Characteristics And Role Of Myeloid-derived Suppressor Cells In Plasmodium Yoelii Infection

BackgroundMalaria,a tropical disease transmitted by the female anopheles mosquito,with a total of more than 200 million infections and 600,000 deaths by 2021,malaria remains a major public health problem today.Of the five genera of Plasmodium that can infect humans,Plasmodium falciparum(P.falciparum)is the absolute dominant strain and the most deadly of them all.P.falciparum is responsible for more than 90%of malaria infections and deaths worldwide.At the beginning of Plasmodium infection,innate immune cells,mainly DCs(Dendritic cells)and macrophages,promote the activation of adaptive immune response by ingesting,processing and presenting Plasmodium-associated antigens and producing cytokines such as IFN-γ and IL-6.Th1 immune response mediated by the activation of CD4+ T cells is the main force against Plasmodium infection is the main force,and the process of regulation of Th1 cells largely determines the outcome of infection.Myeloid-derived suppressor cells(MDSCs)and regulatory T cells(Tregs),which have immunosuppressive capacity,are widely present in malaria infection,and their strong immunomodulatory ability on T cells may be an important reason for Plasmodium immune evasion,which resulting in the inability of the host to effectively clear Plasmodium.MDSCs are a highly heterogeneous population of cells derived from myeloid progenitors that exist as immature cell populations in pathological conditions such as tumors and chronic inflammation,and are mainly characterized by their ability to suppress T-cell responses.In mice,MDSCs can be divided into two subgroups,CD11b+Ly6G+Ly6Clow PMN-MDSCs and CD11b+Ly6Glow Ly6C+ M-MDSCs,based on the expression of Ly6 G and Ly6 C on their surface.MDSCs exert their immunosuppressive ability in different disease models through different mechanisms and are mediated by related subpopulations.MDSCs have been shown to be involved in the suppression of T cell immunity in Plasmodium falciparum infection and to be mutually regulated and promoted with Th17 cells in Plasmodium berghei-infected mice.However,the characteristics and role of MDSCs in Plasmodium yoelii infection in C57BL/6 mice are not clear.ObjectivesMDSCs are involved in the development of tumors and some infectious diseases by exerting immunosuppressive activities,while the regulatory role of MDSCs in Plasmodium infection and the related molecular mechanisms have not been systematically investigated.Elucidating these specific signals can further enrich the biological functions of MDSCs and provide some theoretical basis for malaria prevention and control and target therapy.Methods1.A mouse model of Plasmodium yoelii NSM(P.yoelii-NSM)strain infection was constructed.2.Flow cytometry was applied to detect the distribution of MDSCs after Plasmodium infection and the associated molecular characterization.3.To detect the proliferation of T cells by flow cytometry after sorting out post-infection splenic MDSCs by anti-Gr-1 magnetic beads and co-culture with CFSE-labeled splenocytes.4.To detect the ability of infected splenic MDSCs to secrete cytokines by using flow cytometry and real-time quantitative polymerase chain reaction(RT-qPCR).5.Flow cytometry was applied to study the ability of infected mouse serum to induce MDSCs in vitro.6.The factors responsible for the induction of MDSCs in the serum of infected mice were studied by RT-qPCR and Enzyme Linked Immunosorbent Assay(ELISA).7.To determine the pathway of immunosuppressive activity of MDSCs in Plasmodium infection by measuring arginase activity and Arginase-1(Arg-1)expression in MDSCs after infection.8.The arginase inhibitor Nor-NOHA was used to confirm the immunosuppressive effect of MDSCs mediated by the arginase pathway.9.Detection of molecules associated with proliferation and differentiation and apoptosis in post-infected MDSCs by RT-qPCR.10.Detect apoptosis levels in post-infected MDSCs by flow cytometry.11.Flow cytometry and Western Blot(WB)were used to detect the phosphorylation level of STAT3 in infected-induced MDSCs.12.The role of STAT3 in the regulation of proliferation and function of MDSCs in Plasmodium infection was further confirmed using the phosphorylation inhibitor of STAT3,JSK-124.13.To study the role of MDSCs in Plasmodium infection by specific depletion of MDSCs by anti-Gr-1 antibody.14.To further investigate the role played by MDSCs and the regulation of immune response after Plasmodium infection by depletion of MDSCs with 5-Fluorouracil(5-FU).Results1.A mouse model of Plasmodium infection was successfully constructed.2.On the eighth day of Plasmodium infection,the percentage and absolute number of MDSCs in the spleen,bone marrow,peripheral blood and lung were significantly increased,the expression of programmed cell death 1 ligand 1(PD-L1)was elevated,and the proportion of infection-induced M-MDSCs subpopulation was increased.3.Plasmodium infection-induced MDSCs suppressed T cell proliferation in a concentration-dependent manner.4.The ability of MDSCs to secrete IL-6 and IL-10 was enhanced after infection.5.Infected mouse serum was able to induce the expansion of MDSCs in vitro and inhibit the maturation of other myeloid cells.6.The induction of MDSCs by infected mouse serum was associated with IL-6,IL-10 and GM-CSF.7.Arginase activity and Arg-1 expression were significantly elevated in MDSCs induced by in vivo infection and in MDSCs induced by serum in vitro.8.The arginase inhibitor Nor-NOHA was able to impair the inhibitory ability of MDSCs on T cell proliferation.9.The expression levels of Bcl-2 and HIF-1α were significantly upregulated in MDSCs after infection.10.The level of apoptosis in MDSCs was significantly decreased after infection.11.Both in vivo infection and in vitro serum stimulation induced an increase in STAT3 phosphorylation levels in MDSCs.12.The STAT3 phosphorylation inhibitor JSK-124 inhibited in vitro serum-induced expansion of MDSCs,increased apoptosis of MDSCs,weakened the suppressive activity of MDSCs on T cells,and suppressed arginase activity and Arg-1 and Bcl-2 expression in MDSCs.13.Anti-Gr-1 antibody successfully cleared most MDSCs and promoted recovery from Plasmodium infection.14.5-FU successfully cleared most of the MDSCs and promoted the recovery of Plasmodium infection and immune response.ConclusionsIn this study,we found that Plasmodium infection can promote the process of infection by activating the JAK2/STAT3 signaling pathway to upregulate the expression of the anti-apoptotic gene Bcl-2 and the functional gene Arg-1 in MDSCs,thereby mediating the expansion of MDSCs after infection as well as exerting immunosuppressive activity.