The Role And Mechanism Of B1 And B1a Cells In The Development Of Viral Myocarditis To Dilated Cardiomyopathy

Viral myocarditis(VMC)and virus-associated dilated cardiomyopathy(DCM)are the most common clinical viral heart diseases(VHD)that seriously threaten human health.Some scholars believe that VMC and DCM are actually two stages of a disease.Coxsackie virus B3(CVB3)is the most common cause of VMC.Studies have shown that autoimmune activation is considered to be the main pathogenesis of VMC developing into DCM.B1 cells are innate immune cells,originated in the early stage of embryo,mainly distributed in the serous cavity,and have the ability of self-renewal.B1cells include B1a and B1b cell subsets.B1a cells mainly participate in innate and adaptive immune responses by secreting a variety of cytokines such as natural IgM antibody and interleukin-10(IL-10).Animal experiments and clinical researches suggest that B1 cells,especially B1a cells,are involved in the pathogenesis of a variety of infectious diseases,chronic inflammatory diseases and autoimmune diseases,and play different immunomodulatory roles.Our previous studies have found that pan-B cells and IL-10-producing B cell subsets were involved in the pathogenesis of VMC and DCM.Does B1 cells,as an important B cells subgroup,participate in the pathogenesis of VMC and DCM?What is the role and mechanism in it?It has not been reported yet.In order to further explore the pathogenesis of the development of VMC to DCM and to seek new therapeutic ideas and targets,this study intends to establish a VMC mouse model with B1a cells deficiency,and collect DCM cases for the following three aspects:Part Ⅰ Changes of B1 and B1a cells during the development of viral myocarditis to dilated cardiomyopathy in miceStudy One:Establishment of mouse model of viral myocarditis and dilated cardiomyopathyObjective:To establish VMC and DCM mouse models and to observe the heart changes of mice during the development from VMC to DCM.Methods:One hundred and eighty male BALB/c mice aged 4-5 weeks were randomly divided into viral cardiopathy group(n=120)and control group(n=60).Each group was subdivided into 6 subgroups(1 week,2 weeks,4 weeks,6weeks,8 weeks,24 weeks).Each subgroup of VHD was injected intraperitoneally with CVB3 diluent of cardiomyocyte passage to establish VMC and DCM mouse models,while the control group was intraperitoneally injected with the same amount of phosphate buffer(PBS).At the corresponding time point,the structure and function of mouse heart were evaluated by animal ultrasound,the pathological sections of mouse heart were made,and the histopathological changes of myocardium were observed.Results:Compared with the control group,the left ventricular end-systolic diameter(LVESD)in VHD group was gradually enlarged from the first week after CVB3 infection,accompanied by the decrease of persistent left ventricular short axis shortening fraction(LVFS)and left ventricular ejection fraction(LVEF),and the left ventricular end-diastolic diameter(LVEDD)was gradually enlarged after the 4th week(P<0.05 or P<0.01).At 24 weeks,the above ultrasonic parameters changed most significantly(P<0.05 or P<0.01).Cardiac pathological sections showed that cardiomyocyte swelling,necrosis and interstitial inflammatory cells infiltration appeared in VHD group at the first week,reached the peak and began to appear myocardial interstitial fibrosis at the second week,and the left ventricle was enlarged and myocardial interstitial fibrosis was the most serious at the 24th week.The myocardial pathological score and collagen volume fraction(CVF)of the subgroup from the 1st to 8th week and the 4th to 24th week in the VHD group were significantly higher than those in the control group at the same time(P<0.01).Conclusion:Acute VMC,chronic VMC and DCM mouse models could be established by intraperitoneal injection of myocardial passage CVB3 diluent.During the development of mice from VMC to DCM,myocardial inflammation was the most serious in the acute stage,and myocardial interstitial fibrosis was progressively aggravated,accompanied by progressive left ventricular enlargement and decreased left ventricular systolic function.Study two:Changes of B1 and B1a cells during the development