The Role Of CD4⁺T_EM Cells In The Progression Of Acute Viral Myocarditis To Dilated Cardiomyopathy In Mice

Viral myocarditis（VMC）is an inflammatory disease of the cardiac muscle caused by viral infection.Most patients with VMC terminate the myocardium inflammatory after the acute phase and can recover spontaneously without special treatment.However,some patients may evolve into chronic myocarditis or even dilated cardiomyopathy（DCM）,with poor prognosis.The autoimmune response mainly mediated by CD4⁺T cells plays a key role in the pathogenesis of VMC and DCM.Previous studies mainly focused on the effector CD4⁺T cell subsets.For example,it have been demonstrated that effector Th1 cells and effector Th17 cells as well as their secretion of IFN-γand IL-17A play an important role in promoting the progression of myocarditis to DCM.However,it is worth noting that after the peak of myocardial inflammation,the effector CD4⁺T cell subsets go to apoptosis.Therefore,studies on the effector CD4⁺T cell subsets cannot fully explain the pathogenesis of the progression of acute VMC to DCM.Effector memory CD4⁺T(CD4⁺T_EM)cells are a subset of memory CD4⁺T cells,which can maintain antigen-specific memory.When stimulated by the same antigen again,CD4⁺T_EM cells can proliferate rapidly and secrete Effector cytokines,resulting in a highly effective immune response.In recent years,a large number of studies have shown that CD4⁺T_EM cells play an important role in the pathogenesis of chronic inflammatory diseases and autoimmune diseases.Further studies have confirmed that there are autoantigen-specific memory CD4⁺T cells in a variety of autoimmune diseases mediated by CD4⁺T cells.After being stimulated by autoantigens,these cells can proliferate and secrete a large amount of pro-inflammatory cytokines such as IFN-γand IL-17A.Thus promoting the initiate and development of diseases.In a recent study,CD4⁺T_EMcell subsets dominated the infiltration of CD4⁺T in myocardium of mice with experimental autoimmune myocarditis.Therefore,we hypothesized that CD4⁺T_EM cells may be an important factor in promoting the progression of acute VMC into DCM.In this study,to establish a VMC model,Balb/c mice were infected with heart-passage coxsackievirus B3（CVB3）.And the following three parts were studied.Part I Changes of CD4⁺T_EM cells in the progression of acute viral myocarditis to dilated cardiomyopathy in mice.Study one:Establishment of the viral myocarditis model in mice.Objective:A VMC model was established by CVB3 intraperitoneal injection in Balb/c mice,to observe the changes of cardiac structure and function and myocardial histopathology in different stages of VMC.Methods:Male Balb/c mice with 6 weeks old were randomly divided into infected group and uninfected group.According to the different observation time points,these two groups were divided into 14 days,28 days and 42 days subgroups.Mice in the infected group were intraperitoneally injected with CVB3 virus diluent,0.2 m L/mice.The uninfected group recieced intraperitoneally injection with the same volume of PBS.The intraperitoneal injection day was defined as day 0.When the observation timepoints arrived,the changes of cardiac structure and function were observed by echocardiography.Then the mice were sacrificed.The hearts were taken out,embedded in paraffin,and sectioned.The myocardial pathologic histology changes were observed by HE staining and myocardial pathological scores were calculated.The myocardial interstitial fibrosis was observed by Masson trichromatic staining,and the myocardial fibrosis scores were determined.Results:The echocardiography results showed that compared with the uninfected subgroup,the left ventricular ejection fraction and left ventricular fractional shortening significantly reduced in the infected group at different stages（all P<0.01）,and the left ventricular end-diastolic internal diameter in the infected subgroup at day 42 was significantly increased（P<0.01）.HE staining results