Effect And Mechanism Of TIM-3 Regulating Treg Cells On Lupus Nephritis

Lupus nephritis(LN)is a serious complication of systemic lupus erythematosus(SLE)and an important driver of the morbidity and mortality of SLE.It is also one of the most common secondary glomerular diseases and an important cause of end-stage renal disease in China.Genetic susceptibility,pro-inflammatory and anti-inflammatory cytokines,production of autoantibodies,abnormal lymphocyte subsets,and complement system defects all play a role in the pathogenesis of LN.Regulatory T cells(Treg),CD4+CD25+Foxp3+T cells,are an inhibitory subset of CD4+T cells.Treg cells can inhibit a variety of immune cells and thus play an important role in maintaining immune tolerance.The quantitative and inhibitory defects of Treg cells are involved in the pathogenesis of LN.Targeting Treg cells for LN treatment has great clinical value and become a new target for treating LN.T-cell immunoglobulin and mucin-containing protein-3(TIM-3)is an important checkpoint protein that can be expressed in a variety of immune cells and a variety of histiocytes and affect their function.TIM-3 is also expressed in Treg cells and serves as an important regulator of Treg cells,regulating the differentiation,proliferation,apoptosis,and inhibitory function of Treg cells.The abnormal expression of TIM-3 on the cell surface of Treg cells plays an important role in the progression of various diseases.In LN,it is not clear about the expression of TIM-3 in Treg cells,whether TIM-3 is associated with Treg cell defects in LN,and whether TIM-3 blockade improves or aggravates LN.Elucidation of these issues is very important for exploring the pathogenesis of LN,exploring new targets for the diagnosis and treatment of LN,and developing a new treatment regimen.Therefore,in this study,the regulatory effect of TIM-3 on Treg cells and the disease progression of LN have been explored in LN animal model MRL/lpr mice and in the lymphocytes of the spleen of MRL/lpr mice cultured in vitro,and the regulatory mechanism was further explored.Studies have confirmed that the overexpression of TIM-3in Treg cells may lead to a decrease in the number of Treg cells and functional defects,participating in the pathogenesis of LN.Blocking the TIM-3 signaling pathway can amplify Treg cells and enhance the inhibitory ability of Treg cells through PI3K/Akt signaling pathway to slow the progression of LN disease.The increased expression of TIM-3 on the surface of CD4+T cells and Treg cells in LN is mediated by the activation of m TOR.Part I Bioinformatics analysis of pathogenesis of LNObjective:Bioinformatics methods were used to explore the potential pathogenesis of LN to find more effective therapeutic targets.Methods:The gene expression profiles of LN kidney tissue and normal kidney tissue were obtained from GEO public database.Principal component analysis was performed using R software packages facto Mine R and factoextra,and the differentially expressed genes(DEG)between kidney samples and normal samples were screened using the R software package limma.The DEG was crossed with the LN related genes obtained from the Gene Cards database and Dis Ge NET database to obtain an LN related gene set.Gene ontology biological process(GO-BP)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes Access(KEGG)pathway enrichment analysis of the gene set was performed using the R software package cluster Profiler.A protein interaction network was constructed using the STRING online analysis platform,and the Top10 Hub gene was screened using Cyto Hub.The R software package ggpubr and ggstatsplot software packages were used for correlation analysis between genes.Genes related to TIM-3 were obtained from the STRING database,genes related to Treg cells were obtained from Gene Cards and LN related genes were crossed to obtain possible target genes of Treg cells in LN regulated by TIM-3.KEGG pathway enrichment analysis was performed using the R software package cluster Profiler.Results:A total of 73 LN related genes were screened out from the GEO database,Gene Cards and Dis Ge NET data sets in the present bioinformatics analysis.GO BP showed that most biological processes were involved in immune regulation,including regulation of the immunologic process,production of cytokines,activation of leukocytes/lymphocytes /T cells,and the proliferation of leukocytes/lymphocytes/monocytes and their regulation,and intercellular/leukocyte adhesion.The most important biological process is the immune effect process.After screening the Top10 Hub gene among them,we identified four Hub genes of interest: TIM-3,the Treg cell marker molecule CD25,and Foxp3,and the Treg inhibitory function-related molecule IL-10.Further analysis of the expression levels of these four genes revealed that the expression of TIM-3 was increased in LN kidney tissue,and the TIM-3 expression was significantly and negatively correlated with Foxp3 and IL-10 expressions.TIM-3 may regulate Treg cells in LN through PI3K/Akt signaling pathway,Foxo signaling pathway,cytokine-cytokine receptor interaction,T cell receptor signaling pathway,Th17 cell differentiation,and parasitic,viral,and bacterial infection pathway,etc.Conclusion:TIM-3 may affect Treg cells by regulating the PI3K/Akt signaling pathway,Foxo signaling pathway,cytokine-cytokine receptor interaction,T cell receptor signaling pathway,and parasitic,viral,and bacterial infections pathway,thereby playing an important role in the pathogenesis of LN.Part II Expression of TIM-3 on CD4+T cells and Treg cells in LN mouse model and its effect on Treg cellsObjective:To explore the expression of TIM-3 on CD4+T cells and Treg cells in LN mouse model MRL/lpr and its influence on Treg cells.Methods:The expression of TIM-3 on CD4+T cells and Treg cells in the spleen of 10 weeks old and 20 weeks old LN mice model MRL/lpr and Mp J control model mice was detected by flow cytometry.The