Effects Of B Cells On Myocardial Macrophages In Mice With Viral Myocarditis And Its Mechanism

Viral myocarditis(VMC)is an important cause of cardiac dysfunction.Viral infection is the main cause of viral myocarditis,with Coxsackievirus B3(CVB3)being the most common pathogen.Myocardial damage in viral myocarditis is associated with inflammatory cells of myocardial infiltration.In VMC,macrophages are one of the major inflammatory cells infiltrating myocardial tissue and play an important role in myocardial injury.Recent studies have found that embryonic-derived macrophages(CCR2-macrophages)andmonocyte-derivedmacrophages(CCR2+macrophages)are present in myocardial tissue and play a role in myocardial tissue in myocardial infarction mice and ischemic cardiomyopathy patients.CCR2-macrophages maintain their homeostasis by situ proliferation,and CCR2+macrophages are supplemented by situ proliferation and peripheral blood mononuclear cell infiltration.In acute VMC model,large number of monocytes and macrophages were observed in myocardial tissue.Macrophages from different sources may be involved in the repair process of myocardial injury in VMC.However,the biological characteristics and roles of myocardial CCR2+macrophages and CCR2-macrophages in the pathogenesis of VMC are still unclear.In a mouse model of myocardial infarction,while mononuclear cells in the myocardial tissue of CCR2 knockout mice decreased,myocardial CCR2+macrophage infiltration decreased,and myocardial damage was reduced.Inhibition of monocyte infiltration in myocardial tissue can reduce myocardial CCR2+macrophage infiltration.Our previous study found that B cells aggravate VMC myocardial damage by secreting cytokines.In a mouse model of myocardial infarction,B cells promote the recruitment of monocytes to the site of myocardial lesion by secreting CCL7 and aggravate myocardial damage.B cells can promote myocardial tissue monocyte infiltration by secreting chemokines.B cells may have a role in regulating a subset of cardiac macrophages.However,in VMC,does B cell regulate a subpopulation of cardiac macrophages?What is the mechanism?It is still unclear.Macrophages have the characteristics of functional diversity and plasticity.In different microenvironments,macrophages can differentiate into classical activated macrophages(M1)and selectively activated macrophages(M2).The differentiation of macrophages into M1 and M2 is called polarization.In VMC,myocardial inflammation can be alleviated by inhibiting M1 polarization or promoting M2 polarization.LPS and IFN-?promote M1 polarization by activating the STAT1 signaling pathway.Our previous study found that in VMC,myocardial IFN-?was significantly elevated,while reduced in BKO mice.Therefore,B cells may aggravate myocardial damage by promoting M1polarization in VMC.However,this still needs further research to confirm.Based on the above problems,this study explored the effects of B cells and its mechanism on myocardial macrophages in VMC mice from myocardial macrophage source and macrophage polarization.The research includes three parts:(1)Changes of myocardial macrophage subsets and their antigen presentation ability in mice with viral myocarditis;(2)Effects of B cells on myocardial macrophage subsets in mice with viral myocarditis and its mechanism;(3)Effects of B cells on the polarization of myocardial macrophages in mice with viral myocarditis and its mechanism.Part I Changes of myocardial macrophage subsets and their antigen presentation ability in mice with viral myocarditisStudy One:Establishment of mouse model of viral myocarditisObjective:To establish VMC mouse model by intraperitoneal injection of CVB3 in C57BL/6 mice,and to observe the pathological changes of myocardial tissue in VMC mice at different time points.Methods:Eighty male C57BL/6 mice,5-6 weeks old,were randomly divided into control group(n=40)and VMC group(n=40).Each group was divided into 4 subgroups(1 week,2 weeks,3 weeks,4 weeks),10 in each subgroup.Each mouse in VMC group was intraperitoneally injected with 0.1 ml of CVB3 virus solution,while the control group was injected with 0.1 ml of phosphate buffered saline(PBS).At the corresponding time,the mice were sacrificed,and the heart was stained with hematoxylin&eosin(HE).The pathological changes of myocardial tissue were observed under light microscope,and pathological scores were calculated.Results:In the VMC group,large number of inflammatory cell infiltration,myocardial cell focal necrosis,myocardial cell swelling,and myocardial rupture were observed in the myocardial tissue at 1 week.Subsequently,myocardial inflammatory cell infiltration gradually decreases.The myocardial pathological score of the VMC group was significantly