showed that a large number of inflammatory cells infiltrating with focal necrosis in the myocardium of mice in the infected subgroup at day14.There were still obvious inflammatory cells infiltrating in the myocardium of mice in infected subgroup at day 28 and day 42,but the number of inflammatory cells were reduced whitout obvious necrosis compared with that in the infected subgroup at day 14.The myocardial pathological scores in each infection subgroup were significantly increased（all P<0.01）,with the infected subgroup at day 14 were the highest when compare with infected subgroup at day 28 and day 42（all P<0.01）.Masson trichrome staining showed a large amount of collagen fiber deposition in the myocardial interstitium of infected mice at 28and 42 days.The myocardial fibrosis scores of the infected subgroups were higher than those of the uninfected group（all P<0.05）,and the infection subgroups showed a progressive increase of myocardial fibrosis scores in the 14,28 and 42 day subgroup（all P<0.01）.Conclusion:VMC model is successfully established in Balb/c mice infected by intraperitoneal injection of CVB3.Myocardial tissue inflammation is the most severe with left ventricular systolic disfunction at day 14 after virus infection,indicating the acute stage.At day 28 after virus infection,myocardial tissue inflammation reduced,but obvious myocardial interstitial fibrosis with left ventricular systolic disfunction,which suggest the stage of chronic myocarditis.At day 42 after virus infection,inflammatory cells infiltrating the myocardial tissue,with increased myocardial interstitial fibrosis,decreased systolic function of the left ventricular and significant enlargement of the left ventricle,which suggest the stage of DCM.Study two:Changes of CD4⁺T_EM cells in the progression of acute viral myocarditis to dilated cardiomyopathy in miceObjective:To explore whether CD4⁺T_EM cells are involved in the pathogenesis of the progression of acute VMC to DCM in mice,the changes of CD4⁺T_EM cells were determinded in different organs and tissues of mice at different stages of the progression of acute VMC to DCM.Methods:The experimental animals,grouping and VMC model establishment were the same as Part I study one.Blood samples,the hearts and spleens were collected when each group reached the respective observation timepoints.The proportion of total CD4⁺T cells in leukocyte in myocardium,the proportion of CD4⁺T_EM cells in total CD4⁺T cells in myocardial tissues,peripheral blood and spleens of mice in each subgroup were detected by flow cytometry.Results:Compared with the respective subgroups of uninfected group,the flow cytometry showed that the proportion of total CD4⁺T cells in myocardial tissue of mice in the three infected subgroups were significantly increased（all P<0.01）.In the infected group,the proportion of total CD4⁺T cells in the myocardium of mice in the 14 day subgroup was higher than that in the 28 day subgroup（P<0.01）,and the difference between the 28 day subgroup and the 42day subgroup was not statistically significance（P﹥0.05）.The proportion of CD4⁺T_EM cells in myocardial tissue and peripheral blood of mice in infected subgroups were both higher than those in uninfected subgroups at corresponding time points（all P<0.05）.In the infection group,the proportion of CD4⁺T_EMcells in myocardium and peripheral blood of mice in the 28 day subgroup were both higher than those in the 14 day subgroup（all P<0.05）,and there was no significant difference between the 42 day subgroup and the 28 day subgroup（all P﹥0.05）.The proportion of CD4⁺T_EM cell subsets in total CD4⁺T were（56.19±4.44）%,（67.83±4.51）%and（66.55±4.84）%in myocardium of the infected group at day 14,28 and 42,respectively.The proportion of myocardium infiltrating CD4⁺T_EM cell subsets in total memory CD4⁺T in the 14,28 and 42day subgroup of the infected group was（96.85±1.31）%,（97.85±1.14）%,and（97.89±1.19）%,respectively.The proportion of CD4⁺T_EM cells in spleen of mice in the infected