1,25-Dihydroxyvitamin D3 inhibits the differentiation and migration of T(H)17 cells to protect against experimental autoimmune encephalomyelitis

Abstract

Background: Vitamin D(3), the most physiologically relevant form of vitamin D, is an essential organic compound that has been shown to have a crucial effect on the immune responses. Vitamin D(3) ameliorates the onset of the experimental autoimmune encephalomyelitis (EAE); however, the direct effect of vitamin D(3) on T cells is largely unknown.

Methodology/principal findings: In an in vitro system using cells from mice, the active form of vitamin D(3) (1,25-dihydroxyvitamin D(3)) suppresses both interleukin (IL)-17-producing T cells (T(H)17) and regulatory T cells (Treg) differentiation via a vitamin D receptor signal. The ability of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) to reduce the amount of IL-2 regulates the generation of Treg cells, but not T(H)17 cells. Under T(H)17-polarizing conditions, 1,25(OH)(2)D(3) helps to increase the numbers of IL-10-producing T cells, but 1,25(OH)(2)D(3)'s negative regulation of T(H)17 development is still defined in the IL-10(-/-) T cells. Although the STAT1 signal reciprocally affects the secretion of IL-10 and IL-17, 1,25(OH)(2)D(3) inhibits IL-17 production in STAT1(-/-) T cells. Most interestingly, 1,25(OH)(2)D(3) negatively regulates CCR6 expression which might be essential for T(H)17 cells to enter the central nervous system and initiate EAE.

Conclusions/significance: Our present results in an experimental murine model suggest that 1,25(OH)(2)D(3) can directly regulate T cell differentiation and could be applied in preventive and therapeutic strategies for T(H)17-mediated autoimmune diseases.

Figure 1. 1,25(OH)₂D₃ inhibits the onset of EAE and modulates the composition of T_H cells.

(A) Disease scores are shown for EAE in B6 mice at various time points after subcutaneous immunization with MOG_35–55 peptide in CFA and pertussis toxin. Results shown are mean ± SD. **p<0.01, ***p<0.001, compared with EAE-PBS group. (B) At 20 days after challenge, total mononuclear cells obtained from the brains of MOG_35–55-immunized wild-type mice and vitamin D_3-treated mice and stained with anti-CD4 and anti-CD3 Abs. Data are representative of three independent experiments with at least five mice per group. ***p<0.001, compared with EAE-PBS group. (C) Mononuclear cells from brains or splenocytes were restimulated in vitro with PMA/ionomycin for 5 hr, then stained intracellularly for Foxp3, IL-17A, IL-10, and IFN-γ. Data are representative of three independent experiments with at least five mice per group. *p<0.05, compared with splenocytes of EAE-PBS group.

Figure 2. 1,25(OH)₂D₃ negatively regulates Treg and T_H17 induction in neuro-antigen-specific CD4⁺ T cells.

CD4⁺ T cells isolated from MOG TCR-Tg mice (Vα3.2 and Vβ11 TCR, B6 background) were cultured with MOG_35–55 peptide (25 µg/ml) in the presence of CD3⁺ T cell-depleted splenocytes for 4 days under Treg-polarizing conditions (rTGF-β, 1 ng/ml; anti-IFN-γ, 10 µg/ml; and anti-IL-4, 10 µg/ml) or T_H17-polarizing conditions (rTGF-β, 1 ng/ml; rIL-6, 20 ng/ml; anti-IFN-γ, 10 µg/ml; and anti-IL-4, 10 µg/ml) or T_H1-polarizing conditions (rIL-12, 10 ng/ml; and anti-IL-4, 10 µg/ml) together with 1,25(OH)₂D₃ (VitD, 100 nM). Cells were then stained intracellularly for Foxp3, IL-17, or IFN-γ, respectively. The plots shown are gated on CD4⁺Vα3.2⁺ cells with quadrants drawn based on isotype controls. Data are representative of two independent experiments with at least three mice per group.

Figure 3. 1,25(OH)₂D₃ negatively regulates Treg and T_H17 induction in OVA-specific CD4⁺ T cells.

(A) Naïve CD4⁺ T cells from Rag2^−/− DO11.10 mice (BALB/c background) were cultured with 0.25 µM OVA_323–339 peptide in the presence of CD3⁺ T cell-depleted splenocytes for 4 days under polarizing conditions (Treg, T_H17, or T_H1) together with retinoic acid (RA, 100 nM) or 1,25(OH)₂D₃ (VitD, 100 nM) as described for Figure 2. Then cells were stained intracellularly for Foxp3, IL-17, or IFN-γ, respectively. The plots shown are gated on CD4⁺KJ1-26⁺ cells with quadrants drawn based on isotype controls. The numbers in the quadrants indicate cell percentages (left). Means ± SD of triplicate samples are plotted (right). Data are representative of five independent experiments with at least three mice per group. **p<0.01 compared with each cytokine-alone group. (B) Expression of Foxp3 and IL-17 genes was analyzed by quantitative PCR. Data are representative of five independent experiments with at least three mice per group.

Figure 4. Vitamin D receptor on CD4⁺ T cells is required for regulation of Treg and T_H17 differentiation by 1,25(OH)₂D₃.

