Differential Induction Of Regulatory T Cells In Different Genetic Background And Its Influence On The Susceptibility To CVB3 Myocarditis

Viral myocarditis(VMC) is a kind of clinical common infectious diseases of cardiovascular system.In recent years,the incidence of VMC is increasing in China and western countries,and VMC has become one of the main reasons of sudden death in young people.Moreover,chronic VMC can also lead to dilated cardiomyopathy, which is one of the important reasons of heart transplantation.To date,treatment of VMC is only conservative with no effective etiological therapy due to unelucidated pathologic mechanism.So,it is of important theoretical and practical significance to clarify VMC pathologic mechanism.Viral infection is one of the important reasons for VMC.Among the viruses, Coxsackievirus group B type 3(CVB3) infection accounts for more than half of VMC cases.CVB3 infects myocytes through its receptor(Coxsackievirus-adenovirus receptor,CAR) and coreceptor(Decay accelerating factor,DAF),then replicates in myocytes.Although CVB3 can directly lead to myocytes damage through its encoded Pro2A to change the framework of myocytes or through the binding of caspid VP2 and host proapoptotic protein Siva to induce the apoptosis of myocytes.More and more evidence showed that the most important reason for myocytes damage is the excessive immune response.Woodruff and Woodruff first demonstrated the role of T lymphocytes in the pathogenesis of murine myocarditis using antithymocyte serum or thymectomy and irradiation leading to a decrease in mortality and a reduction in the inflammatory infiltrate after CVB3 infection.Opavsky MA et al.also found the severity of disease was attenuated in CD4 KO mice,confirmed the role of CD4~+ T cells in CVB3 induced myocarditis.Epidemiological studies revealed that although the percentage of CVB3 infection is very high,most people show recessive infection or common cold symptom,only a small group of people progress to viral myocarditis or chronic dilated cardiomyopathy. So,it will help to further understand the mechanism of VMC to explore the reasons why different group of people infected with CVB3 show different clinical prognosis. In the present study,two different mouse strains BALB/c and C57BL/6 were infected with CVB3 and the correlation between the susceptibility of VMC and the ability of virus infection in myocytes of these two mice or immune response induced by CVB3 infection were determined.Results showed that BALB/c mice had a higher susceptibility to VMC and a stronger cellular immune response than C57BL/6 mice after infection,while the infection,replication and translation of CVB3 was similar in myocytes of these two mice which substantiated the role of immune injury in the pathogenesis of VMC.Since regulatory T cells(Tregs) play important roles in immune tolerance and the regulation of anti-viral immune response,the variance of the number or function of Tregs may directly influence the progression of disease.As VMC is mainly due to the immune response-mediated myocytes damage after CVB3 infection,then whether Tregs also play a role in the regulation of anti-CVB3 immune response? Whether the disparate susceptibility to VMC was correlated with the percentage and function of Tregs? And what kind of factors may modulate the percentage and function of Tregs? So,in the present study,the role of Tregs in VMC and the correlation between Tregs and the susceptibility of VMC were studied using two mouse strains which are differently susceptible to CVB3 infection.PartⅠ.The susceptibility of BALB/c and C57BL/6 mice to CVB3 induced myocarditis1.The distinct susceptibility to CVB3 induced VMC between BALB/c and C57BL/6 miceViral myocarditis is characterized with inflammatory cells infiltration in the interstitial tissue of myocardium with the result of myocytes focal necrosis or fibrosis and finally the loss of heart function.It well mimics the characteristics of clinical viral myocarditis using CVB3 infected myocarditis murine model with the indices of serology,pathology and body weight reduced rate as well as mortality.From day 3 postinfection,the body weight of BALB/c mice decreased obviously.On day 7 postinfection,the body weight decreased 20%.While in C57BL/6 mice,on day 3 postinfection,the body weight only slightly reduced and then maintained at this level. The activity of