypes, as well as with immunosuppressive treatments, autoantibody profiles, and monocyte adhesion properties in patients with SSc. Overexpression of CD52 led to decreased levels of CD18 and monocyte adhesion, while knockdown of CD52 increased monocyte adhesion. Experiments with the humanized anti-CD52 monoclonal antibody alemtuzumab in blood samples from healthy controls increased monocyte adhesion and CD11b/CD18 expression, and enhanced type I IFN responses. Monocytic CD52 expression was up-regulated by interleukin-4 (IL-4)/IL-13 via the STAT6 pathway, and was down-regulated by lipopolysaccharide and IFNs α, β, and γ in a JAK1 and histone deacetylase IIa (HDAC IIa)-dependent manner. Conclusion Down-regulation of the antiadhesion CD52 antigen in CD14+ monocytes represents a novel mechanism in the pathogenesis of SSc. Targeting of the IFN-HDAC-CD52 axis in monocytes might represent a new therapeutic option for patients with early SSc.

TGF-beta is a master regulator of fibrosis, driving the differentiation of fibroblasts into apoptosis-resistant myofibroblasts and sustaining the production of extracellular matrix (ECM) components. Here, we identified the nuclear long noncoding RNA (lncRNA) H19X as a master regulator of TGF-beta-driven tissue fibrosis. H19X was consistently upregulated in a wide variety of human fibrotic tissues and diseases and was strongly induced by TGF-beta, particularly in fibroblasts and fibroblast-related cells. Functional experiments following H19X silencing revealed that H19X was an obligatory factor for TGF-beta-induced ECM synthesis as well as differentiation and survival of ECM-producing myofibroblasts. We showed that H19X regulates DDIT4L gene expression, specifically interacting with a region upstream of the DDIT4L gene and changing the chromatin accessibility of a DDIT4L enhancer. These events resulted in transcriptional repression of DDIT4L and, in turn, in increased collagen expression and fibrosis. Our results shed light on key effectors of TGF-beta-induced ECM remodeling and fibrosis.

Background: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown. Methods and Results: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-β. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-β-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis. Conclusion: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-β-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.

Background: Cardiac fibroblasts represent a main stromal cell type in the healthy myocardium. Activation of cardiac fibroblasts has been implicated in the pathogenesis of many heart diseases. Profibrotic stimuli activate fibroblasts, which proliferate and differentiate into pathogenic myofibroblasts causing a fibrotic phenotype in the heart. Cardiac fibroblasts are characterized by production of type I collagen, but non-transgenic methods allowing their identification and isolation require further improvements. Herein, we present a new and simple flow cytometry-based method to identify and isolate cardiac fibroblasts from the murine heart. Methods and Results: Wild-type and reporter mice expressing enhanced green fluorescent protein (EGFP) under the murine alpha1(I) collagen promoter (Col1a1-EGFP) were used in this study. Hearts were harvested and dissociated into single cell suspensions using enzymatic digestion. Cardiac cells were stained with the erythrocyte marker Ter119, the pan-leukocyte marker CD45, the endothelial cell marker CD31 and gp38 (known also as podoplanin). Fibroblasts were defined in a two-color flow cytometry analysis as a lineage-negative (Lin: Ter119−CD45−CD31−) and gp38-positive (gp38+) population. Analysis of hearts isolated from Col1a1-EGFP reporter mice showed that cardiac Lin−gp38+ cells corresponded to type I collagen-producing cells. Lin−gp38+ cells were partially positive for the mesenchymal markers CD44, CD140a, Sca-1 and CD90.2. Sorted Lin−gp38+ cells were successfully expanded in vitro for up to four passages. Lin−gp38+ cells activated by Transforming Growth Factor Beta 1 (TGF-β1) upregulated myofibroblast-specific genes and proteins, developed stress fibers positive for alpha smooth muscle actin (αSMA) and showed increased contractility in the collagen gel contraction assay. Conclusions: Two-color flow cytometry analysis using the selected cell surface antigens allows for the identification of collagen-producing fibroblasts in unaffected mouse hearts without using specific reporter constructs. This strategy opens new perspectives to study the physiology and pathophysiology of cardiac fibroblasts in mouse models.

