| Objective:Study the regulatory effect of STAT3 citrullination mediated by phthalyl arginine deiminase 2(PAD2)on the immunosuppressive function of polymorphonuclear myeloid suppressor cells(PMN-MDSCs),and to explore its molecular mechanism,providing new ideas for further understanding the functional changes of PMN-MDSCs in tumor-bearing mice.Methods:(1)1×106 Lewis cells were injected subcutaneously into C57BL/6 mice to construct tumor-bearing mouse model.MDSCs and their subpopulations(PMNMDSCs and MO-MDSCs)from spleen of tumor-bearing mice were separated by immunomagnetic beads method,and the purity of objective cells was analyzed by flow cytometry(FCM).PADs m RNA expressions of MDSCs were detected by q RT-PCR,and the expressions of PADs and citrullinated proteins in cells were detected by Western blot.The expression levels of PAD2 and PAD4 in PMN-MDSCs or MOMDSCs were detected by Western blot at different time points in the growth of tumorbearing mice.(2)PAD2 or PAD4 in MDSCs subsets were knocked down by transfection siRNA-PAD2 and siRNA-PAD4,respectively.The knockdown efficiency of siRNA was detected by q RT-PCR and Western blot.CD8+T cells were labeled with CFSE dye to establish a proliferation system of CD8+T cells in vitro.MDSCs subsets were added into CD8+T cell proliferation system,and the inhibitory effect of MDSCs subsets on CD8+T cell proliferation was detected by FCM.Colorimetric method was used to detect the activity of PMN-MDSCs and MO-MDSCs immunosuppressive molecules Arg-1,Griess coupling method was used to detect the activity of MO-MDSCs immunosuppressive molecules NO,and H2 DCFDA probe was used to detect the expression level of ROS of PMN-MDSCs immunosuppressive molecules.(3)Western-blot was used to compare the changes in the citrullinated modification level of the total protein of PMN-MDSCs cells before and after PAD2 knockdown,and the possible target molecules of PAD2 were screened out by molecular weight comparison and literature review.The target molecule STAT3 was enriched by immunoprecipitation experiment,the citcitline modification of STAT3 was verified by Western blot technique,and the citrulline modification site of STAT3 was identified by liquid chromatography-mass spectrometry.The combination of PAD2 and STAT3 was detected by immunoprecipitation,and the co-localization of PAD2 and STAT3 was analyzed by immunofluorescence.The changes of citrullination and phosphorylation of STAT3 after PAD2 knockdown were detected by Western blot.The Flag-PAD2,HASTAT3 and Arg1-promotor-Luc plasmids were constructed and transfected into HEK293 T cells to verify the regulation of Arg-1 transcriptional activity by citrullination modification of STAT3 by detecting luciferase activity.(4)Tumor bearing mouse model was established,and mice were divided into siRNA-PAD2 group and siRNA-NC group.siRNA-PAD2 and siRNA-NC group were respectively used to treat PMN-MDSCs in vitro,and then treated PMN-MDSCs were injected locally into the tumor of the above mice at multiple points.Each mouse was injected with 2×106 PMN-MDSCs.The tumor growth process of mice in each group was recorded and tumor growth curve was drawn.On the 28 th day,the tumor bearing mice were sacrificed,stripped of tumor tissue and weighed.The tumor tissues were digested with collagenase and cell suspensions were obtained.Flow cytometry was used to detect the proportion of infiltrated CD8+IFN-γ+CTL cells and CD4+IFN-γ+Th1 cells.Results:(1)The purity of MDSCs from spleen of tumor-bearing mice and its subgroups were all over 90%,which met the requirements of follow-up experiments.The results of q RT-PCR showed that MDSCs from spleen of tumor-bearing mice expressed m RNA of 5 PADs subtypes,among which PAD2 and PAD4 had higher expression levels.MDSCs and their subgroups PMN-MDSCs and MO-MDSCs all expressed PAD2 and PAD4 proteins by Western-blot analysis.In addition,Western-blot analysis showed that the expression levels of PAD2 protein and PAD4 protein in MDSCs from tumor tissue were significantly higher than those of MDSCs from spleen.After MDSCs were treated with tumor tissue culture supernatant(TES),the protein expression levels of PAD2 and PAD4 in cells were significantly increased.The dynamic expression levels of MDSCs and their subsets PAD2 and PAD4 in tumor-bearing mice were further detected by Western-blot.The average relative expression levels of PAD2 protein in spleen MDSCs of tumor bearing mice at the 2nd,3rd and 4th week were 0.73,0.81 and 1.02(P<0.01),the mean relative expression levels of PAD4 protein of MDSCs were 0.77,1.09 and 1.27(P<0.01).The mean relative expression levels of PAD2 protein in PMN-MDSCs were 0.35,0.63 and 1.07(P<0.001),the mean relative expression levels of PAD4 protein of PMN-MDSCs were 0.58,0.83 and 1.06(P<0.05).The mean relative expression levels of PAD2 protein in MOMDSCs were 0.66,1.03 and 1.26(P<0.05),the mean relative expression levels of PAD4 protein of MO-MDSCs were 0.9,1.03 and 1.14(P<0.05).The results showed that PAD2 and PAD4 expression levels of MDSCs and their subsets in tumor-bearing mice were significantly increased during tumor growth.PMN-MDSCs and MO-MDSCs were added to spleen of tumor-bearing mice at different time points in CD8+T cell proliferation system,and the results showed that,The percentages of CD8+T cell proliferation in PMN-MDSCs group at 2,3 and 4 weeks were 52.4±1.9%,42.3±3.72% and 33.1±0.93%(P<0.001);The percentages of CD8+T cell proliferation in MO-MDSCs group were 42.4±3.7%,33.3±1.1% and 26.9±3.4%(P<0.01).The results showed that the immunosuppressive function of PMN-MDSCs and MO-MDSCs in tumor bearing mice was enhanced with the extension of tumor bearing time.