| Viral myocarditis (VMC) is a common heart disease. Coxsackie virus B3 (CVB3) is the leading cause of VMC which could cause sudden death, and may progresses to dilated cardiomyopathy (DCM). Direct viral response, inflammational cytokines and immune-mediated mechanisms have been shown to contribute to the pathogenesis of myocardium injury, including autoimmunity. In past times, a multitude of studies had investigated the role of the Th1/Th2 mediated immune-mediated injury pattern present in animals with VMC. However, this concept could not elucidate the pathogenesis of VMC completely.Th17 cells, a distinct new CD4+T helper lineage, are characteristic of producing IL-17. This new CD4+ subset has been shown to be involved in many kinds of autoimmune diseases in animal models and human. It has been shown that Th17 cells and its crucial effector cytokine, IL-17, may participate in the pathogenesis of VMC. But how the Th17 cells and Th17-related cytokines change during the development of VMC? What's the relationship of those cytokines? Which signal pathway participates in the differentiation of Th17 cells in VMC? No reports have been presented now. In order to elucidate the role of Th17 cells in VMC, and explore the new therapeutical target, VMC was induced in male BALB/c mice by CVB3 peritoneal injection, and the following 4 parts researches were carried out:Part 1: The alteration of Th17 cells and its related cytokines in different stages of murine viral myocarditis.Objective: To investigate the alteration of Th17 cells and its related cytokines (IL-17, IL-23, IL-6, TNF-α) in different stages of murine viral myocarditis, and to investigate their role in VMC.Methods: 78 male, 6 weeks old BALB/c mice were used. BALB/c mice were infected by an intraperitoneal (i.p.) injection of 0.1 ml of PBS containing approximately 100 TCID50 of the CVB3 virus (n=48), and randomly separated into 6 subgroups. Mice inoculated i.p. with 0.1 ml PBS were taken as control (n=30) and separated into 6 subgroups as well. The day when mice were injected i.p was defined as week 0, and myocardial tissues, spleens and serums were harvested 0, 1, 2, 3, 4, and 6 weeks after i.p. The myocardial tissues were stained with hematoxylin & eosin to determine the level of myocardial inflammation and the pathological scores, Flow cytometric analysis was used to evaluate the frequencies of Th17 subsets in spleen. IL-17, IL-6, IL-23, TNF-αmRNA in the myocardium of mice were assessed by semi-RT-PCR. IL-17, IL-6, IL-23, TNF-αprotein from blood serum were evaluated by ELISA. IL-17 protein expression in the myocardium was evaluated by immunohistochemical staining. Results:In VMC group, the pathological scores myocardiums began to increase 1 week after i.p. injection (1.8±0.5), attaining the top on week 2 (2.8±0.4), and began to decrease on week 3. There were statistical difference when compared the pathological scores of VMC group with those of the control group (P<0.05). The proportion of Th17 cells increased on week 1(2.23±0.89%), peaked on week 4(5.00±0.81%), maintaining a higher tendency till week 6(2.35±0.35%). The proportion of Th17 cells in VMC subgroup were higher than those of controls except week 0(P<0.01). No statistical difference were seen when compared the proportion of Th17 cells among control subgroups (P>0.05).IL-17 mRNA expression increased in VMC mice from 1 week after CVB3 infection, reaching the peak on week 4 till week 6. The (optical density) value of IL-17 mRNA comparing with the house keeping gene (β-actin) in VMC mice on different times were higher than those of controls except 0 week (P<0.01). No statistical difference were seen when compared the OD among control subgroups (P>0.05). Immunohistochemistry showed that IL-17 protein began to be expressed in myocardium of VMC mice one week after CVB3 infection, reaching the peak on 4 week, and were still higher than those of controls. The IOD (integrated