Research On The Mechanism Of Histone Lactylation Up-regulating CD38 To Enhance Function Of MDSCs

Objective:To explore the role of Lactic acid(LA)in promoting Myeloid-derived suppressor cells(MDSCs)by up-regulating CD38 expression in Tumor microenvironment(TME).This study provides new ideas and targets for anti-tumor immunotherapy targeting MDSCs.Methods:(1)MDSCs derived from the spleen of tumor-bearing mice were sorted by magnetic beads,and the changes in immunosuppressive function and immunosuppressive molecules of MDSCs treated with lactic acid were detected.Transcriptome sequencing(RNA-seq)analysis was used to screen and verify the differentially expressed genes related to the function of MDSCs after lactic acid treatment,and CD38 was identified as the key gene in lactate-regulated MDSCs function.Gepia website was used to predict the expression of CD38 in lung cancer tissues and its relationship with the survival rate of lung cancer patients.The expression level of CD38 in lung cancer tissues was verified by immunohistochemistry,and the expression level of CD38 in MDSCs in lung cancer tissues was verified by immunofluorescence.In addition,MDSCs in tumor tissues of tumor-bearing mice were sorted by Flow cytometry,and the expression of CD38 in tumor tissues and spleen MDSCs was detected by real-time fluorescent quantitative PCR(qRT-PCR)and flow cytometry(FCM).The expression of CD38 in tumor tissues of mice with different tumor-bearing weeks was also detected by flow cytometry(FCM).(2)To explore the regulation and mechanism of CD38 on the immunosuppressive function of MDSCs.In vitro experiments,CD38 inhibitor CD38 inhibitor 1 or siCD38 was added into the culture system of MDSCs to detect the immunosuppressive function of MDSCs and the changes of immunosuppressive molecules.In vivo,wild-type mice were divided into control group(siNC-MDSCs group)and experimental group(siCD38-MDSCs group).MDSCs transfected with siNC/siCD38 were injected into the tumor on days 14 and 21.The tumor diameter of mice was measured according to the preset time,and the tumor growth curve was drawn.The mice were euthanized when the tumor diameter was about 1 cm,and the proportions of CD4+IFN-γ+ T cells(Thl)and CD8+IFN-γ+ T cells(CTL)in the lymph nodes,spleen and tumor tissues of the tumor bearing mice were detected by FCM.(3)To explore the molecular mechanism of lactic acid up-regulation of CD38 expression.MDSCs from spleen of tumor bearing mice were sorted by immunomagnetic beads.After lactic acid treatment,the lactylation level of total histone and the level of H3K181a(histone H3,lysine site 18)and H4K121a(histone H4,lysine site 12)were detected by Western blot.In order to further verify the regulation of CD38 by histone lactylation,two different glycolysis inhibitors were added into the culture system of MDSCs:After A certain period of treatment with FX11,an inhibitor of lactate dehydrogenase A(LDHA),and 2-DG,an inhibitor of hexokinase(HK),the expression level of CD38 was detected by qRT-PCR,Western blot and FCM.The level of histone lactylation,H3K181a and H4K121a were detected by Western blot.In addition,Chromatin immunoprecipitation qPCR(ChIP-qPCR)was used to detect the enrichment of H3K181a and H4K121a in the transcription initiation region of CD3 8 after lactic acid treatment.(4)Explore histone lactylation modification enzymes that mediate CD38 expression.In order to identify the modified enzyme mediating CD38 promoter histone lactylation,p300-specific inhibitor C646 was added to the culture system of MDSCs or p300 was overexpressed by plasmid transfected with p300.In order to explore the histone delactylation enzymes involved in the regulation of CD38 in MDSCs,HDAC1-3 and SIRT1 inhibitors were added into the culture system of MDSCs,respectively.In order to study the histone lactylation reading protein involved in the regulation of CD38,the BRD4 inhibitor JQ1 was added into the culture system of MDSCs.The changes of CD38 expression and histone lactylation level were detected by qRT-PCR,FCM and Western blot.(5)To explore the mechanism of CD38 regulating the immunosuppressive function of MDSCs.Since CD38 is an enzyme that consumes NAD+,NAD+levels in tumor tissues of mice with different weeks of tumor bearing were first detected by NAD+detection kit.Then,in order to understand the influence of lactic acid on NAD+,lactic acid was added into the culture system of MDSCs for a certain period of time,and NAD+detection kit was used to detect the intracellular NAD+ content.Furthermore,Morpheus online website was used to analyze the expression of NAD+related genes in the RNA-seq results of MDSCs treated with lactic acid.In addition,in order to verify the effect of CD38 on the intracellular NAD+content,after CD38 inhibitor was added into the culture system of MDSCs for a certain period of time,cells were collected and NAD+detection kit was used to detect the intracellular NAD+content.In order to clarify the effect of NAD+on the