Regulatory Function Of IL-10-Producing B Cells Involved In Pathogenesis Of Murine Viral Myocarditis

Viral myocarditis(VMC)is an inflammatory disease of the myocardium caused by a kind of viruses preferring entry to heart.Most of acute VMC recover spontaneously in early phase,but some would deteriorate and develop dilated cardiomyopathy(DCM).The pathogenesis of VMC is not totally illuminated yet.It is shown that T cells play a vital role in pathogenesis of VMC,in which Thl cells are elevated in acute VMC and participate in the cardiac inflammation by IFN-? production,whereas Th17 cells aggravate cardiac damage and promote fibrosis by IL-17 production.Regulatory B cells(Bregs)is a kind of new B cells which have potent regulatory function.One of most important subtypes is IL-10-producing B cells.It depends on IL-10 to regulate other cells,especially effector T cells on cellular level.Regulatory B cells are indentified to restrain immune responses and release inflammation in most autoimmune diseases and inflammatory diseases.However,whether do IL-10-producing B cells exist in VMC and how do these cells function in VMC?There is no report about that.To further study the role of IL-10-producing B cells in VMC,we produced a murine model of VMC which was induced by Coxsackie virus B3(CVB3)to confirm the regulatory function of IL-10-producing B cells involved in the pathogenesis of VMC and offer a new clue to treat VMC.Part 1 The changes of IL-10-producing B cells in the pathogenesis of VMC.Objective:To found out the changes of IL-10-producing B cells and the correlation of IL-10-producing B cells and Th cells in VMC mice.Methods:Male 4-week-old BALB/c mice were divided into VMC group(n=50)and control group(n=30).Each group was divided into 3 subgroups:1w subgroup,2w subgroup and 4w subgroup.There were 12,18,20 mice respectively in different subgroups of VMC group.There were 10 mice in every subgroup of control group.BALB/c mice were inoculated intraperitoneally(i.p.)to induce VMC and 10 mice of them were chosen on the right time.Flow cytometry was applied to study the proportions of spleen IL-10-producing B cells,Th1 cells,Th17 cells and CD1dhiCD5+B cells(one of phenotype of IL-10 producing B cell).HE staining was performed to observe inflammation in myocardium and calculate cardiac pathological scores.Results:1.HE staining in myocardium showed necrosis of cardiac myocytes and infiltration of inflammatory cells on 1 week,then it was more severe on 2 week,finally it was milder on 4 week in VMC mice.It was normal in myocardium of control group.2.In contrast to control group(values were 0 in all),cardiac pathological scores were increased on 1w,2w and 4w(2.30±0.48;2.90±0.74;1.40±0.52.all P<0.01).Meanwhile cardiac pathological scores on 2w were higher than that on 1w(P<0.05)and on 4w(P<0.01).3.Spleen IL-10-producing B cells proportions in each subgroup of VMC group were higher than that of control group(2.70%±0.69%versus 0.90%± 0.36%;3.10%± 0.50%versusl.18%±0.31%;1.37%±0.35%versus 0.97%±0.21%.all P<0.01).Meanwhile spleen IL-10-producing B cells proportions on 1w and 2w were higher than that on 4w(all P<0.01).4.Spleen Thl cells proportions in each subgroup of VMC group were also higher than that of control group(6.62%±1.00%versus 3.44%±1.34%;8.28%±2.04%versus 3.52%+1 ± 115%;5.61%± 1.14%versus 3.97%±1.01%.all P<0.01).Meanwhile,spleen Thl cells proportions on 2w were higher than that on 1w(P<0.05)and on 4w(P<0.01).5.Spleen Th17 cells proportions in each subgroup of VMC group were also higher than that of control group on 1w,2w,4w(2.46%±0.50%versus 0.98%±0.40%,P<0.05;5.35%±1.11%versus 1.12%±0.51%,P<0.01;2.49%± 0.48%versus 0.98%±0.27%,P<0.01).Meanwhile,spleen Th17 cells proportions on 2w were higher than that on 1w and on 4w(all