of viral myocarditis to dilated cardiomyopathy in miceObjective:To observe the changes of the proportion of B1 and B1a cells in abdominal cavity and spleen and the levels of IgM,IgG and interleukin-10(IL-10)in plasma during the development of VMC to DCM in mice.Methods:The experimental animals,grouping methods and model establishment methods are the same as this part of study I.The mice were sacrificed at the corresponding time point,the plasma of the mice was separated,and the peritoneum and spleen cells were also collected.The proportion of B1and B1a cells in peritoneum and spleen was analyzed by flow cytometry,and the levels of plasma IgM,IgG and IL-10 were detected by enzyme-linked immunosorbent assay(ELISA).Results:Compared with the control group,the proportion of B1 and B1a cells in peritoneum and spleen in VHD group increased significantly from the first week after CVB3 infection,reached the peak in the second week,and decreased to the lowest at the 24th week.There was no significant change in B1b cells in peritoneum and spleen compared with the control group(P>0.05).The plasma IgM of VHD group increased and peaked at the first week,then decreased gradually,and decreased after 8 weeks,and the plasma IL-10 increased and peaked at the first week and returned to the level of the control group at the 4th week.The level of plasma IgG increased from the first week,reached the peak at the 4th week,was still higher than that in the control group from the 6th to 8th week,and returned to the same level at the 24th week.Conclusion:The proportion of B1 and B1a cells in peritoneum and spleen of mice in acute stage of VMC increased significantly and reached the peak,with the increase of plasma IgM and IL-10.After chronic phase,the proportion of B1and B1a cells in peritoneum and spleen decreased progressively,and the lowest in DCM phase,and the plasma IgM also decreased.Overall conclusion in part I:During the development of mouse VMC to DCM,the proportion of B1 and B1a cells in peritoneum and spleen increased with the aggravation of myocardial inflammation in the acute stage of VMC,along with the increase of plasma IgM and IL-10 levels,while the proportion of B1 and B1a cells in peritoneum and spleen decreased progressively after the chronic phase of VMC,and the lowest in DCM phase,and the plasma IgM also decreased.Therefore,B1 and B1a cells participated in the whole process of the development of mouse VMC to DCM,in which B1a cells might play a major role in the course of the disease.Part Ⅱ The role and mechanism of B1a cells in acute and chronic viral myocarditis in mice.Study one:The establishment of a mouse model of B1a cell deficiencyObjective:To establish two kinds of mouse models of B1a cell deficiency at birth and adult,and to observe the effects of B1a cell deficiency on mice.Methods:Forty newborn male BALB/c mice were randomly divided into two groups:neonatal B1a cell deficiency group(N-B1D n=20)and neonatal control group(N-CON,n=20).Twenty 7-week-old male BALB/c mice were randomly divided into two groups:adult B1a cell deficiency group(A-B1D,n=10)and adult control group(A-CON,n=10).Repeated intraperitoneal injection of distilled water caused hypoosmotic shock of peritoneal cells and eventually depleted mouse peritoneal B1a cells.The B1a cell deficiency model was established from birth in the N-BID group,while the B1a cell deficiency model was established in the A-B1D group from the age of 4 weeks.The corresponding control group was injected with the same amount of sterile PBS.The proportions of B cells,T cells and macrophages in peritoneum and spleen were detected by flow cytometry at the age of 6 weeks and 12 weeks,respectively.The cardiac structure and function were evaluated by animal ultrasound,and the levels of IgM,IgG and IL-10 in plasma were detected by ELISA.Cardiac pathological sections were made to analyze the histopathological changes of myocardium.Results:Compared with the respective control groups,the proportion of B1and B1a cells in peritoneum and spleen in N-B1D group and A-B1D group decreased significantly,and the levels of plasma IgM and heart IgM and IL-10decreased significantly(P<0.05 or P<0.01).However,the proportion of B1b cells,B2 cells,T cells and F4/80~+macrophages