Background Recently, the authors have shown that Th17, but not Th1 cells, from patients with early rheumatoid arthritis (RA) are potent activators of synovial fibroblasts (RASF) resulting inautocrine IL-17A production. This IL-17A production results in a pro-inflammatory loop, which is characterized by an up-regulation in pro-inflammatory cytokines and cartilage degrading enzymes. The autocrine IL-17A production by Th17 cells is critical for the perseverance of the pro-inflammatory loop, but the mechanism underlying the autocrine IL-17A induction is unclear.
Objectives To investigate the mechanism responsible for the autocrine IL-17A induction upon Th17-RASF interaction.
Methods CD4+CD45RO+CCR6+ (Th17) and CD4+CD45RO+CCR6- (Th1) cells were isolated by FACS sorting from healthy controls and early RA patients. These cells were co-cultured with RASF, in the presence of neutralizing antibodies directed against soluble IL-6R (anti-sIL-6R) and/or IL-1b, and celecoxib. Gene expression profiles were generated and supernatant was collected for cytokine analyses by ELISA.
Results Gene expression analysis revealed that the genes encoding for IL-6 and IL-1b were up-regulated in Th17-RASF cultures. These data were confirmed by ELISA and Q-PCR, respectively. Since IL-1b and IL-6 may be involved in IL-17/Th17 polarization we examined the contribution of these cytokines on the autocrine IL-17A loop.Blockade of IL-1b and IL-6 activity, but not TNF-α significantly suppressed IL-17A production in the co-culture, but the inhibitory effects were limited.Of note, the combination of anti-IL-1b and anti-sIL-6R markedly suppressed IL-17F. No effects were found on IFN-γ.
Interestingly,the genes encoding for cyclo-oxygenase-2 (COX-2) and prostaglandin-E-synthase (PTGES), which are involved in prostaglandin-E2 (PGE2) synthesis, were also up-regulated in Th17-RASF cultures. This was associated with a dramatic increase of PGE2 production in Th17-RASF cultures compared to Th1-RASF cultures. Treatment of Th17-RASF cultures with celecoxib, an inhibitor of cyclo-oxygenase-2 (COX-2) activity, resulted in a significant decrease of IL-17A, but not of IFN-g and TNF-a production. Furthermore, the decrease in IL-17A production caused by celecoxib treatment was much more pronounced compared to neutralizing IL-1 and IL-6 activity. Flow cytometry revealed a decrease in Th17 cells in celecoxib treated co-cultures, while no effects on Th1 or Th2 cells were found. Moreover, celecoxib treatment significantly inhibited the pro-inflammatory loop as less induction of IL-6, IL-8 and MMP-3 production was observed in the Th17-RASF co-cultures.
Conclusions These findings show the critical role of the COX/PGE2 pathway in the autocrine IL-17A production upon Th17 and synovial fibroblast interaction.Inhibition of this pathway down-regulates the pro-inflammatory feedback loop induced by Th17-RASF interaction leading to less production of pro-inflammatory cytokines and destructive mediators.
Disclosure of Interest None Declared
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