Lactic Acid Enhances The Immunosuppressive Function Of MDSCs By Regulating SGK1

Objective: To investigate the molecular mechanism of lactic acid(LA)in tumor microenvironment(TME)regulating the immunosuppressive function of myeloid suppressor cells(MDSCs)through SGK1,and to provide a new strategy and target for anti-tumor immunotherapy against MDSCs.Methods:(1)MDSCs from spleen of lung cancer transplanted mice were sorted by immunomagnetic beads.After treated with lactic acid,genes differentially expressed in MDSCs after treated with lactic acid were screened by whole-genome transcriptional sequencing.Using qRT-PCR and Western blot to verify the sequencing results and confirm SGK1 as a candidate gene for lactic acid to regulate the immunosuppressive function of MDSCs.In vitro experiments,GSK650394,an inhibitor of SGK1,was added to the culture system of MDSCs to detect the inhibitory effect of MDSCs on the proliferation of CD4+ T cells,related indicators of glucose metabolism,and the activity and expression of immunosuppressive effector molecules.(2)The online CpG island prediction software was used to detect the promoter region of SGK1.MDSCs derived from the spleen of tumor-bearing mice were sorted by magnetic beads.Three groups were set up: control group,lactic acid treatment group and pH 6.7 group.The combination of sodium bisulfite sequencing(BSP)and methylation specific PCR(MSP)was used to detect whether lactic acid could down-regulate the DNA methylation level of SGK1.In order to further verify that it is indeed lactic acid that caused the change of SGK1 methylation level.MDSCs isolated from the spleen in tumor-bearing mice were cultured in normoxia and hypoxia culture systems,and after adding FX-11,an inhibitor of lactate dehydrogenase LDHA,the concentration of intracellular lactic acid was detected,and qRT-PCR and MSP detects the mRNA level and methylation level of SGK1.(3)To explore the molecular mechanism of lactate regulating SGK1 methylation level.In vitro immunomagnetic beads were used to sort MDSCs from the spleen of tumor-bearing mice,add lactic acid and culture for 24 hours.Collecting MDSCs to extract total RNA and protein,and detecting the expression level of TET2 by Western blot and qRT-PCR.In addition,siTET2 was added to the culture system to detect the changes of SGK1 mRNA level and methylation level by qRT-PCR and MSP.Chromatin immunocoprecipitationmethod(Ch IP)was used to investigate whether TET2 bound to the transcription start site region of SGK1 after lactic acid treatment,thus affecting the methylation level of SGK1.(4)In order to explore the effect of TET2-regulated SGK1 demethylation on the immunosuppressive function of MDSCs.In vitro experiments,after adding TET2 small interfering RNA to the culture system,the inhibitory function of MDSCs on T cells and the activity of immune effector molecules Arg-1 and iNOS were tested.In vivo experiments we divided wild-type mice into a control group(siNC-MDSCs group)and a siTET2-MDSCs group.MDSCs transfected with siNC/siTET2 and mouse lung cancer Lewis cells were injected subcutaneously at a ratio of 1:1.Observe the growth process of the tumor and record the size of the tumor.When the the tumor was around 1 cm,the mice were euthanized,the tumor were stripped and weighed.The proportion of Th1 and CTL in lymph nodes,spleen and tumor tissues was determined by FCM.Results:(1)Lactic acid could enhance the immunosuppressive function of MDSCs.Whole transcriptome sequencing analysis showed that After lactic acid treatment,the expression of a large number of genes was found to be altered.Among them,the transcription level of SGK1 was significantly increased(P<0.05).After adding GSK650394,an inhibitor of SGK1,to the culture system,the activity and expression levels of immunosuppressive effect molecules Arg-1,iNOS and ROS secreted by MDSCs were significantly reduced(P<0.05).In addition,The ability to take up glucose and to produce lactic acid reduced significantly(P<0.05),and the expression of genes related to the glucose metabolism pathway reduced significantly(P<0.05),suggesting that lactic acid participates in regulating the immunosuppressive function of MDSCs and affects the glucose metabolism level of MDSCs by up-regulating SGK1.(2)It was found online through the website that there are CpG islands and multiple methylation sites in the region of the transcription start site of SGK1.Combined with BSP and MSP testing,it was found that lactic acid could reduce the methylation level of SGK1(P<0.05).Cultivate MDSCs in normoxic and hypoxic culture system and add FX-11.The results show that the intracellular lactic acid is significantly reduced after FX-11 is added.And consistent with the change,the methylation level of SGK1 was also significantly increased,and the mRNA level was significantly reduced(P<0.05),further indicating that lactic acid can indeed reduce the methylation level of SGK1.(3)The expression of TET2 in MDSCs derived from the spleen of tumor-bearing mice was higher than that of TET1 and TET3(P<0.05),and the expression of TET2 was significantly increased after lactic acid treatment(P<0.05).After interference with TET2,the mRNA level of SGK1 decreased(P<0.05)and the methylation level of SGK1 increased,suggesting that lactic acid regulates the demethylation of SGK1 through TET2.The Ch IP experiment shows that TET2 can bind to the transcription initiation site region of SGK1,further indicating that lactic acid can increase the expression of TET2,and TET2 further binds to the transcription initiation site of SGK1 to mediate the demethylation of SGK1.(4)After inhibiting TET2,the immunosuppressive function of MDSCs and the secreted immunosuppressive effector molecules are significantly down-regulated(P<0.05),suggesting that TET2 is involved in regulating the immunosuppressive function of MDSCs.In vivo experiments,the tumor growth in the siTET2-MDSCs group was significantly slowed down,and the volume and weight of the tumor were significantly reduced(P<0.05).The proportion of CTL in the draining lymph nodes in the control group was 6.945±0.145%,and the proportion of CTL in the tumor tissue was 0.525±0.025%;the proportion of CTL in the draining lymph nodes in the siTET2-MDSCs group was 8.385±0.145%.The proportion of CTL in tumor tissue was 0.65±0.01%,which was higher(P<0.05).Conclusion: Lactic acid in the tumor microenvironment enhances the immunosuppressive function of MDSCs through TET2-mediated demethylation of SGK1.