serum Creatine Kinase(CK) and MB isoenzyme of Creatine Kinase (CK-MB) was significantly upregulated and the level of Cardiac Troponin I(cTnI) was also increased in BALB/c mice on day 7 postinfection.While in C57BL/6 mice, they were only slightly increased.The pathological studies,the most direct and important diagnosis criteria,revealed that in BALB/c mice,on day 7 postinfection, white streaks were exudated in heart thoracic surface,H.E showed the focal infiltration of inflammatory cells and the massive necrosis of myocytes.While in C57BL/6 mice,there was no obvious white streaks exudation and only a small amount of inflammatory cells infiltration and no myocytes necrosis.As far as mortality was concerned,the mortality in CVB3 infected BALB/c mice was 75%, which was much higher than that in infected C57BL/6 mice with the mortality of 25%. From the above results,the conclusion could be drawn that BALB/c mice was more susceptible to viral myocarditis than C57BL/6 mice after CVB3 infection.2.The ability of virus infection,replication or translation in myocytes of BALB/c and C57BL/6 miceCVB3 can infect myocytes and replicate in cells,which is the prerequisite to induce VMC.To exclude the possibility that the insusceptibility of C57BL/6 mice to VMC was due to the inability of CVB3 to infect the myocytes of C57BL/6 mice,or due to the low efficiency of CVB3 to replicate or translate viral proteins in C57BL/6 myocytes.We firstly compared the virus loading in the heart tissue of BALB/c and C57BL/6 mice at the early phase(day 3) of CVB3 infection.Results showed that similar virus loading was observed in heart tissue of these two mice.And we next detected the copy of negative strand RNA of CVB3 and got similar results.Then the expression level of virus protein VP1 in CVB3 infected BALB/c and C57BL/6 heart was determined and we found the expression level of VP1 was similar in heart of these two mice at day 3 postinfection.However the virus loading,negative strand RNA copy and VP1 expression level was much higher in BALB/c heart than that in C57BL/6 heart at day 7 postinfection.To exclude the impact of complex in vivo environment on virus infection or replication,we determined the virus loading,copy of negative strand CVB3 RNA and VP1 expression in vitro,and results showed that the virus loading,copy of negative strand RNA and VP1 expression was comparable in CVB3 infected BALB/c and C57BL/6 primary myocytes at early and late phase of infection.In summary,it suggest that the different susceptibility of BALB/c and C57BL/6 mice to VMC induced by CVB3 infection was not due to the different infection and replication of CVB3 in the heart tissue of different murine strains. PartⅡ.The alteration of the percentage and function of Tregs in BALB/c and C57BL/6 mice after CVB3 infection1.Differential immune response in BALB/c and C57BL/6 mice after CVB3 infectionAfter exclusion of the difference in virus infection and replication,the difference in immune response was further observed.In heart local immune response aspect,the number of CD3~+ T cells was twice higher in heart infiltrated cells in BALB/c mice than that in C57BL/6 mice on day 7 postinfection,and the increased T cells was mainly CD4~+ T cells.And we also observed the hyperproliferation of CD4~+ T cells in BALB/c heart,suggesting the excessive local immune response in BALB/c mice.The levels of heart local expressed TNF-αand INF-γwere much higher in BALB/c mice than that in C57BL/6 mice.And we also found the higher level of systemic serum TNF-αand INF-γin BALB/c mice than that in C57BL/6 mice on day 7 postinfection, suggesting the correlation between the excessive cellular immune response and the high susceptibility of viral myocarditis after CVB3 infection.2.The percentage alteration of Tregs in BALB/c and C57BL/6 mice after CVB3 infectionTregs play important roles in controlling excessive immune response and maintaining immune homeostasis,their number and function are closely related with the extent of immune response.In the present study,considering the results that a stronger Th1 immune response was in BALB/c mice on day 7 after CVB3 infection, which led to the heart damage and a relatively weaker Th1 immune response was in C57BL/6 mice,which endowed C57BL/6 mice with insusceptibility to myocarditis, the hypothesis was presented that the different extent of immune response was due to the