Role And Mechanism Of CCR2~+ Macrophages In Viral Myocarditis In Mice

Viral myocarditis(VMC)can develop into dilated cardiomyopathy(DCM),which is one of the important causes of heart failure.The common cause is cardiotropic virus infection,such as Coxsackie virus B(CVB).Most patients are self-limiting,and some patients may develop progressive heart failure or sudden cardiac death.It is not clear which factors determine the prognosis of patients.As for the pathogenesis of VMC,current studies believes that the virus enters the human host through the gastrointestinal tract or respiratory tract,and its progress can be divided into three stages:the first stage,the virus enters the cardiomyocytes,and the replication of the virus directly damages the cardiomyocytes and activates the innate immune response.The second stage,the adaptive immune response was activated.The third stage,the inflammation subsided or developed into DCM.VMC myocardial injury is mainly caused by viral replication,activation of innate immune cells,secretion of cytokines and activation of secondary adaptive immune response.Autoimmune response is considered to be the main mechanism of VMC progressing to DCM.Excessive innate immune response can increase the damage of myocardial cells,expose myocardial myosin and induce autoimmune response.Therefore,the extent of innate immune response may be one of the key factors in the progression of VMC to DCM.Macrophages are innate immune cells,and account for the largest proportion of immune cells in myocardium,accounting for about 10%of non myocardial cells in mice.and infiltrating a large number in the myocardium in the acute stage of VMC.Macrophages are divided into M1 and M2 polarization phenotypes according to their functions.This classification method is to co-culture macrophages with specific cytokines in vitro to define the functions,which is considered too simple.At present,macrophages are divided into CCR2~+and CCR2~-subgroups according to their origin.CCR2~-macrophages are derived from embryonic hematopoietic progenitor cells,express low inflammatory mediators,regulate the development of coronary artery,mediate the regeneration of cardiomyocytes in neonatal mice and some species,and maintain self proliferation in myocardium.CCR2~+macrophages are derived from bone marrow mononuclear cells and secrete pro-inflammatory and chemotactic cytokines.In the non-steady state,CCR2~-macrophages were consumed and could not be replenished in time.CCR2~+macrophages can recruit monocytes through CCR2-CCL2 axis and become the main macrophage subpopulation in myocardium.Reducing the monocyte recruitment by removal of CCL2 can alleviate the remodeling of heart in myocardial ischemia model.In autoimmune myocarditis,silencing CCR2 can improve cardiac function.At present,in the VMC model,the research on macrophages is mainly based on the functional classification of M1 and M2,but the role of CCR2~+macrophages in the pathogenesis of VMC is not fully clear.Based on the above problems,this study explored the role and mechanism of CCR2~+macrophages in the pathogenesis of VMC,including three parts:(1)changes of CCR2~+macrophages in myocardium of VMC mice;(2)effects of CCR2~+macrophages on the pathogenesis of VMC based on CCR2 inhibitors and monocyte adoptions;(3)mechanism of CCR2~+macrophages in the pathogenesis and development of VMC.Part I Changes of CCR2~+macrophages in myocardium of mice with viral myocarditisStudy One Establishment of viral myocarditis model in BALB/c miceObjective:To establish VMC model,BALB/c mice were infected with CVB3 by intraperitoneal injection The pathological score and fibrosis changes of myocardium in VMC mice were observed at different time points.Methods:One hundred male BALB/c mice aged 5-6 weeks were randomly divided into control group(n=50)and VMC group(n=50).Each group was divided into 5 subgroups(1 week,2 weeks,3 weeks,4 weeks,6 weeks),with 10mice in each subgroup.Mice in VMC group were intraperitoneally injected with0.1ml CVB3 virus diluent to establish viral myocarditis model.Each animal in the control group was intraperitoneally injected with 0.1ml phosphate buffer(PBS).The mice were sacrificed at the corresponding time points,and the hearts were taken for hematoxylin&eosin(HE)staining and Masson staining.The changes of myocardial injury and fibrosis were observed under light microscope,and the pathological score was calculated.Results:In VMC group,inflammatory cell infiltration was observed in the myocardium at the first week.In the second week,a large number of inflammatory cells infiltrated,myocardial cells swelled,and the myocardium dissolved and ruptured.At the 3rd,4th and 6th week,the infiltration