The Role And Mechanism Of Myeloid-derived Suppressor Cells (MDSCs) In CVB3-induced Viral Myocarditis

Objective: In this project, we want to detect the role and mechanism ofmyeloid-derived suppressor cells （MDSCs） and its subsets in CVB3-induced differentsexual viral myocarditis.Methods: Firstly, we establish the model of CVB3-induced viral myecarditis indifferent sexual BALB/c mice by intraperitoneal injection, and then we examine the weightchange, serum CK （creatine kinase） activity, heart histopathological analysis, survival rateand virus loading to assess whether the model is successful. Second, we detect the immuneresponse type and the expression of MDSCs and its subsets in peripheral blood, spleen,heart and bone marrow7days post infection. Wright′s stain is used to identify itsmorphous and slenactomy to detect the source of MDSCs in heart. Thirdly, we sort theinfiltrating MDSCs and it subsets from spleen and heart to explore them function in vitroand in vivo. We use Brdu Kit to examine its influence on the proliferzation of T cells invitro and adoptive transfer them to these mice day3postinfection to examine themfunction in vivo. At last, we also explore the mechanism of MDSCs and its subsetsregulating the immune response in viral myecarditis in vitro and in vivo with Real-Time PCR,trans-well, NO detection, FACS analysis and so on.Results:1. Severity of CVB3-induced viral myocarditis in male and female issignificant different: male is more severe than female, while the susceptibility of male andfemale heart to CVB3is no different.2. Real-Time PCR indicates that the expression ofTNF-α, IFN-γ, IL-1β, IL-6, IL-17A and IL-18in male heart is^higher than female, whileIL-4, IL-10and FoxP is less than female.3. The expression of MDSCs in spleen and heartof male and female mice is different: the percentage of MDSCs in the hearts of male miceis5.045±0.362%, compared1.759±0.202%in female mice（P＜0.001）, while thepercentage in spleen is reverse to heart（male:9.975±1.031%vs female:5.258±0.466%, P＜0.001）.4. The subsets of MDSCs in hearts of male and female mice is different too:male is main expressing CD11b⁺LY-6G⁺LY-6C^lowphenotype, liking to granulocytemorphous （PMN-MDSCs）, while female is CD11b⁺LY-6G-LY-6C^highphenotype, liking to monocyte morphous（MO-MDSCs）.5. MDSCs from female mice can significantlysuppress T cells proliferation, while MDSCs from male could not suppress T cellsproliferation. The function of its subsets is also different in male and female mice:CD11b⁺LY-6G-LY-6C^highMDSCs from spleen and heart of female mice could markedlysuppress T cells proliferation, but CD11b⁺LY-6G⁺LY-6C^lowMDSCs from male could nothave this function.6. CD11b⁺LY-6G-LY-6C^highMDSCs from female mice could alleviateCVB3-induced viral myocarditis and not influce the replication of CVB3in heart, whileCD11b⁺LY-6G⁺LY-6C^lowMDSCs from male could not reduce myocarditis.7. In vitro, wefind the suppressive of MDSCs and its subsets to T cells proliferation is reverse bytrans-well, FACS analysis indicates that CD40, IL-10and CD62L is up-regulated inCD11b⁺LY-6G-LY-6C^highMDSCs, compared to CD11b⁺LY-6G⁺LY-6C^lowMDSCs, whileI-A/I-E is up-regulated on CD11b⁺LY-6G⁺LY-6C^lowMDSCs, compare to CD11b⁺LY-6G-LY-6C^highMDSCs. CD80, CD86, F4/80and CD11c is no different betweenCD11b⁺LY-6G⁺LY-6C^lowMDSCs and CD11b⁺LY-6G⁺LY-6C^lowMDSCs. Real-Time PCRresults indicate ARG-1,IL-10and iNOS is increasing in CD11b⁺LY-6G⁺LY-6C^lowMDSCs,compared to CD11b⁺LY-6G⁺LY-6C^lowMDSCs, but the production of NO in suspension isno different between two subsets.8. CD11b⁺LY-6G-LY-6C^highMDSCs from female micecould promote the production of IL-4⁺CD4⁺T cells, IL-10⁺CD4⁺T cells IFN-γ⁺CD4⁺T cellsand FoxP3⁺CD4⁺Treg, but down-regulate the percentage and total number of Th17cells.Conclusions: Severity of CVB3-induced viral myocarditis in male and female issignificant defferent: male is more severe than female. The expression of MDSCs and itssubsets in heart of different sexual BALB/c mice is different and related to CVB3-inducedviral myecarditis. The percentage and total number in female heart is more than male. Malemice heart is mainly expressing CD11b⁺LY-6G⁺LY-6C^lowphenotype subset, while female isCD11b⁺LY-6G-LY-6C^highphenotype subsets. CD11b⁺LY-6G-LY-6C^highMDSCs from femalemice could significantly suppress T cells proliferation and alleviate CVB3-induced viralmyocarditis, while MDSCs and its subsets from male could not suppress T cellsproliferation and reduce myocargitis. CD11b⁺LY-6G-LY-6C^highMDSCs from female micecould promote the production of IL-4⁺CD4⁺T cells, IL-10⁺CD4⁺T cells IFN-γ⁺CD4⁺T cellsand FoxP3⁺CD4⁺Treg, and down-regulate the percentage and total number of Th17cells toreduce myocarditis. The mechanism of CD11b⁺LY-6G-LY-6C^highMDSCs from femalesuppressing T cells proliferation is depend on cell-cell contact way and the possiblemolecule is suppressive co-stimulate molecule CD40. In all, in this research we did not only detect the reason of viral myocarditis in different sexual BALB/c mice, but alsoinvestigate the protective function of MDSCs and its subsets in different sexual VMC,which will become a new strategy and way of prevention and control in viral myocarditis.