Background Cardiac dysfunction is a significant cause of mortality in SSc due to inflammation, vasculopathy and fibrosis. In fibrotic conditions, the excessive number of fibroblasts, myofibroblasts and extracellular matrix in the myocardium may directly or indirectly affect the cardiac conduction system by different mechanisms. Myofibroblasts are the main players in cardiac fibrogenesis, but their origin in SSc remains unknown. Objectives To unravel the role of Fos-related antigen 2 (Fra2) in stromal cell activation, cardiac fibrosis and alteration of the conduction system. Methods Fra2 transgenic (tg) mice and Rag2-/- Fra2 tg mice were studied. Hearts from Fra2 tg and WT mice were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). Cardiac function was assessed by echocardiography, electrocardiogram (ECG) and telemetry. Myocardial stromal (Ter119-CD45-CD31-, hereafter Lin-) gp38+ stromal cells were sorted and cultured in vitro. The cellular phenotype was evaluated by qPCR, α-smooth muscle actin (αSMA) and stress fibre staining, secreted collagens and contraction assay. Results Echocardiography of 20-week-old Fra2 tg mice displayed increased ejection fraction (p=0.02) and fractional shortening (p=0.02) with decreased left ventricle end-diameter and end-volume in systole (p=0.006, p=0.008) and in diastole (p=0.007, p=0.008). ECG in conscious mice revealed lower heart rate (HR) (WT 741 ± 33 BPM, tg 644 ± 37 BPM, p Conclusion Fra2 overexpression and inflammation foster stromal cell-to-myofibroblast differentiation, leading to cardiac fibrosis and defects of the conduction system. Targeting this process might be a therapeutic strategy for SSc patients with disorders of cardiac involvement. Disclosure of Interests Mara Stellato: None declared, Michal Rudnik: None declared, Florian Renoux: None declared, Przemyslaw Blyszczuk: None declared, Elena Osto: None declared, Matthias Dewenter: None declared, Petra Seebeck: None declared, Elena Pachera: 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, Gabriela Kania: None declared

Background During the course of systemic sclerosis (SSc), infiltration of inflammatory cells, including monocytes, into the organs is a major process leading to fatal fibrosis, remodelling and organ dysfunction. CD52 protein is highly expressed on CD4+ T-cells and plays an important role in the modulation of T-cell receptor signalling. Nevertheless, the function of this protein on monocytes is not completely understood. Objectives We aimed to functionally investigate the role of circulating CD14+ monocytes in the course of SSc with a special focus on monocyte adhesion and the influence of CD52 expression. Methods Biopsies from the heart, lungs and skin of SSc patients (n=11, 7, 7 respectively) and healthy controls (HC) (n=10, 7, 9 respectively) were analysed by immunohistochemistry for the presence of CD14+ cells. PolyA RNA sequencing of CD14+ monocytes isolated from peripheral blood of lcSSc (n=5, age=54.4±6.7), dcSSc patients (n=5, age=51.8±7.2) and age- and sex-matched HC (n=5, age=50.8±9.7) was performed using Illumina HiSeq 4000 platform. Differentially expressed genes were computed using DeSEQ2 algorithm. Gene ontology and pathway analysis were performed using Metacore software and ShinyApp. Expression of adhesion molecules was confirmed on the protein level using flow cytometry (HC n=8, SSc n =22). Adhesion of CD14+ monocytes to immobilized ICAM1 and TNFα-stimulated endothelial cells was checked using the 96-well plate adhesion assay (HC n=8, SSc n=22). CD52 regulation in CD14+ monocytes from HC (n=4) was analysed on mRNA level upon stimulation with different concentrations of LPS, IFNγ, IL-4 and IL-13. THP-1 cell lines with overexpression and silencing of CD52 were generated using lentiviral vectors, and further, their adhesion was investigated (n=4). Results Immunochemistry confirmed higher infiltration of CD14+ cells in the heart (p Conclusion This is the first report pointing to an increased adhesion of peripheral blood CD14+ monocytes to ICAM1 and endothelial cells in SSc. Our results suggest the primary activation of monocytes in peripheral blood, which translates into higher organ infiltration in SSc patients. Finally yet importantly, we characterised a novel function of CD52 molecule on monocytes and its possible contribution during the course of the disease. Disclosure of Interests Michal Rudnik: None declared, Mara Stellato: None declared, Przemyslaw Blyszczuk: None declared, Karin Klingel: None declared, Jorg Henes: None declared, Carol Feghali-Bostwick: 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, Gabriela Kania: None declared

