PINK1-dependent Mitophagy Regulates T Cell Differentiation And Its Role In The Development Of Systemic Lupus Erythematosus

Background:Systemic lupus erythematosus(SLE)is a kind of autoimmune disease mainly in women of reproductive age.After activation and differentiation,CD4+T cells participate in the immune response by secreting cytokines and helping B cells to secret antibodies.CD4+T cells in SLE have abnormalities in the phenotype and function of several cell subsets,including Th1,Th2,Th17,Treg,Tfh,etc.Several studies have shown that SLE CD4+ T cells have mitochondrial hyperpolarization and dysfunction compared to healthy controls.Mitophagy is a pathway for healthy cells to clear damaged mitochondria,and the classical mitophagy is mediated by PINK1-Parkin pathway.It is worth noting that mitophagy has not been well-studied in SLE CD4+T cells.Objectives:(1)To investigate the status of mitophagy and the expression of its major molecules PINK1 and Parkin in SLE CD4+ T subsets,and the correlation between mitophagy and the SLEDAI score of disease activity;to explore the mechanism of PINK1 deficiency on T cell function;(2)To investigate the effect of PINK 1 on the differentiation of effector T cells;(3)To investigate whether defective mitophagy will impact the clinical phenotype and immune cells of SLE mouse model and the correlation with type I interferon(IFN).Methods:(1)In this study,we first determined the mitophagy of healthy control(HC)and SLE total CD4+T cells by transmission electron microscopy(TEM),real-time quantitative PCR(RT-qPCR)and western immunoblot(WB)techniques.Mitophagy-specific probes were used to analyze mitophagy in CD4+T cells and its various subsets and further,correlation with SLEDAI.We further transfected siPINK1 to explore the phenotype change of CD4+T cells and the state of Jurkat cells.(2)In vitro induction of T cell subpopulation differentiation was performed by sorting naive CD4+ T cells from wild-type and PINK1 knockout(KO)mice,while T cell colitis mouse model in RAG2 knockout mice was constructed through cell transfer to observe the difference of in vivo T cell differentiation.(3)Topical TLR7 agonist IMQ treatment of WT,PINK1 KO and Parkin KO mice to construct a lupus-like mouse model to observe clinical phenotypic differences at the tissue level and cellular level by ELISA,immunohistochemistry(IHC),and flowcytometry(FCM).Results:(1)Compared to HC,CD4+T cells from SLE patients showed mitochondrial crinkling,reduced mitochondrial DNA,and reduced PINK1 and Parkin at both RNA level and protein level.Further analysis in cell subpopulations revealed the most significant reduction in mitophagy level in Treg cells(HC vs SLE:45512.8000± 3293.87231 vs 26576.8462±2864.59111,P<0.0001).Interference of PINK1 had no significant effect on the differentiation of mouse Treg cells in vitro,but reduced the differentiation of human Treg cells(siNC vs siPINK1:11.68±1.729 vs 9.160± 1.293,P<0.05).After PINK1 interference in Jurkat cells,p-Akt pathway,a critical pathway for Treg function,was impaired and early apoptotic cells were increased,but they could be rescued by glycolysis inhibitor.(2)Higher percentage of differentiation into Th1 cells were observed in PINK1-deficient CD4+T cells in vitro(WT vs KO:9.040 ± 3.771 vs 33.90 ± 7.063,P<0.05)and was consistent with the induction of T-cell colitis in vivo(WT vs KO:5.481±0.8418 vs 15.62±1.270,P<0.001),and a reduced percentage of Th1 cells was observed in CD4+T cells in lupus mice treated with the mitochondrial autophagy agonist urolithin A in vitro.(3)In the IMQ induced lupus mouse model,increased autoantibodies(WT vs KO:14836.60±1761.31 vs 36629.2 ±7113.08,P<0.01)and kidney damage were predominantly observed in Parkin knockout mice while skin damage was more significant in PINK1 knockout mice,but all of these changes were associated with increased expression of the type Ⅰ IFN-related protein STING in the tissues.At the cellular level,Parkin KO and PINK1 KO mice showed opposite trends in type Ⅰ IFN-secreting cells,plasmacytoid dendritic cells(WT vs Parkin KO:3.27± 0.5567 vs 5.037±0.3268,P<0.05;WT vs PINK1 KO:1.490±0.2796 vs 1.250±0.2774,P>0.05).Conclusions:(1)The deficiency of PINK 1-dependent mitophagy was present in CD4+T cells of SLE patients,and especially,Treg cells.PINK1 can affect the phenotype,p-Akt pathway and early apoptosis of human Treg,and glycolysis inhibitor may be an effective antagonist.(2)T cell differentiation in vitro and in vivo suggested that PINK1 KO results in promoted Th1 cell differentiation.(3)The inconsistent clinical phenotypes of PINK1 KO and Parkin KO mice after induction of lupus-like model by IMQ suggests that those two mitophagy-related proteins have cellular and tissue heterogeneity,and regulating mitophagy to treat SLE requires more data of tissue-and gene-specific studies.