Roles And Mechanisms Of Tim-3 In Regulating Glucose Metabolism Of Macrophages

BackgroundThe abnormal phenotype of macrophages is closely related to various immune-related diseases,such as infection,autoimmune diseases and tumors which are undermining the people's quality of life and expected lifespan.The upregulated pro-inflammatory phenotype of macrophages is the main cause for autoimmune diseases,and the downregulated pro-inflammatory phenotype is another main cause for worsening infectious diseases and tumors progression.Therefore,precise regulation of macrophage phenotypes to maintain normal immunity is an effective and feasible strategy.Amounts of studies have shown that the macrophage phenotypes depend on the way of glucose metabolism.Exogenous inducers,such as LPS and GM-CSF,could transform macrophages to proinflammatory phenotype,secreting a large number of inflammatory cytokines such as TNF-?,IL-1?,and shifting their metabolic pathway to glycolysis;while IL-4 and IL-10 could transform them to an anti-inflammatory phenotype with a large number of tissue repair and angiogenic factors,such as TGF-?.IL-10,and shifting their metabolic pathway to fatty acid oxidation and oxidative phosphorylation.Inducing macrophage phenotypic transformation through regulating the metabolic pathways has important theoretical and practical significance.T cell immunoglobulin and mucin domain 3(Tim-3)is one kind of transmembrane protein expressed on the membranes of macrophages,DC cells,NK cells,NKT cells,and bronchial epithelial cells.Tim-3 is considered to be a new immune checkpoint for inhibit T cell activation by inducing apoptosis and maintain immune tolerance in vivo.Previous laboratory studies have shown that through the stimulation of LPS,macrophages with differentially expressed Tim-3,would be induced into two distinct phenotypes,the pro-inflammatory and the anti-inflammatory phenotype.Since the macrophages phenotype is closely related to its glucose metabolism,we are trying to answer whether Tim-3 could regulate the secretion of macrophage cytokines through shifting its metabolism pathways.ObjectivesThis paper attempts to answer the following three scientific questions,based on the previous lab data and background above:1.To determine the effect of Tim-3 on glucose metabolism in macrophages in vitro;2.To clarify the regulation mechanism of Tim-3 on macrophage glucose metabolism in vivo and in vitro;3.To elucidate the signaling pathway by which Tim-3 regulates macrophage glucose metabolism.Methods & Results 1.Tim-3 inhibits macrophage glycolysis.1.1 In order to understand the effect of Tim-3 on the glucose metabolism of macrophages,we first tried to analyze the effect of Tim-3 on the pH value of macrophage culture medium.Raw 264.7 mouse macrophage cell line was stimulated by the laboratory-prepared Tim-3 signal blocker,to detect the change of pH value of cell culture supernatant after 0,6,12,24,36 and 48 h,respectively.The pH value of 12?24?36?48h decreased significantly,and the 24 h decreased most significantly.Further,the CIK-8 and FACS techniques ruled out the possibility that Tim-3 promotes macrophage proliferation and apoptosis.Therefore,we assume that the decrease in pH of the culture medium after the blocking of Tim-3 is caused by increased lactation,The glucose is the raw material,and the lactate is the end product of the glycolytic pathway.So in the next phase,the raw materials and final products of the glycolysis pathway are to be tested.1.2 Using 0,1,2,4?g of Tim-3 agonists and 0,5,10,20?g Tim-3 blockers to stimulate Raw 264.7 24 h before detecting the lactate content changes in cell culture medium,the result showed that Tim-3 downregulates the lactate level in a concentration-dependent manners.1.3 The glucose uptake capabilities of Raw 264.7 with Tim-3 consistently knock-downed,Raw 264.7 stimulated with Tim-3 agonist and blocker are detected,the result showed the Tim-3 downregulates the macrophage glucose uptake capability.2.Tim-3 inhibits the expression of macrophage glycolytic-related molecules,thereby inhibiting its pro-inflammatory phenotype.2.1 Tim-3 inhibits GLUT1 mRNA and protein expression in macrophages.Glucose transporter(GLUT1)'s function is to transport glucose into the cytoplasm through the cell membrane.Western blot and Q-PCR were used to verify the differential expression of GLUT1 gene and protein levels in the Raw264.7 samples with Tim-3 agonist and blocker.The results showed that the gene and protein levels of GLUT1 were significantly upregulated in macrophages after blocking or knocking down Tim-3.2.2 Tim-3 inhibits gene expression of macrophage GAPDH.Recent researches showed that when macrophages transformed into a pro-inflammatory phenotype,the glycolytic pathway is enhanced,GAPDH activity is increased,and the post-transcriptional regulation of TNF-? mRNA is attenuated,and the protein production of TNF-? is increased.The mRNA and protein levels of GAPDH and TNF-? were detected