Bahja Ahmed Abdi

OP0213 Macrophages from a scleroderma subgroup with higher skin scores express activation markers and induce fibroblasts in co-culture

Background Scleroderma (SSc) is characterized by pathological fibrosis. The mechanisms by which fibrosis occurs in SSc are not fully understood. Alternatively activated M2-like macrophages are associated with fibrosis and have been found to have an important role in pathological fibrosis in humans. Therefore, there is interest in elucidating their role in SSc. M2 macrophages express mannose receptor CD206 and are known to secrete a number of soluble factors to establish a pro-fibrotic milieu when present in damaged tissues. Furthermore, we have shown adenosine tri-phosphate (ATP) concentration is increased in the skin of patients with SSc. Within the extra-cellular environment, ATP is a Damage-Associated Molecular Pattern (DAMP), binding the P2X class of purinergic receptors. Such mechanisms may contribute to SSc pathology. Objectives In this study, we explore the relationship of macrophage CD206 and P2X7 expression to Rodnan Skin Score. The role of these cells in establishing fibrosis was also examined in vitro. Methods 17 SSc patients and 9 controls were consented and their skin score recorded. Macrophages were derived from peripheral blood mononuclear cells (PBMCs) and identified through CD14 expression by FACS. CD206 and P2X7 co-expression was quantified. CD206 immunofluorescence of skin biopsies was also performed. Macrophages were co-cultured with 8x104 and 2x104 fibroblasts in a collagen matrix and within a monolayer respectively. Collagen gel contraction was quantified as a measure of fibrotic activity. CTGF and Collagen mRNA expression from gel matrices and cellular monolayers was quantified by qPCR. Results CD206 and P2X7 expression is higher on SSc PBMC-derived macrophages (mean fluorescence 776.1 SD=409.1, 724.4 SD=455.3) compared to healthy controls (mean fluorescence 632.2 SD=73.7, 472.9 SD=25.4). There is significant correlation of CD206 expression to P2X7 expression (p<0.001, r2=0.76) and CD206 expression is significantly correlated to Rodnan skin score (p<0.05, r2=0.26). P2X7 expression is positively correlated to skin score. Double positive P2X7 and CD206 cells were seen in a subgroup with higher skin scores. Healthy fibroblasts co-cultured with scleroderma macrophages showed increased collagen mRNA by qPCR compared to co-culture with healthy macrophages (p<0.01). CTGF mRNA was positively correlated with macrophage P2X7 (r2=0.23) and CD206 (r2=0.81) expression. Preliminary work suggests contraction of collagen discs in fibroblast and macrophage co-culture is increased with SSc macrophages compared to healthy controls. Conclusions Data indicates a correlation between disease severity and CD206 expression by macrophages. Upregulation of CTGF and collagen expression in fibroblasts co-cultured with macrophages expressing high CD206 suggests a role for these cells in pathogenic fibrosis. The co-expression of high levels of P2X7 with CD206 also indicates a possible role for the purinergic pathway in SSc fibrosis. Future work will examine the mechanism of macrophage-fibroblast cross-talk and investigate the effect of inhibitors of CD206. Acknowledgements Rosetrees Trust Arthritis Research UK Scleroderma and Raynauds UK Royal Free Charity Disclosure of Interest None declared

OP0242 Role of MRTF-A Pathway in Scleroderma-Related Fibrosis

Background MRTF-A is a 120kDa transcription factor widely expressed and normally sequestered in the cytosol by binding to G actin. Following actin polymerisation downstream of Rho signalling, or mechanosensing, MRTF-A is released and functions as a signalling molecule partnering serum response factor (SRF) and influencing gene expression via CARG elements in the promoter region of genes. Genes expressing CARG like elements induced by MRTF-A/SRF include CTGF and type I collagen. MRTF-A is also implicated in vascular remodelling and epithelial to mesenchyme transition (EMT). The MRTF-A/SRF axis is highly relevant to fibrosis in systemic sclerosis (SSc). Methods MRTF-A signal transduction was studied in healthy control and SSc fibroblasts. The MRTF-A/SRF small molecule inhibitor CCG1423 was used to block MRTF-A in vitro. SSc fibrosis responses were modelled by collagen gel contraction, CTGF, and type I collagen expression. MRTF-A signalling was assayed by Western blotting of nuclear and cytoplasmic extracts. Wound healing and fibrosis was studied in an MRTF-A knockout mouse (Ollsen lab) and wild type controls. Immunochemistry looking for nuclear localisation of MRTF-A was used to determine presence of active signalling in SSc involved skin biopsy material and healthy control tissue. Results SSc fibroblasts showed enhanced nuclear localisation of MRTF-A at 8 hours following exposure to TGFβ (4ng/ml) not seen in healthy control fibroblasts. Immunochemistry of SSc skin biopsy material revealed enhanced nuclear localisation in dermal fibroblast like cells, keratinocytes within the epidermis, as well as in perivascular cells (pericytes). Following excisional wounding (4mm punch biopsy, basal wound area 12.6 mm2) MRTF-A mice wounds failed to close normally and increased in size during days 1-7, wound area decreasing by day 11. When compared to wild type controls MRTF-A knockout wounds were enlarged at day 7 (wild type area 6mm2, knockout area 12.4 mm2, p<0.03), and at day 11 (wild type area 0.42 mm2, knockout area 3.4 mm2, p<0.01). Day 11 wounds were extracted and found to abnormal showing reduced scar formation, and abnormal vasculogenesis. Small blood vessels within the granulation tissue were dilated, and exhibited extravasation of red blood cells. Gel contraction by wild type fibroblasts was enhanced by TGFβ and blocked by CCG1423 1μM (basal conditions mean gel mass 0.176g, TGFβ treated 0.118g, TGFβ+CCG1423 0.238g, p<0.002). Dermal fibroblasts from MRTF-A knockout mice showed reduced basal gel contraction, and impaired response to TGFβ,(basal conditions mean gel mass 0.349g, TGFβ treated 0.259g, TGFβ+CCG1423 0.313g (p<0.05 basal vs wild type). Studies of belomycin induced skin fibrosis are ongoing. Conclusions MRTF-A signalling is abnormal in SSc involved skin, enhanced in multiple cell types, as well as in SSc fibroblasts cultured in vitro. MRTF-A knockout mice fail to contract wounds adequately and show reduced scar formation, as well as abnormal vasculogenesis. Multiple pro-fibrosis pathways converge on MRTF-A including response to stiffness of the extracellular matrix, profibrosis growth factor stimulation, as well as transition of epithelial cells and perivascular cells to mesenchymal cells. CCG1423 and its derivatives are potential anti-fibrotics to benefit SSc fibrosis. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3676