Investigation On The Effect Of TREM-1in The Pathogenesis Of Acute Viral Myocarditis

Acute viral myocarditis(AVMC) is a common disease of cardiovascular system, especially among adolescents and young adults. Although most patients have good prognosis, about25%of them will evolve into dilated cardiomyopathy(DCM) which has a high death rate. Because of the lack of understanding of its pathogenesis, there are few effective treatments to preventing deterioration.The basic pathogenesis of myocarditis is immune inflammatory response which is induced by persistent virus infection and will lead to apoptosis or necrosis of myocardiocytes. The main characteristics of the inflammation caused by virus infection is the release of cytokines and the infiltration of inflammatory cells, such as macrophages, NK cells and so on, which is an important defense method of our body, helping in the clearance of pathogen. However, there always resist in AVMC excessive inflammatory response, which aggravate pathological injury of the myocardium.TREM-1is an important medium for the inspiration and amplification of the inflammatory. It promotes cytokines secretion, inducing monocytes and neutrophils to secrete cytokines such as TNF-α, IL-2, IFN-γ, and so on. It also induces neutrophils to release peroxidase. By these means, TREM-1leads to the enhancement and amplification of the inflammation, and finally causes an "excessive" inflammation in AVMC.Whether TREM-1plays a role in the incidence of AVMC deteriorating myocyte injury through excessive inflammatory, there has not clear. Our laboratory’s previous study has found a high expression of TREM-1in the myocardium of AVMC mice. And we found that the expression amount of TREM-1is in positive correlation of the pathological score of the myocardium and the heart function. Inhibiting the activation of TREM-1will decrease the amount of IL-1and TNF-α, lessen the pathological score, ease myocyte apoptosis, and improve heart function. As previous studies shows that TREM-1regularly expressed on myeloid cells such as neutrophils and macrophages, and macrophage is one of the important inflammatory cells for the pathogenesis of AVMC, therefore, this study will investigate whether TREM-1express on macrophages infiltrating in the myocardium, and will study the effect of TREM-1on cytokine secretion of the macrophages and the injury of myocytes in vitro.Part I Location of TREM-1Expression in the myocardium on AVMCObjective To detect the expression of TREM-1in the myocardium of the CVB3infected AVMC mice.Methods BALB/c mice were single-infected with CVB3to establish the experimental model of acute viral myocarditis (n=10), normal controls were prepared with same volume of DMEM injection without CVB3(n=8). Animals were killed at day7, HE dyeing were performed to observe the histopathology changes of the myocardium; Immunohistochemistry (IHC) dyeing were used to detect TREM-1expression in myocardium; double-labeling immunoflurescence technology and Confocal microscope were implied to detect the expression of TREM-1and macrophges’marker CD68in myocardium, using the merging technology to compare the expressions of the two proteins.Results Compared with control group, the VMC group showed obvious inflammatory cell infiltration and myocyte necrosis. IHC detect TREM-1expression in myocardium of AVMC mice, and confocal detecting confirms that most of the TREM-1expression was in coincidence with CD68expression.Conclusion We have successfully established the experimental model of VMC in murine by single-infection of CVB3. TREM-1expression exists in myocardium of AVMC mice, which is largely expressed on infiltrated macrophages.Part II Effect of TREM-1on CVB3-caused macrophages infection and myocytes injuryObjective To determine whether the expression of TREM-1(Triggering Receptor Expressed on Myeloid cells-1) increases in macrophages after Coxsackievirus B3infection, therefore study the effect of TREM-1on cytokines secretion by macrophages and myocytes injury.Methods We cultured macrophages in vitro, use CVB3to infect the cells at different time, measured the amount of TREM-1mRNA using Real-time PCR, detected the expression of TREM-1and DAP-12Protein by Western blot; then use LP-17to block TREM-1activation, measured the changes of TNF-a secretion of macrophages by ELISA, assessed the vitality and the apoptosis degree of cardiomyocytes by CCK8and Annexin V-FITC after the myocytes was cultured with the supernatant of macrophages.Similarly, we cultured cardiomyocytes in vitro, use CVB3to infect the cells at different time, measured the amount of TREM-1mRNA using Real-time PCR, detected the expression of TREM-1and DAP-12Protein by Western blot; then use LP-17to block TREM-1activation, compared with pure virus infection, we detected changes of the amount of CVB3RNA in myocytes, assessed the vitality of myocytes by CCK8method.Results We found that TREM-1mRNA amount was much higher at4h,8h, and12h after CVB3infection, and the latter two had a significant difference(p<0.01), the protein level was also significantly higher at16h and24h (p<0.01), The protein level of DAP-12, a direct downstream signaling molecule of TREM-1, also increased remarkably at24h and48h(p<0.01). Macrophages secreted less amount of TNF-a when using TREM-1blocker LP-17before CVB3infection (p<0.01). When cultured with supernatant of macrophages, cardiomyocytes in LP-17intervention group showed better vitality and less apoptosis than the virus group (p<0.01).After CVB3infection, TREM-1mRNA amount in myocytes was increased at2h(p<0.01), the protein level was also higher at8h,12h, and24h, the peak is at12h, the protein level of DAP-12also increased at8h,12h, and24h; after12hs infection of CVB3, we detected positive TREM-1expression in myocytes by immunoflurescence; when using TREM-1blocker LP-17, there showed less amount of CVB3RNA in myocytes of (p<0.01), and myocytes in the intervention group showed better vitality than pure CVB3infection(p<0.01).Conclusion After CVB3infection, expression of TREM-1on macrophages and cardiomyocytes increases, blockade of TREM-1activation will inhibit macrophages to secrete cytokines, decrease the replication of CVB3in the myocardium, and alleviate myocytes’injury and apoptosis.