optical density) value of IL-17 protein in VMC mice at different times were higher than those of controls, except 0 week (P<0.01). No statistical difference were seen when comparing IOD among the subgroups of control mice (P>0.05). IL-17 protein levels increased in the serum of VMC mice from the first to the sixth week. After reaching the highest level on the fourth week, IL-17 slightly declined on the sixth week. The IL-17 protein in the serum of VMC mice at different times were higher than those of controls except 0 week (P<0.01). No statistical difference were seen when comparing IL-17 protein level among the subgroups of the control mice (P>0.05). IL-23 mRNA expression and IL-23 protein levels in the serum of VMC mice showed the same trend as the IL-17, manifested by increasing on 1 week, peaking on 4 week, and decreasing on 6 week. IL-6, TNF-αmRNA expression in the myocardium of VMC and protein levels in the serum of VMC increased on 1 week, peaked on 2 week, and maintained a high level till 6 week. The OD values of IL-6, TNF-αmRNA in the myocardium of VMC and protein levels in the serum of VMC at different times were higher than those of controls, except 0 week (P<0.05).Condusion:The expression of Th17 cells and its related cytokines (IL-17, IL-23, IL-6, TNF-α) increased 1-6 weeks after CVB3 infection, which suggested that Th17 cells and its related cytokines may involve in the pathogenesis of VMC.Part 2: Therapeutic Efficacy of IL-17 Neutralization in Murine Coxsackievirus B3-Induced Viral MyocarditisObjective: To investigate the effect of IL-I7 antagonism in vivo in VMC mice so that to establish the pathogenesis role of Th17 in VMC.Methods: 24 male 6 weeks old BALB/c mice were infected by i.p. injection of 0.1 ml 100 TCID50 CVB3. Followed by injection, the mice were injected intraperitoneally with 100μg/0.1ml IL-17 monoclonal antibody (n=8, IL-17mAb group), or 100μg/0.1ml isotype control immunoglobulin (Ig) G2AAb (n=8, isotype group), or 0.1ml PBS (n=8, PBS group) on day 4, 7 and 10. In addition, 8 uninfected and without any intervention mice were assigned as the normal control (n=8, normal group). All surviving animals were sacrificed on day 14 after CVB3 infection, Kaplan-Meier curve was used to evaluate the survive rate, HE staining were used to assessment the pathological changes of myocardium, IL-17, IL-6, TNF-αmRNA of the myocardium were assessed by semi-quantitative RT-PCR. Systemic IL-17, IL-6, and TNF-αlevel were measured by enzyme-linked immunosorbent assay, and local myocardium IL-17 expression was analyzed using immunohistochemical staining. Flow cytometric analysis was used to evaluate the frequencies of Th17 subsets in CD4+T cells.Results:Results showed that neutralization of IL-17 with anti-IL-17 can ameliorate survive rate, decrease HW/BW value, reduce the inflammational infiltration in heart, and decrease the pathological scores. IL-17 monoclonal antibody can decrease the serum IL-17 level, without declining the IL-17, IL-6 and TNF-αmRNA transcript level and serum IL-6, TNF-αlevel. The differentiation and proliferation of the Th17 cells were unchanged.Condusion:Anti-IL-17 treatment can improve the survival rate, alleviate the severity of myocardium, which indicated that Th17/IL-17 involve in the development of VMC.Part 3: The effect of cytokines on spleen Th17 cells of VMC miceObjective: To investigate the effect of cytokines on the proliferation of differentiated Th17 cells.Methods: 6 weeks old male BALB/c mice were infected by intraperitoneal injection of 100 TCID50 of the CVB3 virus to induce VMC. Spleen CD4+ T cells were purified using a CD4+ T cell isolation kit from mice 1 week after CVB3 injection. Isolated CD4+ T cells were activated for 5 d with 10 ng/ml of recombinant TGF-β(10ng/ml) , or TGF-β(10ng/ml)+ IL-6(20ng/ml), or TGF-β(10ng/ml)+ IL-6(20ng/ml)+ rIL-23 (10ng/ml), or IL-23(10ng/ml). IL-17 and STAT3 mRNA of the cultured cells was assessed by