Background and objectives Low serum vitamin D associates with chronic inflammatory diseases such as rheumatoid arthritis (RA), thus vitamin-D supplementation has been suggested for their treatment. T-cells have a pathogenic role in RA. We have shown that active vitamin-D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has potent anti-inflammatory effects on T-cells. However the efficacy of vitamin-D in an inflammatory setting is unclear. This study was designed to assess the effect of 1,25(OH)2D3 upon inflammatory responses by CD4+T-cells from the blood and synovial fluid (SF) of RA patients.
Methods Ethical approval was granted by Solihull Research Ethics Committee. Patients who fulfilled 1987 ACR criteria for RA and age-matched healthy controls were recruited with informed consent. CD4+T-cell responses to 100nM 1,25(OH)2D3 were assessed by flow cytometry after stimulation. Mononuclear cells from blood and SF were stimulated with antiCD3 and magnetically isolated naïve (CD45-RA) and memory (CD45-RO) CD4+T cells were stimulated with monocytes+antiCD3. Regulators of 1,25(OH)2D3signalling were measured in purified T-cells by qPCR before and after stimulation with antiCD3/CD28 beads. Significance was tested by Wilcoxon analysis.
Results 1,25(OH)2D3 significantly reduced IL-17 and IFNy production by T-cells from the blood of RA patients and healthy controls. By contrast, SF T-cells from RA patients, showed reduced suppression of IL-17 by 1,25(OH)2D3 and no inhibition of IFNγ. 98(±1.8)% of SF T-cells were memory compared to 49(±14)% for blood. Unlike SF T-cells, 1,25(OH)2D3 suppressed IFNγ production by memory T-cells from blood. However across several targets: IL-17, IFNγ, IL-21, CTLA-4 and FoxP3, naive cells showed greater sensitivity than memory cells to 1,25(OH)2D3. Interestingly, the vitamin D receptor (VDR) was elevated in SF memory T-cells compared to equivalent cells from blood, suggesting that lack of VDR does not explain the diminished responsiveness of SF T-cells. Furthermore, stimulation, increased VDR, RXR and the VDR co-enhancer/co-repressor ratio to similar levels in T-cells from both sites and suppressed their expression of the 1,25(OH)2D3-inactivating enzyme, CYP24A1 equally.
Conclusion Stimulation primes CD4+T-cells from RA patient blood and SF to respond to 1,25(OH)2D3. However, SF T-cells are less sensitive to 1,25(OH)2D3. This is only partially explained by their shift to memory status. Such insensitivity of joint T-cells to 1,25(OH)2D3 has important implications regarding the use of vitamin D to treat RA.
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