higher than that of the control group(P<0.01);the myocardial pathological score of VMC was the highest at 1week.Conclusions:The mouse VMC model is successfully established by intraperitoneal injection of CVB3 into C57BL/6 mice.Myocardial local inflammatory injury is most obvious at 1 week after CVB3 infection.Study Two:Changes of myocardial macrophage subsets and their antigen presentation ability in mice with viral myocarditisObjective:To observe the changes of myocardial macrophage subsets and differential expression of costimulatory molecules in myocardial macrophage subsets during myocardial pathological changes in VMC.Methods:A total of 144 male C57BL/6 mice,5-6 weeks old,were randomly divided into control group(n=64)and VMC group(n=80).Each group was divided into 4 subgroups(1 week,2 week,3 week,4 week),16 in each subgroup of the control group,and 20 in each subgroup of the VMC group.The VMC group was intraperitoneally injected with 0.1 ml of CVB3 virus solution,and the control group was intraperitoneally injected with 0.1 ml of PBS.The mice were sacrificed at the corresponding time points to retain the heart.Flow cytometry was used to detect the expression changes of cardiac CCR2+macrophages(CCR2~+F4/80~+CD45~+)andCCR2-macrophages(CCR2~-F4/80~+CD45~+)and the expression of CD40,CD80,CD86 and MHC-II on cardiac CCR2+macrophages and CCR2-macrophages.Results:Compared with the control group,the proportion of CCR2+macrophages in VMC group was significantly increased(P<0.01),reached the highest in 1-week subgroup,then gradually decreased,and still higher than the control group in 4-week subgroup(P<0.01).The proportion of CCR2-macrophages in VMC group was significantly lower than that in control group(P<0.01),which was the lowest in 1 week,then gradually increased,and still lower than that in control group in 4 weeks(P<0.01).In VMC,CD80 and CD40 were mainly expressed on the surface of CCR2+macrophages,while CD86 and MHC-II were expressed in both CCR2+macrophages and CCR2-macrophages.In the VMC group,the expression of CD80,CD40,CD86and MHC-II on CCR2+macrophages was significantly higher than that in control group(P<0.01),and the highest expression was in 1-week subgroup(36.29±2.14,55.98±3.26,29.66±2.56 and 79.75±3.83,respectively).Then,it will fall to the lowest in 4-week subgroup,which is 12.75±0.96,11.13±1.73,13.46±1.76 and 65.08±2.50.In the VMC,the expression of CD80 on CCR2-macrophages was significantly higher than that of the control group in1-week subgroup(2.46±0.08 vs 1.69±0.18,P<0.01),but there was no significant difference between VMC subgroups(P>0.05).The expression of CD86 on CCR2-macrophages in VMC group was significantly higher than that in the control group(P<0.01).The expression of CD86 on CCR2-macrophages was the lowest at 1-week subgroup(10.80±1.9),and the highest at 3-week subgroup(16.66±0.83).The expression of CD40 on CCR2-macrophages in VMC group was higher than that in control group at 1-week subgroup and 2-week subgroup(P<0.01).The CD40 expression was highest at 1-week subgroup(1.78±0.27),and then decreased to the level of control group at 3 weeks.The expression of MHC-II on CCR2-macrophages was significantly lower than that of the control group(P<0.01),and decreased to the lowest at 1-week subgroup(19.00±4.80),then gradually increased,and the highest expression at 4-week subgroup(35.05±2.57),but still significantly lower than the control group(P<0.01).Conclusions:In VMC mice,myocardial CCR2+macrophages significantly elevate and peak at 1 week.The CCR2-macrophages decrease significantly and lowest at 1 week.As myocardial inflammation gradually recover,the CCR2+macrophages gradually decrease,while CCR2-macrophages gradually increase.The antigen presentation function of CCR2+macrophages is activated more strongly than CCR2-macrophages.Overall conclusion in part 1:In VMC mice,when myocardial inflammation is most severe,myocardial CCR2+macrophages is significantly elevated to the highest,while myocardial CCR2-macrophages is reduced to the lowest.As myocardial inflammation gradually recovere,the CCR2+macrophage gradually decrease,while CCR2-macrophages gradually increase.The antigen presentation function of CCR2+macrophages is activated more strongly than CCR2-macrophages.Part?Effects of B cells on myocardial macrophage subsets in mice with viral myocarditis and its mechanismStudy One:Effects of B cells on myocardial macrophage subsets in mice with viral myocarditisObjective:To observe the effect of B cells on myocardial macrophage subsets in mice with viral myocarditis.Methods:Sixteen male C57BL/C mice,5-6 weeks old,were randomly divided into control group and WT group,8 mice in each group.Sixteen male B cell-deficient(BKO)mice of 5-6 weeks old