subgroup at 14 days was lower than that in the respective uninfected subgroup（P<0.01）,but the proportion of the infected subgroup at28 days and 42 days was higher than that in the respective uninfected subgroup（all P<0.05）.In the infection group,the proportion of CD4⁺T_EM cells in the spleen of mice in the 28 day subgroup was higher than that in the 14 day subgroup（P<0.01）,and there was no significant difference between the 42 day subgroup and the 28 day subgroup（P﹥0.05）.Conclusion:The proportions of total CD4⁺T cells infiltrating in myocardial tissue of infected group are significantly increased at different stages.Among them,CD4⁺T_EM cell subsets are the majority,and almost all memory CD4⁺T cells are CD4⁺T_EM cell subsets.During the progression of acute VMC to chronic myocarditis,the proportion of CD4⁺T_EM cell subsets increased in myocardial tissue,peripheral blood and spleen,which remaine at a high level at DCM stage.These results suggest that CD4⁺T_EM cells involve in the pathogenesis of acute VMC progression to DCM in mice.Part I Conclusion:Mice infected with CVB3 show acute VMC at day 14,chronic myocarditis at day 28,and DCM at day 42.During the evolution from acute VMC to DCM in mice,the myocardial infiltrating CD4⁺T_EM cells increases significantly,suggesting that CD4⁺T_EM cells involve in the pathogenesis of the acute VMC progression to DCM in mice.Part II Mechanism of CD4⁺T_EM cells in the progression of acute viral myocarditis to dilated cardiomyopathy in mice.Study one:The Changes of proinflammatory cytokines secreted by CD4⁺T_EMcells in myocardial tissue during the progression of acute viral myocarditis to dilated cardiomyopathy in mice.Objective:To investigate the role of CD4⁺T_EM cells in the progression of acute VMC to DCM,the changes of the secretion of pro-inflammatory cytokines IFN-γand IL-17A by memory CD4⁺T cells were detected in the myocardial tissue of mice during the progression of acute VMC to DCM.Methods:The experimental animals,grouping and VMC model establishment were the same as Part I of Study one.When each subgroup reached the respective observation points,the mice were sacrificed and their hearts were taken.The proportions of IFN-γand IL-17A secreted by memory CD4⁺T cells in myocardial tissues were detected by flow cytometry.Results:The proportions of IFN-γsecreted by memory CD4⁺T cells in the three infected subgroups were significantly higher than that in uninfected subgroups（all P<0.01）.In the infection group,the proportion of IFN-γsecreted by memory CD4⁺T cells in the 28 day subgroup was lower than that in the 14day subgroup（P<0.05）,while the proportion in the 42 day subgroup was higher than that in the 28 day subgroup（P<0.01）.The proportion of memory CD4⁺T cells secreting IFN-γwere higher than that of non-memory CD4⁺T cells in all the three subgroups of infection group（all P<0.01）.The proportions of IL-17A secreted by memory CD4⁺T cells in all of the infected subgroups were significantly higher than that in uninfected subgroups（all P<0.05）.In the infection group,the proportion of IL-17A secreted by memory CD4⁺T cells in the 28 day subgroup was higher than that in the 14 day subgroup（P<0.01）,while that in the 42 day subgroup was lower than that in the 28 day subgroup（P<0.01）.The proportion of memory CD4⁺T cells secreting IL-17A were higher than that of non-memory CD4⁺T cells in the three subgroups of the infected group（all P<0.01）.Conclusion:Proinflammatory cytokines IFN-γand IL-17A secreted by memory CD4⁺T cells are significantly increased during the progression of acute VMC to DCM,and IFN-γand IL-17A secreted by CD4⁺T cells are mainly derived from the memory CD4⁺T cell subpopulation,considering that almost all memory CD4⁺T cells are CD4⁺T_EM cell subsets in the inflame heart which have been demonstrated in Part I study two,thus suggesting that CD4⁺T_EM cells may play an important role by secreting IFN-γand IL-17A in the pathogenesis of acute VMC progression to DCM.Study two:The effect of myocardial