Roles And Mechanisms Of Rapamycin On Myeloid-derived Suppressor Cells Regulation In Acute Graft-versus-host Disease

IntroductionMyeloid-derived suppressor cells(MDSCs)paly protective effect in acute graft-versus-host disease(aGVHD)regulation after allogenic hematopoietic stem cell transplantation(allo-HSCT).mTOR inhibitor rapamycin(RAPA)has also been demonstrated to be an effective immunosuppressor in the management of aGVHD.However,the relationship between RAPA and MDSCs in aGVHD models is unclear.Meanwhile,the effect of RAPA on different subgroups of MDSCs is also less well described.In this study,first,we established stable murine bone marrow transplantation(BMT)model.We analysised the change of quantity and function of MDSCs in peripheral blood and spleen of mice after BMT.Then we study the effect of RAPA treatment on MDSCs in vivo of the aGVHD model.Finally,how RAPA regulating different subgroups of MDSCs was researched and the related mechanisms were further studied.Our research can reveal novel mechanisms of function regulation of MDSCs and provide novel clues for aGVHD prevention and treatment.Pat1 Quantity and function change of MDSCs in vivo after BMTAim:This part will establish the murine models of BMT and aGVHD and then detect quantity and function.change of MDSCs in peripheral blood and spleen of.mice after BMT.The aim is to analysis the relationship between MDSCs and aGVHD.It can provide the clue to clarify the regulating effect of MDSCs on aGVHD.Methods:After establishing the murine models of BMT and aGVHD,we analysised aGVHD symptoms,weight curve,HE staining and anti-CD3 immunohistochemical staining of histologic section to evaluate the severity of aGVHD.We used flow cytometry(FCM)to analyze total CD11b+Gr1+ cells and two subsets of MDSCs on different time point(2 weeks,4 weeks post-BMT)in different groups(no aGVHD group,mild aGVHD group,severe aGVHD group).Then we isolated MDSCs from bone marrow cells of recipients in no aGVHD group and aGVHD group on different time point(2 weeks,4 weeks post-BMT)and cocultured with activated spleen cells of naive B6 mice for 3.5 days in vitro.The proliferation of splenocytes were detected by flow cytometry using CFSE-based assay.Furthermore,we compared regulatory T cells(Tregs)induction function of MDSCs derived from different groups.MDSCs isolated frombone marrow of different groups were cocultured with splenic CD4+T cells in vitro.Five days later,the proportion of Tregs were determined by FCM assays.Results:Balb/c mice received condition regimen of 8Gy X-ray irradiation.Then BM-TCD cells or BM-TCD plus T cells were infused to establish no aGVHD and aGVHD models,respectively.The amount of T cells determined severity of aGVHD.The results of chimerism testing on day 7 after BMT showed fully implanted in all groups.Loss of weight,alopecia,diarrhea and reduced activity could be ovserved in aGVHD group from about 16 days after BMT.We found tissue destruction and inflammatory cells infiltration in liver,lung and intestine in aGVHD group.In BM-TCD recipients,CD11b+Gr1+cells and two subsets of MDSCs accumulated in the spleen and blood at week 2 but declined gradually to the level of about 20%at week 4.But in aGVHD groups,the proportion sustained at a high level at week 4.The proportions of MDSCs in spleen were positively correlated to the severity of aGVHD.The immunosuppression function of MDSCs were impaired when aGVHD occurred.Besides,MDSCs from aGVHD models induced lower proportion of Tregs in vitro.Conclusion:We found that the proportions of MDSCs in spleen were positively correlated to the severity of aGVHD.The immunosuppression function and Treg indction ability of MDSCs were impaired in aGVHD models.Part 2 Roles and mechanisms of RAPA on MDSCs in aGVHD models Aim:In part 1 we establised the mice models of BMT and aGVHD.The aim of this part is to investigate the roles and mechanisms of RAPA treatment on MDSCs in aGVHD models.Methods:Balb/c recipient mice were divided into four groups:BM-TCD,BM-TCD plus T cells,RAPA treatment and vehicle treatment.We compared aGVHD severity,weight curve and anti-CD3 immunohistochemical staining of histologic section between four groups to investigate whether RAPA can be effective to improve the severity of aGVHD.To investigate the impact of RAPA on MDSCs in vivo,we used FCM to analyze total CD11b+Gr1+ cells and two subsets of MDSCs on different time point after BMT.We next investigated impact of RAPA on the suppression function of G-MDSCs in BMT models using CFSE-based assay.mRNA level of immunologic suppressor factors were detected by qPCR in G-MDSCs.To determine whether Argl and iNOS play key role in G-MDSCs suppression function,we applied L-NMMA,an inhibitor of iNOS,and nor-NOHA,an inhibitor of Argl,to the co-culture system and then analysis the suppression function using CFSE-based assay.Furthermore,we compared Tregs induction function of G-MDSCs derived from four groups in vitro.G-MDSCs isolated from bone marrow of different groups were cocultured with splenic CD4+T cells in vitro.Five days later,the proportion of Tregs were