The Role And Mechanism Of Mitochondrial Dysfunction Of Regulatory T Cells In The Pathogenesis Of Sj(?)gren’s Syndrome

Objective:By comparing the level of Treg cells with high suppressive potential(GARP,LAP),Treg cells proliferation ability(Ki67)and DNA content(DRAQ5),Treg cells mitochondrial membrane potential(JC-1)and the mitochondrial oxidative phosphorylation(OXPHOS)functional molecules(MO,MG,PGC1α,TFAM)between primary sj(?)gren’s syndrome(pSS)patients and health controls(HCs),and analyzing the correlation between OXPHOS functional molecules and the other indicators,we aimed to explore whether immune tolerance deficiency in pSS is related to the dysfunctional OXPHOS of Treg cells and to clarify the role of abnormal OXPHOS in the pathogenesis of pSS.Methods:Thirty newly diagnosed pSS patients(1 male,29 female,median age 58.63 years)diagnosed in our hospital from July 2019 to January 2020,and 5 healthy people with matching age and gender(5 females,Mean age 59.40 years)were included in the study.The expression of GARP,LAP,Ki67,DRAQ5,JC-1,MO,MG,PGC1αand TFAM in Treg cells was detected by flow cytometry.The Mann-Whitney U test was used to compare the differences of the above indicators between pSS and HCs,and the Spearman correlation test was used to analyze the correlation between mitochondrial OXPHOS functional molecules and other indicators.Results:1.Compared with HCs,the level of GARP~+LAP~+Treg cells in pSS was significantly reduced[7.30(6.24,8.81)vs.2.20(0.40,3.30),P<0.001],and the level of GARP~-LAP~+Treg[5.20(4.03,5.91)vs.3.80(2.40,7.90),P=0.310]and GARP~+LAP~-Treg cells[10.66(6.75,14.73)vs.6.60(2.33,10.40),P=0.150]also showed a downward trend,with no statistical significance.2.Compared with HCs,the level of proliferating Treg cells in pSS[12.94(10.64,16.04)vs.5.27(3.70,11.20),P=0.018]and the corresponding DNA content[12067.00(7870.50,13423.50)vs.6221.00(5774.00,7119.00),P=0.001]were significantly reduced,and there was no significant difference in non-proliferating Treg cells.3.Compared with HCs,the level of JC-1 aggregates in the mitochondria of Treg cells in pSS did not show any significant difference,and the level of JC-1 monomer[7.40(5.25,8.20)vs.9.78(7.98,12.50),P=0.040]was elevated significantly,and the ratio of aggregates/monomer[1.59(1.05,2.57)vs.0.87(0.73,0.95),P=0.005]decreased significantly.4.Compared with HCs,the MFI of MO[1046.00(725.50,1212.00)vs.790.00(590.50,918.00),P=0.047]and TFAM[1203.00(1188.50,1232.50)vs.578.00(470.00,637.50),P=0.001]in the mitochondria of Treg cells in pSS were significantly reduced,while MG and PGC1αhad no significant difference.Further,the MFI of MO[1041.00(886.00,1366.00)vs.677.00(366.50,823.00),P=0.002],MG[9313.00(8783.50,9351.00)vs.6249.00(3873.00,8653.00),P=0.040]and TFAM[1485.00(1398.50,1552.00)vs.878.00(728.00,1051.50),P=0.001]in proliferating Treg cells in pSS were significantly lower than these in HCs,and the MFI of MO[835.00(701.50,1050.50)vs.667.00(300.00,776.50),P=0.034]and TFAM[1250.00(1242.00,1264.50)vs.599.00(485.00,658.50),P=0.001]in non-proliferating Treg cells in pSS were also significantly reduced compared with HCs.5.The expressions of MO,MG and TFAM in Treg cells were positively correlated with the level of GARP~+LAP~+Treg cells(r=0.509,P=0.008;r=0.402,P=0.042;r=0.496,P=0.010),and the expression of TFAM was also related to the level of GARP~-LAP~+Treg cells positively(r=0.534,P=0.005).6.The expression of MO in Treg cells was positively correlated with the content of JC-1 aggregates(r=0.651,P<0.001),and the expression of TFAM was negatively correlated with the content of JC-1 monomers(r=-0.503,P=0.009),but correlated with the ratio of aggregates/monomer positively(r=0.433,P=0.027).7.The expression of MO and TFAM in proliferating Treg cells were positively correlated with the level of proliferating Treg cells(r=0.411,P=0.037;r=0.593,P=0.001)and the corresponding DNA content(r=0.477,P=0.014;r=0.481,P=0.013).Conclusion:Compared with HCs,the mitochondrial OXPHOS of Treg cells in pSS was impaired.OXPHOS dysfunction could decrease the number and suppressive function of Treg cells by reducing the proliferation rate of Treg cells,reducing the mitochondrial membrane potential of Treg cells to induce apoptosis and reducing the number of GARP~+LAP~+Treg cells,which eventually leads to immune tolerance deficiency in pSS.