Lactic Acid Promoting Glycolysis Of MDSCs And Enhancing Its Immunosuppressive Function

Objective:To investigate the regulation of lactic acid(LA)on the metabolism,immunosuppressive function and other biological behaviors of myeloid-derived suppressor cells(MDSCs)in tumor microenvironment,which provide new ideas and strategies for the immune escape treatment of tumors.Methods:(1)Immunomagnetic beads were used to sort out MDSCs derived from tumor tissue of lung cancer xenograft tumor mice and MDSCs derived from spleen of lung cancer xenograft tumor mice.The glycolysis activity of MDSCs were detected by real time quantitative PCR(qRT-PCR)and glucose and lactate detection kits.The immunosuppressive function and the expression level of immunosuppressive effector molecules of MDSCs were detected by flow cytometry(FCM)and arginase(Arg-1)and nitric oxide(NO)detection kits.(2)Tumor cell conditioned medium(TCCM)was used to simulate tumor microenvironment,and the effects of TCCM on glycolysis activity,and immunosuppressive function were detected.After blocking the glycolysis of MDSCs with glycolysis inhibitor(2-DG),we observed the regulation of TCCM on the glycolysis activity,and immunosuppressive function of MDSCs.(3)In order to determine the molecules that play a role in the tumor microenvironment,the immunomagnetic beads were used to sort out MDSCs.Lactic acid was added to the culture system,and the glycolysis activity of MDSCs was detected by qRT-PCR,glucose and lactate detection kits.The expression levels of immunosuppressive effector molecules of MDSCs were detected by FCM,arginase and nitric oxide detection kits.The inhibitory effect of MDSCs on the proliferation of CD4~+T cells was detected by CFSE staining.The number of viable cells was counted by trypan blue staining.And the apoptosis and expression of surface molecules CD40,CD80,CD86,MHC-II and F4/80 of MDSCs were detected by FCM.(4)In order to eliminate the influence of the acidic environment caused by the addition of lactic acid into the culture system,the pH of the culture solution was adjusted to 6.7 by hydrochloric acid,and the MDSCs were cultured with the culture solution of pH 6.7 to detect the expression level of the key enzyme of the glycolysis of MDSCs.And the glucose uptake,lactic acid production,immunosuppressive function,cell survival,apoptosis and expression of surface molecules CD40,CD80,CD86,MHC-II and F4/80 were also detected.In the case of 2-DG inhibition of the glycolysis of MDSCs,the regulation of lactic acid on the glycolysis activity and immunosuppressive function of MDSCs was examined.(5)In order to explore the mechanism of regulation of lactic acid on MDSCs,qRT-PCR was used to detect the mRNA expression levels of monocarboxylate transporter proteins(MCTs)in cells stimulated by lactic acid,and the protein level of MCTs was detected by Western blot.At the same time,in order to analyze the intracellular mechanism of lactic acid action,Western blot was used to detect the changes of PI3K/AKT/mTOR signaling pathway and the protein level of glucose transporter 1(GLUT1).Results:(1)Compared with spleen-derived MDSCs,the expression levels of glycolysis-related protein mRNA were significantly increased(p<0.05),and the expression levels of immunosuppressive molecules Arg-1,iNOS and ROS were increased(p<0.05).It is suggested that the tumor-derived MDSCs are more active than the spleen-derived MDSCs of the glycolysis activity,and the immunosuppressive function is also stronger.Based on this phenomenon,we explore the mechanism.(2)Compared with the untreated group,the expression levels of key enzymes and related protein mRNA of MDSCs in the TCCM treatment group were significantly increased(p<0.05),and the glucose uptake and lactic acid production were significantly increased(p<0.05).The inhibitory effect of MDSCs on CD4~+T cell proliferation was enhanced,and the secretion of immunosuppressive effector molecules was also significantly increased(p<0.05),which suggesting that TCCM can up-regulate the glycolysis activity and immunosuppressive function of MDSCs,and the differences are consistent between MDSCs in tumor tissues and spleen tissues.After adding 2-DG,the glycolysis of MDSCs was inhibited(p<0.05).TCCM could not enhance the inhibitory effect of MDSCs on CD4~+T cell proliferation and the secretion of immunosuppressive effector molecules,suggesting that TCCM may regulate the immunosuppressive function of MDSCs by affecting its glycolysis pathway.(3)When lactic acid was added to the in vitro culture system of MDSCs,the expression levels of key enzymes and related protein mRNA of MDSCs were significantly increased(p<0.05).Glucose uptake and lactic acid production were significantly increased(p<0.05).The inhibitory effect of MDSCs on CD4~+T cell proliferation was enhanced,and the secretion of immunosuppressive effector molecules was also significantly increased(p<0.05).The number of viable cells of MDSCs was increased(p<0.05).The proportion of apoptotic cells was decreased(p<0.05).The expression levels of cell surface molecules CD40,CD80,CD86,MHC-II and F4/80 were decreased(p<0.05).However,in the culture system of PH6.7,these phenomena no longer exist,suggesting that lactic acid can regulate the glycolysis activity and immunosuppressive function and survival,apoptosis,and maturity of MDSCs in the tumor microenvironment,and this regulation is caused by the lactic acid itself rather than the acidic environment caused by it.In the in vitro culture system of MDSCs,2-DG was added to inhibit the glycolysis of MDSCs,and then the lactic acid was added.The various biological behaviors of the above MDSCs did not change,suggesting that the regulation of lactic acid on MDSCs may be achieved by affecting its glycolysis activity.(4)After lactic acid was applied to MDSCs,the mRNA and protein levels of MCTs were significantly increased(p<0.05).PI3K protein expression was increased,and AKT phosphorylation and mTOR phosphorylation were also enhanced(p<0.05).In the end the GLUT1 protein expression level was increased significantly(p<0.05).It is suggested that after lactic acid acts on MDSCs,the expression of lactic acid transporter on the membrane surface is increased,and the intracellular PI3K/AKT/mTOR pathway is activated,which increases the expression of glucose transporter on the cell membrane surface,allowing more glucose to be transported into the body,enhancing the cell's glycolysis activity.Conclusion:(1)Compared with spleen-derived MDSCs,tumor-derived MDSCs are more active in glycolysis activity and have stronger immunosuppressive effects.(2)After the lactic acid in the tumor microenvironment is transported to the intracellular space through MCTs,the PT3K/AKT/mTOR pathway is activated,and the expression of GLUT1 is enhanced to up-regulate the glycolysis activity of MDSCs.(3)Lactic acid in the tumor microenvironment enhances its immunosuppressive function by up-regulating the glycolysis activity.