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. 2017 Nov;152(3):414-424.
doi: 10.1111/imm.12776. Epub 2017 Jul 10.

1,25-dihydroxyvitamin D3 -induced dendritic cells suppress experimental autoimmune encephalomyelitis by increasing proportions of the regulatory lymphocytes and reducing T helper type 1 and type 17 cells

Zhongxiang Xie  1 , Jingtao Chen  2 , Chao Zheng  1 , Jing Wu  2 , Yun Cheng  1 , Shan Zhu  2 , Chenhong Lin  3 , Qingqing Cao  1 , Jie Zhu  1   3 , Tao Jin  1
Affiliations

1,25-dihydroxyvitamin D3 -induced dendritic cells suppress experimental autoimmune encephalomyelitis by increasing proportions of the regulatory lymphocytes and reducing T helper type 1 and type 17 cells

Zhongxiang Xie et al. Immunology. 2017 Nov.

Abstract

Dendritic cells (DCs), a bridge for innate and adaptive immune responses, play a key role in the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Administration of tolerogenic DCs has been used as an immunotherapy in autoimmune diseases. Deficiency of vitamin D is an environmental risk factor of MS. In this study, we induced tolerogenic DCs by 1,25-dihydroxyvitamin D3 and transferred the tolerogenic DCs (VD3 -DCs) into EAE mice by adoptive transfer. We found that VD3 -DCs inhibited the infiltrations of T helper type 1 (Th1) and Th17 cells into spinal cord and increased the proportions of regulatory T cells (CD4+ CD25+ Foxp3+ ), CD4+ IL-10+ T cells and regulatory B cells (CD19+ CD5+ CD1d+ ) in peripheral immune organs, which resulted in attenuated EAE. However, the proportions of T helper type 1 (Th1) and Th17 cells in spleen and lymph nodes and the levels of pro-inflammatory cytokines and IgG in serum also increased after transfer of VD3 -DCs. We conclude that transfer of VD3 -DCs suppressed EAE by increasing proportions of regulatory T cells, CD4+ IL-10+ T cells and regulatory B cells in spleen and reducing infiltration of Th1 and Th17 cells into spinal cord, which suggests a possible immunotherapy method using VD3 -DCs in MS.

Keywords: 1,25-dihydroxyvitamin D3; experimental autoimmune encephalomyelitis; multiple sclerosis; tolerogenic dendritic cells.