(2)After siRNA was used to knock down PAD2 or PAD4 in MDSCs subsets,FCM was used to detect the inhibition of CD8+T cell proliferation.The results showed that the proliferation rate of CD8+T cells in PMN-MDSCs with PAD2 knockdown was 52.3±2.5%,that in PMN-MDSCs with PAD4 knockdown was 40.4±1.4% and that in the control group was 39.8±1.2%,indicating that the immunosuppressant function of PMN-MDSCs with PAD2 knockdown was significantly decreased.There was no significant change in immunosuppressive function of PMN-MDSCs with PAD4 knockdown.The proliferation rate of CD8+T cells in MO-MDSCs with PAD2 knockdown was 40.5±2.9%,38.3±0.6% in MO-MDSCs with PAD4 knockdown and 35.9±3.7% in the control group,indicating no significant changes in immunosuppressive function of MO-MDSCs with PAD2 knockdown or PAD4 knockdown.After PAD2 knockdown,m RNA and protein levels of immunosuppressive molecule Arg-1 and enzyme activity levels of PMN-MDSCs were significantly decreased,and the m RNA transcription level of Arg-1 was decreased by 75%(P<0.01),protein expression level decreased by 40%(P<0.01),the enzyme activity decreased by 30%(P<0.01),these results indicate that PAD2 in PMN-MDSCs can regulate Arg-1 transcription and protein expression.The expression level of ROS,another important immunosuppressive molecule of PMN-MDSCs,did not change significantly.CD8+T cell proliferation rate did not change significantly in other groups,and PAD4 knockdown of PMN-MDSCs immunosuppressive molecules Arg-1 and ROS did not change significantly(P>0.05),PAD2 and PAD4 knockdown MO-MDSCs immunosuppressive molecules Arg-1 and NO had no significant changes(P>0.05).(3)In order to find the molecular mechanism of PAD2 regulation of Arg-1 in PMN-MDSCs,we used siRNA to knock down PAD2 protein in PMN-MDSCs,and Western blot detection showed that the level of intracellular protein citrullination was significantly reduced.Moreover,the modification level of citrullination of proteins with molecular weight above 55 k Da was significantly changed.After further analysis of key molecules in MDSCs regulating Arg-1 transcription,we speculate that STAT3 may be the target molecule catalyzed by PAD2.Next,we confirmed the citrullinated modification of STAT3 molecule by immunoprecipitation experiment.Subsequently,mass spectrometry was used to analyze citrullination modification sites of STAT3.The results showed that 13 arginine residues of STAT3 were citrullinated.The co-location of PAD2 and STAT3 was observed by immunoprecipitation,and the co-location of PAD2 and STAT3 was observed by immunofluorescence.Finally,after PAD2 knockdown,the citrullination modification level of STAT3 was reduced.These results indicate that PAD2 in PMN-MDSCs can catalyze citrullination of arginine residues on STAT3.Since the activation and function of STAT3 are mainly related to phosphorylation modification,Western blot was used to detect the phosphorylation modification level of STAT3 after PAD2 knockdown,and it was found that the phosphorylation modification level of STAT3 did not change significantly.Luciferase reporter gene assay in HEK293 T cells showed a 2.5 fold increase in transcriptional activity in the Arg-1 promoter region after PAD2 co-transfection with STAT3(P<0.001),indicating that citrullination of STAT3 can promote Arg-1 transcription.(4)The adoptive transfer of PMN-MDSCs to tumor-bearing mice showed that the ability of PMN-MDSCs to promote tumor growth was significantly weakened after PAD2 knockdown,which was manifested as a slowing of tumor growth(P<0.05)and reduced tumor weight(P<0.05).The ratio of CTL cells to Th1 cells in tumor tissues was measured by FCM.The results showed that the ratio of CD8+IFN-γ+CTL cells and CD4+IFN-γ+Th1 cells in siRNA-PAD2 group was higher than that in control group(P<0.05).The proportion of CD8+IFN-γ+CTL cells and CD4+IFN-γ+Th1 cells in siRNA-PAD2 group was 6.408±0.581% and 7.283±0.807% respectively.The proportion of CD8+IFN-γ+CTL cells and CD4+IFN-γ+Th1 cells in control group was 5.28±0.361% and 5.73±0.392% respectively.Conclusion:In the process of tumor development in tumor-bearing mice,PAD2 expression of PMN-MDSCs is increased,which promotes citrullination modification of STAT3,increases Arg-1 transcription,improves the immunosuppressor function of PMNMDSCs. |
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