The Role And Mechanism Of B Cells In Murine Viral Myocarditis

Viral myocarditis(VMC)is an inflammatory heart disease caused by cardiotropic virus infection and is one of the major causes of sudden death and heart failure in young people under 40 years old.The main pathogenesis of VMC is related to the direct invasion of virus in myocardium and the inflammatory immune response induced by the activation of inflammatory cytokines.Although existing studies have shown that T cells play an important role in VMC,especially Th1 and Th17,studies on T cells have not fully explained the pathogenesis of VMC.B cell is another important immune cell,which mainly regulates humoral immune response.The main functions of B cells are antibodies production,antigen presentation and cytokines production.Classically,the studies about B cells primarily focus on the production of immunoglobulins.Currently,more and more studies have shown that antibody-independent functions of B cells play a more important role in autoimmune diseases.Our previous studies have shown that regulatory B cells(Breg,one of the subgroups of B cells)regulate immune response in VMC.However,the role and mechanism of B cells in VMC have not been reported.In order to further understand the role and the mechanism of B cells in VMC,to further clarify the pathogenesis of VMC and guiding clinical treatment,we induced a murine model of VMC with C57BL/6 mice by Coxsackie virus B3(CVB3)intraperitoneal(i.p.)injection,and the following three parts researches were carried out:Part 1 Changes of immunological characteristics of B cells in murine viral myocarditis Study OneObjective: we induced a murine model of VMC with C57BL/6 mice by CVB3 i.p.injection and observed the myocardial pathological changes of VMC mice at different time points.Methods: Male aged 4 weeks C57BL/6 mice(n=72)were randomly divided into VMC group(n=40)and control group(n=32).Both groups were divided into 1,2,3,and 4 weeks subgroups.There were ten mice in each subgroup in VMC group and were eight mice in each subgroup in control group.C57BL/6 mice were infected intraperitoneally with CVB3 as VMC group,and were injected intraperitoneally with an equal volume of phosphate-buffered saline(PBS)as control group.Heart tissues were harvested at 1,2,3,and 4 weeks after i.p injection.Hematoxylin & eosin(HE)staining was used to determine the myocardial pathological changes and myocardial pathological scores were calculated.Results Myocardial inflammation was the most severe at the first week after CVB3 infection,showing cardiomyocyte swelling,massive infiltration of inflammatory cells and focal necrosis,and inflammation gradually decreased after the second week.The results of myocardial pathological scores showed that myocardial pathological scores were significantly increased and reached the peak at the first week after i.p.injection,then decreased at the second week in VMC group.There was a significant difference compared with the control group(P<0.01).Conclusion: The VMC model is successfully established and the myocardial inflammation is the most severe at the first week after CVB3 infection.Study TwoObjective: To observe the changes of B cells in antibodies production,antigen presentation and cytokines production during the pathogenesis of murine VMC,and to explore the changes and significance of B cells immunological characteristics in VMC.Methods: Study objects and grouping were the same with the study one.Serum Ig A,IgM and Ig G concentrations were measured by ELISA.The total number of B cells,the expressions of CD40,CD69,CD80,CD86,and MHC-II in B cells and the frequencies of TNF-?,IL-6,and IL-17 in B cells were detected by flow cytometry.Results In VMC group,the serum levels of IgG and IgM,the expressions of CD40,CD69,CD80,and MHC-II in B cells all increased significantly(P<0.01),reached the peak at 1st week(P<0.01),and declined gradually at the 2nd week(P<0.01),but were still higher than those in the control group at the 4th week(P<0.01).There was also no significant difference both serum IgA levels and the total number of B cells between control group and VMC group(P>0.05).However,the expression of CD86 in B cells began to decline at the 1st week(P<0.01),decreased significantly at the 2nd week(P<0.01),gradually increased at the 3rd week(P<0.01),peaked at the 4th week(P>0.05).The frequencies of TNF-?,IL-6 and IL-17 in B cells increased significantly at the first week(P<0.01),gradually decreased at the second week(P<0.01),and decreased to the level of the control group at the fourth week(P>0.05).The frequencies of TNF-? and IL-6 in B cells in VMC group were significantly higher than those in control group at 1st week,2nd week,and 3rd week(P<0.01).But the frequency of IL-4 in B cells was significantly lower in VMC group than that in control group at 1st and 2nd week(P<0.01),and there was no significant difference between VMC group and control group at 3rd and 4th week(P>0.05).Conclusion: The immunoglobulins production,antigen-presenting ability,and anti-inflammatory cytokines production by B cells all increase significantly and peak in the acute phase of VMC.Overall conclusion in part 1: B cells are activated in the acute phase of VMC.The immunoglobulins production,antigen-presenting ability,and proinflammatory cytokines production by B cells all increase significantly and peak in the acute phase of VMC,suggesting that B cells may participate in the pathogenesis of VMC.Part 2 The role of B cells in murine viral myocarditis Study OneObjective: To observe the effect of B cells knockout BKO on myocardial injury and to explore the role of B cells in VMC.Methods: Sixteen wild type WT C57BL/6 mice were divided into control and WT groups,eight mice in each group;eight BKO mice were in BKO group;sixteen severe combined immune deficiency SCID mice were divided into SCID and SCID + B cells groups,eight mice in each group.All mice were