The effect of vitamin D on the differentiation of Th17 cells -

Abstract

Background: Th17 cells play an important role in the pathogenesis of Multiple Sclerosis, a demyelinating neurodegenerative disease of the central nervous system, by the secretion of several pro-inflammatory cytokines. The active form of Vitamin D, 1α,25-dihydroxyvitamin D3, has several anti-inflammatory properties. Studies have shown its negative effect on Th1 and Th17 responses and its positive effect on regulatory T-cells and Th2 cellular responses. The aim of this study was to investigate the effect of 1α,25-dihydroxyvitamin D3 on the differentiation of naïve CD4+ T cells into Th17 cells ex-vivo in healthy controls, and to characterize the phenotype of Th17 cells and its modulation by vitamin D. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Isopaque density gradient centrifugation from collected blood samples or Leukopacks obtained from healthy donors. Naïve CD4+ T helper cells were isolated by microbead negative selection. The cells were then cultured in Th17 polarizing conditions in the presence of the cytokines: rIL-6, rTGF-b, rIL-1 and rIL-23, in the presence or absence of 1α,25-Dihydroxyvitamin D3 at a 10 nM concentration. On the 6th day of culture, cells were stained with antibodies conjugated to different fluorochromes against IL-17, IFNγ, TNFα, IL-10, CCR6, CD161, CCR4, CD25, RORγt and Foxp3. The acquisition of stained cells was done on a fluorescence-activated cell sorter (FACSAria III SORP) analyzer. The secretion of IL17, IFNγ, TNFα, IL-6, IL-2, IL-4, and IL-10 by the cells was quantitatively measured by ELISA and cytometric bead array (CBA).Results: Results obtained from healthy controls showed an effect for 1α,25-dihydroxyvitamin D3 on Th17 polarization as measured by the frequency of IL-17+ CD4+ T cells by flow cytometry. Vitamin D also decreased the frequency of RORγt+, IFNγ+, TNFα+, CCR6+, CD161+ and CCR4+ cells, all associated with the pro-inflammatory phenotype of Th17 cells.