| Background and objective :Multiple sclerosis(MS) is an autoimmune disease of the central nervous system(CNS). It is generally accepted that dysregulated immune responses participate in the development of MS. So far, there are no effective therapies of MS. Experimental autoimmune encephalomyelitis(EAE) is a widely accepted animal model of MS that has been used to study the pathophysiology and therapy of MS. Dendritic cell(DC) are a group of professional, antigen-presenting immune cells, which can regulate immune responses and maintain the balance between tolerance and immunity. The purpose of this study was exploring the role of 1,25-dihydoxyvitamin D3 induced tolerogenic dendritic cell(t DC) in EAE.Methods:1. EAE, induce in C57BL/6 mice, were immunized with MOG35-55 emulsified in complete Freund’s adjuvant(CFA) containing 4mg/m L of mycobacterium tuberculosis H37 RA. Meanwhile, pertussis toxin(PTX) was intraperitoneally injected at day 0 and 2 post immunization.2. Bone marrow derived monocytes were cultured in medium including IL-4 and GM-CSF. At day 3, 5 and 7, half of fresh medium were refreshed. In order to obtain t DC, the medium was supplemented with 1,25-dihydroxyvitamin D3. At day 7, lipopolysaccharide(LPS) was added to induce DC maturation for 24 h. At day 8, we measured the surface expressions of MHCII, CD86, CD80 and CD83 of DC with a flow cytometer and the data were analyzed using Flow Jo 7.6 software.3. At day 8, DC/t DC were harvested and then cultured with MOG35-55 for 4 hours. After washed with PBS, DC/t DC were intravenously injected into EAE mice at days 10,13, and 16. Then we compared the clinical scores of 4different groups-the blank control group, the negative control group, the normal DC-treated group, the 1,25(OH)2D3-induced t DC-treated group.4. At the peak of disease, mice from 4 different groups were sacrificed.Tissue of spinal cord were stained by Haematoxylin & Eosin(HE) and observed.5. At the peak of disease, the negative control group, the normal DCtreated group, and the 1,25(OH)2D3-induced t DC-treated group mice were sacrificed. The levels of serum IL-6、IL-17 were detected by CBA according to the manufacturer’s instructions.Results:1. We successful induced the EAE mice. The onset of the disease was at day 12~14 post immunization and the peak of the disease at day 20 post immunization2. The percentage of CD11c+ DC in t DC is 95.9% while that in DC is90.0%. Compared to DC, the expression of MHC-II and CD86 in t DC decreased(P=0.045 and P=0.028). At the same time, the expression of MHC-II and CD86 in LPS-treated t DC was lower than that in LPS-treated DC(P=0.024 and P=0.038).3. Adoptive transfer of both the normal bone marrow-derived DC and the1,25(OH)2D3-induced t DC could ameliorate the severity of the clinical signs comparing to negative control group, while t DC lead to a more significant decrease in the clinical scores.4. It showed an infiltration of inflammation at different groups except the blank control group through Haematoxylin & Eosin(HE) staining. The normal DC-treated group and the 1,25(OH)2D3-induced t DC-treated group were less severity than the negative control group, while t DC-treated group was more significant.5. The levels of serum IL-6 and IL-17 in the negative control group were higher than that in the normal DC-treated group and the 1,25(OH)2D3-induced t DC-treated group, but this difference did not achieve statistical significance.Conclusion:1. We successfully induced EAE through immunization with MOG35-55peptide/CFA;2. In vitro, 1,25-dihydroxyvitamin D3 could efficiently induce t DC;3. Intravenous transfer of 1,25-dihydroxyvitamin D3 induced t DC ameliorated the clinical course of EAE;4. 1,25-dihydroxyvitamin D3 induced t DC could decrease the inflammatory cell infiltration in spinal cord. |
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