determined by FCM assays.Results:RAPA treatment group showed reduced weight loss,alopecia and less tissue destruction and inflammatory cells infiltration in liver,lung and intestine.In BM-TCD recipients,CD11b+Gr1+cells and two subsets of MDSCs accumulated in the spleen and blood at week 2 but declined gradually to the level of about 20%at week 4.But in recipients of BM-TCD plus T cells and vehicle treatment group,the proportion of CD11b+Gr1+cells sustained at a high level.RAPA treatment significantly induced more CD11b+Gr1+cells,especially G-MDSCs accumulation in recipients spleens and were significantly higher than other three groups on week 4.G-MDSCs isolated from BM-TCD recipients significantly inhibited T cell proliferation.The immunosuppression function was impaired when aGVHD occurred.RAPA treatment could restore the suppression role of G-MDSCs.mRNA level of iNOS and Argl were significantly up-regulated expression in G-MDSCs of RAPA treatment group.Both L-NMMA and nor-NOHA reduced the immunosuppressive activity efficiently.The Treg induction results were similar with the immunosuppression function.G-MDSCs from aGVHD models induced lower proportion of Tregs in vitro.But after RAPA treatment,the ability was recovered.Conclusion:Our findings for the first time demonstrate that in vivo administration of RAPA results in the expansion and function enhancement of G-MDSCs in murinemodel of aGVHD.RAPA treatment can enhance the suppression function of G-MDSCsvia Argl and iNOS up-regulation and can enhance the Treg induction ability of G-MDSCs at later stage after BMT.Part 3 Roles and mechanisms of RAPA on subgroups of MDSCs in vitroAim:To further explore roles and mechanisms of RAPA on bone marrow CD11b+Gr1+cells and two subgroups of MDSCs in vitroMethods:In order to further explore the effects of RAPA on MDSCs,RAPA was usedto treat CD11b+Gr1+ cells and two subgroups of MDSCs separated from naive B6 bone marrow cells directly in vitro.After pretreated with rapamycin,CD11b+Gr1+ cells or subgroups of MDSCs were cocultured with activated spleen cells of naive B6 mice for 3.5 days.The proliferation of splenocytes were detected by flow cytometry.Then we investigated the therapeutic capacity of RAPA-treated G-MDSCs in the models of aGVHD.G-MDSCs pretreated with RAPA or not were co-injected with the allogeneic transplant and then the survival situation were observed.To further explore the possible mechanisms underlying the influence of RAPA-treated G-MDSCs on alloreactive T cell responses,T cells were harvested from G-MDSCs and T cell coculture system.Percentage of apoptotic T cells and CD4+T cells differentiation were assessed through FCM.We also examined Thl cytokine(IL-2,IFN-γ and TNF-α)and Th2 cytokines(IL-4,IL-6,and IL-10)secretion in supernatant after 3.5 days co-culture using CBA assay.Then we analyzed Tregs induction ability of G-MDSCs before or after RAPA treatment.The effects of RAPA on expression of immunologic suppressor factors in G-MDSCs were detected by qPCR.Colorimetric method,ELISA,CM-H2DCFDA assay were used to detect the concentration of Argl,PGE2 in cultural supernatant and level of intracellular ROS,separately.Results:Pretreatment with RAPA for 4 hours could significantly induce the immunosuppression ability of CD11b+Gr1+ cells obtained from bone marrow cells of naive B6 mice.Both CD4+ and CD8+ T cell proliferation coule be inhibited remarkably.We found that naive G-MDSCs without RAPA treatment failed to inhibit T cell proliferation in contrast to M-MDSCs,which displayed powerful suppression function.However,after pretreated with RAPA,G-MDSCs gained a notable suppression function,while the suppression function of M-MDSCs were reduced.The in vivo study showed reduced mortality and lower aGVHD scores were observed after co-injection of G-MDSCs,while the mortality has further decline in the group received RAPA-treated G-MDSCs.To further explore the possible mechanisms,we found there was no difference in the incidence of both early apoptosis and late apoptosis of T cells between systems in which T cells were cocultured with RAPA-treated or untreated G-MDSCs.But RAPA-treated G-MDSCs displayed.a significant reduction of both Thl and Th2 differentiation during the coculture period and the production of Thl and Th2 cytokines were remarkably reduced in RAPA-treated G-MDSCs co-culture system.In the Treg induction study,we found RAPA-treated G-MDSCs presented higher ability to induce Tregs in vitro.mRNA level of iNOS and Argl were down-regulated expression in G-MDSCs in RAPA pretreatment group.There is no difference in concentration of Argl and level of intracellular ROS between RAPA-treated and untreated groups.But concentration of PGE2 in cultural supernatant decreased in RAPA-treated group.Conclusion:We found for the first time that RAPA can induce a strong immunosuppression function of mice bone marrow G-MDSCs in vitro,but has an totally contrary effect on M-MDSCs.RAPA-treated G-MDSCs do not increase the incidence of T cell death but markedly inhibit Thl and Th2 differentiation and promote Tregs induction meanwhile.