The Effect And Mechanism Of CCL20on Th17Cells Infiltration Into Myocardium In Cvb3Induced Acute Myocarditis

Viral myocarditis(VMC) is a common cardiovascular disease, whose morbidity in recent years has been on the rise. Viral myocarditis is an autoimmune disease followed by viral infection and can develop into dilated cardiomyopathy(DCM). Viral Myocarditis can also induce cardiac insufficiency and refractory arrhythmia, with serious influence to the health of patient. It is the common cause of cardiac sudden death. The pathogenesis of viral myocarditis includes the direct damage of the virus, the immune response of the body, apoptosis and myocardial fibrosis. Some researches show that, the immune response induced by T cells play a leading role in the pathogenesis of viral myocarditis, secondly the local damage of the virus. Among the sudden death induced by viral myocarditis, T helper cells(Th) and the abnormal expression of cytokines are critical causes. Th17cell is a new subset of CD4+T cells. Th17cells secrete specifically interleukin-17(IL-17). Th17cells contribute to the pathogenesis of cardiovascular diseases, especially viral myocarditis and dilated cardiomyopathy. CCL20(macrophage inflammatory protein, MIP-3a) is a inflammatory cytokine, which can be secreted by endothelial cells, macrophages and fibroblasts inducing by IL-1β、TNF-α IFN-y and LPS(lipopolysaccharide). Its receptor is CCR6. CCL20can induce leukocytes to participate in the mechanism of inflammatory.So we can suppose that whether Th17cells can be recruited to infiltration into myocardial tissue. This research intended to clarify the effect of Th17cells in the pathogenesis of viral myocarditis by study on the effect of CCL20on Th17cells in acute viral myocarditis in vivo and in vitro. Part1The effect of CCL20on Th17cells in acute viral myocarditis in vivoMaterials and Methods96male BALB/c mice aged4weeks were randomly divided into four groups:(1) AVMC group (n=24):mice were treated with0.2ml of RPMI-1640medium containing approximately105PFU of CVB3and100μl saline intraperitoneally on day0;(2) CCL20mAb group (n=24):mice were treated with0.2ml CVB3and100μg rat anti-mouse CCL20mAb one hour later intraperitoneally on day0;(3) Isotype control group (n=24):mice were treated with0.2ml CVB3and100μg isotype control immunoglobulin (Ig) GlAb one hour later intraperitoneally on day0;(4) Normal group (n=24):mice were not treated with anything. On day3,5,7and10of the experiment, six animals were randomly selected to be killed separately for blood and heart. The amount of serum cardiac troponin (cTn) T and CCL20were quantitively measured by Elecsys (Roche Diagnostics GmbH) and Elisa kits. The ventricular tissue was stained with H&E to observe the histopathology. The expression of IL-17was evaluated by immunohistochemistry. The percentages of intracardiac Th17cells in AVMC mice were detected by flow cytometry analysis.Results The ratios of HW/BW (heart weight to body weight), serum cTnT level and the pathological scores of heart sections in AVMC, CCL20mAb and isotype control groups were elevated significantly compared with those in normal group on days3,5,7and10(p<0.05). The CCL20level in the hearts in AVMC, CCL20mAb and isotype control groups were increased significantly with those in normal group on days3,5,7and10(p<0.05). And the expression of CCL20in CCL20mAb group was reduced compared with those in AVMC and isotype control groups (p<0.05). The CCL20-secreting cells appeared on day5, and culminated on day7. On days5,7and10, the numbers of IL-17-secreting cells increased significantly in AVMC, CCL20mAb and isotype control groups compared with those in normal group (p<0.05). But they were reduced in CCL20mAb group compared with those in AVMC and isotype control groups (p<0.01). Moreover, it revealed that the percentages of intracardiac CD4+Th17cells were obviously higher in AVMC, CCL20mAb and isotype control groups than those in normal group on days7and10(p<0.01). And the elevations were attenuated in CCL20mAb group compared with those in AVMC and isotype control groups (p<0.01).Conclusions Th17cells and IL-17play an important role in the mechanism of acute viral myocarditis. CCL20is involved in the mechanism of acute viral myocarditis by promoting the effect of Th17cells. And CCL20mAb can obviously reduce the pathological reactions through neutralization with CCL20. Consequently CCL20may be the new target for treatment of acute viral myocarditis. Part2The effect of CCL20on Th17cells in acute viral myocarditis in vitroMaterials and Methods Neonatal cardiomyocytes and cardiac fibroblasts were isolated from1-3days BALB/c mice. Myocardial endothelial cells were isolated from the hearts of7-day-old BALB/c mice. The splenic CD4+T cells were purified by negative selection from AVMC mice. The CCL20mRNA and protein in neonatal cardiomyocytes and cardiac fibroblasts was evaluated with the stimulation of TNF-a, IFN-y, IL-1, IL-6or IL-17.(1) Cell arrest assay:Myocardial endothelial cells were seeded on the basolateral side of the upper chamber in24-hole transwell (0.4μm pore, Corning). Neonatal cardiac fibroblasts were added in the lower chamber. Two days later, Th17cells stained with FITC-labeled anti-mouse CD4antibody were loaded onto the upper chamber. After1hour of incubation, non-adherent cells were removed by washing with PBS. Adherent cells were counted under fluorescence microscopy. The endothelial cells on the upper chamber were gathered for the expression of ICAM-1and VCAM-1by real-time PCR.(2) Cell migration assay:Cardiac fibroblasts were seeded in the lower chamber of transwell (5μm pore, Corning). Th17cells stained with FITC-labeled anti-mouse CD4antibody were loaded onto the upper chamber. After eight hours, the number of cells migrating into the lower chamber was counted under fluorescence microscopy. The expressions of CCR6were tested by flow cytometry.(4) Cell differentiation assay:The purified CD4+T cells were co-cultured with or without cardiac fibroblasts. The percentages of Th17cells were detected by flow cytometry.Results With the stimulation of TNF-a, IFN-y, IL-1, IL-6or IL-17, cardiac fibroblasts have high expression of CCL20mRNA and protein, but cardiomyocytes have lower expression of CCL20mRNA and protein than cardiac fibroblast.(1) Cell arrest assay:The numbers of Th17cells adhering to myocardial endothelial cells in the upper chamber were increased in TNF-a, TNF-a+CCL20mAb and isotype control groups compared with saline group (p<0.01). But the arrest of Th17cells were alleviated in TNF-a+CCL20mAb group compared with the other two groups (p<0.01). The mRNA levels of ICAM-1and VCAM-1in endothelial cells were elevated in the culture system treated with TNF-a, but neutralization by CCL20mAb could reduce the expression of the two adhesion molecules.(2) Cell migration assay:The numbers of Th17cells migrating into the lower chamber were higher in TNF-a, TNF-a+CCL20mAb and isotype control groups than saline group. The numbers were reduced in TNF-a+CCL20mAb group compared with the other two groups (p<0.01). But there was no significantly difference in the expressions of CCR6. Further experiment showed that Th17cells migrations in the CCR6mAb group were far less than those in saline and isotype control groups.(3) Cell differentiation:The percentages of CD4+T cells differentiating to Th17cells were increased in co-culture system.Conclusions With the stimulation of the inflammatory cytokines like TNF-a, cardiac fibroblasts can promote the infiltration of Th17cells into myocardium including adhesion and migration by secreting CCL20. But the differentiation of Th17cells is not related to secreting CCL20fibroblasts.