A6.321,25(OH)2D3 suppresses the pro-inflammatory TH17-RASF feedback loop via IL-4-dependent and -independent mechanisms

Background and objectivesVitamin D has suppressive effects on autoimmune diseases, such as rheumatoid arthritis (RA). Within these diseases, Th17 cells are considered to play a crucial role in the processes underlying chronic inflammation. We have shown that Th17 cells from RA patients induce a pro-inflammatory feedback loop upon interaction with RA synovial fibroblasts (RASF). Interestingly, 1,25(OH)2D3 is able to inhibit this feedback loop by modulating Th17 cell activity. However the molecular mechanisms underlying this inhibition by vitamin D are currently unclear.Materials and methodsCD4+CD45RO+ (memory) and CCR6+ memory T-helper cells were sorted from peripheral blood of patients with early RA and healthy volunteers. They were cultured for three days in the presence or absence of 1,25(OH)2D3 together with synovial fibroblasts from RA patients. The expression of cytokines and transcription factors of interest was analysed using microarray based gene expression profiling, flow cytometry, ELISA and/or RT-PCR.ResultsIn the presence of 1,25(OH)2D3 the pro-inflammatory cytokines IL-17A, IL-17F and IL-22 were inhibited. Also the expression of Th17 signature genes like RORgt and IL-23R was reduced. On the other hand we find an increase in IL-4 and IL-10 expression.Interestingly neutralisation of IL-4 partly reversed the effect of 1,25(OH)2D3 on the inhibition of IL-17A, IL22 and ROR gamma t expression. In addition, the inhibition of IL-17F by 1,25(OH)2D3 was almost completely absent when IL-4 was blocked. In contrast to IL-4, IL-10 neutralisation had limited effects in these cultures.Because the effect of 1,25(OH)2D3 is only partially dependent on IL-4, we examined factors that could play a role independent of IL-4. Gene expression profiling revealed that two transcription factors that are known to play a role in Th17 differentiation, EOMES and IRF8, are upregulated by 1,25(OH)2D3. EOMES and IRF8 are direct regulators of RORgt expression. Blocking IL-4 does not affect this up regulation, indicating that EOMES and IRF8 might be important in the IL-4 independent regulating of Th17 polarisation by 1,25(OH)2D3.ConclusionsFrom these findings, we conclude that 1,25(OH)2D3 inhibits the pro-inflammatory feedback loop between Th17 cells and synovial fibroblast. This modulation is partly dependent on up regulation of IL-4. IL-4 independent mechanisms may include the down-regulation of RORgt expression via up regulation of IRF8 and EOMES.