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OP0213 Macrophages from a scleroderma subgroup with higher skin scores express activation markers and induce fibroblasts in co-culture
  1. J King,
  2. D Sleep,
  3. Y Sohrabi,
  4. A Tam,
  5. BA Abdi,
  6. D Abraham,
  7. C Denton,
  8. R Stratton
  1. Centre for Rheumatology and Connective Tissue Diseases, Royal Free Hospital, London, United Kingdom

Abstract

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 Raynaud's UK

Royal Free Charity

Disclosure of Interest None declared

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