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FRI0355 Il-17a up-regulation in peripheral blood mononuclear cells co-cultured with autologous skin fibroblasts is associated with down-regulation of pro-fibrotic mediators and increased fibroblast apoptosis
  1. S Vettori1,
  2. G Barra2,
  3. G Pasquale3,
  4. L Pellecchia1,
  5. L Vicedomini1,
  6. R De Palma1,
  7. G Valentini1
  1. 1Internal and Experimental Medicine “F. Magrassi”, University of Campania “Luigi Vanvitelli”, Naples
  2. 2Internal and Experimental Medicine “F. Magrassi”, University of Campania “Luigi Vanvitelli”, Napoli
  3. 3Internal and Experimental Medicine “F. Magrassi”, University of Campania “Luigi Vanvitelli”, Naples, Italy

Abstract

Background IL-17A has been implicated in the pathogenesis of systemic sclerosis (SSc) (1). We previously showed that skewed peripheral blood mononuclear cells (PBMCs) from SSc patients can induce Fas-mediated apoptosis in co-cultured autologous skin fibrobalsts (2).

Objectives We therefore aimed to investigate IL-17A expression and effects in these co-cultures.

Methods PBMCs and skin fibroblasts from 5 dcSSc patients with disease duration <3 years were co-cultured up to 10 days in presence of hrIL-2 [20 U/ml] in a 1:10 ratio, as previously described. IL17A, IL17RA, CXCL1, CCL2, CCL3, TGFBR2, SMAD3, CTGF, COL1A1, COL3A1 mRNA expression was assessed by Sybr Green real-time PCR. Chemokine production was further investigated at the protein level by multiple suspension immunoassay. In subset experiments, co-cultures were treated with either IL-17A or IL-17A plus anti-IL17 receptor A neutralizing monoclonal antobodies (anti-IL-17RA mAb), then cells were stained with Annexin V, anti-IL17RA, and anti-FAS antibodies and were investigated by flow-citometry.

Results IL17A mRNA in co-cultured PBMCs was increased by 11.5 fold (p<0.01), and IL17RA by 4.3 fold (p<0.05) in co-cultured fibroblasts. CXCL-11, CCL2, and CCL3 were also up-regulated at both mRNA (11.9 fold, 773.3 fold, and 29 fold, respectively; p<0.05) and protein level (8.9 fold, 11.2 fold, and 252.4 fold, respectively; p<0.05). Profibrotic mediators, such as COL1A1, COL3A1, and CTGF mRNA expression in co-cultured fibroblasts was reduced to 0.33 fold, 0.24 fold, and 0.31 fold, respectively (p<0.05). This effects were associated with mRNA down-regulation of two key effectors of TGF-β signaling, TGFBR2 and SMAD3 to 0.59 and 0.79 fold, respectively. At flow cytometry analysis, we observed a reduction in co-cultured fibroblasts apoptosis by adding IL-17RA neutralizing mAb to IL-17A treated cells (39% to 16.8%; p<0.05), as compared to controls treated with IL-17A and isotype controls. Moreover, IL17RA mAb addition also reduced Fas expression in co-cultured fibroblasts as compared to IL-17A treated cells (47.7% to 10.6%; p<0.05).

Conclusions Our results support the role of IL-17A in the pathogenesis of SSc. Furthermore, here we first show that IL-17A up-regulation in co-cultured PBMCs might play antifibrotic effects in autologous skin fibroblasts and might be implicated in fibroblast apoptosis, interphering with the FAS/FASL pathway.

References

  1. Brembilla NC, Chizzolini C. T cell abnormalities in systemic sclerosis with a focus on Th17 cells. Eur Cytokine Netw 2012; 23: 128–39.

  2. De Palma R et al. Peripheral T cells from patients with early systemic sclerosis kill autologous fibroblasts in co-culture: is T-cell response aimed to play a protective role? Rheumatology (Oxford) 2010; 49: 1257–66.

References

Disclosure of Interest None declared

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