approximately 4-6 weeks old male mice.In SCID+B cells group: B cells isolated from the spleens in VMC mice were injected intraperitoneally into SCID.Three days after the i.p.injection,the SCID mice were inoculated with CVB3.Control group mice were injected intraperitoneally with PBS,whereas the others groups mice were injected intraperitoneally with CVB3 to establish the VMC model.HE staining was used to observe the myocardial pathological changes in each group and calculate the myocardial pathological scores.Serum troponin T level was measured by ELISA.Results: Myocardial pathological scores in BKO and SCID groups were significantly lower than those in WT group and SCID+B cells group(P<0.01).There was significant difference between BKO group and WT group(P<0.01),and also as between BKO group and SCID+B cells group(P<0.01).The myocardial pathological scores in SCID group were lower than those in WT group or in SCID+ B cells group(P<0.01).However,There were no difference between BKO group and SCID group or WT group and SCID+B cells group(P>0.05).The result of serum troponin T level was consistent with the myocardial pathological scores in different groups.Conclusion: B cells play a pathogenic role in VMC independent of T cells.B cell-derived cytokines play a pathogenic role in VMC.Study 2Objective: To investigate the effect of B cell deletion on IFN-,IL-4,IL-17 and IL-22.Methods: Eight WT C57BL/6 mice were as WT group and eight BKO mice were as BKO groups,respectively.Mice were infected intraperitoneally with CVB3 to establish VMC model.Surviving mice were euthanized on day 7.Heart and blood samples were collected aseptically.Serum IFN-?,IL-4,IL-17 and IL-22 levels were measured by ELISA.The mRNA expressions of IFN-?,IL-4,IL-17 and IL-22 were measured by Real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-qPCR).Results: The serum levels of IFN-? and IL-17 were reduced in BKO group compared to the WT group(P<0.01),However,the serum level of IL-4 were higher than that in WT group(P<0.01).There was no significant difference between IL-22 serum level in BKO and WT groups(P>0.05).The data for RT-qPCR were consistent with ELISA results.Conclusion: In VMC,B cells promote the production of IFN-? and IL-17,and inhibit the production of IL-4.Overall conclusion in part 2: B cells play a pathogenic role independent of T cells,and may affect Th cell response.Part 3 Effect of B cells on Th cell differentiation in murine viral myocarditis Study OneObjective: To observe the changes of Th1,Th2,Th17 and Th22 cells and their related transcription factors such as T-bet,GATA-3,ROR?t and AHR mRNA expressions after B cells deletion.Methods: Eight WT C57BL/6 mice were as WT group and eight BKO mice were as BKO group,respectively.Mice were infected intraperitoneally with CVB3 to establish VMC model.Heart tissues and spleens were collected aseptically at 1 week after CVB3 infection.The frequencies of Th1,Th2,Th17,and Th22 cells in splenic cells were measured by flow cytometry.The mRNA expressions of T-bet,GATA-3,ROR?t,and AHR in the heart tissues were measured by RT-q PCR.Results: The frequencies of Th1 and Th17 cells were significantly reduced in BKO group compared to WT group(P<0.01),but the frequency of Th2 cells was higher than that in WT group(P<0.01),and there was no significant difference between the frequency of Th22 cells in BKO and WT groups(P>0.05).The data for RT-qPCR were consistent with flow cytometry results.Conclusion: B cells may promote Th1 and Th17 cell differentiation and limit Th2 cell differentiation in VMC.Study TwoObjective: To verify the effect of B cells on Th cell differentiation in murine VMC in vitro experiments.Methods: The study objects and animals grouping were the same with the study one of this part.The spleens were collected aseptically at 1 week after CVB3 infection.B cells were isolated from the speens of VMC mice,and naive CD4+ T cells were isolated from the speens of BKO group mice and WT group mice using magnetic beads.The Th cell differentiation was induced in vitro.The experiments in vitro were divided into three groups:(1)WT group: naive CD4+ T cells were isolated from the speens of WT group mice;(2)BKO group: naive CD4+ T cells were isolated from the speens of BKO group mice;(3)BKO + B cells group: naive CD4+ T cells isolated from the speens of BKO mice were co-cultured with B cells isolated from the speens of VMC mice.The frequencies of Th1,Th2,Th17,and Th22 cells were measured by flow cytometry;the levels of IFN-?,IL-4,IL-17 and IL-22 in the supernatant were measured by ELISA.The mRNA expressions of T-bet,GATA-3,ROR?t,and AHR were measured by RT-q PCR.Results: The frequencies of Th1 and Th17 cells were significantly reduced in BKO group compared to WT and BKO+B cells groups(P<0.01),but the frequency of Th2 cells was higher in BKO group than that in WT and BKO+B cells groups(P<0.01),and there was no significant difference in the frequency of Th22 cells among three groups(P>0.05).The data for ELISA and RT-qPCR were consistent with flow cytometry results.Conclusion: The results suggest that B cells promote Th1 cell and Th17 cell differentiation and inhibit Th2 cell differentiation in vitro.Overall conclusion in part 3: B cells promote Th1 and Th17 cell differentiation,and limit Th2 cell differentiation in VMC.The full text summary:(1)B cells are activated obviously in the acute phase of VMC.The immunoglobulins producing by B cells increase significantly and peak in the acute phase of VMC.Both the abilities of antigens presentation and cytokines producing by B cells are significantly enhanced in the acute phase of VMC.(2)B cells play a pathogenic role in VMC independent of T cells.B cell-derived cytokines promote myocardial injury in VMC.B cells promote the productions of IFN-? and IL-17,and inhibit the production of IL-4 in VMC.(3)B cells promote Th1 and Th17 cell differentiation,and limit Th2 cell differentiation in VMC.