Background Fos like 2 (Fosl-2) is a transcription factor belonging to the AP-1 transcription complex. We have recently described a Fosl-2 transgenic (tg) mouse model which develops a multi-organ inflammatory and autoimmune phenotype. In these mice, we have characterized a decrease in regulatory T cells (Tregs), which preceded the activation of T cells and the development of inflammation. Objectives To analyze how Fosl-2 reduces the Treg population and triggers autoimmunity in Fosl-2 tg mice. Methods We used previously generated Fosl-2 tg overexpressing and Fosl-2 T cell specific knock out (Fosl-2 ko) mice. For mixed bone marrow transfer experiments, lethally irradiated recipients received a one to one mix of Fosl-2 tg (CD45.2) and wt (CD45.1) bone marrow to create Fosl-2 tg-wt chimera. We addressed the contribution of Tregs to the inflammatory phenotype by co-transferring Fosl-2 tg CD4 T cells and wt Tregs to Rag2-/- recipients. Treg and T cell populations were analyzed by flow cytometry and inflammation was addressed by CD45 staining on paraffin-embedded sections. RNA-sequencing was used to compare wt, Fosl-2 tg and Fosl-2 ko CD4 T cells after 24 hours of stimulation with anti-CD3 (2μg/ml) and anti-CD28 (2μg/ml). Results We first addressed whether Fosl-2 affected Tregs in a cell intrinsic way using mixed bone marrow experiments. In these animals, the CD45.2 Fosl-2 tg CD4 T cells showed a much lower proportion of Tregs compared to the CD45.1 wt population, both in the spleen (0.73%±0.15 vs 31.6%±3.6, P=0.062) and thymus (1.3%±1.04 vs 3.23%±0.78, P=0.001). This demonstrates that Fosl-2 overexpression represses Treg development in a cell intrinsic way. In T cell transfer experiments, Rag2-/- mice receiving 106 Fosl-2 tg CD4 cells developed lung inflammation 5 weeks after transfer, confirming that T cells are inducers of inflammation in Fosl-2 tg mice. Moreover, co-transfer of either 3*105 or 106 wt Treg cells resulted in a dose dependent reduction of inflammation. These data indicated that the decrease in the Treg population in Fosl-2 tg mice is responsible for the induction of inflammation. We then analysed Fosl-2 transcriptional targets in T cells by RNA-seq. Using a fold change > 1.5 and False Discovery Rate (FDR) of 0.05, we identified 191 differentially expressed genes in both Fosl-2 tg and Fosl-2 ko cells compared to wt. Interestingly, one of the top target genes of Fosl-2 was FoxP3. This unbiased approach thus revealed that FoxP3 expression is repressed by Fosl-2, with a 6.5 fold reduction in Fosl-2 tg cells and a 2.5 fold increase in Fosl-2 ko cells. This effect was confirmed on the protein level with a reduction in FoxP3 induction in Fosl-2 tg cells treated with TGFβ. The repression of Treg development observed in Fosl-2 tg mice could thus be explained by a direct transcriptional control of FoxP3 expression. Additionally, we found that Fosl-2 repressed a set of genes important for Tregs and other T helper cells. This included Nr4a2, a transcription factor involved in Treg development, IRF8 and Eomes, two genes involved in the polarization of Th1 and Th17 cells, or Ccl1 a chemokine important for Treg homeostasis. Conclusion Fosl-2 is involved in the control of FoxP3 expression in T cells. Through this, overexpression of Fosl-2 represses Treg development and induces a Treg dependent autoimmune phenotype in mice. This mechanism could thus be involved in the pathogenesis of autoimmune diseases and might represent a therapeutic target to modulate the Treg population. Disclosure of Interests Florian Renoux: None declared, Mara Stellato: None declared, Alexander Vogetseder: None declared, Riyung Huang: None declared, Arun Subramaniam: None declared, Przemyslaw Blyszczuk: None declared, Jorg Distler: None declared, Gabriela Kania: None declared, Onur Boyman: 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

