25-hydroxyvitamin D3 conversion by dendritic cells and T cells drives 1,25-dihydroxyvitamin D3 mediated anti-inflammatory CD4+ T cell responses

Abstract

Background: Low vitamin D status is associated with an increased risk of autoimmune diseases, including rheumatoid arthritis. Thus, understanding how vitamin D modifies immune reactions holds therapeutic potential. We have shown that 1,25(OH)2D3, the active form of vitamin D, acts directly upon human CD4+ T cells, suppressing inflammatory cytokines (IL-17, IL-21, IFNγ and IL-22) whilst enhancing regulatory-markers (CTLA-4, CD25, FoxP3 and IL-10). However, the short half-life of 1,25(OH) 2D3 and its low serum level imply that local conversion of 25(OH)D3, the major circulating form of vitamin D, by 1α hydroxylase (CYP27B1), is necessary for immune regulation in-vivo. Thus, we have studied the effect of 25(OH)D3 upon T cell responses. In view of the critical role of CTLA-4 in immune-regulation and its strong sensitivity to 1,25(OH) 2D3, we have also investigated CTLA-4 function in vitamin D-modified immune responses. Lastly, in considering a use for vitamin D in inflammatory arthritis treatment, we analysed its effects upon T cells from synovitis patients. Methods: CD4+CD25- T cells were purified from human peripheral blood (PB) and stimulated with LPS-matured allogenic dendritic cells (DCs) plus antiCD3 or with antiCD3/CD28 beads in the presence and absence of 25(OH)D3. FoxP3, CTLA-4 and cytokines were measured by flow cytometry and CYP27B1 by qPCR. In function assays, CTLA-4 was blocked with antiCTLA-4 and T cell division monitored by CFSE. DC expression of CD80 and CD86 was assessed by flow cytometry. Synovitis patients were recruited from Birmingham City Hospital. Their PB and synovial fluid (SF) mononuclear cells were stimulated with antiCD3 in the presence and absence of 1,25(OH)2D3 and cytokines examined by flow cytometry. Results: Stimulation of T cells by DCs in the presence of 25(OH)D3 led to strong suppression of IL-17 and IFNγ but up-regulation of CTLA-4 and CTLA-4+FoxP3+ frequencies. By contrast, in the absence of DCs, 25(OH)D3 caused modest CTLA-4 induction and IFNγ suppression (P<0.05 for all). These differences corresponded with CYP27B1 levels, as maturation induced much higher CYP27B1 in DCs than arose in T cells (P<0.05). CTLA-4 blockade overcame 1,25(OH)2D3- mediated suppression of T cell division in DC stimulations and prevented down-regulation of co-stimulatory CD80 and CD86 on DCs. PB T cells from synovitis patients responded normally to 1,25(OH)2D3 with suppressed IL-17 and IFN- (P<0.0001). 1,25(OH)2D3 also reduced SF IL-17 (P<0.05) but not IFNγ. Conclusions: We have shown that DCs can efficiently convert 25(OH)D3 to drive 1,25(OH)2D3-modified T cell responses and that up-regulation of CTLA-4 is mechanistically important in immune suppression by vitamin D. Thus, 25(OH)D3 supplement could be useful in the treatment of conditions such as RA. This is supported by our finding that T cells from synovitis patients can respond to 1,25(OH)2D3.