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FRI0522 Peripheral Blood Mononuclear Cells Co-Cultured with Autologous Skin Fibroblasts Up-Regulate IL-17A and Play Anti-Fibrotic Effects in Systemic Sclerosis
  1. S. Vettori,
  2. G. Pasquale,
  3. M. Iudici,
  4. G. Cuomo,
  5. B. Russo,
  6. G. Barra,
  7. R. De Palma,
  8. G. Valentini
  1. Department Of Internal and Experimental Medicine, Second University of Naples, Naples, Italy

Abstract

Background IL-17A has been recently implicated in the pathogenesis of systemic sclerosis (SSc).

Objectives Therefore, we explored its expression and effects in peripheral blood mononuclear cells (PBMCs) co-cultured with autologous skin fibroblasts.

Methods PBMCs and autologous skin fibroblasts from 5 patients with early (disease duration <3 years) diffuse (dc) SSc were co-cultured in presence of IL-2 20U/ml in a 1:10 ratio. Separate cultures of PBMCs with IL-2 20U/ml and unstimulated fibroblasts were used as controls. The expression of IL17A, IL1B, IL4, and TGFB1 mRNA was analyzed in co-cultured and control PBMCs after 10 days by real-time PCR. The expression of IL17RA, CCL2, CCL3, CXCL1, COL1A1, COL3A1, CTGF, TGFBR2, and SMAD3 mRNA was analyzed in co-cultured and control fibroblasts. Chemokine production was further investigated at the protein level in culture surnatants by multiplex suspension fluorescence-based immunoassay.

Results Real-time PCR analysis showed an increased expression of IL17A in co-cultured PBMCs by 11.5 fold (p<0.01). No significant difference was found in the expression of IL1B, IL4, and TGFB1 between co-cultured and control PBMCs. Consistently with IL17A up-regulation in co-cultured PBMCs, mRNA levels of IL17RA were increased by 4.3 fold in co-cultured fibroblasts (p<0.05). In order to clarify whether this up-regulation ensued in the formation of a functional receptor, we analyzed the expression of CCL2, CCL3, and CXCL1, which are IL-17A target genes, in co-cultured fibroblasts. Indeed, CCL2, CCL3, and CXCL1 were up-regulated by 11.9 fold, 773.26 fold, and 29 fold respectively (p<0.05). This induction was confirmed at the protein level in surnatants from co-cultures (CCL-2 25014 pg/ml; CCL-3 2227 pg/ml; CXCL-1 605.8 pg/ml) compared to control PBMCs (CCL-2 2821 pg/ml; CCL-3 199.1 pg/ml; CXCL-1 2.4 pg/ml) and control fibroblasts (CCL-2 2894 pg/ml; CCL-3 21.56 pg/ml; CXCL-1 3.1 pg/ml) (p<0.05).

Lastly, we investigated the effects of co-cultured PBMCs on the expression of pro-fibrotic genes in fibroblasts. We found that COL1A1, COL3A1, and CTGF mRNAs were down-regulated by 0.33 fold, 0.24 fold, and 0.31 fold respectively (p<0.05). In addition, we also found a down-regulation by 0.78 fold of TGFBR2 and by 0.79 of SMAD3, suggesting that co-cultured PBMCs may interfere with the TGF-beta pathway hyperactivity in SSc fibroblasts.

Conclusions Here we show for the first time that PBMCs from early dcSSc co-cultured with autologous skin fibroblasts over-express IL17A and exert anti-fibrotic effects in vitro. The simultaneous up-regulation of IL17RA and IL-17A target genes in the co-cultured fibroblasts suggests that IL-17A pathway is active in early dcSSc fibroblasts. These findings are paralleled by the down-regulation of TGF-beta signalling components. We previously showed that in co-cultures performed with PBMCs and autologous skin fibroblasts from early dcSSc patients T cells are expanded and kill the autologous fibroblasts. Taken together, these novel data support the hypothesis that immune system may be primarily aimed to control fibroblast activation in the early phases of the disease, potentially opening new therapeutic approaches in SSc.

Disclosure of Interest None declared

DOI 10.1136/annrheumdis-2014-eular.5910

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