A P300 Mediated Acetylation Of C/EBP? Controls MDSCs Immunosuppression Function

Objective:In the following research,we revealed the role of P300 protein and CCAAT/enhancer-binding protein beta(C/EBP?)acetylation in the regulation of suppressive activity of myeloid-derived suppressor cells(MDSCs)and discussed the possible molecular mechanism.We tried to provide some new experimental evidence for immunotherapeutics programs targeting MDSCs.Methods:(1)First,we subcutaneously injected Lewis cells to construct a lung cancer transplanted tumor model.MDSCs derived from the spleen of tumor-bearing mice(TB-spleen-derived MDSCs)were isolated and purified by immunomagnetic beads method.The purity of MDSCs was determined by FCM.Then we used Co-Immunoprecipitation(CO-IP) technique to detect the C/EBP?acetylation level in TB-spleen-derived MDSCs.CO-IP was used to detect whether C/EBP?and P300 were combined with each other.At the same time,immunofluorescence technique was used to detect whether C/EBP?and P300 are co-localized.After treatment of P300 specific inhibitor C646,type I histone deacetylases inhibitors(HDACi)CI994,HDAC1,2,3 specific inhibitors Pyroxamid,Santecruzamate A,RGFP966 for 24 h,CO-IP was used to detect the C/EBP?acetylation in TB-spleen-derived MDSCs.(2)Western-blot technology was used to detect the protein expression level of P300 in WT-spleen-derived MDSCs and TB-spleen-derived MDSCs.And CO-IP was used to detect the C/EBP?acetylation in MDSCs derived from different sources at the same time.WT-spleen-derived MDSCs were cultured with tumor supernatant from lewis cell for 48hours to detect P300 expression.We added mouse IL-6R?antibody to the culture system to block IL-6 signal,then added tumor culture supernatant.After that,we detected P300expression again.TB-spleen-derived MDSCs were treated with IL-6 at different concentrations for 48 h.After that,Western-blot was used to detect the expression of P300in MDSCs,while CO-IP was used to detect the C/EBP?acetylation in MDSCs.(3)C646-treated MDSCs were added to the CD4~+T cell proliferation culture system and co-cultured for 3 days.To observe the immunosuppressive function of MDSCs on CD4~+T cells,we detected the proliferation ability of CD4~+T cells by CFSE staining.TB-spleen-derived MDSCs were treated with P300 specific inhibitor C646 for 24h.After C646 treatment,qRT-PCR was used to detect transcription levels of MDSCs effector molecules Arg-1,iNOS.Arginase Assay kit was used to detect Arg-1 activity.Bone marrow cells from na?ve mice were differentiated in vitro into MDSCs for 3 days,cultured with 20ng/ml IL-6 and 20ng/ml GM-CSF.P300 inhibitor C646 was added on the first day simultaneously.Cells of each group were harvested every day to measure the Arg-1 activity.Mouse differentiated MDSCs were further stained for flow cytometry analysis on the third day.In addition,GV366-Cebpb-HA and GV141-Ep300-Flag overexpression vectors were constructed and co-transfected with the reporter gene vector GV238-Arg1-promoter-Luc into HEK293T cells to detect luciferase activity and verify the C/EBP?regulation of target gene Arg-1 in the presence of P300.(4)WT mice were divided into PBS group,MDSCs group,DMSO-MDSCs group and C646-MDSCs group,and were injected with 1×10~6 Lewis cells.After the tumors grew to the size of soybeans at Day8,gemcitabine was pretreated for 1 week to eliminate endogenous MDSCs.Then,according to different groups,each mouse was injected with PBS,1×10~6 MDSCs,1×10~6 DMSO-treated MDSCs and 1×10~6 C646-treated MDSCs at multiple points in the tumor tissues.We constantly monitored the tumor growth of different groups.Finally,the mice were sacrificed on the 28th day after the subcutaneous injection of lewis cells,and tumor nodules were completely stripped for weighing.Results:(1)The sorting purity of TB-spleen-derived MDSCs was above 90%,which is enough for later experiments.C/EBP?was acetylated in TB-spleen-derived MDSCs.Becides,the CO-IP test and immunofluorescence technique confirmed that C/EBP?and P300 combined with each other in TB-spleen-derived MDSCs.After treatment with P300 inhibitor C646for 24 hours,CO-IP technology detected a decrease of C/EBP?acetylation in TB-spleen-derived MDSCs.After treatment with Type I HDAC inhibitor CI994 or HDAC2 inhibitor Sanacruzamate A for 24 hours,CO-IP technology detected an increase of C/EBP?acetylation in TB-spleen-derived MDSCs.The results showed that C/EBP?was acetylated in TB-spleen-derived MDSCs,and may be regulated by P300 and HDAC2.(2)Compared with WT-spleen-derived MDSCs,the expression of P300 in TB-spleen-derived MDSCs was increased,accompanied by the upregulation of C/EBP?acetylation.We used the tumor supernatant from lewis cells to treat WT-spleen-derived MDSCs for 48 hours,and the expression of P300 was increased.However,after using mouse IL-6R?antibody,lewis tumor cell culture supernatant was added and P300 expression no longer significantly increased.After IL-6 treatment for 48 hours,the expression of P300 in TB-spleen-derived MDSCs was increased,accompanied by the upregulation of C/EBP?acetylation.The above results indicated that IL-6 from the tumor microenvironment can upregulate the expression of P300 in TB-spleen-derived MDSCs,possibly regulating C/EBP?acetylation.(3)C646-pretreated TB-spleen-derived MDSCs were co-cultured with CD4~+T cells for 3days.CFSE staining showed that CD4~+T cells of C646-pretreated group had a higher proliferation capacity than the DMSO control group.The result indicated that after specifically inhibiting P300 in MDSCs,the immunosuppressive effect of MDSCs to prevent CD4~+T cell proliferation was attenuated.Inhibition of P300 with C646 decreased mRNA levels of Arg-1 and decreased Arg-1 activity in MDSCs.The inhibition of P300 in vitro-generated MDSCs did not change whether total MDSCs frequencies nor frequencies of PMN-MDSCs and M-MDSCs,but Arg-1 activity was decreased significantly.In addition,with database prediction and literature search,we predicted that C/EBP?bound to the promoter region of the target gene Arg-1 and directly regulated the transcription of Arg-1.The luciferase reporter gene experiment showed that P300 promoted the transcriptional activation activity of C/EBP?,which in turn promoted the transcription of the target gene Arg-1.(4)Compared with DMSO-MDSCs control group,the development of tumors in C646-MDSCs group was delayed,and the volume and weight of tumors were reduced.Conclusion:C/EBP?is acetylated in MDSCs,regulated by acetyltransferase P300 and deacetyltransferases HDAC2.Tumor-derived IL-6 induced P300 expression,promoting C/EBP?acetylation.P300 regulated C/EBP?acetylation,thereby upregulating Arg-1transcription and affecting immunosuppressive function of MDSCs.