The Effect Of Resveratrol On The Number And Function Of G-MDSCs And Its Role In Anti-tumor Immunity

Objective:Granulocytic myeloid-derived suppressor cells(G-MDSCs)will accumulate extensively and inhibit the anti-tumor immune response of the host during the development of tumor.Resveratrol is a natural plant antitoxin with various biological activities.Its mechanisms of resistance on tumors are multidirectional,but its effect on G-MDSCs is not yet clear.To determine whether resveratrol can attenuate tumor growth by suppressing the expansion of G-MDSCs in a murine Lewis lung carcinoma model;splenic G-MDSCs were stimulated with resveratrol in vitro,to explore the effect of resveratrol on the apoptosis and migration of G-MDSCs,to observe the changes of immunosuppressive function of G-MDSCs and investigate the role of STAT3.Method:(1)Lewis lung carcinoma(LLC)tumor-bearing mice models were established by subcutaneous injection of Lewis cells.And then,the mice were randomized into 2groups,they were orally treated with PBS and resveratrol respectively every day for 3 weeks.To observe the growth of tumor,the volumes and weights of tumors were measured and calculated;survival of mice was also monitored every 2 days;after sacrificing the mice,the proportions of total MDSCs,G-MDSCs,M-MDSCs and CD8+T cells in spleens,tumors and draining lymph nodes were examined by flow cytometry(FCM).(2)Murine G-MDSCs which were sorted from the splenocytes of LLC tumor-bearing mice by magnetic activated cell sorting(MACS)were treated with resveratrol in various concentrations in vitro,then flow cytometry was conducted to test the apoptosis of G-MDSCs by Annexin-V-APC/7-AAD dual-labeling technique;the expressions of Bcl-2,Bax,caspase3 and cleaved-caspase3 were also analyzed by Western-blot.(3)Lewis cells were stimulated with resveratrol in indicated concentrations for 48 h in vitro,after that,Lewis cells were sequentially cultured with serum-free DMEM medium for 24 h,and the transwell assay was conducted to explore the role of LLC cell culture supernatant modified by resveratrol in inducing the migration of G-MDSCs.(4)After treatment of resveratrol in vitro,the protein levels of Arg-1 and the phosphorylation of STAT3 in sorted G-MDSCs were determined by Western-blot;FCM was used to examine the expressions of ROS in G-MDSCs;CFSE and ELISA were performed respectively to detect the proliferation of CD8+T cells and its production of Granzyme B.Results:(1)Compared with the control group,the tumor growth was significantly slower after resveratrol treatment,especially at day 16,day 18 and day 20(P<0.05);tumor weights were decreased(P<0.01);mice in experimental group survived longer(P<0.05)after resveratrol administration for 3 weeks;the results of FCM indicated that the percentages of total MDSCs infiltrated in spleens of LLC tumor-bearing mice showed no marked difference,while its proportions in tumor tissues decreased(P<0.05),the percentages of G-MDSCs both existed in spleens and in tumor tissues dropped in the presence of resveratrol therapy(P<0.01,P<0.05),but the proportions of M-MDSCs showed no significant difference;the frequencies of CD8+T cells in spleens,tumor tissues and draining lymph nodes were significantly higher than those in control group(P<0.05,P<0.01,P<0.01).(2)Culturing G-MDSCs sorted from LLC tumor-bearing mice with resveratrol in different concentrations for 6h and 24 h separately,the apoptotic program of G-MDSCs was triggered;when the concentration of resveratrol equaled 100?M,the early and late apoptosis of G-MDSCs in experimental group were(21.27±0.844)% and(29.93±1.870)%,which were respectively higher than(11.86±1.648)% and(13.01±1.286)% in control group;the results of immunoblotting showed a significantly downregulation in Bcl-2 of G-MDSCs and upregulation in Bax,cleaved-caspase3 in resveratrol treatment group.(3)Comparing the resveratrol group with control group,the transwell assay revealed that LLC cell culture supernatant modified by resveratrol dreamtically inhibited the migration of G-MDSCs in a concentration-dependent manner.(4)Compared with the control group,the results of Western-blot displayed that the protein levels of Arg-1 and the expressions of phosphorylated STAT3 in G-MDSCs were strikingly declined after resveratrol exposure,especially at dose of 100?M;the analysis of FCM showed that the production of ROS was significantly decreased(P<0.001),the proliferation of CD8+T cells was enhanced(P<0.05)and its secretion of Granzyme B was markedly upregulated(P<0.05).Conclusion:(1)Treatment with resveratrol significantly delays LLC tumor progression,which is associated with the effect of resveratrol on inhibiting G-MDSCs expansion.(2)Resveratrol suppresses G-MDSCs survival directly in vitro by inducing its apoptosis.(3)Resveratrol lessens G-MDSCs migration to tumor tissues indirectly in vitro by modifying the tumor microenviroment.(4)Resveratrol impedes G-MDSCs immunosuppressive function through reducing phosphorylation of STAT3 in vitro.