P075 Pro-inflammatory and anti-fibrotic potential of Th17 cells on human dermal fibroblast from SSc individuals

Abstract

IntroductionIn systemic sclerosis (SSc), inappropriate T cell responses are thought to participate in initiating events ultimately leading to excessive extracellular matrix deposition and fibrosis.We recently demonstrated that the frequency of Th17 cells is significantly increased in SSc compared to healthy donors (HD). However, the role exerted by Th17 cells on the disease course is still poorly understood.Our purpose was to assess whether Th17 cells and IL-17A could promote inflammatory and fibrotic responses in dermal fibroblasts from HD and SSc. MethodsDermal fibroblasts were obtained from incisional biopsies of affected skin from 8 SSc and abdominoplasty surgery from 8 age- and sex-matched HD. Th17 cell clones were generated from the peripheral blood of HD upon enrichment of cells expressing CCR4/CCR6 and CD161.The cytokine production by T cell clones was assessed by FACS analysis and profiled in supernatants (SN) using multiplex beads immunoassay.Pro-inflammatory chemokines, MMP-1 and type I collagen production by IL-17A- or Th17 clone SN-treated fibroblasts was quantified by ELISA and RIA. Relative changes in steady-state gene transcription levels were assessed by real-time PCR on total mRNA from untreated vs treated-fibroblasts.IL-17 neutralizing antibody was used to confirm the specificity of the effects.Phosphorylation events induced by IL-17A were investigated by western blotting and the signaling pathways involved assessed using selective signaling protein inhibitors. ResultsIL-17A increased CCL2/MCP-1 (p<0.01), MMP-1 (p<0.01) and CXCL8/IL-8 (p<0.01) production by HD and SSc fibroblasts in a dose-dependent manner, while having no effect on type I collagen.Consistently, IL-17A increased the mRNA levels of CCL2, IL-8 and MMP-1 with no change in COL1A1 and COL1A2 mRNA. The production of CCL2/MCP-1, CXCL8/IL-8 and MMP-1 were all partially inhibited by the specific MEKK and PI3K inhibitors, indicating a wide participation of these pathways in transducing IL-17A signals.In addition, the production of CCL2/MCP-1 and CXCL8/IL-8 induced by IL-17A required NF-kB and p38 signaling, while the production of MMP-1 was dependent on JNK and AP-1 pathways.SN of activated Th17 clones strongly enhanced CCL2/MCP-1, CXCL8/IL-8 and MMP-1 production by both HD and SSc fibroblasts while inhibiting collagen synthesis.The neutralization of IL-17A in T cell clone SN reduced CCL2/MCP-1, CXCL8/IL-8 and MMP-1 and tended to enhance collagen production, thus proving the role of IL-17A in Th17 clone effects.In these SN, IL-17A had a synergistic activity with TNF as shown by simultaneous TNF/IL-17 blockade.Consistently, IL-17A/TNF co-treated fibroblasts had an enhanced production of CCL2/MCP-1, CXCL8/IL-8 and MMP-1 when compared to those subjected to each cytokine independently.Of interest, the simultaneous blockade of TNF and IL-17A in Th17 clone SN resulted in enhanced type I collagen production, specifically in SSc fibroblasts. ConclusionOur findings demonstrate that Th17 and IL-17A elicit in vitro pro-inflammatory responses and Th17 cells inhibit collagen production in human fibroblasts. Thus, the increased Th17 cell number observed in SSc may impact on the inflammatory component of the disease and may actually have a protective role against fibrosis.