in peritoneum and spleen,plasma IgG and IL-10 levels,myocardial pathological scores of LVESD,LVDDD,LVFS,LVEF,and CVF were not affected(P>0.05).Conclusion:Peritoneal hypoosmotic shock could deplete B1a cells in the peritoneum and spleen of mice without affecting other immune cells.Therefore,this method could successfully establish B1a cell defect models in newborn and adult mice.B1a cell deficiency could reduce the levels of plasma IgM and heart IgM and IL-10 in mice,but had no significant effect on the structure,function and histopathology of mouse heart.Study two:The role and mechanism of B1a cells in acute viral myocarditis in miceObjective:To investigate the effect of B1a cells on acute VMC in mice and its mechanism.Methods:Seventy newborn male BALB/c mice were randomly divided into four groups:neonatal B1a cell deficiency group(N-B1D,n=20),neonatal B1a cell deficiency+B1a cell adoption group(B1a+B1D,n=20),VMC control group(VMC-CON,n=20)and blank control group(CON,n=10).The model of B1a cell deficiency in N-B1D group and B1a+B1D group was established by peritoneal hypotonic shock method from birth.At 4 weeks old,except CON group,the other three groups were inoculated intraperitoneally with myocardial passage CVB3 to establish VMC model.Three days before the establishment of VMC mouse model,the peritoneal B1a cells of healthy BALB/c mice were adoptive transferred in B1a+B1D group,and then intraperitoneal injection of distilled water was stopped.At the second week of VMC,animal ultrasound was used to evaluate the cardiac structure and function,ELISA was used to detect the levels of plasma IgM,IgG and IL-10,and cardiac pathological sections were made to analyze myocardial histopathology.Results:Compared with CON group,LVESD in N-B1D group,B1A+B1D group and VMC-CON group increased significantly,LVFS and LVEF decreased significantly,plasma IgM,IgG and IL-10 levels increased,cardiac pathological score and CVF increased,myocardial IgM and IL-10 expression increased(P<0.05 or P<0.01).Compared with B1a+B1D and VMC-CON group,the LVESD of B1a cell deficient N-B1D group was the highest,the LVFS and LVEF were the lowest,the cardiac pathological score was the highest,and the levels of plasma IgM and cardiac IgM and IL-10 were relatively decreased(P<0.05 or P<0.01).However,there was no significant difference in the above parameters between B1a+B1D group and VMC-CON group.Conclusion:In acute VMC mice with B1a cell deficiency,the levels of IgM in plasma and IgM and IL-10 in heart was relatively decreased,myocardial inflammation was aggravated and left ventricular systolic function was decreased.The above changes in the acute phase of VMC in B1a deficient mice could be reversed by adoption B1a cells.Therefore,B1a cells in the acute phase of mouse VMC might play a protective role by regulating the expression of IgM in plasma and IgM and IL-10 in heart,reducing myocardial inflammation and preventing left ventricular enlargement and decrease of systolic function.Study three:The role and mechanism of B1a cells in chronic viral myocarditis in miceObjective:To explore the role and possible mechanism of B1a cells in chronic VMC mice.Methods:Twenty-five 4-week-old male BALB/c mice were randomly divided into two groups:adult B1a cell deficiency group(A-B1D,n=15)and adult VMC control group(A-VMC,n=10).Forty newborn male BALB/c mice were randomly divided into two groups:neonatal B1a cell deficiency group(N-B1D,n=20)and neonatal B1a cell deficiency+B1a cell adoption group(B1a+B1D,n=20).Myocardial passage CVB3 was used to establish VMC model in A-B1D group and A-VMC group.After three weeks,B1a cell deficiency model was established in A-B1D group,while the same amount of PBS was injected in A-VMC group.These two groups of mice were used to observe the effect of B1a cell deficiency in chronic phase of VMC on mice.The model of B1a cell deficiency was established in N-B1D group and B1a+B1D group from birth,and the VMC model was established at 4 weeks old.At the end of VMC 3-week,B1a+B1D mice were adoptive transferred the peritoneal B1a cells from healthy BALB/c mice.These two groups of mice were used to observe the effects of B1a cell deficient mice on mice in the chronic phase of VMC.At the 8th week of VMC,the