self-antigen stimulation on the response of CD4⁺T_EM cells in vitro.Objective:To investigate whether CD4⁺T_EM cells can response to heart self-antigen stimulation,CD4⁺T_EM cells obtained from DCM mice or contol mice spleens were co-cultured with dendritic cells loaded with heart self-antigen in vitro,and the proliferation of CD4⁺T_EM cells was detected as well as the protein concentrations of IL-17 and IFN-γin supernatant.Methods:Male Balb/c mice with 6 weeks old were randomly divided into DCM group and control group.The DCM group mice were intraperitoneally injected with CVB3 virus dilution,0.2 m L/mice.Mice in the control group were intraperitoneally injected with the same volume of PBS.The day of intraperitoneal injection was defined as Day 0,and the mice in each group were sacrificed and their spleens were collected at day 42 after injection.Splenic DCM CD4⁺T_EM cells and control CD4⁺T_EM cells were sorted by flow cytometry respectively,and then co-cultured with actived dendritic cells（DC）loaded with heart self-antigen.Four groups were set up:（1）control CD4⁺T_EM+PBS culture group,（2）control CD4⁺T_EM+DC co-culture group,（3）DCM CD4⁺T_EM+PBS culture group,and（4）DCM CD4⁺T_EM+DC co-culture group.The proliferation rate of CD4⁺T_EM cells was determined by flow cytometry,and the protein concentrations of IL-17 and IFN-γin the cultured supernatant were determined by ELISA.Results:The proliferation rate of CD4⁺T_EM cells in DCM CD4⁺T_EM+DC co-culture group was significantly higher than that in DCM CD4⁺T_EM+PBS co-culture group（P<0.01）and control CD4⁺T_EM+DC co-culture group（P<0.01）.There was no significant difference in the proliferation rate of CD4⁺T_EMcells among the control CD4⁺T_EM+PBS culture group,the control CD4⁺T_EM+DC co-culture group and the DCM CD4⁺T_EM+PBS culture group（all P﹥0.05）.The concentrations of IL-17 and IFN-γin supernatant of DCM CD4⁺T_EM+DC co-culture group were significantly higher than those of DCM CD4⁺T_EM+PBS co-culture group（all P<0.05）.Conclusion:DCM mice splenic CD4⁺T_EM cells can proliferate and secrete IL-17 and IFN-γin co-culture with actived dendritic cells loaded with heart self-antigen,suggesting that CD4⁺T_EM cells formed during the progression of acute VMC to DCM can response to myocardial self-antigen stimulation and thus have specificity of heart self-antigen.Part II Conclusion:CD4⁺T_EM cells of DCM mice can proliferate and secrete IL-17 and IFN-γafter being stimulated by heart self-antigen in vitro.In addition,the amount of IL-17A and IFN-γsecreted by CD4⁺T_EM cells in mice during the progression of acute VMC to DCM is significantly increased,suggesting that CD4⁺T_EM cells formed during the evolution of mouse VMC to DCM are specific for myocardial self-antigen.Heart self-antigen specific CD4⁺T_EM cells and their pro-inflammatory cytokines IL-17A and IFN-γmay promote the progression of acute VMC to DCM by mediating persistent hear-specific autoimmune pathological injury.Part III The effect of CD4⁺T_EM cells on myocardial injury in mice.Objective:To investigate the effect of CD4⁺T_EM cells on myocardial injury in mice,Splenic CD4⁺T_EM cells sorted from DCM mice and uninfected control mice were injected respectively into syngeneic male Balb/c na（?）ve mice,changes in cardiac histopathology as well as cardiac structure and function of the recipient mice were assessed after 28 days of adoptive transfer.Methods:The experimental animals,grouping and VMC model constructing were the same as the Part II study two.DCM CD4⁺T_EM cells and control CD4⁺T_EM cells were sorted by flow cytometry.Male Balb/c na（?）ve mice with 6 weeks old were randomly divided into DCM CD4⁺T_EM group and control CD4⁺T_EM group.Cells were injected intravenously in the lateral tail vein.The mice in the DCM CD4⁺T_EM group mice received DCM CD4⁺T_EM cells,while control CD4⁺T_EM group mice received control CD4⁺T_EM cells.The day of intravenous injection was defined as Day 0.After 28 days of observation,echocardiography was performed.The