Background Previous studies indicated monocyte-derived cells as important players in the development of multiple organ fibrosis. Although changes in monocyte’s phenotypes as well as an increased infiltration into fibrotic organs were reported in systemic sclerosis (SSc), the detailed role of these cells in multi-organ fibrogenesis remains unclear. Objectives We aimed to characterise a contribution of circulating CD14+ monocytes in the disease course in limited cutaneous (lc) and diffuse cutaneous (dc) SSc. Methods CD14+ monocytes were isolated from peripheral blood of lcSSc (n=5, age=54.4±6.7), dcSSc patients (n=5, age=51.8±7.2) and age- and sex-matched healthy controls (HC) (n=5, age=50.8±9.7). Total RNA was isolated and polyA libraries were prepared using TruSeq Stranded mRNA kit. Next Generation Sequencing was performed using Illumina HiSeq 4000 platform. Differentially expressed genes were computed using DeSEQ2 algorithm. Principal Component Analysis (PCA) was accomplished as well as pathway analysis using Metacore software. mRNA levels of top targets were confirmed by qPCR. Results We detected 1440 differentially expressed genes between dcSSc vs HC and 225 between lcSSc and HC respectively (p≤0.01; log2 ratio ≥0.5, figure 1). Among those, in dcSSc 1076 were upregulated (e.g. MMP9, IL1R2, FLT3, MIF, TLR9) and 364 were downregulated (e.g. TGFBR1, CD44, CD244, HLA-DRA, HLA-G). In lcSSc 160 transcripts were upregulated (e.g. CCL2, WNT5B, MMP17) and 65 were downregulated (e.g. KLF11, IRAK2). We identified 123 commonly deregulated genes between SSc subgroups (e.g. CCL3, CD14, IL27, MMP17). Principal component analysis showed close clustering within SSc subgroups and clear separation from healthy controls. Pathway analysis revealed alterations in several biological processes important in fibrogenesis including antigen presentation, MIF-induced immune responses, TGF-β, NOTCH and WNT signalling pathways. qPCR analysis further confirmed differences in gene expression on mRNA level (n HC=8, n SSc=25, p≤0.05). Conclusions To our knowledge, this is the first global transcriptome analysis of peripheral blood CD14+ monocytes in SSc. Our results suggest an initial activation of monocytes in peripheral blood, which might be further translated into novel cellular biomarker of the disease and potentially used for distinguishing between responders and non-responders to a novel treatment in future clinical trials. Disclosure of Interest M. Rudnik: None declared, M. Stellato: None declared, P. Blyszczuk: None declared, O. Distler Grant/research support from: Actelion, Bayer, Boehringer Ingelheim, Mitsubishi Tanabe Pharma and Roche, Consultant for: Actelion, Bayer, BiogenIdec, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Sinoxa and UCB, G. Kania Grant/research support from: Bayer Pharma AG, Actelion

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

Background Animal studies indicated bone marrow-derived cells as a source of pathological myofibroblasts in multiple organ fibrosis such as lungs, heart, skin and kidney. Monocytes are implicated in the pathogenesis of systemic sclerosis (SSc), however detailed role of specific monocyte subsets in multi-organ fibrogenesis in SSc remains unclear. Objectives We aimed to determine the role and the contribution of circulating monocytes in the onset and progression of multi-organ fibrosis in SSc. Methods Endomyocardial biopsies (n=10) from SSc patients and healthy controls were screened by immunohistochemistry. CD14+ monocytes isolated from peripheral blood of SSc patients and healthy donors were differentiated towards a myofibroblast phenotype by stimulation with TGF-β1, IL-4, IL-10 and IL-13. In addition, CD14+ monocytes were co-cultured in 2D and 3D models with dermal fibroblasts originating from SSc patients or healthy subjects, or with adult cardiac fibroblasts. TGF-β signalling was blocked by SD208 and A83–01 inhibitors. Profibrotic gene expression and protein secretion were evaluated by qPCR, Western blot, protein array, immunofluorescence and ELISA. Results The myocardia of SSc patients revealed the presence of CD45-expressing infiltrates, an extended collagen I deposition and the presence of CD14-expressing elongated cells in the fibrotic tissue. Stimulated monocytes acquired a myofibroblast-like phenotype with increased expression of collagen I (p Conclusions Here we demonstrated the capability of peripheral blood monocytes to differentiate towards the functional