The Role Of Regulatory T Cells To Treat Experimental Autoimmune Myositis

Objective:1. To develop a modified model for human idiopathic polymyosits;2. To observe the proportion of CD4~+CD25~+Foxp3~+Tregs with CD4~+T cells inperipheral blood and spleen mononuclear cells of experimental autoimmunemyositis (EAM), explore the relationship of regulatory T cells in autoimmunemyositis;3. To observe the clinical manifestations and EMG changes with CD4~+CD25~+Foxp3~+Tregs reinfusion treatment EAM mice in order to explore thetherapeutic value of CD4~+CD25~+Foxp3~+Tregs in inflammatory myopathies.To investigate its therapy mechanisms through analyze expression of PD-1andCTLA-4in spleen CD4~+CD25~+Foxp3~+Tregs before and after EAM micetreatment using flow cytometry, expression of IL-10and TGF-β in peripheralblood using double antibody sandwich ELISA method.Methods:1. EAM induction by guinea pig skeletal homogenates: Six to eight-week-oldBALB/c mice were divided randomly into two groups: control group (n=10)were immunized by PBS and complete Freund adjuvant (CFA) for six times;EAM group (n=10) were immunized by guinea pig skeletal homogenates andCFA by the same protocol.Clinical manifestations was observed. Serum muscleenzymes spectrum analysis were dected by Roche E170automatic biochemicalanalyzer, Muscles electrophysiological changes were dected by keypoint EMGevoked potential system.2. The animals were randomly divided into two groups: control group (n=5)without any treatment, EAM group (n=5).The proportion of CD4~+CD25~+Foxp3~+Tregs with CD4~+T cells in peripheral blood and spleen mononuclearcells was measured by flow cytometry.3. Sufficient quantities of CD4~+CD25~+Foxp3~+Tregs were separated fromnormal BALC/c mice spleen through MACS. We analyzed expression of PD-1and CTLA-4in spleen CD4~+CD25~+Foxp3~+Tregs before and after EAMmice treatment using flow cytometry, expression of IL-10and TGF-β inperipheral blood using double antibody sandwich ELISA method. In the sametime, we also observed the changes of clinical manifestations of experimentalanimals and electrophysiological changes by EMG.Results:1. The clinical score of EAM group were1.65±0.64.We analyzed total eighteenmice including EAM group (n=8) and control group (n=10). Theconcentrations of CK, AST and ALT were significantly increased in EAMgroup compared with control group (P <0.05), while CKMb was nosignificantly increased between two groups (P>0.05).Re-contraction amplitude,light contraction duration and amplitude of motor units in fore and hind limbsof EAM group were significantly reduced than control group (P <0.05), onlylight contraction amplitude in hind legs was no significant difference betweentwo groups (P>0.05). The light contraction polyphasic motor unit in EAMgroup increased significantly than control group.2.The proportion of CD4~+CD25~+Foxp3~+Tregs with CD4~+T cells in peripheralblood and spleen mononuclear cells of EAM group was8.0±3.94and4.7±2.15,while the proportion in control group was15.5±6.87and8.6±3.98.EAM group was significantly reduced with control group (P <0.05).3. The clinical score of EAM mice after treatment was0.95±0.24, which wassignificantly improved comparing with1.12±0.38, its four weeks immuneclinical score. The electrophysiological changes of EAM mice after treatmentinclude the Re-contraction amplitude and light contraction duration in fore andhind limbs were increased significantly than before treatment (P <0.05), lightcontraction amplitude of motor units in fore and hind limbs were no significantdifference before and after treatment (P>0.05), The light contraction polyphasicmotor unit was no significant difference before and after treatment (P>0.05).The expression of IL-10and TGF-β in peripheral blood were significantlyincreased after treatment than before treatment (P <0.05).The expression ofPD-1and CTLA-4in spleen CD4~+CD25~+Foxp3~+Tregs were significantlyincreased after treatment than before treatment (P <0.05).Conclusions: 1.Developed more ideal animal model for human IPM;2. Treg cells play an important role in maintaining immune balance whichprovides a new idea for research and treatment of the idiopathic inflammatorymyopathies.3. CD4~+CD25~+Foxp3~+Tregs reinfusion have therapeutic effect in EAM mice. Itplay a therapeutic role by increase IL-10and TGF-β in peripheral blood,PD-1and CTLA-4in spleen CD4~+CD25~+Foxp3~+Tregs.