The Mechanism Of CD4~+CD25~+Treg Cells In Tolerance Of Rat Experimental Autoimmune Encephalomyelitis

Objective:Increasing studies have demonstrated that multiple sclerosis is a chronic immune-inflammatory disease, and myelin basic protein (MBP) active autorecative effector T cells, pass through the blood brain barrier, and contribute to demyelination in the central nervous system. Nasal administration of MBP or the peptide68-86, which was identified as a candidate auto antigen in CNS, was shown to induce tolerance and suppress development of experimental autoimmune encephalomyelitis. In this study, we explored the effect of nasal MBP68-86on EAE as well as the cellular and molecular mechanisms leading to demyelination responses, and study the role of CD4+CD25+regulatory T cells Foxp3mRNA and the cytokine IL-17A、TGF-β1in pathogenesis of immune tolerance of EAE.Methods:6-8weeks female rats were divided into three groups: tolerant group, immunized control group, and blank control group. The rats in the tolerant and immunized control group were nasally administrated MBP68-86and PBS daily for10consecutive days. The Blank control group was administrated equal PBS+CFA. One day after the final nasal MBP68.86administration, rats were immunized by subcutaneous injection of200μg of MBP68-86+CFA+PTX. We evaluated the weight and neurological severity scores of the rats daily;16days later the rat were sacrificed, isolate the spleen and prepare spleen lymphocyte quickly. Histological examinations were performed on the sections of brain with the aid of hematoxylin-eosin. The number of CD4+CD25+Tregs in spleens was detected using FACS analysis. The mRNA expression of Foxp3in splenocyte were analyzed by Real-Time-PCR. The cytokine levels of IL-17A, TGF-β1in splenocyte were analyzed by ELISA kitsResults:(1)We found nasal administration of MBP68-86suppress encephalo-myelitis, compared to immunized control rats, tolerant rats prolonged eclipse period(P<0.05), decreased morbility、clinical severity and neurological scores (P<0.05);(2) EAE control group rats were found with obvious pathological changes. at peak stage, HE stain showed vasculitis throughout the CNS, the vascular dilatation and perivascular infiltration of inflammatory cells looked severely, while the neuropathological evaluation of tolerant group rats was slightly, almost no inflammatory lesion in the blank control group;(3) The ratio of spleen CD4+CD25+T cells/CD4+T cells was higher in tolerant group than in EAE control group (P<0.05), and in blank control gruop(P<0.05);(4) The expression level of Foxp3mRNA in spleen cells was significantly higher in tolerant group than in EAE group (P<0.05),and in blank control group(P<0.05);(5) The serum levels of IL-17A were lower in tolerant group than in EAE group(p<0.05), and in blank control gruop(P<0.05),while the levels of TGF-β1were higher in tolerant group than in EAE group (P<0.05), and in blank control gruop(P<0.05).Conclusion:Our data suggested that multiple low dose nasal administration of encephalitogenic MBP68-8can inhibit EAE development, together with increasing the ratio of CD4+CD25+Tregs subgroups, and up-regulation of Foxp3mRNAexpression and TGF-β1, and down-regulation of IL-17A, which might play an important role in the pathogenesis of Treg-mediated EAE tolerance.