myofibroblast phenotype, designating these cells as one of the potential sources of pathological tissue myofibroblasts in SSc. Additionally, these cells sustained pro-fibrotic cytokines secretion, highlighting their important regulatory functions in the fibrogenesis in SSc. Further studies of monocytes involvement in SSc might lead to novel treatment strategies. Disclosure of Interest M. Rudnik: None declared, M. Stellato: None declared, V. Milleret: None declared, P. Blyszczuk: None declared, B. Maurer Grant/research support from: AbbVie, Protagen, EMDO, Novartis. Congress support from Pfizer, Roche, Actelion. Patent licensed: mir-29 for the treatment of systemic sclerosis, K. Klingel: None declared, J. Henes: None declared, K. Sotlar: None declared, M. Ehrbar: None declared, O. Distler Grant/research support from: Actelion, Bayer, Boehringer Ingelheim, Pfizer, Sanofi. Patent licensed: mir-29 for the treatment of systemic sclerosis, 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, G. Kania Grant/research support from: Bayer

Background The majority of Systemic Sclerosis (SSc) patients have subclinical primary cardiac involvement, which resembles the inflammatory dilated cardiomyopathy (iDCM) with inflammation and fibrosis. Firstly, cellular progenitors of pathological myofibroblasts remain undescribed. Secondly, autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts Objectives To unravel the role of Fos-related antigen 2 (Fra2)/autophagy crosstalk in TGF-β-driven myocardial fibrosis in SSc Methods Genetically modified mice overexpressing Fra2 were used. Endomyocardial biopsies (EMBs) from SSc/iDCM patients and hearts from Fra2 tg and control mice were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). Murine myocardial gp38+ (podoplanin+) stromal cells were sorted and stimulated in vitro with TGF-β. The cellular phenotype was assessed by qPCR, IF, stress fiber staining, SIRCOL and contraction assay on sorted cells. The antisense oligonucleotide GapmeR was used to knock-down Fra2 Results The myocardium of SSc/iDCM patients (n=10) showed the typical phenotype of iDCM: increased fibrotic area with extended collagen deposition and enlarged number of pathological myofibroblasts, expressing Fra2 and, interestingly, the autophagy markers LC3B and Atg5. Similarly, Fra2 tg mice showed higher expression of profibrotic markers: αSMA, vimentin and collagen I compared to control mice (n=5), as well as the expression of the autophagy markers LC3B and Beclin in fibrotic regions. Among cardiac stromal cells (Ter119-CD45-CD31-Sca1+CD29+) the frequency of gp38+ cells was significantly higher in Fra2 myocardium compared to control mice. The majority of gp38+ cells co-expressed αSMA, vimentin and collagen I together with autophagy markers (LC3B and Beclin, n=3). Following in vitro stimulation with TGF-β, gp38+ cells entered fibroblast-to-myofibroblast transition characterized by increased mRNA and protein levels of αSMA, collagen I, fibronectin (n=3–6), αSMA-fiber and stress-fiber formation (n=3), increased cell proliferation (n=4; p=0.04) and contraction capability (n=2; p TGF-β stimulation of control gp38+ cells induced the expression of LC3B, Beclin and Atg5 at mRNA and protein level (n=3–5). In contrast, TGF-β inhibition caused the downregulation of these markers (n=3). In addition, Fra2 silencing resulted in a decreased differentiation capability of gp38+ cells: mRNA and protein levels of the profibrotic genes αSMA and collagen I were significantly downregulated (n=5; p=0.007). Moreover, Fra2 downregulation impaired the secretion of collagens (n=4; p Conclusions The TGF-β/Fra2 axis fosters an enhanced autophagy flow, leading in turn to the stromal-to-myofibroblast transition. Therefore, targeting this process might be a therapeutic strategy to abrogate fatal cardiac remodeling in SSc Disclosure of Interest M. Stellato: None declared, M. Rudnik: None declared, F. Renoux: None declared, E. Pachera: None declared, D. Kayalar: None declared, K. Sotlar: None declared, K. Klingel: None declared, J. Henes: None declared, P. Blyszczuk: 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, G. Kania Grant/research support from: Bayer AG

