The Mechanism Of FOXP3~+CD4~+CD25~+Treg Cells In Pathogenesis And Tolerance In Experimental Autoimmune Myasthenia Gravis

PARTⅠTHE ROLE OF FOXP3 AND REGULATORY T CELLS IN PATHOGENESIS OF PASSIVE TRANSFERRED MYASTHENIA GRAVIS IN YOUNG MICEObjective: To study the role of Foxp3+CD4+CD25+ regulatory T cells (Foxp3+CD4+CD25+ Tregs),Foxp3mRNA and correlated cytokine IL-2,IFN-γin pathogenesis of passive transferred myasthenia gravis (PTMG) by detecting their expressions in a PTMG model.Methods: Sixteen C57BL/6 young female mice were divided into model group and control group.A PTMG model was established by injecting Ringer's buffer solution containing 1.0mg/kg mAb35 into abdominal cavity.Mice in control group were injected with Ringer's buffer solution not containing mAb35.Levels of CD4+CD25+T cells and Foxp3+CD4+CD25+Tregs in spleen cells of mice were measured by flow cytometry.Expression of Foxp3mRNA in spleen cells was detected by real-time fluorescent quantitative polymerase chain reaction assay (RT-FQ-PCR). Serum levels of interleukin-2 and interferon-γwere detected by ELISA.Results: The ratio of CD4+CD25+T cells and CD4+T cells was higher in model group[(8.82±0.74)%] than in control group[(9.89±0.88)%] (P<0.05).The ratio of Foxp3+CD4+CD25+Tregs and CD4+CD25+T cells was significantly lower in model group[(6.83±1.18) % ]than in control group[(8.38±0.76)%], (P<0.01).The expression level of Foxp3 mRNA in spleen cells was significantly lower in model group(0.47±0.30) than in control group[(1.53±1.19)(P<0.05). The serum levels of IL– 2 and IFN–γwere higher in model group(24.41±13.83 and 142.31±6.05ng/L)than in control group(12.09±2.96 and 109.36±3.64ng/L)(P<0.05).The change in the number of Foxp3+CD4+CD25+Tregs was positively correlated with that of CD4+CD25+T cells between model and control groups (r=0.864 vs r=0.977, P<0.01).Conclusion: Our study found that down-regulation of Foxp3 expression could decrease the expression of Foxp3+CD4+CD25+Tregs subgroups, and up-regulation of IL-2 and IFN–γ, which might play an important role in the pathogenesis of Foxp3-mediated PTMG. PARTⅡINTERVENTION MECHANISM OF A DUAL ANALOGUE ON FOXP3 AND REGULATORY T CELLS IN PASSIVE TRANSFERRED MYASTHENIA GRAVIS IN YOUNG MICEObjective: To study the prophylactic effects of nasal tolerance with a dual analogue, Lys262-Ala207, on the mouse model of passive transferred myasthenia gravis (PTMG) and the underlying mechanism. Young mice were treated with a specific toleragen-dual analogue (Lys262-Ala207) intranasally to observe its effect on the invasion process of mice model and the clinical symptoms,to assess its clinical effects,to measure the levels of Foxp3+CD4+CD25+Tregs and Foxp3mRNA, and to explore the cell immunologic intervention function and regulatory mechanism of Foxp3 on nasal tolerance with Lys262-Ala207 on PTMG.Methods: Mouse model of PTMG was established by intraperitoneal injection of mAb35. Twenty four C57BL/6 young female mice were divided equally into three groups: Tolerance group (T),model control group (MC)and control group (C).Lys262-Ala207 or PBS was given nasally before 10 days of immunization for 10 days. Clinical symptoms were evaluated after immunization. Serum level of acetylcholine receptor antibody IgG was detected using ELISA. Levels of CD4+CD25+T cells and Foxp3+CD4+CD25+Tregs in spleen cells were measured by flow cytometry. Expression of Foxp3mRNA in spleen cells was detected by RT-FQ-PCR. Serum level of IL-2 and IFN-γwas detected by ELISA.Results:(1)Compared with group MC, the clinical symptoms were improved in mice from group T, but that of group T did not return to normal. (2) The positive rate of the repetitive nerve stimulation test in group T was significantly lower than that in group MC. The serum levels of AChRAb IgG in group T decreased compared with that in group MC ( P <0.01), Both the test items in group T higher than that in group C.(3)The ratio of CD4+CD25+T cells and CD4+T cells in group T increased compared with that in group MC (P<0.05); The ratio of Foxp3+CD4+CD25+Tregs and CD4+CD25+T cells was higher in group T than that in group MC (P<0.01); The expression lever of Foxp3 mRNA in spleen cells was higher in group T than in group MC (P<0.05); All the test items in group T lower than that in group C. The serum levels of IL-2 and IFN-γwere lower in group T than that of group MC, the serum levels of IL-2 and IFN-γin group T were also higher than that of group C.Conclusions: Our study found that nasal administration with Lys262-Ala207 could increase the level of Foxp3mRNA, further caused the expression of Foxp3+CD4+CD25+Tregs subgroups to increase obviously,which was close to normal level, The nasal administration with Lys262-Ala207 ameliorated muscular weakness in PTMG young mice, related cytokines IL-2 and IFN-γtransforms to the normal condition correspondingly. All this indicated that:1. Nasal administration with Lys262-Ala207 had some preventive effects on PTMG young mice;2. One of prophylactic mechanisms of dual analogues might be due to up-regulation of Foxp3 mRNA expression;3. Foxp3+CD4+CD25+Tregs played a crucial role in controlling myasthenia.Therefore, Foxp3 has the possibility to be a target gene for MG treatment.