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
Authors: S Vettori, G Barra, G Pasquale, L Pellecchia, L Vicedomini, R De Palma, G Valentini
Journal: Annals of the Rheumatic Diseases (2017)

<h3>Background</h3> 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). <h3>Objectives</h3> We therefore aimed to investigate IL-17A expression and effects in these co-cultures. <h3>Methods</h3> PBMCs and skin fibroblasts from 5 dcSSc patients with disease duration &lt;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 <i>plus</i> 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. <h3>Results</h3> IL17A mRNA in co-cultured PBMCs was increased by 11.5 fold (p&lt;0.01), and IL17RA by 4.3 fold (p&lt;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&lt;0.05) and protein level (8.9 fold, 11.2 fold, and 252.4 fold, respectively; p&lt;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&lt;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&lt;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&lt;0.05). <h3>Conclusions</h3> 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. <h3>References</h3> Brembilla NC, Chizzolini C. T cell abnormalities in systemic sclerosis with a focus on Th17 cells. Eur Cytokine Netw 2012; 23: 128–39. 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. <h3>Disclosure of Interest</h3> None declared