P<0.01).6.Spleen CD1dhiCD5~+B cells proportions in each subgroup of VMC group were higher than that of control group on 1w,2w,4w(5.32%±1.20%versus 2.10%± 0.66%;5.56%±1.03%versus 2.70%±0.63%;4.85%±1.02%versus 2.85%±0.69%.all P<0.01),whereas there were on differences between the 3 subgroups of VMC group(P>0.05).Conclusions:A murine model of VMC is induced by CVB3 successfully.Spleen IL-10-producing B cells proportions are increased on 1w,2w and 4w of VMC,presenting a first-high-post-low trend.Spleen CD1dhiCD5~+B cells proportions maintain to be high in VMC mice.Spleen Th1 and Th17 cells proportions are increased on 1w,2w and 4w of VMC,presenting a peak on 2w.IL-10-producing B cells proportions,Th1 cells and Th17 cells proportions parallel with the pathogenesis conditions of VMC.Part 2 The regulation of IL-10-producing B cells on Th1 and Th17 cells in VMC mice1.Objective:To investigate the adoptive effect of IL-10-producing B cells on VMC in T and B cell-deficient mice.Methods:Male 4-week-old SCID mice(n=40)were randomly divided into treated group(n=20)and control group(n=20).CVB3 were i.p.to SCID mice to induce VMC.In treated group,injections of IL-10-producing B cells from acute VMC mice were conducted by tail vein before induction.In control group,physiological salines were injected into SCID mice as controls.The mean survival time was observed during 14 days in VMC mice and the survival mice(n=8)were randomly chosen on day 14 to observe cardiac lesions and calculate cardiac pathological scores.Results:Between treated groups and control group,there were no differences in cardiac pathological scores(3.28±0.28 versus 3.47±0.24,P>0.05)and in mean survival time(8.70d±0.57d versus 7.93d±0.65d,P>0.05).Conclusions:The adoptions of IL-10-producing B cells do not alleviate cardiac lesions and prolong the mean survival time in T and B cell-deficient VMC mice.2.Objective:To investigate the adoptive effect of IL-10-producing B cells on B cell-deficient VMC mice.Methods:Male 4-week-old SCID mice(n=60)were randomly divided into 3 groups:naive group(n=20),sensitized group(n=20)and control group(n=20).SCID mice were injected CD4+T cells by tail veins,at the same time,CVB3were i.p.to produce a B cell-deficient VMC mice model.IL-10-producing B cells from normal BALB/c mice(naive group)or from acute VMC mice induced by CVB3(sensitized group)were adopted 1 day before the induction of VMC.Physiological saline was injected as controls.The mean survival time was observed during 14 days in VMC mice and the survival mice(n=10)were randomly chosen on day 14 to observe cardiac lesions,calculate cardiac pathological scores,measure mRNA expression levels of the transcription factors T-bet of Th1 cells and ROR?t of Th17 cells by RT-PCR and the proportions of spleen Thl cells and Th17 cells by flow cytometry.Results:1.In contrast to control group,the cardiac pathological scores of naive group did not change(2.12± 1.07 versus 2.8710.85,P>0.05),whereas that of sensitized group were significantly decreased(1.21±0.60 versus 2.87±0.85,P<0.01).2.In contrast to control group,the mean survival time of naive group did not change(12.50d±0.70d versus 11.05d±0.68d,P>0.05),whereas that of sensitized group was prolonged(13.4d±0.24d versus 11.05d±0.68d,P<0.05).3.Compared with control group,the spleen Th1 and Th1 7 cells proportions of naive group did not change(4.10%±1.74%versus5.83%± 2.02%;2.24%± 1.34%versus 2.92%±1.3207%.all P>0.05),whereas that of sensitized group significantly decreased(3.20%± 2.31%versus5.83%±2.02%,P<0.01;1.35%±1.24%versus2.92%±1.32%,P<0.05).4.Compared with control group,the mRNA expression level of transcription factor T-bet of Thl cells and RORyt of Th17 cells in myocardium of naive group showed no change(3.76± 1.49 versus4.62±2.60;1.55±0.90 