The Role And Mechanism Of PGK1 In The Regulation Of Th17 Cell Metabolism In Autoimmune Myocarditis

Objective: Autoimmune response is an important mechanism in the pathogenesis of myocarditis.The T helper 17(Th17)cells are the main driver of cardiac-specific autoimmunity.Glycolysis is an essential pathway for Th17 cell differentiation.Phosphoglycerate kinase 1(PGK1)has been reported in the past to regulate glycolysis in tumor cells,but the role of PGK1 on Th17 cells and myocarditis is unclear.The aim of this study is to investigate whether PGK1 mediates the pathogenesis of myocarditis by regulating Th17 cell differentiation through glycolytic metabolism.Methods:Single-cell sequencing analysis was performed to assess the pathway and gene expression of glycolysis in experimental autoimmune myocarditis(EAM)mice of cardiac infiltration Th17 cells.The EAM model was established by subcutaneous injection of cardiac α-myosin heavy chain(MHC)peptide and adjuvant.The expression and localization of PGK1 were detected by real-time quantitative polymerase chain reaction(RT-q PCR),immunohistochemistry,and immunofluorescence.EAM mice were given oral PGK1 inhibitor NG52(100 mg/kg/d).Mice were observed for changes in cardiac appearance and heart/body weight ratio.Hematoxylin-eosin(HE)and Masson staining were performed to assess the extent of inflammatory injury and fibrosis.Echocardiography was used to assess the cardiac function.Flow cytometry was performed to detect changes in CD4+ T,Th17,and Treg cells in the heart and spleen of mice.RT-q PCR was performed to detect level of inflammatory factor expression in cardiac tissues.EAM mouse-derived CD4+ T cells were isolated and activated,then treated with NG52.Flow cytometry was used to observe changes in cell activation and proliferation.RT-q PCR was performed to detect changes in cytokine expression.Na?ve CD4+ T cells were isolated and induced to differentiate to Th17 cells,and treated with NG52 to observe changes of IL-17A+ cell ratio and Th17-related cytokines and transcription factors by flow cytometry and RT-q PCR,respectively.Fluorescent glucose analogue uptake assay,RT-q PCR,Seahorse glycolysis rate analysis was performed to observe the changes of glycolytic capacity of Th17 cells.The co-localization and phosphorylation regulation of PGK1 and pyruvate dehydrogenase kinase 1(PDHK1)were detected by immunofluorescence and western blot.The regulation of reactive oxygen species(ROS)by PGK1 was detected by DCFDA and mito SOX staining.N-Acetyl-Lcysteine(NAC)was co-treated with NG52 to observe the recovery effect of ROS scavenging on Th17 differentiation.The CD4+ T cells from patients with myocarditis were isolated and treated with NG52.The localization of PGK1 was observed by immunofluorescence,and the changes of cell activation,proliferation,and Th17 differentiation were observed by flow cytometry.Results: There was an enrichment of glycolytic pathway and high expression of PGK1 in Th17 cells infiltrating in the heart of EAM mice.PGK1 expression was increased in EAM mouse heart tissue and inflammatory cells and was localized to CD4+ T and Th17 cells.Oral administration of PGK1 inhibitor NG52 reduced heart/body weight ratio(4.23 ± 0.51 vs 5.26 ± 0.54,P < 0.001 vs Vehicle),attenuated cardiac inflammatory injury and fibrosis,and increased left ventricular ejection fraction(68.00 ± 8.82% vs 51.26 ± 16.52%,P < 0.05 vs Vehicle)and short-axis shortening(37.3 ± 6.33% vs 26.2 ± 9.72%,P < 0.05 vs Vehicle)in the hearts of EAM mice.Oral NG52 reduced the number and ratio of cardiac CD4+ T cells and Th17 cells and decreased the expression of cardiac inflammatory factors in EAM.Oral NG52 also increased the proportion of splenic na?ve CD4+ T cells and decreased the proportion of mature CD4+ T cells,as well as inhibited Th17 cell development and increased the proportion of Treg cells.NG52 directly inhibited the activation,proliferation,and cytokine secretion of CD4+ T cells and suppressed Th17 cell differentiation in vitro.Mechanistically,NG52 inhibited glucose uptake,glycolysis-related genes expression,basal glycolytic rate,and complementary glycolytic rate of Th17 cells.Meanwhile,PGK1 was co-expressed with PDHK1 on Th17 cells.NG52 inhibited phosphorylation of PDHK1 and promoted mitochondrial ROS production,Scavenging ROS with NAC recovered the damage caused by NG52 on Th17 differentiation.Finally,NG52-treatment inhibited the activation,proliferation,and Th17 differentiation of CD4+ T cells of myocarditis patients.Conclusion: PGK1 is highly expressed in Th17 cells of EAM mice.PGK1 mediates Th17 cell differentiation by promoting glycolysis and inhibiting ROS production,thereby participating in the pathogenesis of myocarditis.PGK1 may become a new target for myocarditis therapy.