were divided into BKO group and BKO+B cell group,8 mice in each group.BKO+B cell group:Magnetic beads were used to sort spleen B cells from WT group mice.The B cells were intraperitoneally injected into BKO mice one day before intraperitoneal injection of CVB3.The WT group,BKO group and BKO+B cell group were intraperitoneally injected with 0.1 ml of CVB3 virus solution,and the control group was intraperitoneally injected with 0.1 ml of PBS.One week after CVB3infection,mice in each group were sacrificed.The myocardial tissue was taken for HE staining and the myocardial pathological score was calculated.Flow cytometry was used to detect the changes of frequency of CCR2+macrophage(CCR2~+F4/80~+CD45~+)and CCR2-macrophage(CCR2~-F4/80~+CD45~+)in each group.Results:There was no inflammatory cell infiltration in myocardial tissue of the control mice.Myocardial tissues of WT group,BKO group and BKO+B cell group showed different degrees of inflammatory cell infiltration,focal cardiomyocyte swelling and necrosis.The myocardial pathological scores of BKO group(1.54±0.21)and BKO+B cell group(2.09±0.34)were significantly lower than that of WT group(3.08±0.37)(P<0.001),while the pathological score of BKO group was significantly lower than BKO+B cell group(P<0.01).The proportion of CCR2+macrophages in BKO group was significantly lower than that in WT group(P<0.01),while the proportion of CCR2+macrophages in BKO+B cell group was higher than that in the BKO group(P<0.05).The proportion of CCR2-macrophages in BKO group was significantly higher than that in WT group(P<0.01).The proportion of CCR2-macrophages in BKO+B cells group was significantly lower than that in BKO group(P<0.05).Conclusions:In VMC,B cells aggravate myocardial inflammation;B cells promote myocardial tissue CCR2+macrophages infiltration and inhibit myocardial tissue CCR2-macrophages infiltration.Study Two:Effects of B cells on myocardial monocyte infiltration and monocyte chemotactic factors in mice with viral myocarditisObjective:To observe the effects of B cells on the infiltration of monocytes and the expression of CCL2 and CCL7 in myocardium of VMC miceMethods:Experimental animals,grouping methods,and model building methods were the same as in Study One.Flow cytometry was used to detect the expression changes of myocardial CCR2+monocytes(CCR2~+Ly6G~-CD11b~+)at one week after CVB3 infection.The spleen B cells from control mice and VMC mice were sorted by magnetic beads and cultured for 48 hours in vitro to collect B cell supernatants.The expressions of CCL2 and CCL7 proteins in B cell culture supernatants were detected by ELISA.The expressions of CCL2 and CCL7 mRNA in B cells and myocardial tissues were detected by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).Results:Compared with control group,the frequency of CCR2+monocytes in WT group were significantly higher than that in control group(P<0.01).The frequency of CCR2+monocytes in BKO group was significantly lower than that in WT group(P<0.01),while that in BKO+B cell group was significantly higher than BKO group(P<0.01).The expression of CCL2 mRNA and CCL7 mRNA in myocardial tissue of WT group was significantly higher than that of control group(P<0.01).The expression of CCL2 mRNA and CCL7mRNA in BKO group was significantly lower than that in WT group(P<0.01),while BKO+B cell group was higher than BKO group(P<0.01).The levels of CCL2 mRNA and CCL7 mRNA in spleen B cells of VMC group were significantly higher than those of control group(P<0.01).The levels of CCL2and CCL7 in supernatant of spleen B cells in VMC group were significantly higher than those in control group(P<0.01).The levels of CCL2 mRNA and CCL7 mRNA in spleen B cells of VMC group were significantly higher than those of control group(P<0.01).The levels of CCL2 and CCL7 in supernatant of spleen B cells in VMC group were significantly higher than those in control group(P<0.01).Conclusions:In VMC mice,B cells secrete CCL2 and CCL7,increase the expressions of CCL2 and CCL7 in myocardial tissue,and promote the infiltration of CCR2+monocytes in myocardial tissue.Overall conclusion in part?:In VMC mice,B cells secrete CCL2 and CCL7,increase the expression of CCL2 and CCL7 in myocardial tissue,promote the infiltration of mononuclear cells in myocardial tissue,increase the infiltration of myocardial CCR2+macrophages,and aggravate myocardial injury.Part III Effects of B cells on the polarization of myocardial macrophages in mice with viral myocarditis and its mechanismStudy One:Effects of B cells on polarization of myocardial macrophages in mice with viral myocarditisObjective:To observe the effect of B cells on myocardial macrophage polarization in VMC mice in vivo.Methods:Sixteen male C57BL/C