different percentage and function of Tregs in two strain mice.Under normal conditions,the percentage of Tregs(CD4~+CD25~+Foxp3~+) was very low,only accounts for 5%to 10%.While in the process of immune response,the number of Tregs significantly increased through self-proliferation or the transformation from effector T cells in order to control the extent of immune response. In the present study,from day 4 postinfection,the percentage of Tregs in C57BL/6 peripheral blood was significantly increased,and accounted for 20%of CD4~+ T cells on day 7 postinfection.While in BALB/c mice,the number of Tregs was only slightly increased and the percentage of Tregs was much lower than that in C57BL/6 mice on day 7 postinfection.In heart infiltrated cells,the percentage of Tregs was much higher in C57BL/6 mice than that in BALB/c mice.RT-PCR assay also revealed that Foxp3 expression of heart infiltrated cells was higher in C57BL/6 mice than that in BALB/c mice.3.The comparison of Tregs function between BALB/c and C57BL/6 mice after CVB3 infectionApart from the percentage of Tregs,the extent of Tregs inhibitory function also directly affects the ability of controlling immune response.In the present study,Tregs in spleen were isolated and purified in CVB3 infected mice and observed that Tregs derived from BALB/c and C57BL/6 mice both could effectively inhibit the proliferation of effector T cells in vitro,moreover,the extent of inhibitory function was related with Tregs percentage.The above data showed that the percentage of Tregs in peripheral blood or in heart was much higher in C57BL/6 mice after CVB3 infection than that in BALB/c mice,strongly suggests that the percentage of Tregs was closely correlated with the susceptibility of viral myocarditis.PartⅢ.The study on the relationship between Tregs and viral myocarditisThe above two parts showed that the susceptibility of BALB/c to viral myocarditis was higher than that of C57BL/6 mice,the reason was probably that the percentage of Tregs in BALB/c mice was much lower than that in C57BL/6 mice after infection.In order to confirm the role of Tregs in viral myocarditis,transfer experiment was performed to artificially control the percentage of Tregs and the role of Tregs in VMC was explored.1.The influence of Tregs transfer on the susceptibility to VMC of BALB/c miceThe insufficiency of Tregs in BALB/c mice could not control the excessive immune response,which led to the susceptibility to viral myocarditis,then whether the increase of Tregs could protect mice from myocarditis.In the present study,Tregs and effector cells with high purity were sorted using FACS and then transferred to CVB3 infected BALB/c nu/nu mice(day 3 postinfection).When only effector cells were transferred,viral myocarditis was developed in mice with typical serological and pathological features on day 7 postinfection.While when Tregs and effector cells were cotransferred,the mice did not develop viral myocarditis,which suggests the protective role of Tregs in viral myocarditis.2.The influence of Tregs depletion on the susceptibility to VMC of C57BL/6 miceIn order to confirm the role of Tregs in viral myocarditis,whether the susceptibility to viral myocarditis would increase when Tregs were deleted in C57BL/6 mice was observed.Treg-depleted effector cells were purified and transferred into CVB3 infected C57BL/6 nu/nu mice(day 3 postinfection).On day 7 postinfection,the serological and pathological results showed that the susceptibility to viral myocarditis did not increase although Tregs were deleted in transferred effector cells.It was not in line with our hypothesis that the susceptibility of C57BL/6 mice to viral myocarditis did not increase when Tregs were deleted.In order to explain this phenomenon,after the effector cells were transferred,the spleen cells and heart infiltrated cells in C57BL/6 nu/nu mice were isolated and assayed by FACS.It was found that more than 20%of CD4~+ cells expressed CD25 and more than 10% expressed Foxp3 in spleen.In heart infiltrated cells,more than 90%of CD4~+ cells expressed CD25 and more than 15%of CD4~+ cells expressed Foxp3.So it was probably that the transferred CD4~+CD25~- T cells were induced into CD4~+CD25~+Fopx3~+ T cells in C57BL/6 nu/nu mice and thus protected the mice from viral myocarditis.The above results showed that transfer of Tregs could protect BALB/c mice from CVB3 