Vitamin D suppresses Th17 cytokine production by inducing C/EBP homologous protein (CHOP) expression

Abstract

Vitamin D has been shown to have immunomodulatory function, but the molecular basis for it has not been well understood. In this study, we found that vitamin D receptor expression was induced in a CD4+ effector T cell lineage, Th17 cells, which required the transcription factors, RORα, RORγt, and STAT3. Treatment of mice with an active ligand of vitamin D receptor (VDR), 1,25-dihydroxyvitamin D(3) (1,25D3), ameliorated experimental autoimmune encephalomyelitis, accompanied with reduced IL-17 and IL-17F expression. In vitro, treatment of CD4+ T cells with the physiological doses of 1,25D3 preferentially inhibited cytokine production by Th17 cells, in a VDR-dependent manner, without affecting the expression of transcription factors or surface molecules. Moreover, at these concentrations, cytokine expression was suppressed only at protein but not at mRNA levels. Stimulation of Th17 cells with 1,25D3, in a concentration-dependent manner, induced the expression of C/EBP homologous protein (CHOP), a molecule involved in endoplasmic reticulum stress and translational inhibition. In addition, overexpression of CHOP in developing Th17 cells suppressed their cytokine production. Our results suggest a novel, post-transcriptional mechanism whereby Th17 cytokines are inhibited by VDR, which may underscore future therapeutic usage of vitamin D in treatment of autoimmune diseases.

FIGURE 1.

Differential expression of Vdr in the subsets of CD4+ T cells. A, mRNAs were isolated from different subsets of CD4+ T cells and subjected to real-time RT-PCR analysis. Relative mRNA expression was compared by setting the lowest expression amount to an arbitrary value of 1. iTreg, inducible Treg cells; nTreg, natural Treg cells. B, naive CD4+ T cells were cultured with TGFβ and IL-6. mRNAs were isolated at each time point and subjected to real-time RT-PCR. C, WT and RORα-, RORγt-, and STAT3-deficient mice were differentiated into Th17 cells, and mRNAs from these cells were subjected to real-time RT-PCR analysis. Data represent the means ± S.E. of at least three independent experiments.

FIGURE 2.

1,25D3 treatment protected mice from development of EAE. A, clinical scores of control and 1,25D3-treated mice after EAE immunization. B, C, splenocytes from mice with EAE mice were collected and restimulated with MOG peptide for 3 days. The culture supernatants were analyzed by ELISA. Data represent the means ± S.E. of three independent experiments and at least four mice in each group.

FIGURE 3.

Suppressive effect of 1,25D3 is VDR-dependent, and cytokine production is independent from transcriptional inhibition. A, flow cytometry plots of naive CD4+ T cells after culture in Th17-polarizing conditions for 5 days. Cells were activated with PMA/Ionomycin for 4 h and then stained for IL-17 and IFNγ. B, measurement of IL-17, IL-17F, and IL-22 in the supernatants of the cultures described in A. C, real-time RT-PCR analysis of the Rorc, Rora, and Il23r genes from T cells after culture under Th17-polarizing conditions. D, comparison of ELISA versus mRNA expression levels of Il17, Il17f, and Il22. Data represent the means ± S.E. of at least three independent experiments.

FIGURE 4.

CHOP is induced upon 1,25D3 treatments in Th17 cells, and overexpression of CHOP on Th17 cells leads to inhibition of cytokine production. A, naive CD4+ T cells were treated with increasing concentrations of 1,25D3 under Th17-inducing conditions. Cells were collected at day 5, lysates were analyzed by SDS-PAGE gel, and CHOP was detected using anti-CHOP antibody. B, naive CD4+ T cells were cultured under Th17-inducing conditions with or without retroviral overexpression of CHOP. Cells were stained at day 4 (*, p < 0.05). C, GFP-positive and -negative cells were sorted, and mRNAs were isolated for gene expression analysis by real-time RT-PCR. The sorted cells were cultured overnight with anti-CD3, and culture supernatants were subjected to ELISA. Data represent the means ± S.E. of at least three independent experiments. n/s, not significant.