Experimental Study Of HDAC2 Regulating The Immunosuppressive Function Of Mdscs In Tumor-bearing Mice

Objective:In this study,we tried to explore the effect of histone deacetylase 2(HDAC2)on the immunosuppressive function of myeloid-derived suppressor cells(MDSCs)and investigate its possible mechanism.We aimed to provide new ideas for understanding the biological function of MDSCs.Methods:(1)We used immunomagnetic bead sorting method to sort wild-type mice and Lewis tumor-bearing mice from spleen CD11b~+Gr1~+MDSCs and detected MDSCs’purity by flow cytometry.Western blot was used to detect the expression of HDAC2in the spleen MDSCs of wild-type mice and Lewis tumor-bearing mice.At the same time,we used western blot to detect the expression of HDAC2 in the spleen PMN-MDSCs and M-MDSCs of Lewis tumor-bearing mice.(2)In order to explore whether HDAC2 has a regulatory effect on the immunosuppressive function of MDSCs,we set four HDAC2 specific inhibitor——santacruzamate A concentrations:0μM,5μM,10μM,15μM.MDSCs were treated with different concentrations of santacruzamate A for 24 hours,and then detected the activity of Arg-1 and content of NO by colorimetry and griess method,respectively.After determining the concentration of santacruzamate A based on the results of the first two experiments,MDSCs were treated with this concentration of santacruzamate A for 24 hours and co-cultured with CD4~+T cells for 72 hours.At last,we used CFSE fluorescence staining method to detect the proliferation of CD4~+T cells.(3)The histone interacting with HDAC2 was found in the Bio Grid database,and the immunoprecipitation test was used to verify whether HDAC2 binds to the histone in MDSCs.After MDSCs were treated with santacruzamate A,the levels of histone acetylation sites were detected by Western Blot.Previous literature has shown that histone H4 can regulate gene transcription of the functional molecule c-Myc of MDSCs.After MDSCs were treated with santacruzamate A,c-Myc gene transcription levels were detected by real-time fluorescent quantitative reverse transcription PCR(q RT-PCR),and protein levels were detected by Western Blot.(4)To understand the effect of MDSCs treated with HDAC2 inhibitor on tumor cells in vivo,two groups were set up:the DMSO treatment group and santacruzamate A treatment group.The MDSCs from the spleen of Lewis tumor-bearing mice were sorted by immunomagnetic beads method.MDSCs were treated with DMSO and santacruzamate A for 24 hours,respectively.The mice in the control group were subcutaneously injected with a cell mixture of 1×10~6 Lewis cells and 1×10~6DMSO treated MDSCs for 24 hours.The mice in the santacruzamate A group were subcutaneously injected with a cell mixture of 1×10~6 cells and 1×10~6 santacruzamate A treated MDSCs for 24 hours.The tumor growth process of the mice was observed.After 24 days,the mice were sacrificed to strip the tumor and weighed.We used to flow cytometry to detect the ratio of CD3~+CD4~+IFN-γ~+T(Th1)cells and CD3~+CD8~+IFN-γ~+T(CTL)cells in the local draining lymph nodes,spleen and tumor tissues of the two groups of mice.Results:(1)The purity of CD11b~+Gr1~+MDSCs from spleen of Lewis tumor-bearing mice selected by immunomagnetic bead method was higher than 95%,which met the requirements of subsequent experiments.HDAC2 was highly expressed in spleen-derived MDSCs from Lewis tumor-bearing mice compared with wild-type mice,while HDAC2 was not expressed in wild-type mice.Both PMN-MDSCs and M-MDSCs from spleen-derived Lewis tumor-bearing mice expressed HDAC2.(2)After treating MDSCs with different concentrations of santacruzamate A for 24hours,the activity of Arg-1 was significantly reduced and it was the most significant at 15μM(p<0.001).The NO content and expression of ROS did not change.15μM santacruzamate A treated MDSCs for 24 hours and co-cultured with CD4~+T cells,the proliferation ability of CD4~+T cells was higher than the control group,indicating that the reduction of HDAC2 activity would inhibit the immunosuppressive function of MDSCs,that is,HDAC2 would enhance the immunosuppressive function of MDSCs.(3)Co-immunoprecipitation experiment proved that HDAC2 binds to histone H4 in MDSCs.After MDSCs were treated by 15μM santacruzamate A for 24 hours,the acetylation levels of H4K5 and H4K16 were significantly increased compared with MDSCs treated by DMSO.The reduction of HDAC2 activity enhanced the acetylation of H4K5 and H4K16,indicating that HDAC2 deacetylated histone H4K5and H4K16 in MDSCs.The q RT-PCR results showed that c-Myc transcription level was significantly decreased after treated with 15μM santacruzamate A.Western Blot results showed that the c-Myc protein level was also significantly down-regulated after MDSCs treated with 15μM santacruzamate A.(4)Compared with the control group,mice injected with MDSCs treated with 15μM santacruzamate A for 24h and Lewis mixed cells had significantly delayed tumor growth and reduced tumor weight(P<0.001).Compared with the control group,the proportion of Th1 cells in tumor tissue in santacruzamate A group was significantly increased(P<0.05),while the proportion of Th1 cells in drainage lymph nodes and spleen was not significantly changed.The proportion of CTL cells in drainage lymph nodes and tumor tissue was significantly increased in santacruzamate A group (P<0.05),while the proportion of CTL cells in spleen was not significantly changed.Conclusions:HDAC2 was expressed in MDSCs derived from spleen of Lewis tumor-bearing mice.Inhibitor of HDAC2 could decrease the Arg-1 activity and immunosuppressive function of MDSCs,and the mechanism might be related to the enhancement of H4 histone acetylation level.