| Viral heart disease (VHD) is a common cardiac disease, characterized by myocardial inflammation due to virus infection and including viral myocarditis (VMC) and dilated cardiomyopathy (DCM). Coxsackievirus B (CVB) is regarded as the primary pathogen in human VMC. Since the persistence of viral infection and immune homeostasis exist, some patients with VMC may progress to chronic myocarditis and DCM. It is believed that VMC and DCM are perceived as different stages of the same disease, VHD. The main characteristic of progress from VCM to DCM is extensive myocardial fibrosis. However, the machanism of myocardial fibrosis remain uncertain.Th22 cells are a subset of CD4+ effector T cells that primarily secrete interleukin-22 (IL-22). These cells do not express IL-17, IL-4, or IFN-y. More and more researches indicatied that IL-22-secrecting Th22 cells play a key role in the pathological mechanism of inflammation and autoimmune diseases. Our previous studies have found that Th22 cells and IL-22 showed critical anti-inflammatory and antiviral activity in disease development of CVB3-induced acute viral myocarditis (AVMC) mice.However, whether do IL-22-secrecting Th22 cells participate in the progress from CVB3-induced mice VMC to DCM is still unclear. The role of Th22 cells and the mechanism of myocardial fibrosis in these courses are uncertain. Our present study attempted to detect the role of IL-22-secrecting Th22 cells in myocardial fibrosis of VHD and provided new insights into the potential therapeutic target of DCM. Therefore, the next 3 parts researches were performed:Part 1:The alteration of myocardial fibrosis indicators in different stages of VHDThis part included the following two sections:1. Induction of VHD, including AVMC, chronic myocarditis and DCM; 2. The alteration of myocardial fibrosis indicators in different stages of VHD.One hundred male BALB/c mice were divided into four groups randomly: 20 in the AVMC group; 20 in chronic myocarditis group; 20 in DCM group; and the rest 30 mice as a PBS injection control group. BALB/c mice were infected by intraperitoneal injection (i.p) of 100 μl PBS containing approximately-106 PFUs of the virus for establishing AVMC and injected once every 4 weeks in increments of 10μl in a total of three doses for establishing the model of chronic myocarditis and six doses for establishing the model of DCM. All surviving animals in AVMC group and 10 control mice were sacrificed by the end of week 2 (14 days) after infection. In chronic myocarditis and DCM groups, surviving mice and 10 control mice at the same time points were sacrificed by the end of week 12 and 24 (84 days and 148 days), respectively. Myocardial histopathologic images in AVMC, chronic myocarditis and DCM were evaluated by hematoxylin-eosin (HE) staining, and myocardial fibrosis images were evaluated by Masson’s trichrome staining. Cardiac expressions of collagen type I-A1 (COL1-A1), collagen type III-A1 (COL3-A1), matrix metalloproteinase-9 (MMP9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were investigated by real time-polymerase chain reaction (RT-PCR), and the levels of circulating protein were measured by enzyme linked immunosorbent assay (ELISA).In sections of cardiac tissues from AVMC mice, we observed larger numbers of inflammatory cells and necrosis, and destruction of myocardial fibers was detected. There were decreasing inflammatory cells and increasing myocardial collagen fibers at the stage of chronic myocarditis with compensatory hypertrophy of myocardial cells, and necrotic myocardial cells replaced by fibrous tissue gradually. In DCM mice, myocardial fibers were increased dramatically disorganized, with no inflammatory cell infiltration. The severity of myocardial fibrosis significantly increased accompanied by up-regulation of collagen volume fraction (CVF), COL1-A1, COLA3-A1, MMP9 and ratio of MMP9/TIMP-1 but down-regulation of TIMP-1 in the progression from AVMC to DCM.As a result, we have established the model VHD successfully, and the severity of myocardial fibrosis significantly increased in the progress from AVMC to DCM.Part 2:The alteration of IL-22-secreting Th22 cells and their related cytokines IL-22, IL-22 receptor (IL-22R) in murine VHD.In