mice were sacrificed after blood collection.The hearts were taken,embedded in paraffin and sectioned.HE staining and Masson trichromatic staining were performed.The expression of CVB3 m RNA in DCM CD4⁺T_EM cells and control CD4⁺T_EM cells were detected by RT-PCR.Results:The results of echocardiography showed that compared with the control CD4⁺T_EM group,the left ventricular end-systolic internal diameter diameter of DCM CD4⁺T_EM group were significantly increased（P<0.05）,while the left ventricular ejection fraction and left ventricular fractional shortening of DCM CD4⁺T_EM group were significantly decreased（all P<0.05）.HE staining showed significant inflammatory cell infiltration in myocardium of mice in the DCM CD4⁺T_EM group,and the myocardial pathological score was significantly increased（P<0.01）.Masson trichromatic staining showed significant collagen fiber deposition in the myocardial interstitium of mice treated with DCM CD4⁺T_EM,and myocardial fibrosis scores significantly increased（P<0.01）.No abnormality was found by the myocardial histopathological examination in CD4⁺T_EM group.The level of serum CK-MB concentration in DCM CD4⁺T_EMgroup was significantly increased（P<0.05）.RT-PCR results showed that compared with negative control,there was no significant difference in CVB3m RNA expression in splenic CD4⁺T_EM cells obtained from DCM mice and splenic CD4⁺T_EM cells obtained from control mice（all P>0.05）.Conclusion:Splenic CD4⁺T_EM cells sorted from DCM mice can lead to obviously inflammatory cell infiltration,increase deposition of myocardial collagen fibers and serum CK-MB levels,and decrease in cardiac left ventricular ejection fraction and left ventricular fractional shortening,suggesting that splenic CD4⁺T_EM cells obtained from DCM mice can induce chronic myocardial inflammation,leading to myocardial injury,aggravated myocardial interstitial fibrosis and cardiac systolic disfunction,thus indicating CD4⁺T_EM cells paly an important role in driving acute VMC to DCM in mice.In summary,there are four important findings in this research:（1）Mice infected with CVB3 show acute VMC at day 14,chronic myocarditis at day 28,and DCM at day 42 post infection.During the progression of acute VMC to DCM in mice,the myocardial infiltrating CD4⁺T_EM cells increase significantly,suggesting that CD4⁺T_EM cells involve in the pathogenesis of the acute VMC progression to DCM in mice.（2）The amount of proinflammatory cytokines IFN-γand IL-17A secreted by memory CD4⁺T cells are significantly increased during the progression of acute VMC to DCM,and IFN-γand IL-17A secreted by CD4⁺T cells are mainly derived from the memory CD4⁺T cell subpopulation,considering that almost all memory CD4⁺T cells are CD4⁺T_EM cell subsets in the inflame heart which have been demonstrated in Part I study two,thus suggesting that CD4⁺T_EM cells may play an important role by secreting IFN-γand IL-17A in the pathogenesis of acute VMC progression to DCM.（3）CD4⁺T_EM cells of DCM mice can proliferate and secrete IL-17 and IFN-γafter being stimulated by heart self-antigen in vitro,suggesting that CD4⁺T_EM cells formed in the progression of acute VMC to DCM are specific for myocardial self-antigen.Heart self-antigen specific CD4⁺T_EM cells and their pro-inflammatory cytokines IL-17A and IFN-γmay promote the progression of acute VMC to DCM by mediating persistent hear-specific autoimmune pathological injury.（4）Splenic CD4⁺T_EM cells sorted from DCM mice can lead to obviously inflammatory cell infiltration,increase deposition of myocardial collagen fibers and serum CK-MB levels,and decrease in cardiac left ventricular ejection fraction as well as left ventricular fractional shortening,suggesting that splenic CD4⁺T_EM cells obtained from DCM mice can induce chronic myocardial inflammation,leading to myocardial injury,aggravated myocardial interstitial fibrosis and cardiac systolic disfunction,thus indicating CD4⁺T_EM cells paly an important role in driving acute VMC to DCM in mice.