induced myocarditis.And in C57BL/6 mice,Tregs could be induced from CD4~+CD25~- effector T cells after CVB3 infection,which endows the mice with insusceptibility to viral myocarditis.PartⅣ.The differential induction of Tregs in CVB3 infected BALB/c and C57BL/6 miceThe above results showed that CD4~+CD25~+Foxp3~+ Tregs could be induced from CD4~+CD25~- T cells when Tregs deleted effector cells were transferred into CVB3 infected C57BL/6 nu/nu mice.Then the reason deserved to be further studied.It has been reported that immature or semi-mature dendritic cells(DCs) could induce Tregs. So,DCs in BALB/c and C57BL/6 mice were isolated and the maturity alteration was observed,and the ability of DCs to induce Tregs in vitro was also detected.1.The alteration of the maturity of DCs in CVB3 infected BALB/c and C57BL/6 miceMature DCs could induce the activation and proliferation of effector T cells,and play a positive role in the regulation of immune response.However,Immature DCs usually induced the apoptosis or immune anergy of the effector cells.It has been reported that immature DCs could induce the transformation of CD4~+CD25~- T cells to Tregs,which played an important negative immune regulation.In the present study, the expression intensity of MHC-Ⅱ,CD80 and CD86 was detected in CD11c~+ DCs in the spleens of BALB/c and C57BL/6 mice on day 0,day3 and day7 after CVB3 infection.It was shown that on day3 postinfection,the expression intensity significantly increased in BALB/c mice,and on day7,it reached the higher level. However,in C57BL/6 mice,the expression intensity was not obviously increased and maintained at the pre-infection level.Immature DCs could induce the production of Tregs through the secretion of IL-10,TGF-βand other cytokines,so the ability of the secretion of IL-10 and TGF-βin splenic DCs of BALB/c and C57BL/6 mice after CVB3 infection was detected by FACS.The results showed that on day7 postinfection,the percentage of DCs secreting IL-10 in spleen of BALB/c mice was significantly decreased,while in C57BL/6 mice, the DCs percentage increased to the double of that before the infection.Although the percentage of DCs which secreting TGF-βwas both increased in CVB3 infected BALB/c and C57BL/6 mice,there was no significant difference between these two mice.Then DCs were purified and cultured in vitro,and the production of IL-10 and TGF-βin the supernatant was detected.Result showed that CVB3 infection decreased the production of IL-10 of DCs in BALB/c while it increased the production of IL-10 of C57BL/6 DCs.And the level of IL-10 in the supematant of DCs from CVB3 infected C57BL/6 mice was higher than that of DCs from CVB3 infected BALB/c mice.2.The induction of Tregs induced by immature DCs in vitroTo further confirm the role of DCs in the induction of Tregs,splenic CD11c~+ DCs and CD4~+CD25~- T cells were isolated and purified,then were cocultured for 7 days in vitro,the percentage of Foxp3~+ Tregs were detected by FACS.It was shown that DCs from CVB3 infected C57BL/6 mice exhibited stronger inductive ability to Tregs than that from CVB3 infected BALB/c mice.When anti-IL-10 antibody was added in the system,DCs inductive ability was significantly weakened,indicate that DCs induct the production of Tregs partly through IL-10 in CVB3 infected C57BL/6 mice.While anti-TGF-βantibody had little effects on the induction of Tregs.In conclusion,the present study confirmed the protective role of Tregs in viral myocarditis,and for the first time,presented the idea that the different susceptibility of BALB/c and C57BL/6 mice to CVB3 induced myocarditis was due to the different number of Tregs postinfection,and not due to the difference of virus infection and replication.Further study revealed that the different Tregs number was caused by stronger ability of DCs from CVB3 infected C57BL/6 mice to induce Tregs.The study clarified the reason for different susceptibility to VMC in different mice strains, further elucidated the mechanism of the pathogenesis of viral myocarditis.Moreover, it provided the experimental basis for the different clinical prognosis of different groups of CVB3 infected people.And further research will focus on whether it is always the same when these two strains of mice were infected with other virus and what kind of factors may lead to the different maturity of DCs in BALB/c and C57BL/6 mice after CVB3 infection.