Aims Myocardial fibrosis critically contributes to cardiac dysfunction in inflammatory dilated cardiomyopathy (iDCM). Activation of transforming growth factor-β (TGF-β) signalling is a key-step in promoting tissue remodelling and fibrosis in iDCM. Downstream mechanisms controlling these processes, remain elusive. Methods and results Experimental autoimmune myocarditis (EAM) was induced in BALB/c mice with heart-specific antigen and adjuvant. Using heart-inflammatory precursors, as well as mouse and human cardiac fibroblasts, we demonstrated rapid secretion of Wnt proteins and activation of Wnt/β-catenin pathway in response to TGF-β signalling. Inactivation of extracellular Wnt with secreted Frizzled-related protein 2 (sFRP2) or inhibition of Wnt secretion with Wnt-C59 prevented TGF-β-mediated transformation of inflammatory precursors and cardiac fibroblasts into pathogenic myofibroblasts. Inhibition of T-cell factor (TCF)/β-catenin-mediated transcription with ICG-001 or genetic loss of β-catenin also prevented TGF-β-induced myofibroblasts formation. Furthermore, blocking of Smad-independent TGF-β-activated kinase 1 (TAK1) pathway completely abrogated TGF-β-induced Wnt secretion. Activation of Wnt pathway in the absence of TGF-β, however, failed to transform precursors into myofibroblasts. The critical role of Wnt axis for cardiac fibrosis in iDCM is also supported by elevated Wnt-1/Wnt-5a levels in human samples from hearts with myocarditis. Accordingly, and as an in vivo proof of principle, inhibition of Wnt secretion or TCF/β-catenin-mediated transcription abrogated the development of post-inflammatory fibrosis in EAM. Conclusion We identified TAK1-mediated rapid Wnt protein secretion as a novel downstream key mechanism of TGF-β-mediated myofibroblast differentiation and myocardial fibrosis progression in human and mouse myocarditis. Thus, pharmacological targeting of Wnts might represent a promising therapeutic approach against iDCM in the future., European Heart Journal, 38 (18), ISSN:1522-9645, ISSN:0195-668X

Background Decreased number or altered function of regulatory T cells (Tregs) has been reported in many inflammatory rheumatic diseases, and Tregs are considered promising therapeutic targets for autoimmune diseases. It is thus of high importance to delineate the pathways controlling Tregs biology and the onset of autoimmunity. Fra2 is a transcription factor belonging to the Fos family proteins which takes part in the AP-1 transcription complex. The role of Fra2 in Tregs is so far unknown. Objectives To characterize the potential role of Fra2 in controlling Tregs and autoimmunity in Fra2 transgenic mice. Methods Fra2 transgenic mice were generated, in which the Fra2 transcription factor is ubiquitously overexpressed under the control of an MHCI promotor. T lymphocyte populations were analyzed by flow cytometry, and pathological manifestations in multiple organs by histology. Bone marrow cells were transferred into lethally irradiated recipients to create Fra2-wt chimeric mice. Results At 3 weeks of age, Fra2 mice showed a striking decrease of the Treg population (CD4 + CD25 + FoxP3 + in wt and Fra2 littermates spleens: 11.35 and 5.75% of CD4 + , respectively, p=0.0005, Mann-Whitney test, n=6). The strong decrease in Tregs was stable over time and also observed in CD4 + single positive thymocytes, indicating that Fra2 mice have a defect in natural Treg development. Interestingly, from 7 weeks on, we could also detect the appearance of activated T cells (CD4 + and CD8 + , with CD62L high CD44 high CD127 low profile) which represented up to 60% of total T cells by 16 weeks of age. While the phenotype of young mice was limited to decreased Tregs, macroscopic and histological observations in 16 week-old mice showed the presence of an extensive multi-organ autoimmune phenotype (see figure). Perivascular inflammatory infiltrates, containing T cells, B cells and numerous granulocytes were observed in the lung and liver. Extensive inflammation and fibrosis were observed in the thymus. Spleens were also enlarged with pronounced extramedullary haematopoiesis. Fra2 mice also developed dermatitis on eyelids and back-skin, with an increase in dermal and epidermal thickness. Finally, duodenum was enlarged due to acute inflammation and reactive hyperplasia. To understand whether the effect of Fra2 is T cell intrinsic or extrinsic, we also performed bone marrow transfer experiments in which irradiated wt recipient mice