| Part 1 The role of glucose metabolism in regulating the function of CD4+T cells in primary Sj(?)gren’s syndromeBackground and objectives:Primary Sjogren’s syndrome(pSS)is a common autoimmune disease,mainly manifested as xerostomia and xerophthalmia caused by involvement of exocrine gland.Hyperactivity of CD4+T cells plays an important role in the pathogenesis of pSS,which can cause tissue damage directly by secreting a variety of cytokines or contribute to local inflammation by activating other immune cells.However,the underlying mechanism of hyperactivity of CD4+T cells in pSS patients has not been clarified.The metabolic state of CD4+T cells plays an important role in their activation and effector function,and the dysfunction mediated by abnormal glucose metabolism of CD4+T cells is involved in the pathogenesis of various autoimmune diseases.However,whether CD4+T cells in pSS patients exhibited glucose metabolic abnormalities and its potential role in the hyperactivity of CD4+T cells remains to be further explored.Therefore,this study aims to explore the mechanism of glucose metabolism in regulating the hyperactivation of CD4+T cells and the role of key molecules and metabolites in the pathogenesis of pSS,which will help to understand the pathogenesis of pSS and explore new therapeutic targets.Methods:In this study,64 pSS patients were included from the Department of Rheumatology and Immunology of Peking Union Medical College Hospital,and 58 ageand gender-matched healthy individuals were enrolled as healthy controls(HC).The peripheral blood mononuclear cells of the experimental subjects were isolated and CD4+T cells were isolated and purified by immunomagnetic cell sorting,and then stimulated and activated in vitro for 72 hours.Seahorse XF energy metabolism detection system was used to detect the metabolic phenotype of CD4+T cells;flow cytometry was used to detect the effector function and differentiation of CD4+T cells and the level of intracellular ROS;transcriptome sequencing,PCR and Western blot were performed to detect the expression of genes related to glycolysis pathway.Results:The level of aerobic glycolysis in activated CD4+T cells from pSS patients was significantly higher than that from HC,with a greater elevation in patients seropositive for anti-SSA and SSB antibody compared with those seropositive for anti-SSA antibody alone.While the level of oxidative phosphorylation of CD4+T cells in the two groups had no significant difference.Compatible with their metabolic phenotype,the production of IFNy and IL-17A of CD4+T cells in patients with pSS were also higher than those in HC,and the glycolysis inhibitor 2-DG could significantly inhibit the secretion of IFN-γ and IL-17A by CD4+ T cells in patients with pSS.2-DG could also inhibit the differentiation of naive CD4+T cells into Thl and Th17 cells.While metformin,an inhibitor of the oxidative phosphorylation pathway,had no such effect.The results of RNA sequencing,PCR and Western blot showed that the signaling molecules,protein kinases and key enzymes related to the aerobic glycolysis pathway,which included CD3E,CD28,PI3K,AKT,mTOR,MYC,LDHA,PFKFB3,PFKFB4,had higher expression in CD4+T cells from pSS patients.And the KEGG pathway analysis revealed that the upregulated differential expressed genes were highly associated with the TCR signaling pathway.Inhibition of mTOR by rapamycin also significantly suppressed the effector functions of CD4+T cells.In addition,the activity of LDHA and LDHA-mediated ROS production also increased significantly in CD4+T cells from pSS patients.LDHA inhibitor FX-11 could reduce the level of intracellular ROS and at the same time inhibit the secretion of IFN-γ and IL-17A by CD4+T cells from pSS patients.Meanwhile,ROS inhibitor NAC could also significantly inhibit the effector function of CD4+T cells.Conclusion:CD4+T cells from pSS patients undergo metabolic reprogramming after activation,and the hyperactivation of the aerobic glycolysis pathway may be involved in the dysfunction of CD4+T cells.TCR/CD28 signaling pathway and PI3K/AKT/mTOR signaling pathway may play an important role in activating the aerobic glycolysis of CD4+T cells from pSS patients.LDHA-mediated ROS generation may also contribute to the hyperactivity of CD4+T cells from pSS patients.Glycolysis pathway inhibitor 2-DG,mTOR inhibitor rapamycin,LDHA inhibitor FX-11,and ROS inhibitor NAC can significantly reverse the hyperactivity of CD4+T cells from pSS patients,suggesting that the key molecules