of inflammatory cells decreased gradually.The pathological score of VMC group increased significantly in the first week,reached the peak in the second week,and gradually decreased in the third week,but always higher than that of the control group(all P<0.01).Masson staining showed that fibrosis appeared in the first week.At the 6th week,the degree of fibrosis was the most serious,and the cells arranged disorderly and the intercellular space was irregular.Myocardial cells were destroyed and replaced by blue bundle collagen fibers.Conclusions:The viral myocarditis model was successfully established in BALB/c mice infected with myocardial passage CVB3.The degree of myocardial inflammation was the most severe at 2 weeks after infection,and fibrosis was the most significant at 6 weeks.Study Two Changes of CCR2~+macrophages in myocardium of mice with viral myocarditisObjective:To observe the changes of CCR2~+macrophages and related cytokines in myocardium of BALB/c mice with viral myocarditis.Methods:The research object,grouping and modeling method are the same as study one.There were 30 mice in each subgroup of control and VMC group.The mice were sacrificed and the heart was taken at the corresponding time point.The percentage of CCR2~+macrophages(F4/80~+CCR2~+)in leukocytes(CD45~+)(CCR2~+macrophages/leukocytes),the percentage of CCR2~+macrophages(F4/80~+CCR2~+)in total macrophages(F4/80~+)(CCR2~+macrophages/total macrophages),and the proportion of M1 and M2 polarization phenotypes of CCR2~+macrophages were detected by flow cytometry.The m RNA expressions of IL-1β,IL-6,TNF-α,TGF-βand CCL2 were detected by reverse transcription PCR(RT-PCR).Results:(1)The ratio of CCR2~+macrophages/leukocytes in VMC group was significantly higher than that in control group from 1 to 3 weeks(all P<0.01),but there was no significant difference between VMC and control group at 4 and6 weeks(all P>0.05).(2)The ratio of CCR2~+macrophages/total macrophages in each subgroup of VMC was significantly higher than that in the control group(all P<0.01).The ratio of CCR2~+macrophages/total macrophages was the highest in the third week,which was higher than that in other subgroups of VMC(all P<0.01).(3)The percentage of M1 phenotype in CCR2~+macrophages peaked in the first week,decreased in the second week,increased again in the third week,and decreased in the fourth and sixth weeks.It was significantly increased in the first and third weeks compared with the control group(all P<0.01),but there was no difference in the second,fourth and sixth weeks(all P>0.05).The percentage of M2 phenotype in CCR2~+macrophages decreased to the lowest level in the first week(P<0.01),and there was no significant difference between the the 2nd week and the control group(P>0.05).It decreased again in the third week(P<0.01),and gradually increased in the fourth and sixth weeks,but it was still lower than the control group(all P<0.01).(4)Compared with the control group,the expression of IL-1βand IL-6 m RNA in VMC group was significantly increased at 1 to 3 weeks(all P<0.01),but there was no significant difference at 4 and 6 weeks(all P>0.05).The m RNA levels of TNF-αand CCL2increased significantly in the first week(all P<0.01),and decreased rapidly to the normal level in the second week(all P>0.05).There was no significant difference in the expression of TGF-βm RNA between VMC group and control group at each time point(all P>0.05).Conclusions:CCR2~+macrophages are involved in the development of viral myocarditis in BALB/c mice.Overall conclusion of part 1:After CVB3 infected,the degree of myocardial inflammation was the most severe at 2 weeks,and the fibrosis was the most significant at 6 weeks.CCR2~+macrophages are involved in the development of viral myocarditis in BALB/c mice.Part II Role of CCR2+macrophages in viral myocarditis in miceObjective:To observe the effect of CCR2~+macrophages on the occurrence and development of viral myocarditis in BALB/c mice based on CCR2 inhibitors and monocyte adoptions.Methods:150 male BALB/c mice aged 5-6 weeks were randomly divided into control group,VMC group,CCR2 inhibitor group,Ly6C~+group and Ly6C~-group,with 30 mice in each group.Mice in VMC group,CCR2 inhibitor group,Ly6C~+group and Ly6C~-group were intraperitoneally injected with 0.1ml of CVB3virus dilution,while mice in control group were intraperitoneally injected with0.1ml PBS.CCR2 inhibitor group:CCR2 inhibitor,30mg/kg/d,was given intraperitoneal injection from day 1 to day 14.The day of CVB3 injection is day0.Ly6C~+group:CD11b~+Lin(CD90.2/CD49R/CD49b/Ly-6G)~-F4/80~-Ly6C~+monocytes were isolated from the spleen of VMC mice by flow cytometry on the third day after CVB3 injection,and were