Purified naïve CD4⁺ T cells from wild-type (WT) or VDR^−/− (KO) mice of B6 background were cultured with APCs from WT or VDR^−/− mice in the presence of 1 µg/ml anti-CD3 mAb for 4 days under Treg-polarizing conditions (rTGF-β, 1 ng/ml; anti-IFN-γ, 10 µg/ml; and anti-IL-4, 10 µg/ml) or T_H17-polarizing conditions (rTGF-β, 1 ng/ml; rIL-6, 20 ng/ml; anti-IFN-γ, 10 µg/ml; and anti-IL-4, 10 µg/ml). (A) Foxp3 expression in gated CD3⁺CD4⁺ cells was analyzed by flow cytometry. (B) For the IL-17A staining, CD4⁺ T cells were restimulated with PMA/ionomycin for 5 hr. Numbers beside quadrants indicate percentages of positive cells in each quadrant. Data are representative of three independent experiments with at least three mice per group. ***p<0.001 compared with cytokine-alone group.

Figure 5. Exogenous IL-2 recovers the decreased Treg but not T_H17 generation by 1,25(OH)₂D₃.

Naïve CD4⁺ T cells from Rag2^−/− DO11.10 mice (BALB/c background) were cultured with 0.25 µM OVA_323–339 peptide in the presence of CD3⁺ T cell-depleted splenocytes for 4 days. (A) Under Treg-polarizing conditions with 1,25(OH)₂D₃ (0.1, 1, 10, and 100 nM), culture supernatants were analyzed for IL-2 production by ELISA. (B) Under Treg-polarizing conditions, IL-2 cytokine was added in 1,25(OH)₂D₃-treated groups in a dose-dependent manner (IL-2: 0.1, 1, 10, and 20 ng/ml); 4 days later the CD4⁺ T cells were stained intracellularly for Foxp3. (C) Under T_H17-polarizing conditions, 1,25(OH)₂D₃ (100 nM) and IL-2 (0.1, 1, 10, and 20 ng/ml) were added. The average frequency of IL-17A⁺ T cells in gated CD4⁺KJ1-26⁺ cells is shown. Means ± SD of triplicate samples are plotted. Data are representative of three independent experiments with at least three mice per group. *p<0.05, ***p<0.001 compared with cytokine-alone group.

Figure 6. 1,25(OH)₂D₃ and TGF-β plus IL-6 partially induce IL-10 production, but IL-10 is not involved in the inhibitory mechanism of vitamin D₃.

(A and B) Naïve CD4⁺ T cells from Rag2^−/− DO11.10 mice (BALB/c background) were cultured with OVA_323–339 peptides in the presence of the indicated cytokines with and without 1,25(OH)₂D₃ (100 nM) for 4 days. The average frequency of IL-10-producing cells is shown. (C) The dose-dependent effect of IL-6 on IL-10 production in CD4⁺ T cells induced by TGF-β and 1,25(OH)₂D₃ was determined by titrated doses of IL-6 (0.1, 1, 10, and 100 ng/ml). (D) IL-10 production by CD4⁺ T cells cocultured with CD3-depleted splenocytes and OVA_323–339 peptide was determined by titrated doses of 1,25(OH)₂D₃ (0.1, 1, 10, and 100 nM). (E) Average frequency of IL-10⁺cells among CD3⁺CD4⁺ cells as determined by flow cytometry after treatment with IL-27 and/or other indicated cytokines with or without 1,25(OH)₂D₃. Plots show mean ± SD of triplicate samples. *p<0.05, **p<0.01, ***p<0.001 compared with medium alone. (F) To analyze the effect of autocrine IL-10 on T_H17 differentiation, we used IL-10^−/− mice of C57BL/6 background. Naïve CD4⁺ T cells isolated from IL-10^−/− or IL-10^+/+ mice were stimulated with anti-CD3 mAb in the presence of the indicated condition for 4 days. IL-17 production in CD4⁺ T cells was analyzed by flow cytometry. Data are representative of three independent experiments with at least three mice per group.

Figure 7. The inhibitory mechanism of 1,25(OH)₂D₃ is independent of the STAT1 signal.

Naïve CD4⁺ T cells from STAT1^+/+ and STAT1^−/− mice (B6 background) were cultured with anti-CD3 Abs (1 µg/ml) in the presence of CD3-depleted splenocytes for 4 days under various cytokine treatment conditions (IL-27, 10 ng/ml; TGF-β, 1 ng/ml; IL-6, 20 ng/ml; anti-IFN-γ, 10 mg/ml; or anti-IL-4, 10 mg/ml) with 1,25(OH)₂D₃ (100 nM) and then stained intracellularly for IL-17A and IL-10. Data are representative of three independent experiments with at least three mice per group. **p<0.01 compared with cytokine-alone group.

Figure 8. 1,25(OH)₂D₃ inhibits the expression of the CCR6 molecule in activated T cells.

(A) Flow cytometry analysis of CCR6 expression on activated T cells under T_H17-polarizing conditions (as described for Figure 2). Data are representative of three independent experiments with at least three mice per group. *p<0.05 compared with cytokine-alone group. (B) MIP-3α/CCL20 was added to the lower chamber and in vitro-generated T_H17 cells were applied to the upper chamber well. Two hours later, cells in the lower chamber were counted. Plots are mean ± SD of triplicate samples. Data are representative of two independent experiments with at least three mice per group. **p<0.01.