received Fra2 transgenic bone marrow. Interestingly, chimera mice displayed a decreased percentage of Tregs in blood, spleen, but also thymus, confirming an intrinsic role of Fra2 in natural Treg development. Conclusions These data suggest that Fra2 overexpression inhibits natural Treg development, resulting in 1: diminished Tregs, 2: activation of effector T cells and 3: development of inflammation in multiple organs. This is the first evidence for a role of Fra2 in controlling Treg development and autoimmunity. This murine model also provides a unique opportunity to delineate the function of the Fra2 pathway in T cells and Tregs and its impact on autoimmunity. Disclosure of Interest F. Renoux: None declared, M. Stellato: None declared, E. Pachera: None declared, D. Impellizzieri: None declared, C. Dees: None declared, J. Distler: None declared, G. Kania: None declared, O. Boyman: None declared, O. Distler Grant/research support from: Bayer, Sanofi, Ergonex, Boehringer Ingelheim, Actelion, Pfizer, Consultant for: 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, EpiPharm, Ergonex, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, MedImmune, Pharmacyclics, Pfizer, Serodapharm, Sinoxa

Background Heart involvement in patients with systemic sclerosis (SSc) resembles the inflammatory dilated cardiomyopathy (iDCM) phenotype with predominance of inflammation, fibrosis, vasculopathy and heart dysfunction. Animal studies of iDCM indicated bone marrow originated cells as a major source of pathological myofibroblasts. MicroRNAs are key regulators of immune cell function and are involved in many cardiac pathological processes. However, their roles in monocyte differentiation and fibrogenesis are unclear. Objectives To determine the role of circulating monocytes and microRNAs in the onset and progression of myocardial fibrosis in SSc, we examined monocyte differentiation and their microRNAs expression profile in SSc. Methods Endomyocardial biopsies from SSc/iDCM patients and healthy controls were screened by immunohistochemistry. CD14+ monocytes isolated from peripheral blood of SSc patients and healthy donors were differentiated towards the myofibroblast phenotype by stimulation with TGF-β1, IL-4, IL-10 and IL-13. In addition, CD14+ monocytes were co-cultured with dermal fibroblasts originated from SSc patients and healthy subjects. After 7 days, myofibroblast gene expression and cytokines secretion profile were evaluated. MicroRNA candidates were selected using DIANA-TarBase v7.0 and TargetScan Release 7.0 and further analysed by qPCR. Results Myocardium of SSc/iDCM patients (n=10) revealed extensive fibrosis and accumulation of inflammatory cells including CD14+ monocytes. Moreover, fibrotic myocardium from SSc patients exhibited the presence of CD14+/Fra-2+ monocyte-derived fibroblast-like cells. Bioinformatical analysis indicated several potential microRNAs being involved in monocyte differentiation, and qPCR confirmed predicted candidates. We observed decreased basal levels of let-7i (Mann-Whitney U test, p=0.005), miR-10b (p=0.005), miR-21 (p=0.005), miR-29a (p=0.005), miR-155 (p=0.002), miR-199a (p=0.008) and miR-544a (p=0.002), and increased levels of miR-29b (p=0.03) in circulating CD14+ monocytes isolated from SSc patients (n=10) compared to healthy controls (n=4). Stimulated monocytes acquired a myofibroblast-like phenotype with increased expression of collagen I, fibronectin, α smooth muscle actin and Fra-2 in comparison to untreated cells. Similarly, CD14+ monocytes exposed to dermal fibroblasts acquired myofibroblast features. CD14+ monocytes from SSc patients were characterised by higher production of IP-10, MIP-3α, LIF and NT-3. The process of monocyte to myofibroblast differentiation employed Fra-2/TGF-β signalling. Inhibition of the canonical SMAD-dependent pathway with TGFβR1 inhibitors resulted in the abrogation of monocyte-to-myofibroblast differentiation. Conclusions Here we demonstrated the capability of peripheral blood monocytes to differentiate towards the myofibroblast phenotype, indicating these cells as one of the potential sources of pathological tissue myofibroblasts in SSc. Different miRNA expression profiles in SSc monocytes indicate a primary activation state. Further studies of miRNAs regulation pathways might lead to novel treatment strategies, particularly for heart involvement. Disclosure of Interest M. Rudnik: None declared, M. Stellato: None declared, P. Blyszczuk: None declared, E. Pachera: None declared, R. Dobrota: None declared, B. Maurer: None declared, K. Klingel: None declared, J. Henes: None declared, K. Sotlar: None declared, O. Distler Grant/research support from: Bayer, Sanofi, Ergonex, Boehringer Ingelheim, Actelion, Pfizer, Consultant for: 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, EpiPharm, Ergonex, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, MedImmune, Pharmacyclics, Pfizer, Serodapharm, Sinoxa, G. Kania: None declared