versus2.67± 1.92.all P>0.05),whereas that of sensitized group were lower(1.31±1.28 versus4.62±2.60,P<0.01;1.30±0.35 versus 2.67±1.92,P<0.05).Conclusions:Immune adoption of IL-10-producing B cells from acute VMC mice would alleviate cardiac lesions and prolong the mean survival time by which downregulate mRNA expression level of transcription factor T-bet and RORyt,lead to lower Th1 cells and Th17 cells proportions,whereas adoption of IL-10-producing B cells from normal mice could not.It is suggested that IL-10-producing B cells limit cardiac lesions in VMC mice by which downregulate transcription factors T-bet and RORyt to decrease Thl cells and Th17 cells proportions.Part 3 The effect of IL-10-producing B cells on pathogenic process ofVMC miceObjective:To study the effect of the adoptive effect of IL-10-producing B cells on myocardial damage and prognosis on different time point of VMC.Methods:Male 4-week-old BALB/c mice(n=40)were divided into treated group(n=20)and control group(n=20).CVB3 was i.p.to BALB/c mice to induce VMC model.In treated group,IL-10-producing B cells from acute VMC mice were adoptively transferred into BABL/c mice 1 day before CVB3 induction or on day 3 or day 7 of VMC.Physiological saline was injected as controls.The survival time was observed during 14 days and the survival mice(n=10)were randomly chosen on day 14 to observe cardiac lesions,calculate cardiac pathological scores,measure spleen Th1 cells and Th17 cells proportions by flow cytometry.Results:1.Adoptive transfer of IL-10 producing B cell before VMC prolonged the mean survival time(13.80d±0.11d versus 11.80d±0.62d,P<0.05).Cardiac pathological scores were lower than that of control group(2.08±0.54 versus2.98±0.55,P<0.01).Spleen Th1 and Th17 cells proportions significantly decreased(4.57%±2.04%versus8.71%±2.29%;3.58%±0.57%versus5.36%±1.08%.all P<0.01).2.Adoptive transfer of IL-10 producing B cell on 3 days of VMC did not prolong the mean survival time(12.95d±0.37d versus 11.75d±0.64d,P>0.05).However,compared with control group,cardiac pathological scores were lower(2.08±0.54versus2.94±0.30,P<0.01);spleen Th1 and Th17 cells proportions significantly decreased(5.23%± 1.94%versus8.17%±2.21%;3.35%±0.62%versus5.14%± 1.20%.all P<0.01).3.Adoptive transfer of IL-10 producing B cell on 7 days of VMC did not prolong the mean survival time(12.55d±0.49d versus 12.20d±0.57d,P>0.05).There were no differences between treated group and control group in cardiac pathological scores(2.47±0.66 versus3.01±0.56,P>0.05),and spleen Th1 and Th17 cells(4.60%±1.97%versus6.27%±2.02%;4.03%±0.60%versus4.87%±1.18%.all P>0.05).Conclusions:The adoption of IL-10-producing B cells before VMC induction downregulate Thl1and Th17 cells proportions to reduce cardiac inflammation lesions and prolong the survival time;the adoption of IL-10 producing B cells on day 3 of VMC downregulate Thl and Th17 cells proportions to alleviate cardiac inflammation but do not prolong the survival time;the adoption of IL-10-producing B cells on day 7 of VMC do not downregulate Th1 and Th17 cells proportions,alleviate-cardiac inflammation lesions and prolong the survival time.In summary,we suggested that:1.Spleen IL-10-producing B cells are elevated on 1w,2w and 4w in VMC mice,presenting a first-high-post-low trend.Spleen IL-10-producing B cells proportions,Th1 cells and Th17 cells proportions parallel with the pathogenesis conditions of VMC.2.IL-10-producing B cells,on T cell-dependent,limit inflammatory responses in VMC by which downregulate mRNA expression levels of transcription factors T-bet and RORyt to lower Th1 and Th17 proportions.3.The adoption of IL-10 before VMC induced and on early day of VMC could downregulate Th1 and Th17 cells proportions to reduce cardiac inflammation in VMC mice.