semi-quantitative RT-PCR. IL-17 protein level in the culture supernatants were measured by enzyme-linked immunosorbent assay, Flow cytometric analysis was used to evaluate the frequencies of Th17 subsets in cultured CD4+T cells.Results:Compared with the TGF-βor TGF-β+ IL-6, rIL-23 could promote the expansion of Th17 cells (P<0.05) Corresponding, IL-17 mRNA expression in the cultured cells and IL-17 protein levels in the culture supernatants increased after rIL-23 stimulation. But administration of TGF-βor TGF-β+IL-6 neither promote the expansion of Th17 cells nor increase IL-17 mRNA expression in the cultured cells or IL-17 protein levels in the culture supernatants after stimulation.(P>0.05).Condusion:rIL-23 can promote the proliferation of the differentiated Th17 cells, wherever TGF-βwith or without IL-6 have no effect on the proliferation of the differentiated Th17 cellsPart 4: The role of STAT3 signal pathway in the differentiation of Th17 cells in viral myocarditisObjective: To investigate the expression of STAT3 pathway and its effect on the differentiation of Th17 cells in mice with VMC. Methods: VMC mice were induced as mentioned above. The expression of STAT3 mRNA in myocardium was assessed by semi-quantitative RT-PCR.p-STAT3 protein level in the myocardium was measured by Western Blot. We used S3I-201, a STAT3-specific inhibitor, to test whether regulation of STAT3 could be partly responsible for IL-17 diminution. Spleen CD4+ T cells were purified using a CD4+ T cell isolation kit from mice 1 week after CVB3 injection. Isolated CD4+ T cells were activatedd with or without 100μM of S3I-201. Flow cytometric analysis was used to evaluate the frequencies of Th17 subsets, IL-17 and STAT3 mRNA of the cultured cells were assessed by semi-quantitative RT-PCR. IL-17 protein level in the culture supernatants were measured by enzyme-linked immunosorbent assay,Results:The expression of the STAT3 mRNA was clearly elevated from the first week to the sixth week. The expression of this gene in controls remained constant throughout the procedure. Immunohistochemistry showed that p-STAT3 protein began to express in myocardium of VMC mice one week after CVB3 infection, reaching the top on 4 week, and still higher than those of controls on 6 week. The IOD of p-STAT3 in the myocardium of VMC at different times were higher than those of controls, except 0 week (P<0.05). No statistical difference were seen when comparing the IOD among subgroups of control mice (P>0.05). S3I-201 decreased the expansion of Th17 cells (5.02±0.72% vs. 3.85±0.69%, P<0.05). Correspondingly, IL-17 and STAT3 mRNA expression in the cultured cells and IL-17 protein levels in the culture supernatants decreased after S3I-201 inhibition.Condusion : The STAT3 pathway was activated in VMC mice. STAT3-specific inhibitor, S3I-201, could inhibit the proliferation of the differentiated Th17 cellsIn summary, our research suggestes that:(1)During the development of VMC in mouse model, the proportion of Th17 cells increase from 1-6 weeks, accompanied by the mRNA up-regulating and proteins increasing of its related cytokines, including IL-17, IL-23, IL-6, and TNF-α. The proportion of Th17 cells, IL-17, IL-23 mRNA and proteins expression reached the peak on week 4. IL-6, TNF-αmRNA and proteins expression reached the peak on week 2. These findings indicate that Th17 cells might be involved in the pathogenesis of VMC. (2) Neutralization of IL-17 with a monoclonal antibody starting after onset of VMC can ameliorate myocarditis and improve the survive rate. (3) IL-23 is the imperative factor to maintain the proliferation of the differentiated spleen Th17 cells in VMC mice. TGF-βwith or without IL-6 could not make the differentiated Th17 cells proliferate. (4) Signal transduction mediated by STAT3 might be involved in the differentiation and proliferation of Th17 cell in VMC mice model.
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