and metabolites of aerobic glycolysis pathway are potential therapeutic targets for pSS.Part 2 Alteration of CD226/TIGIT immune checkpoint on T cells in the pathogenesis of primary Sj(?)gren’s syndromeBackground and objectives:Primary Sjogren’s syndrome(pSS)is a systemic autoimmune disease characterized by lymphocytic infiltration of exocrine gland,with a majority of T cell infiltration.Hyperactivity of T cells plays an important role in the pathogenesis of pSS,which mediates local inflammation by production of a variety of proinflammatory cytokines or cytotoxic granules,causing structural damage and dysfunction of the exocrine gland.However,the mechanism of hyperactivity of T cells in pSS patients remains poorly understood.As a newly discovered immune checkpoint pathway,CD226/TIGIT plays a key role in regulating the immune function of T cells.The hyperactivation of immune cells mediated by the imbalance of CD226/TIGIT pathway is involved in the pathogenesis of various autoimmune diseases,but its role in the pathogenesis of pSS remains to be further studied.Therefore,this study aims to explore the role of CD226/TIGIT in the pathogenesis of pSS,which is of great significance for indepth understanding of the pathogenesis of pSS and the search for new therapeutic targets.Methods:35 patients with pSS were enrolled from the Department of Rheumatology and Immunology of Peking Union Medical College Hospital,16 patients with systemic lupus erythematosus(SLE)and 14 patients with rheumatoid arthritis(RA)were also enrolled as disease controls(DC);and 33 age-and gender-matched healthy volunteers were included as healthy controls(HC).CD4+T cells were isolated and purified by immunomagnetic cell sorting;CD226/TIGIT phenotype,activation and effector function of T cells were detected by flow cytometry,and immunofluorescence was performed to detect the expression of CD226 and TIGIT on CD4+T cells of salivary gland from pSS patients.In addition,the clinical data of pSS patients were also collected for correlation analysis with the CD226/TIGIT phenotype of T cells.Results:The frequencies of CD4+CD226+T cells,CD4+TIGIT+T cells,CD8+CD226+T cells and CD8+TIGIT+T cells were significantly higher in patients with pSS than in HC(56.05± 9.82%vs.50.14 ±11.71%,p<0.05:40.15 ± 9.01%vs.26.35 ± 6.99%,p<0.0001;68.08 ± 16.87%、vs.51.98 ± 13.27%;p<0.001;54.67± 15.42%vs.39.70 ± 11.94%,p<0.01).Meanwhile,the frequencies of CD226 and TIGIT expressing CD4+T cells were also significantly higher in patients with pSS than in DC(56.05 ± 9.82%vs.44.0 ± 15.53%,p<0.05;40.15 ± 9.01%vs.33.76 ± 10.81%,p<0.05),but there was no difference between pSS and DC concerning the frequencies of CD226 and TIGIT expressing CD8+T cells(68.08 ± 16.87%vs.64.53 ± 20.07%,p>0.05;54.67 ± 15.42%vs.50.77 ± 17.18%,p>0.05).Among them,the frequencies of TIGIT/CD226 expressing CD4+T cells closely correlated with pSS disease activity:the percentages of CD4+CD226+and CD4+TIGIT+T cells were significantly higher in the active pSS than the inactive pSS(58.63 ± 11.02%vs.52.01± 5.88%,p<0.05;40.73 ± 9.04%vs.35.11± 8.77%,p<0.05).Besides,the proportion of CD4+TIGIT+T cells positively correlated with the ESR(p<0.01,r=0.61).Further in vitro analysis revealed that CD4+CD226+T cells exerted superior effector function than the CD4+CD226-counterparts in pSS(percentage of IFN-γ+cells:35.9±8.36%vs.10.47 ±5.86%,p<0.001;percentage of TNF-α+cells:81.1±7.93%vs.39.74± 12.64%,p<0.001),which was also seen in HC.TIGIT was preferably expressed on activated cells both in pSS and HC(percentage of HLA-DR+cells:14.70 ± 6.93%vs.5.89± 5.43%,p<0.001;6.69 ±4.26%vs.2.16 ± 1.30%,p<0.05).However,in pSS,CD4+TIGIT+T cells showed enhanced effector function than the CD4+TIGIT-T cells(percentage of IFN-γ+ cells:28.70 ± 8.80%vs.15.40 ± 7.70%,p<0.05;percentage of TNF-α+ cells:66.46 ± 11.11%vs.48.08 ± 14.20%,p<0.001),while the effector function of CD4+TIGIT+T cells was comparable with CD4+TIGIT-T cells in HC(percentage of IFN-γ+ cells:20.08 ± 5.23%vs.16.02 ± 5.15%,p>0.05;percentage of TNF-α+cells:56.94 ± 16.24%vs.52.48 ±13.91%,p>0.05).Moreover,a large number of CD4+CD226+T cells and CD4+TIGIT+T cells were found in the salivary gland of pSS patients.Conclusion:CD226/TIGIT immune checkpoint molecules were over-expressed on T cells in pSS.Proportional and functional alteration of CD226/TIGIT expressing CD4+T cells may be involved in the pathogenesis of pSS and be a potential novel therapeutic target for the disease. |
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