transferred into Ly6C~+group mice via tail vein.Ly6C~-group:CD11b~+Lin(CD90.2/CD49R/CD49b/Ly-6G)~-F4/80~-Ly6C~-monocytes were transferred into Ly6C~-group mice by the same method.The number of adoptive cells in each mouse is about 1*10~5.The left ventricular ejection fraction(LVEF)and Left ventricular fractional shortening(LVFS)were measured by small animal ultrasound system at 2 and 6 weeks.The hearts of mice in each group were taken for HE and Masson staining at 2w and 6w respectively.The ratio of CCR2~+macrophages to leukocytes and the ratio of CCR2~+macrophages to total macrophages were detected by flow cytometry.The m RNA expressions of IL-1β,IL-6,TNF-α,TGF-βand CCL2 were detected by RT-PCR.Results:(1)The LVEF and LVFS of VMC group,Ly6C~+group and Ly6C~-group were significantly lower than those of control group at 2 and 6 weeks(all P<0.05).There was no significant difference among the three groups(all P>0.05).LVEF and LVFS of CCR2 inhibitor group were similar to those of control group at 2 and 6 weeks(all P>0.05).(2)Different degrees of inflammatory cell infiltration and myocardial swelling and necrosis were observed in all groups.The pathological scores were higher than those in the control group(all P<0.01).Among them,CCR2 inhibitor group had the mildest inflammation,which was significantly milder than the other three model groups(all P<0.01),while VMC group,Ly6C~+group and Ly6C~-group had the same degree of inflammation(all P>0.05).(3)Masson staining showed that a small amount of collagen fibers were expressed in myocardial interstitial space of CCR2 inhibitor group,and the degree of fibrosis was significantly milder than that of VMC group.The degree of myocardial fibrosis in VMC group,Ly6C~+group and Ly6C~-group was similar.(4)At 2 weeks,the ratio of CCR2~+macrophages/leukocytes in the CCR2 inhibitor group was significantly lower than that in the control group and VMC group(all P<0.01).The VMC group,Ly6C~+group and Ly6C~-group were significantly higher than the control group(all P<0.01),and there was no significant difference among the three groups(all P>0.05).At 6 weeks,there was no significant difference in CCR2~+macrophages/leukocytes ratio between the model groups and the control group.(5)At 2 weeks,the ratio of CCR2~+macrophages/total macrophages in the CCR2 inhibitor group was higher than that in the control group and lower than that in the VMC group,but the differences were not statistically significant(all P>0.05).The ratio of CCR2~+macrophages to total macrophages in VMC group,Ly6C~+group and Ly6C~-group was significantly higher than that in control group(all P<0.01),and there was no significant difference among the three groups(all P>0.05).At 6 weeks,the ratio of CCR2~+macrophages to total macrophages in the CCR2 inhibitor group was significantly lower than that in the control group(P<0.01).(6)Compared with the VMC group,the myocardial IL-1βand IL-6 of the CCR2 inhibitor group were significantly reduced at 2 weeks(all P<0.01).There was no significant difference at 6 weeks(all P>0.05).There were no significant difference in myocardial TGF-β,TNF-αand CCL2 between CCR2 inhibitor group and VMC group at 2 and 6 weeks(all P>0.05)Conclusions:The reduction of CCR2~+macrophages can reduce the severity of myocardial injury and fibrosis of VMC,improve cardiac function,and protect CCR2~-macrophages.Adoptive transfer Ly6C~+and Ly6C~-monocytes from the spleen of mice could not increase the number of macrophages in the myocardium,nor could they change the severity of VMC mice.Part III Mechanism of CCR2~+macrophages in mice with viral myocarditisStudy One Mechanism of CCR2~+macrophages in mice with viral myocarditisObjective:To investigate the mechanism of CCR2~+macrophages on myocardial injury and fibrosis in BALB/c mice with viral myocarditis.Methods:Ninety male BALB/c mice aged 5-6 weeks were randomly divided into control group,VMC group and CCR2 inhibitor group with 30 mice in each group.The modeling method is the same as the second part.The hearts and spleens of mice in each group were taken at 2 and 6 weeks.The expression of IL-1β,IL-6,TNF-α,TGF-βand CCL2 secreted by myocardial CCR2~+macrophages in the VMC and the control group were detected by flow cytometry.The changes of CD4~+and CD8~+T cells were detected by immunohistochemistry.The changes of CD45~+CD4~+T cells and CD45~+CD8~+T cells were detected by flow cytometry.The expression of CVB3 m RNA was detected by RT-PCR.Results:(1)The expression of IL-1β,IL-6,TNF-αand CCL2 secreted by CCR2~+macrophages in VMC group were significantly increased at 2 weeks compared with the control group(all P<0.01).At 6 weeks,the secretion of IL-6,TGF-βand CCL2 increased compared with the control group(all P<0.05).