by Q-PCR,Western Blot and ELISA,respectively,after the increase of Tim-3 signal activation and the stimulation of macrophages by Tim-3 blockers.The results showed that after blocking the Tim-3,the mRNA level of GAPDH in macrophages was significantly enhanced,and the mRNA and protein levels of TNF-? were also significantly increased.After induced by the Tim-3 agonist,the above results were reversed,and the effects are all concentration-dependent.2.3 HK2 can promote the mRNA and protein expression of TNF-? and IL-1? in macrophages.Tim-3 inhibits the production of TNF-? and IL-1? by inhibiting the mRNA and protein levels of HK2.HK2 is the first rate-limiting enzyme in the glycolytic pathway.We used FACS,Western Blot,QPCR,ELISA and other techniques in different cell systems(including: Tim-3 high expression,low expression of Jurkat cell line,Tim-3KO and Tim-3TG mouse peritoneal macrophages),Tim-3 knockdown of the Raw 264.7 cell line and its control,Tim-3 agonist and blocking of the Raw 264.7 cell line),verified that Tim-3 can inhibit the expression of HK2 mRNA and protein levels in macrophages.Subsequently,by knocking down the HK2 gene expression of macrophages,we verified that the down-regulation of HK2 expression led to the attenuation of macrophage glucose uptake ability and lactate secretion capacity and inflammatory cytokines related to macrophage glycolysis,such as TNF.The gene and protein levels of-? and IL-1? are also significantly reduced.2.4 In the Listeria infection model,Tim-3 could inhibit the expression of HK2 protein in the liver and the protein expression of TNF-? and IL-1? in peripheral blood.We established a model of Listeria infection in Tim-3 transgenic mice(Tim-3 TG)and Tim-3 myeloid knockout mice(Tim-3 KO).The liver tissue was subjected to HK2 protein immunofluorescence and peripheral blood was taken to detect the protein levels of TNF-? and IL-1?.The results showed that the fluorescence density of HK2 protein in liver of Tim-3KO mice was significantly stronger than that of Tim-3TG group,and the protein levels of TNF-? and IL-1? in peripheral blood of Tim-3 KO mice were also significantly higher than Tim-3 TG group.3.Tim-3 regulates the glycolytic pathway activity and pro-inflammatory phenotype of macrophages by inhibiting the STAT1 pathway involved in HK2 transcription.Since the early proof has shown that the blockade of Tim-3 can promote the activity of STAT1 pathway,and hence upregulate the expression of proinflammatory phenotypes of macrophages.So we inhibit the Raw 264.7 STAT1 pathway with STAT1 inhibitor Fludarabine after Tim-3 blockade was introduced,and we found:3.1 When STAT1 pathway was inhibited,the expression of HK2 gene and protein was not significantly increased after the administration of Tim-3 blocker,while the expression of HK2 gene and protein in the control group was significantly enhanced.That is,the activity of the STAT1 pathway can promote macrophage HK2 mRNA and protein expression,and the Tim-3 signal can block this activation process.3.2 After the blocking of Tim-3,the STAT1 signaling pathway was activated,and the glucose uptake capacity and lactate secretion capacity of macrophages were significantly enhanced.The expression of the cytokines TNF-? and IL-1?,which are related to the glycolytic pathway,is enhanced,and this process is significantly inhibited when STAT1 inhibitors are used.3.3 To further clarify the regulatory mechanism between STAT1 signaling pathway and HK2 protein,we established the HK2 promoter dual luciferase reporter system,transfecting the plasmid with HK2 reporter gene,STAT1 plasmid and Tim-3 plasmid into HEK 293 T cells.It was found that STAT1 significantly increased the transcription level of HK2,and Tim-3 over-expression inhibited the transcriptional process of HK2 activated by STAT1.This experiment revealed that Tim-3 inhibits the transcriptional activation of HK2 by STAT1.ConclusionsTim-3 is a new generation of immunoregulatory target following CTLA-4 and PD-1,which is essential for maintaining the body's immune homeostasis.The dysregulation of phenotypic expression of macrophages plays an important role in promoting the occurrence and development of various immune-related diseases such as infections,tumors and autoimmune diseases.Therefore,regulation of macrophage phenotype by Tim-3 is a promising intervention.And the metabolism shifts of macrophages are directly related to their phenotypic expression.Therefore,from the perspective of macrophage metabolism,the influence of Tim-3 on its metabolic mode and its molecular mechanism are clarified for the regulation of Tim-3 regulated macrophages.The innovation of this study is that we have first confirmed the role of Tim-3 in regulating the activity of macroglycolytic pathway,and further elucidated the glycolysis-related molecules and signaling pathways of Tim-3 targeting,providing new aspects and strategies for the feasibility and further clinical intervention of immune-related disease.