mice,aged 5-6 weeks,were divided into control group and WT group,8 in each group.Sixteen male BKO mice,aged 5-6 weeks,were divided into BKO group and BKO+B cell group,8 in each group.BKO+B cell group:Magnetic beads were used to sort spleen B cells from WT group mice,and sorted B cells were intraperitoneally injected into BKO mice one day before intraperitoneal injection of CVB3.The mice in WT group,the BKO group and BKO+B cell group were intraperitoneally injected with 0.1ml of CVB3 dilution solution,and the control group mice were intraperitoneally injected with 0.1 ml of PBS.On day 7,each group of mice was sacrificed.The frequency of M2 macrophages(CD206~+F4/80~+CD11b~+,Arg1~+F4/80~+CD11b~+)and M1 macrophages(NOS~+F4/80~+CD11b~+)in myocardium of each group were detected by flow cytometry.Results:The M2 frequency of myocardial tissue in BKO group was significantly higher than that in WT group(P<0.01).The M2 frequency of myocardial tissue in BKO+B cell group was significantly lower than that in BKO group(P<0.01).The M1 frequency of myocardial tissue in BKO group was significantly lower than that in WT group(P<0.01).The M1 frequency of myocardial tissue in BKO+B cell group was significantly higher than that in BKO group(P<0.05).Conclusions:In VMC,B cells promote M1 polarization and inhibit the M2polarization in myocardium.Study Two:Role of STAT1 Signal Transduction Pathway in B Cells Promoting M1 PolarizationObjective:To observe the role of the STAT1 signal transduction pathway in B cell-promoting M1 polarization.Methods:Experimental animals,grouping methods and model building methods were the same as in Study One.Flow cytometry was used to detect the phosphorylated STAT1 levels in myocardial macrophages of each group.The spleen B cells of mice in control group and WT group were sorted by magnetic beads and cultured for 48 hours in vitro to collect B cell supernatants.Bone marrow-derived macrophages were induced to M2 in vitro.B cell culture supernatants were co-cultured with M2.The specific groups were:1 control group:Supernatant of spleen B cells from control mice+M2;2 VMC group:Supernatant of spleen B cells from VMC mice+M2;3 VMC+fludarabine group:Supernatant of spleen B cells from VMC mice+fludarabine+M2.The frequency of M2(CD206~+F4/80~+CD11b~+)was detected by flow cytometry.The levels of TNF-?,IL-12,IL-1?and IL-10 were detected by ELISA.Expression of TNF-??IL-12?IL-1?,IL-10 and NOS mRNA in macrophages were detected by RT-PCR.Results:The expression of phosphorylated STAT1 in myocardial macrophages of BKO group was significantly lower than that of WT group(P<0.01).The expression of phosphorylated STAT1 in BKO+B cell group was significantly higher than that in BKO group(P<0.05).The frequency of M2 in bone marrow-derived macrophages was 98.4%in vitro,and that in VMC group was significantly lower than that in Control group(P<0.01).The frequency of M2 in VMC+fludarabine group was significantly higher than that in VMC group(P<0.01).The concentrations of IL-12,TNF-?and IL-1?in the culture supernatant of VMC group were significantly higher than those in the control group and VMC+fludarabine group(both P<0.01).The concentration of IL-10in the culture supernatant of VMC group was significantly lower than that in the supernatant of control group and VMC+fludarabine group(both P<0.01).The expression levels of IL-12,TNF-?,IL-1?and NOS mRNA in macrophage of VMC group were significantly higher than those of control group and VMC+fludarabine group(P<0.01).Conclusions:In VMC mice,B cells promote the activation of STAT1signaling pathway in myocardial macrophages;B cells promote myocardial M1polarization via the STAT1 signal transduction pathway.Overall conclusions in part?:In VMC mice,B cells promote myocardial M1 polarization,and that related to the STAT1 signal transduction pathway.In Summary,our research indicated that:(1)During pathogenesis of VMC mice,when myocardial local inflammation injury is most obvious 1 week after virus infection,myocardial CCR2+ acrophage is significantly elevated to the highest,while myocardial CR2-macrophages is reduced to the lowest.As myocardial inflammation gradually recovere,the CCR2+macrophage gradually decrease,while CCR2-macrophage gradually increase.(2)In VMC mice,the antigen presentation function of CCR2+macrophages is activated more strongly than CCR2-macrophages.(3)In VMC mice,B cells secrete CCL2 and CCL7,increase the expression of CCL2 and CCL7 in myocardial tissue,promote the infiltration of mononuclear cells in myocardial tissue,increase the infiltration of myocardial CCR2+macrophages,and aggravate myocardial injury.(4)In VMC mice,B cells promote myocardial M1 polarization,and that related to the STAT1 signal transduction pathway.