Background Cardiac dysfunction is a significant cause of the high mortality in systemic sclerosis (SSc). Heart involvement in SSc patients resembles inflammatory dilated cardiomyopathy (iDCM) with inflammation and fibrosis. Myofibroblasts are the main players in cardiac fibrogenesis, but their origin remains unknown. Objectives Here, we aim to determine the role of specific myocardial stromal cell populations in myocardial remodeling in SSc. Methods The Fos-related antigen 2 (Fra2) tg mouse model of SSc/iDCM was studied. Immunohistochemistry (IHC) and immunofluorescence (IF) were performed on endomyocardial biopsies (EMBs) from SSc/iDCM patients (n=10) and on hearts from Fra2 tg mice (n=5). Flow cytometry analysis was used to identify subsets of myocardial stromal cells (Ter119 – CD45 – CD31 – Sca1 + CD29 + ). Different stromal cell subsets were sorted, cultured and stimulated with TGFβ1. The differentiation potential was assessed by qPCR, IF, stress fiber staining, SIRCOL and contraction assay on sorted cells. The antisense oligonucleotide GapmeR was used to downregulate Fra2. Results Fra2 tg mice showed increased CD45 + leukocyte infiltrates and massive collagen deposition in the heart tissue similarly to the myocardium of SSc/iDCM patients. Moreover, the myocardium of Fra2 tg mice revealed increased expression of pro-fibrotic markers such as αSMA, vimentin, collagen I and fibronectin compared to wild type mice. Four myocardial stromal cell populations were identified: gp38 + CD90.2 – , gp38 + CD90.2 + , gp38 – CD90.2 + and gp38 – CD90.2 – . The frequency of gp38 + CD90.2 – (single positive) cells and gp38 + CD90.2 + (double positive) cells was significantly higher in Fra2 myocardium compared to control mice (p=0.009; n=11). Importantly, in the myocardium of Fra2 tg mice, the majority of gp38 + cells co-expressed αSMA, vimentin, collagen and fibronectin indicating that myocardial gp38 + stromal cells might proliferate and/or differentiate towards the myofibroblast phenotype. Myocardial single and double positive stromal cells were cultured in vitro. After TGFβ1 stimulation, both cell populations up-regulated αSMA mRNA levels. Importantly, stromal cells from Fra2 tg mice showed the presence of αSMA fibers and stress fibers even without TGFβ1 stimulation as well as an increased contraction capability compared to control cells. These findings indicate that Fra2 overexpression might trigger the differentiation of these cells. Consequently, Fra2 silencing resulted in a decreased differentiation capability of gp38 + stromal cells: mRNA levels of the pro-fibrotic genes αSMA and collagen I were significantly downregulated (p=0.0075 and p=0.0073; n=5). Moreover, Fra2 downregulation impaired the secretion of collagens and the formation of αSMA fibers. Conclusions We suggest that cardiac gp38 + stromal cell populations might serve as a cellular source of pathological myofibroblasts playing a pivotal role in TGFβ/Fra2-driven myocardial remodeling in SSc. A better understanding of the mechanisms triggering myocardial dysfunction in SSc might be helpful in developing novel and effective therapies. Disclosure of Interest M. Stellato: None declared, M. Rudnik: None declared, F. Renoux: None declared, E. Pachera: None declared, K. Sotlar: None declared, K. Klingel: None declared, J. Henes: None declared, P. Blyszczuk: None declared, O. Distler Grant/research support from: Bayer, Sanofi, Ergonex, Boehringer Ingelheim, Actelion, Pfizer, Consultant for: from 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, EpiPharm, Ergonex, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, MedImmune, Pharmacyclics, Pfizer, Serodapharm, Sinoxa, G. Kania: None declared