immunosuppressive function of MDSCs,after MDSCs were treated with a certain concentration of NAD+precursor NMN for a certain period of time,cells were collected to detect the intracellular NAD+ content,immunosuppressive function and immunosuppressive molecule changes of MDSCs.Results:(1)The inhibitory effect of MDSCs on CD4+T cell proliferation was significantly enhanced in lactic acid treatment group.The activity of immunosuppressive molecule Argl(Argininase 1)and the expression of ROS were significantly increased(P<0.05),suggesting that lactic acid could enhance the immunosuppressive function of MDSCs.The results of RNA-seq showed that,compared with the control group,the expression of CD38 in lactic acid treatment group was significantly increased(P<0.05).In addition,the expression of CD38 in MDSCs in tumor tissue of tumor-bearing mice was significantly higher than that in spleen and increased with the extension of tumor bearing time(P<0.05),suggesting that lactic acid in TME may enhance the immunosuppressive function of MDSCs by up-regulating CD3 8.(2)Compared with the control group,the inhibitory effect of MDSCs on CD4+T cell proliferation was significantly decreased after addition of CD38 inhibitor CD38 inhibitor 1 or transfection with siCD38,and inhibition of CD38 could partially relieve the inhibitory effect of MDSCs on CD4+T cell proliferation caused by lactic acid.It is suggested that the enhancement of immunosuppressive function of MDSCs by lactic acid is partly mediated by CD38.In addition,compared with the control group,the expression of immunosuppressive molecule Argl of MDSCs was significantly decreased after addition of CD38 inhibitor CD38 inhibitor 1 or siCD38(P<0.05).The in vivo experimental results showed that compared with the siNC-MDSCs group,the spleen size,tumor volume and weight of siCD38-MDSCs group were significantly reduced,and the proportions of CD4+IFN-γ+ T cells and CD8+IFN-γ+ T cells in tumor tissue were significantly increased(P<0.05).It is suggested that CD38 in MDSCs has certain damage to the anti-tumor immunity of tumor-bearing mice,and interference with CD38 in MDSCs can delay the tumor progression in mice.(3)After lactic acid was added into the culture system of MDSCs,the lactylation of total histone protein,H3K181a and H4K121a levels were significantly increased,and the mRNA level and protein level of CD38 were significantly increased(P<0.05).In line with this,after the addition of glycolysis inhibitors FX-11 and 2-DG,the lactylation level of total histone protein,the levels of H3K181a and H4K121a were significantly decreased,and the mRNA level and protein level of CD38 were also significantly decreased(P<0.05).It suggested that lactic acid might up-regulate the expression of CD3 8 by histone lactylation.ChIP-qPCR results showed that H3K 181a was enriched in the transcription initiation region of CD38 after lactic acid treatment.It indicated that lactic acid could up-regulate the expression level of CD38 through H3K181a.(4)When C646 was added into the culture system of MDSCs,the lactylation level of histone was significantly decreased,and the expression of CD38 was also significantly decreased(P<0.05).Overexpression of p300 could promote the lactylation level of histone and the expression of CD38(P<0.05),suggesting that p300 mediated the lactylation level of CD38 histone.The CD38 level and histone lactylation level of MDSCs were significantly increased after MS275 was added into the culture system of MDSCs(P<0.05).The levels of histone lactylation and CD38 were decreased after NAM treatment(P<0.05).Therefore,HD AC 1-3 is a delactylation enzyme that regulates CD38 expression in MDSCs.After JQ1 treatment,the level of histone lactylation and the expression of CD38 in MDSCs were significantly decreased(P<0.05),suggesting that BRD4 may be the reading protein of histone lactylation at the CD38 promoter.(5)The content of NAD+in tumor tissue of 5-week tumor bearing mice was significantly lower than that of 3-week tumor bearing mice(P<0.05),suggesting that the level of NAD+in TME decreased with the progression of tumor.After lactic acid treatment,the content of NAD+in MDSCs decreased.The results of bioinformatics analysis showed that CD38 of NAD+related genes had the most significant changes after lactic acid treatment of MDSCs,suggesting that CD38 is the most important enzyme causing NAD+consumption under lactic acid.In addition,the intracellular NAD+content of MDSCs was decreased after CD38 inhibitor treatment.After treatment with NMN,the inhibitory ability of MDSCs on CD4+T cells was decreased and the activity of immunosuppressive molecule Argl of MDSCs was significantly decreased(P<0.05),suggesting that a higher level of NAD+was not conducive to the immunosuppressive function of MDSCs.Conclusion:In the tumor microenvironment,lactic acid can up-regulate CD38 expression through H3K181a,thereby depleting intracellular NAD+and enhancing the immunosuppressive function of MDSCs.