The Role And Mechanism Of 1, 25-dihydroxyvitamin D3 Induced Tolerogenic Dendritic Cells In Experimental Autoimmune Encephalomyelitis

Background and objectiveMultiple sclerosis(MS) is the most common autoimmune disease in the central nervous system(CNS) characterized by inflammation and demyelination. Autoreactive CD4+ T cells, which targeting self-antigen, including Th1 and Th17 cells play essential roles in the pathogenesis of MS. There is no known cure treatment for MS. Tolerogenic DCs could efficiently induce regulatory T cells and suppress autoreactive T cells. Subsequently, they suppress immune response, induce immune tolerance. 1, 25-dihydroxyvitamin D3(VD3), the active form of vitamin D, activities potent effects on the immune system, capably induce tolerogenic DCs where it favoring the induction of regulatory T cells(Treg). Thus, VD3 induced toleorgenic DCs(VD3-DCs) would have therapeutic value for MS. Experimental autoimmune encephalomyelitis(EAE) is a classic animal model for studying MS. In this study, we investigated the role of VD3-DCs in mice EAE model. MethodsThe EAE mice model was induced by subcutaneous injection of 200 ug MOG35-55 emulsified with Complete Freund′s Adjuvant(CFA) followed by intraperitoneal injection pertussis toxin(PTX). Bone marrow derived monocytes from healthy mice were cultured in medium containing IL-4 and GM-CSF for 8 days. In addition, 1, 25-dihydroxyvitamin D3(10-8M) was added to the medium for VD3-induced-DCs. In order to stimulate maturation for DCs and VD3-DCs, LPS was added for 24 h on the day 7 of incubation. On day 8, flow cytometry was conducted by using the following antibodies: anti-MHC-II, anti-CD86, anti-CD80 and anti-CD83. The molecules expression on DCs and VD3-DCs, as well that expression on LPS matured DCs and VD3-DCs were compared. Mean influence intensity represent the quality of molecule expression.VD3-DCs were collected on day 8. Then these cells were cultured with MOG peptide at 20ug/ml for 4 hours. The VD3-DCs were intravenously injected into EAE mice on day 10, 13, and 16 post immunization after washed with PBS. While EAE mice treated with PBS were used as control. The spinal cords were collected on day 20-post immunization. The infiltration of Th1(CD4+T-bet+) and Th17(CD4+ROR-γt+) cells in spinal cord was analyzed by immunofluorescence. On day 20-post immunization, lymphnodes and spleen cells were isolated from mice. Cells were then cultured with MOG peptide(10ug/ml) for 24 hours. Treg(CD4+CD25+Foxp3+), Th1(CD4+IFN-γ+), Th17(CD4+IL-17A+), and CD4+IL-10+ from lymphnodes and spleen were analyzed by flow cytometry.All experiments were repeated at least three times. Data were expressed as mean ± standard deviation(SD), comparisons between two groups was performed by Student’s t-test. One-way analysis of variance(ANOVA) with Dunnett’s multiple comparisons test was applied to compare the mean values between three groups. For clinical score comparisons between different EAE groups, continuous analysis of variance were conducted. For all statistical tests, P < 0.05 was considered to be significant. ResultUsing MOG35-55 emulsified with CFA followed by PTX intraperitoneal injection successfully induced the EAE mice model. The 1, 25(OH)2D3 could efficiently induced tolerogenic DCs characterize by low expression of MHC-II and CD86(P < 0.001). After incubated with MOG peptide, VD3-DCs could decrease the clinical score of EAE. The infiltration of Th1(CD4+T-bet+) and Th17(CD4+ROR-γt+) cells in spinal cords were lower in EAE mice treated with VD3-DCs than control groups(P < 0.001). Similarly, the infiltration of Th1 and Th17 in VD3-DCs treated mice was also lower than DCs treated mice(P < 0.001). When compared with PBS-treated group, the percentage of Th1 and Th17 in lymphnodes and spleen were significantly increased in VD3-DCs treated group(P < 0.05); however, it was notable that the percentage of Tregs and CD4+IL-10+ T cells in lymphnodes and spleen of VD3-DCs treated group were higher(P < 0.05). Conclusion:MOG35-55 emulsified with CFA followed by PTX intraperitoneally injection could induce EAE in C57BL/6 mice. And the 1, 25(OH)2D3 induced tolerogenic DCs could ameliorate the symptoms of EAE mice when adoptive transfer. The therapeutic effect of VD3-DCs on EAE is via decreasing the infiltration of Th1 and Th17 cells in spinal cord, as well as by elevating the percentage of Tregs and CD4+IL-10+ T cells in lymphnodes and spleen. Therefore, VD3-DCs could be a potential approach for treating MS.