Comparative effect of 25(OH)D3 and 1,25(OH)2D3 on Th17 cell differentiation☆
Introduction
Vitamin D plays an important role in the regulation of calcium homeostasis and bone metabolism, and has been shown to play a critical role in modulating immune function. Consistent with this role, the vitamin D receptor (VDR) is expressed on antigen presenting cells (APC) such as macrophages and dendritic cells, as well as on CD4+ and CD8+ T lymphocytes [1], [2]. CD4+ T cells from VDR KO mice have a heightened propensity to produce IFN-γ and IL-17A than their wild type counterparts, with consequent increased ability to differentiate into Th1 and Th17 cells [3], the pathogenic cells in autoimmune diseases including multiple sclerosis (MS) [4].
Vitamin D deficiency increases the risk of autoimmune diseases [5]. Serum concentration of 25(OH)D3 is more than 1000-fold higher (50–250 nM) than its active metabolite 1,25(OH)2D3 (48–156 pM) and serum levels fluctuate depending on nutrition and sunlight exposure, whereas 1,25(OH)2D3 levels do not vary among individuals, even vitamin D deficient subjects can show normal 1, 25(OH)2D3 levels [6].
Animal studies have shown a suppressive role for vitamin D on autoimmunity via inhibitory effects on Th17 effector responses [7], [8], [9]. While previous studies have addressed the effect of the active form of vitamin D on Th17 cells by examining individual cytokine and gene expression, there are few studies that systematically address the effect of vitamin D on pathogenic markers of Th17 cells and their cytokine expression profile by multiparametric surface staining of human T cells. Here, we investigated in parallel the effect of 1,25(OH)2D3 and 25(OH)D3 on the differentiation and cytokine production of primary CD4+ T cells isolated from normal individuals under non-polarizing and Th17 polarizing conditions. We show that both forms of vitamin D reduced the expression of pathogenic Th17 markers and decreased their ability to secrete inflammatory cytokines (IL-17A, IFN-γ). Moreover, under the influence of vitamin D, activated CD4 + T cells showed a higher frequency of CD4+ CD25hiCTLA-4+ FOXP3+ cells which had a suppressive function when tested in vitro against activated responder cells. Both vitamin D hormones increased VDR mRNA expression in Th17 polarized CD4+ T cells, and translocated equally to the nucleus.
Section snippets
Cell culture and immunophenotyping
CD4+ T helper cells were obtained from healthy leukopack donors at the American University of Beirut Medical Center after obtaining Institution Review Board approval. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Isopaque density gradient separation and subsequently frozen and stored in liquid nitrogen. CD4+ T cells were purified from frozen-thawed PBMCs using MACS separation (human CD4+ T Cell Isolation Kit, Miltenyi) by depletion of non-target cells (negative selection).
1,25(OH)2D3 and 25(OH)D3 decreased the secretion of pro-inflammatory cytokines and increased the secretion of anti-inflammatory IL-10 in Th17 cells
We investigated the effect of 1,25(OH)2 D3 and 25(OH)D3 in non-polarized activated CD4+ T cells and under Th17 polarizing conditions. Based on our optimization experiments, as well as evidence from literature, we used 10 nM 1,25(OH)2 D3 and 250 nM 25(OH)D3 in our culture conditions [9], [10], [11], [12].
Using Cytometric Bead Array (CBA), concentrations of IL-17A, IFN-γ, TNFα, and IL-10 were determined following 5 days of cell culture. 1,25(OH)2D3 and 25(OH)D3 significantly inhibited the secretion
Discussion
In this study, we present a comprehensive evaluation of the immunomodulatory effects of 1,25(OH)2D3 and 25(OH)D3 on human CD4+ T cells differentiated under non-polarizing and Th17 polarizing conditions. Human T cells express the VDR at a low level but this expression increases after activation [21], [22]. VDRs are part of the steroid hormone nuclear receptor family, in the presence of 1,25(OH)2D3 the VDR recruits the nuclear receptor (the retinoid X receptor) and they bind to vitamin D response
Conclusion
Our study characterizes fully for the first time the effect of vitamin D on Th17 pathogenic subset, and shows that both 25(OH)D3 and 1,25(OH)2D3 modulate Th17 effector responses and enhance regulatory function of CD4 + T cells. These findings suggest a potential protective effect for vitamin D by modulating pathogenic effector T cell responses.
The following is the supplementary data related to this article.
Acknowledgements
We thank Maria Esmerian for technical assistance in flow cytometry.
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