this part, two sections are following:1. The alteration of spleen Th22 subsets in VHD; 2. The expression of Th22 cells relative cytokines (IL-22, IL-22R) in AVMC, chronic myocarditis and DCM mice.The mice of AVMC, chronic myocarditis, DCM groups and control mice at the same time points were sacrificed by the end of week 2, week 12, week 24, respectively. Heart and spleen were removed and blood was harvested to obtain plasma aseptically. The percentage of splenic Th22 cells was detected by flow cytometry, the level of plasma IL-22 was measured by ELISA, cardiac IL-22 receptor (IL-22R) expression was observed by immunohistochemistry.The percentage of Th22 cells increased in mice with AVMC, chronic myocarditis and DCM, relative to control mice at the same time points. Higer level of plasma IL-22 in mice with VHD, including AVMC, chronic myocarditis and DCM markedly was detected as compared to control mice. The expression of IL-22R was increased in the mice of AVMC, chronic myocarditis and DCM groups. Spearman rank correlation coefficients showed that the percentage of Th22 cells positively correlated with circulating IL-22 and cardiac IL-22R.As a result, our data showed that higher percentage of splenic Th22 cells, plasma IL-22 and myocardial IL-22R in VHD mice. Th22 cells and related cytokines IL-22, IL-22R play a important role in the progress of VHD.Part 3:Neutralization of IL-22 in VMC and chronic myocarditisIn this part, three sections are following:1. The influence of anti-IL-22 Ab to IL-22 and IL-22R; 2. The effect of anti-interleukin-22 antibody (anti-IL-22 Ab) for the severity of AVMC and chronic myocarditis; 3. The effect of anti-IL-22 Ab to the myocardial fibrosis indicators in the stage of AVMC and chronic myocarditis.A total of 60 mice infected with CVB3 were randomly divided into two groups:week 2 group and week 12 group. Each mouse group was separated into three subgroups:i.p. administration of anti-IL-22 Ab (n=10, anti-IL-22 Ab subgroup); normal IgG control (n=10, IgG control subgroup) and PBS (n=10, PBS subgroup). The survival rates of each mouse group were recorded and plasma, hearts and spleens of surviving mice were harvested on day 14 in week 2 group and day 84 in week 12 group. The survival rates of each mouse group were recorded. Myocardial histopathologic images were evaluated by HE staining, and myocardial fibrosis images were evaluated by Masson’s trichrome staining. The percentage of splenic Th22 cells was detected by flow cytometry, the level of plasma IL-22 was measured by ELISA, cardiac IL-22 receptor (IL-22R) expression was observed by immunohistochemistry. Then we detectd the indicators of myocardial fibrosis by RT-PCR and ELISA in the stage of AVMC and chronic myocarditis after the treatment of anti-IL-22Ab.Compared to IgG and PBS treatment subgroups, the survive rate of mice treated with anti-IL-22 Ab at week 2 and 12 was significant lower. Higher CVF was detected in mice receiving anti-IL-22 Ab at week 12, the increased level of CVF was similar to DCM mouse group (0.30±0.07 vs.0.24±0.09, p>0.05). The percentage of Th22 cells, IL-22, IL-22R in mice treated with anti-IL-22 Ab was lower than IgG and PBS-treated subgroups. Up-regulation of COL1-A1, COLA3-A1, MMP9 and ratio of MMP9/TIMP-1 but down-regulation of TIMP-lin the anti-IL-22 Ab subgroup, compared with IgG and PBS treatment subgroups. Statistically significant difference in the levels of survival rate, CVF, indicators of myocardial fibrosis between IgG control and PBS subgroups was not been investigated on week 2 and week 12.As a result, neutralization of IL-22 decreased the survival rate of AVMC and chronic myocarditis mice and exacerbated myocardial fibrosis with down-regulation ofthe percentage of splenic Th22 cells, circulating IL-22 and myocardial IL-22R expression.In summary, our research indicated that:Our study demonstrated that the up-regulations of IL-22-secrecting Th22 cells may play an important part in the pathogenesis of CVB3-induced mice AVMC, chronic myocarditis and DCM by inhibiting myocardial fibrosis.IL-22 may serve as a myocardium-protective cytokine by means of decreasing COL1-A1, COL3-A1, MMP9 and increasing TIMP-1. |
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