(2)Immunohistochemistry showed that the infiltration of CD4~+T and CD8~+T cells in VMC group was significantly higher than that in control group and CCR2inhibitor group at 2 weeks.At 6 weeks,the number of CD4~+T and CD8~+T in myocardium of mice in each group was scarce.(3)The CD4~+T cells and CD8~+T cells in spleen of VMC group increased at 2 and 6 weeks(all P<0.01),and were significantly higher at 2 weeks than at 6 weeks(P<0.01).CD4~+and CD8~+T cells in spleen of mice in CCR2 inhibitor group increased at 2 and 6 weeks(all P<0.05),but there was no difference between the two groups(all P>0.05).Compared with VMC group,the expression of CD4~+T and CD8~+T cells in CCR2inhibitor group was significantly lower than that in VMC group at 2 weeks(all P<0.01).At 6 weeks,there was no significant difference between the two groups(P>0.05).(4)The expression of CVB3 in VMC group and CCR2 inhibitor group at 2 weeks was significantly higher than that at 6 weeks(all P<0.01).The expression of myocardial CVB3 in the CCR2 inhibitor group was significantly lower than that in the VMC group at 2 weeks(P<0.01).At 6 weeks,there was no statistical difference between the VMC group and the CCR2 inhibitor group(P>0.05)Conclusions:(1)In the development of viral myocarditis in BALB/c mice,myocardial CCR2~+macrophages participate in the process of myocarditis injury and fibrosis by secreting cytokines such as IL-1β,IL-6,TNF-α,TGF-βand CCL2.(2)Myocardial CCR2~+macrophages participate in myocardial injury of VMC mice by activating T cells.Study Two Role of STAT3 signal transduction pathway in cytokine secretion of CCR2~+macrophagesObjective:To investigate the role of STAT3 signal transduction pathway in the secretion of cytokines by CCR2~+macrophages.Methods:40 male BALB/c mice aged 5-6 weeks were randomly divided into control group and VMC group with 20 mice in each group.The expression of p STAT3 in CCR2~+macrophages was detected by flow cytometry at 2 weeks.Bone marrow stem cells from femur and tibia of BALB/c mice were induced into macrophages.They were divided into three groups:control group,LPS group and LPS+STAT3 inhibitor group.The expression of cytokines IL-1β,IL6,TNF-α,TGF-βsecreted by macrophages and the proportion of M1 and M2phenotypes were detected by flow cytometry.Results:(1)The expression of p STAT3 of CCR2~+macrophages in VMC group was higher than that in control group(P<0.01).(2)The expression of IL-1βsecreted by macrophages in the LPS group was higher than that in the control group(P<0.01).The LPS+STAT3 inhibitor group was also higher than the control group(P<0.05),but lower than the LPS group(P<0.05).The expression of TNF-αsecreted by macrophages in the LPS group and LPS+STAT3 inhibitor group was significantly higher than that in the control group(all P<0.01),and there was no statistical difference between the two groups(P>0.05).The expression of of IL-6 and TGF-βsecreted by macrophages in the LPS group was lower than that in the LPS+STAT3 inhibitor group(all P<0.05).(3)The proportion of M1 macrophages in the LPS group was higher than that in the control group(P<0.05),and was also significantly higher than that in the LPS+STAT3 inhibitor group(P<0.01).The LPS+STAT3 inhibitor group was comparable to the control group(P>0.05).The proportion of M2 macrophages in the LPS group was lower than that in the control group(P<0.05),and significantly lower than that in the LPS+STAT3 inhibitor group(P<0.01).The LPS+STAT3 inhibitor group was comparable to the control group(P>0.05).Conclusions:Macrophages can express pro-inflammatory phenotype and secrete pro-inflammatory cytokines by activating STAT3 signaling pathway.Overall conclusion of part III:Myocardial CCR2~+macrophages participate in the process of myocardial inflammation and fibrosis by secreting pro-inflammatory and pro-fibrotic cytokines and activating T cells.Macrophage can secrete proinflammatory cytokines by activating STAT3 signaling pathwayOur research indicated that:(1)In the VMC model of BALB/c mice,the degree of myocardial inflammation was the most severe at 2 weeks after virus infection,and the degree of fibrosis was the most significant at 6 weeks.CCR2~+ macrophages increased in 1 to 3 weeks,and participated in the development of VMC in BALB/c mice(2)Reducing the number of cardiac CCR2~+macrophages in VMC mice can reduce the degree of myocardial injury and fibrosis,improve cardiac function,and protect cardiac CCR2~-macrophages.(3)CCR2~+macrophages participate in the process of myocarditis injury and fibrosis by secreting proinflammatory and fibrogenic cytokines and activating T cells.Macrophage can secrete proinflammatory cytokines by activating STAT3 signaling pathway.