The Effects Of Mitophagy On The Regulatory T Cells Function In Patients With Myasthenia Gravis

Objective:To determine whether the regulatory T cells(Treg,CD4+CD25+T)have abnormal mitophagy and the relation with the function of Treg cells in patients with myasthenia gravis(MG)by observing the mitophagy in Treg of peripheral blood from MG patients;to adjust in vitro the mitophagy state of Treg cells by using rapamycin(Repa)and 3-methyladenine(3-MA),respectively;to observe the effect of different mitophagy on the function of Treg cells;to explore the abnormal mitophagy in Treg cells involved in cell-mediated immune mechanism of MG patients.Methods:1)Fifteen MG patients who met the inclusion criteria according to clinical diagnosis were enrolled as experimental group,15 healthy volunteers were served as normal control group.2)The peripheral blood(30 ml)from brachial vein of MG patients was extracted,mononuclear cells were isolated by density centrifiugation,then CD4+T and CD4+CD25+Treg cells from mononuclear cells were separaed by magnetic cell sorting,and the purity of the two kinds of cell was identified by flow cytometry.3)The obtained Treg cells were divided into four groups:Normal group(N),Myasthenia gravis group(MG),Myasthenia gravis Treg cells were cultured in Repa group(Repa),3-MA-induced culture for 48h group(3-MA).4)Intracellular mitophagy were observed by electron microscopic in CD4+CD25+Treg cells obtained from N and MG.Repa group and 3-MA group Treg cells were also observed and then compared to MG group.5)Intracellular fusion of mitochondria with lysosomes were observed by confocal microscopy in CD4+CD25+ Treg cells obtained from N and MG,Repa group and 3-MA group Treg cells were also observed and then compared to MG group.6)CD4+CD25+Treg cells were collected from N,MG,Repa and 3-MA groups and the expression level of autophagy protein LC3-Ⅱ was detected by Western Blot.7)JC-10 fluorescent probe labeled CD4+CD25+Treg cells from N,MG,Repa group,3-MA group,then mitochondrial membrane potential in each group were detected by flow cytometry.8)After Carboxyfluorescein succinimidyl ester(CFDA-SE,CFSE)labeled,normal CD4+T cells were stimulated by IL2,CD3 and CD28.Then the labeled cells were co-cultured with N,MG,Repa and 3-MA CD4+CD25+Treg cells for 5 days.The inhibition of proliferation of CD4+CD25+Treg cells to normal CD4+T cells was detected by flow cytometry.Results:1)Purity of target cells:After cell separation by magnetic beads,the purity of obtained target cells by flow cytometry was very high:CD4+ T cell was 95%,CD4+CD25+ Treg cell was(92.0±3.1)%.2)Mitophagy(transmission electron):The autophagy was observed under electron microscope.The mitophagy of MG group(19.20±5.49)was less than normal group(25.60±7.81,P<0.05),mitophagy of Repa group was increased(26.33 ± 3.50,P<0.05,and 3-MA group was decreased(8.27±2.12,P<0.05)than MG group.3)Mitophagy(confocal microscope):The ratio of fusion cells to total cells was observed under confocal.The fusion of mitochondria(green)with lysosome(red)of MG group(0.321±0.085)was decreased(P<0.05)than normal group(0.603 ± 0.133),Repa group was significantly increased(0.495 ± 0.139,P<0.05)and 3-MA groupwas decreased(0.237 ± 0.828,P<0.05)than MG group.4)The expression level of LC3-Ⅱ:The autophagy protein LC3-Ⅱ was significantly increased(P<0.05)in MG group(0.297 ± 0.065)than normal group(0.504 ± 0.108)by Western Blot,the Repa group was increased(0.561 ±0.115,P<0.05)and 3-MA group was decreased(0.146 ± 0.490,P<0.05)than MG group.5)Mitochondrial membrane potential of Treg cells:JC-10 labeled cell flow cytometry showed that MG group(9.28 ± 2.09%)was significantly decresed(P<0.05)than normal group(2.73 ± 0.617%),Repa group was increased(3.21 ± 1.09%,P<0.05)and 3-MA group was decreased(11.37 ± 3.02%,P<0.05)than MG group.6)The inhibition of Treg cells on the proliferation of normal CD4+T cells:The proliferation of MG patients(26.82 ± 6.25)was significantly lower than Normal group(36.49-5.94,P<0.05),Repa group(40.18±4.82)was higher than MG group(P<0.05),3-MA group(20.81 ± 6.13)was lower than MG group(P<0.05).Conclusions:The mitophagy and function of Treg cells are decline in peripheral blood from MG patients;Repa induced increase of mitophagy and function of Treg cells in vitro;3-MA inhibit mitophagy and function of Treg cells in vitro.It suggested that mitophagy in Treg cells was closely related with their functional defect and could lead to cellular immunological pathogenesis.