proportion of Treg cells in CD4+T cells with different TIM-3expression levels and Treg cell inhibition function related molecules,such as cytotoxic T lymphocyte associated antigen-4(CTLA-4)、IL-10、GATA binding protein-3(GATA-3)and TGF-β in Treg cells with different TIM-3 expression levels were detected by flow cytometry.Results:In the LN mouse model,the expression levels of TIM-3 on the surface of CD4+T cells and Treg cells were significantly higher than those in the normal control mice,and the expression tended to increase with the disease progression,especially the increase of TIM-3highcells.In LN mice,the expression levels of CTLA-4 and IL-10 in TIM-3low Treg cells were increased compared with those in TIM-3-Treg cells.However,with the further increase of TIM-3 expression,the expression levels of CTLA-4 and IL-10 in TIM-3highTreg cells were decreased compared with those in TIM-3lowTreg cells.The proportion of Treg cells in TIM-3low CD4+ T cells was higher than that in TIM-3-CD4+T cells,whereas the proportion of Treg cells in TIM-3high CD4+ T cells was significantly lower than that in TIM-3lowCD4+T cells.Conclusion:The overexpression of TIM-3 on CD4+T cells and Treg cells(the increase of TIM-3highCD4+T cells and TIM-3highTreg cells)may lead to the decrease in the number and functional defects of Treg cells and participate in the pathogenesis of LN.Part III Effects of blocking TIM-3 signaling pathway on Treg cells and disease progression in LN mouse modelObjective:To investigate the regulatory effect of TIM-3 on Treg cells and its influence on LN disease progression in LN mice.Methods:Eight weeks old female MRL/lpr mice were randomly divided into the anti-TIM-3group and Ig G isotype control group.Anti-TIM-3 mice were intraperitoneally injected with the anti-TIM-3 antibody at 100 μg,twice a week for 10 weeks.Mice in the Ig G isotype group received an equivalent dose of a murine Ig G2 a isotype control.24 h urine was collected every other week using mouse metabolism cages.The mice were sacrificed after10 weeks and tissue and blood samples were collected.The levels of blood creatinine,urea nitrogen,and urine protein in mice were detected by the kit.The pathological changes in kidney tissues in each group were observed after HE,PAS,and MASSON staining.Flow cytometry was used to detect the number of Treg cells in the spleen,peripheral blood,kidney draining lymph nodes,and kidney as well as the expression levels of CTLA-4,IL-10,GATA-3 and TGF-β in Treg cells.ELISA was used to detect the level of anti-double stranded DNA Ig G antibody in mice,and immunohistochemistry was used to detect C3 deposition in kidney tissues.Results:After TIM-3 blockade,the proportion of Treg cells in LN mice was increased,the expression of CTLA-4 and IL-10 in Treg cells was increased,the urinary protein,anti-double stranded DNA Ig G antibody,and blood urea nitrogen levels in LN mice were decreased,the weight of spleen was decreased,and the pathological damage of kidney tissues and C3 deposition were alleviated.Conclusion:TIM-3 plays an important role in the pathogenesis of LN by regulating the Treg cells in LN.Blocking the TIM-3 signaling pathway can slow the progression of LN disease by expanding Treg cells and enhancing their inhibitory ability.TIM-3 may be a potential therapeutic target for the treatment of LN.Part IV Effects and mechanisms of blocking TIM-3 signaling pathway on Treg cells in splenic lymphocytes of LN mice in vitroObjective:To investigate the effect of TIM-3 on Treg cells and on PI3K/Akt signaling pathway in splenic lymphocytes of LN mice in vitro.Methods:The splenic lymphocytes of LN mice were isolated and the treatment group and the control group were set up.Anti-TIM-3 antibody(10μg/L)was added to the treatment group,and an equal amount of Ig G isotype control group was added to the control group.The number of Treg cells,the expression levels of CTLA-4 and IL-10 in Treg cells and the p-Akt level in CD4+T cells were detected by flow cytometry.Results:Blocking the TIM-3 signaling in the splenic lymphocytes of LN mice in vitro could expand the proportion of Treg cells and increase the expression of CTLA-4 and IL-10.P-Akt level in CD4+T cells also decreased following TIM-3 blockade.Conclusion:TIM-3 regulates the number and function of Treg cells through PI3K/Akt signaling pathway in LN.Part V Increased TIM-3 expression in LN mediated by m TORObjective:To verify whether the increased expression of TIM-3 on the surface of CD4+T cells and Treg cells in LN is mediated by the m TOR pathway.Methods:(1)The splenic lymphocytes of LN mice were isolated,and the rapamycin group and the control group were set up.The rapamycin group was added with rapamycin(10 nmol/L),and the control group was added with an equal volume of PBS.The expression levels of TIM-3 on the surface of CD4+T cells and Treg cells were detected by flow cytometry.(2)Twelve weeks old MRL/lpr mice were randomly divided into two groups:rapamycin group and PBS control group.The mice in the rapamycin group were intraperitoneally injected with rapamycin solution at 1mg/kg,once every other day for 12 weeks.Control mice received an equal volume of PBS.At 24 weeks of age,the mice were sacrificed and the tissue and blood samples were collected.The expression levels of CD4+T cells and Treg cell surface TIM-3 in the spleen,peripheral blood,kidney draining lymph nodes and kidney were detected by flow cytometry.Results:Blocking the m TOR signaling pathway with rapamycin in the splenic lymphocytes of LN mice in vitro reduced the expression of TIM-3 on the surface of CD4+T cells and Treg cells.The expression of TIM-3 on CD4+T cells and Treg cells in the spleen,peripheral blood,kidney draining lymph nodes,and kidney significantly reduced after blocking the m TOR signaling pathway in LN mice.Conclusion:The increased expression of TIM-3 on the surface of CD4+T cells and Treg cells in LN is mediated by the activation of m TOR.