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Figures

Figure 1
Figure 1
The expression of MHC‐II, CD86, CD83 and CD80 molecules on dendritic cells (DCs) and DCs treated with 1,25‐dihydroxyvitamin D3 (VD 3‐DCs). VD 3‐DCs and DCs were harvested on day 8 post‐culture and flow cytometry was applied to detect the expression of MHC‐II, CD86, CD83 and CD80 on VD 3‐DCs and DCs as described in the Materials and methods. (a) VD 3‐DCs express lower levels of MHC‐II, CD86 and CD83 molecules than DCs. (b) After stimulation with lipopolysaccharide (LPS), VD 3‐DCs still express lower levels of MHC‐II, CD86 and CD83 molecules than DCs. Experiments were replicated three times and data are shown as the mean ± SD. Two‐tailed Student's t‐test was used. ns, no significant difference; *P < 0·05; **P < 0·01; ***P < 0·001 (n = 10). [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
Dendritic cells treated with 1,25‐dihydroxyvitamin D3 (VD 3‐DCs) alleviate clinical symptoms and reduce spinal cord damage in experimental autoimmune encephalomyelitis (EAE) (models were induced in female C57BL/6 mice). (a) The clinical score of EAE after adoptive transfer of VD 3‐DCs and DCs. VD 3‐DCs and DCs reduce the severity of EAE. (b) Kaplan–Meier survival analysis comparing the onset time of the VD 3‐DCs group, DCs group and controls. VD 3‐DCs postpone the onset of EAE significantly. (c) Inflammatory cell infiltration and structural damage to the spinal cord are more severe in controls than in the VD 3‐DCs group and DCs group. Experiments were replicated three times (n = 10). [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
Dendritic cells treated with 1,25‐dihydroxyvitamin D3 (VD 3‐DCs) decrease the infiltration of T helper type 1 (Th1) and Th17 cells in spinal cord. On day 20 post‐immunization (p.i.), mice with experimental autoimmune encephalomyelitis (EAE) (induced in female C57BL/6 mice) were killed and their spinal cords were sliced into samples. The cells on lumbar spinal cord sections were stained using primary antibodies [CD4 (red), T‐bet (green) and ROR‐γt (green)] and secondary antibodies (goat anti‐rat AF‐647, goat anti‐mouse AF‐488 and goat anti‐rabbit AF‐488). Arrows represent the double‐positive cells (emit both red and green fluorescence), which are Th1 (a) and Th17 (b) cells. The numbers of Th1 and Th17 cells were analysed. Experiments were replicated three times and data are shown as the mean ± SD. One‐way analysis of variance followed by Bonferroni's post‐test was used. ***P < 0·001 (n = 10). [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
Dendritic cells treated with 1,25‐dihydroxyvitamin D3 (VD 3‐DCs) increase the proportion of Treg, CD4+ IL‐10+, Th1 and Th17 cells in the spleen and the lymph nodes. On day 20 post‐immunization (p.i.), mice with experimental autoimmune encephalomyelitis (EAE) (induced in female C57BL/6 mice) were killed, and cells were isolated from spleen and lymph nodes and analysed using flow cytometry as described in the Materials and methods. Flow cytometry pseudocolour images and bar charts of the proportions of Treg cells (CD4+ CD25+ FoxP3+) (a, b), CD4+ IL‐10+ cells (c, d), Th1 cells (CD4+ IFN‐γ +) (e, f) and Th17 cells (CD4+ IL‐17A+) (g, h) are shown. Experiments were replicated three times and data are shown as the mean ± SD. One‐way analysis of variance followed by Bonferroni's post‐test was used. ns, no significant difference; *P < 0·05; **P < 0·01 (n = 10). [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 5
Figure 5
Dendritic cells treated with 1,25‐dihydroxyvitamin D3 (VD 3‐DCs) increase the levels of interleukin‐17A (IL‐17A) and tumour necrosis factor (TNF). On day 20 post‐immunization (p.i.), mice with experimental autoimmune encephalomyelitis (EAE) (induced in female C57BL/6 mice) were killed and serum was obtained after centrifuging at 2092 g for 10 min. CBA kits were used to detect the secretion of cytokines in serum. Results show that the secretion of TNF (a) increases in both the VD 3‐DCs group and the DCs group and IL‐17A (b) only increases significantly in the VD 3‐DCs group. The secretion of interferon‐γ (IFN‐γ) (c) decreases significantly in the DCs group. The differences of secretion of IL‐6 (d), IL‐10 (e) and IL‐4 (f) are not significant. Experiments were replicated three times and data are shown as the mean ± SD. One‐way analysis of variance followed by Bonferroni's post‐test was used. ns, no significant difference; *P < 0·05; **P < 0·01 (n = 6).
Figure 6
Figure 6
Dendritic cells treated with 1,25‐dihydroxyvitamin D3 (VD 3‐DCs) increase the proportion of Breg cells in spleen and the secretion of IgG in serum. On day 20 p.i., experimental autoimmune encephalomyelitis (EAE) mice (induced by female C57BL/6 mice) were killed, cells were isolated from spleen and lymph nodes of mice and serum was obtained after centrifugation of blood at 2092 g for 10 min. Cells were analysed using flow cytometry as described in the Materials and methods and the levels of IgG were evaluated using ELISA kits according to the manufacturer's instructions. In spleen (a), the proportion of Breg cells increases in the VD 3‐DCs group whereas the proportion of it decreases in the DCs group. In lymph nodes (b), the proportions of Breg cells have a slight increase in both the VD 3‐DCs group and the DCs group. The level of IgG increases in the VD 3‐DCs group significantly. But the differences between the DCs group and the controls as well as the VD 3‐DCs group and the DCs group are not significant (c). Experiments were replicated three times and data are shown as the mean ± SD. One‐way analysis of variance followed by Bonferroni's post‐test was used. ns, no significant difference; *P < 0·05; **P < 0·01 (n = 6). [Colour figure can be viewed at wileyonlinelibrary.com]
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