cardiac structure and function of mice were evaluated by ultrasound,the levels of plasma IgM,IgG and IL-10 were detected by ELISA,and myocardial histological changes were analyzed.Results:Compared with A-VMC control group,A-B1D group with B1a cells deficiency had higher LVESD,lower LVFS and LVEF,higher CVF,and lower plasma IgM,cardiac IgM and IL-10 levels(P<0.05 or P<0.01).Compared with N-B1D control group,LVESD and LVEDD decreased,LVFS and LVEF increased,CVF decreased,plasma IgM and heart IgM,IL-10 levels increased significantly in B1a+B1D group after adoptive transfer B1a cells(P<0.05 or P<0.01).Conclusion:The levels of IgM in plasma and IgM and IL-10 in heart decreased,myocardial fibrosis increased and left ventricular systolic function decreased in mice with B1a cell deficiency in chronic phase of VMC.Mice with B1a cell deficiency could reverse the above changes after adoptive transfer B1a cells in the chronic phase of VMC.Therefore,B1a cells in the chronic phase of mouse VMC might play a protective role by regulating the expression of IgM in plasma and IgM and IL-10 in heart,inhibiting myocardial interstitial fibrosis and preventing the decrease of left ventricular dilatation and systolic function.Overall conclusion in part Ⅱ:The deficiency of B1a cells in acute and chronic phase of VMC in mice resulted in the decrease of the levels of IgM in plasma and heart,the aggravation of myocardial inflammation,the increase of myocardial interstitial fibrosis and the dysfunction of left ventricular systolic function.Therefore,B1a cells might reduce myocardial inflammation and inhibit ventricular remodeling and protect left ventricular systolic function by regulating the levels of IgM in plasma and heart IgM and IL-10 in VMC mice,and play a protective role in the whole process of development from VMC to DCM in mice.Part Ⅲ Changes and clinical significance of B1 cells in peripheral blood of patients with dilated cardiomyopathyObjective:To observe the changes of B1 cells in peripheral blood of patients with DCM and its clinical significance.Methods:Twenty-seven patients with primary DCM(DCM group),18patients with heart failure(HF group)and 21 healthy subjects(HC group)were included in this study.The proportion of B1 cells in peripheral blood was analyzed by multi-color flow cytometry.The levels of plasma IgM,IgG and IL-10 were measured by ELISA.In addition,clinical data such as N-terminal pro-brain natriuretic peptide(NT-pro BNP),high sensitivity C-reactive protein(hs-CRP)and LVEF were collected.Results:The proportion of B1 cells in DCM group was significantly lower than that in HF and HC groups.There was no significant difference in plasma IgM,IgG and IL-10 levels among the three groups.The proportion of B1 cells in peripheral blood of patients with DCM was positively correlated with LVEF and negatively correlated with NT-pro BNP.Conclusion:The proportion of B1 cells in peripheral blood of patients with DCM was decreased,and the proportion of B1 cells was closely related to the severity of the disease,suggesting that B1 cells may be involved in the pathogenesis of DCM.Summary of the full text:1.A series of mouse models of acute VMC,chronic VMC and DCM could be established by intraperitoneal inoculation of myocardial passage CVB3 diluent.2.During the development of mouse VMC to DCM,the proportion of B1 and B1a cells in the peritoneum and spleen increased with the aggravation of myocardial inflammation in the acute stage of VMC,decreased gradually in the chronic stage,and was the lowest in the DCM phase.This indicates that B1 and B1a cells participated in the whole process of mouse VMC developing to DCM,and B1a cells might play a major role in the course of disease.3.B1a cells might reduce myocardial inflammation,inhibited myocardial interstitial fibrosis and protected left ventricular systolic function by regulating the levels of IgM in plasma and heart IgM and IL-10 in VMC mice,and played a protective role in the whole process of development from VMC to DCM in mice.4.The proportion of B1 cells in peripheral blood of patients with DCM was decreased,and the proportion of B1 cells was closely related to the severity of the disease,suggesting that B1 cells might be involved in the pathogenesis of DCM.