Influence Of CpG ODN On Acute Coxsackievirus B3-indcued Myocarditis In Mice

Viral myocarditis was one of the major reasons in causing acute heart failture ofhuman. Such studies suggest that myocarditis was a major cause of sudden,unexpected death (accounting for approximately20percent of cases) in adults lessthan40years of age. Coxsackievirus B3was one of the major pathogen in causingviral myocarditis. It could be divided to three distinct phases of murine viralmycarditis. Firstly, in the early phase of the infection, CVB3could stimulate amountsof immune cells (macrophage, dendritic cell, NK cell et.al) activation through TLRssignal pathway. These immune cells could release a serial of cytokines includingTNF-α, IFN-γ, IL-1βand IL-2. Secondly, on day6to7after the infection, the adaptiveimmune response would be activated to induce the immune cell infiltration in themyocardium and the peak usually appeared at7to14days after virus infection.Finally, the patients who have developed the chronic phase of myocarditis wouldoccurred the cardiac remodeling and fibrosis leading to the long-term tissuedegeneration, even develop to dilated cardiomyopathy and congestive heart failure.The innate immune system acts as the first line of defense against viral infection.It is very important to control the CVB3infection. It has been demonstrated that NKcells could inhibit the CVB3replication, reduce the myocarditis in mice. In recentyears, synthetic CpG oligodeoxynucleotides (CpG ODNs), as TLR9agonists, havebeen found to induce anti-viral immune responses in viral infections. CpG ODNs playthe effect through activating NK cells, stimulating plasmacytoid dendritic cells torelease IFN. It has been proved that CpG ODNs could play the effect on preventionand treatment with the infection disease including virus infection.To study the effect of CpG ODN on CVB3-induced myocarditis, we establishedthe murine model by intraperitoneally injecting the CVB3. Using the model, we studied theeffect and mechanisms of YW002, a C-type CpG ODN, on the CVB3-inducedmyocarditis.1. Establishment of the mouse model of acute CVB3-induced myocarditisTo study the effect of YW002on the acute CVB3-indcued myocarditis in mice, we established the model in BALB/C mice infected with different TCID50of CVB3(0.5×104TCID50、1×104TCID50、1.5×104TCID50) by intraperitoneally injection.According to recorde the growth state, body weight and the heart-pathologicalchanges of mice, we decided to inject the mice with1×104TCID50CVB3to establishthe model.2. Effect of different time after the infection on the CVB3-induced myocarditisTo better understanding the phase of CVB3-induced myocarditis, on day4andday10after the infection, the pathological changes of the heart, the CVB3content inheart-tissue and the anti-CVB3neutralizing antibody were detected. On day4, we candetect numbers of CVB3and little lymphocytic infiltrations in the hearts, the level ofanti-CVB3neutralizing antibody is very low; on day10, we can detect morelymphocytic infiltrations in the hearts, higher level of anti-CVB3neutralizingantibody, but we rarely detect the CVB3in the hearts.3. Age-related susceptibility of the mice to CVB3-induced myocarditisBecause the CVB3-induced myocarditis is related with age in human, we decideto detect the response of different old (2-,4-and6-week) mice to CVB3infection andstudied the mechanism. After being infected with CVB3, the mice were observedevery day for recording the the growth status, body weight and survival rate. On day4after the infection, heart rate, CVB3content in heart-and pancreas-tissue and the tissuepathological changes were respectively detected. On day10after the infection, the heartrate, heart-and pancreas-tissue pathological changes and the level of anti-CVB3neutralizing antibody were respectively detected. Compared with4-and6-week oldmice,2-week old mice had the lowest mortability, weight loss, CVB3content inheart-tissues and the highest level of anti-CVB3neutralizing antibody. In addition, onday4after the infection, the CVB3content in pancreas-tissues of2-week old mice wasthe lowest compared with that in pancreas-tissues of4-and6-week old mice and the2-week old mice developed slightest pancreas lesion. Noticeably,2-week old mice diddevelop obvious pancreatitis at day4after CVB3infection and the pancreatitis wasbecoming extinct at day10. While, at the two time points,4-and6-week old mice alldeveloped obvious pancreatitis.4. Effect of ODNs on acute CVB3-induced myocarditis in miceFirstly, one hour after the infection, we respectively chose two types ODNs (aC-type ODN and an inhibitory ODN) to inject (i.p.) the mice at10μg per mouse for six times in one-day interval. According to recorde the growth state, body weight andthe heart-pathological changes of mice, we found that the mice treatment with eitherODN developed more serious myocarditis. Then, before the infection, we chose toinject the mice with the ODNs at10μg per mouse to continuously treat the mice forthree times. We found that either ODN didn,t influence the body weight of mice andthe pathological changes.5. Effect of prophylactic application YW002on acute CVB3-induced myocarditisin miceTo study the effect and the mechasim of pretreatment with YW002on acuteCVB3-induced myocarditis in mice and taking into account the repeated applicationCpG ODN would aggravate the auoimmune myocarditis, we decided that24hourbefore CVB3challenge, the mice were intraperitoneally inject with YW002at20μg permouse through intraperitoneal (i.p) injection, intramuscular (i.m) injection orsubcutaneous (s.c) injection. The mice were observed every day for recording the thegrowth status, survival rate and body weight. On day10after the infection, we detectedthe heart rate and pathological changes in the mouse hearts. The results showed that afterthe infection, one mouse was death in each group. Prophylactic application YW002(20μgper mouse) through s.cinjection significantly reduced the body weight of mice infectedwith CVB3and aggravated heart lession, but it did not influence the the heart rate.Then we chose the i.p injection pathway as the emaple to study the mechanism bydetecting the CVB3content on day4after the infection and the level of anti-CVB3neutralizing antibody in serum on day10. We found that prophylactic applicationYW002could not inhibit the CVB3replication in heart, significantly reduced thelevel of anti-CVB3neutralizing antibody.6. Effect of prophylactic application YW002on acute CVB3-induced myocarditisin different age miceNext, we studied the effect of prophylactic application YW002on acuteCVB3-induced myocarditis in different age mice. At the same time, taken intoaccount the negative effect caused by injecting10g YW002to each mouse, wedecided to redo the experiment by reducing the amount of YW002into10g permouse. The mice were observed every day for recording the the growth status, bodyweight and survival rate. On day10after the infection, we detected the heart rate andpathological changes in the mouse hearts. The results showed that prophylactic application YW002(10g per mouse) did not lessen the CVB3-induced myocarditis,but significantly increased the survival rate of mice.Then we studied the mechanism by detecting the CVB3content on day4after theinfection and the level of anti-CVB3neutralizing antibody in serum on day10. Theresults showed that prophylactic application YW002did not influence CVB3contentand the level of anti-CVB3neutralizing antibody in the serum of2-week old miceinfected with CVB3. In4-and6-week old mice, prophylactic application YW002significant inhibited the CVB3replication in heart-tissues and increased the level ofanti-CVB3neutralizing antibody.6. Effect of prophylactic application YW002on the expression of TLRs in thehearts and the level of IFNγ in the serumIn order to detect the effect of TLRs on the CVB3-induced myocarditis, wedetected the TLR4and TLR9mRNA expression in the hearts of CVB3infected mice.We found that YW002at both10μg and20μg/mouse up-regulated the TLR9mRNAin the heart tissue of CVB3-infected mice dramatically, while YW002at20μg/mousewas more potent than YW002at10μg/mouse. In addition, we also detected the levelof IFNγ in the serum. We found that YW002at both10μg and20μg/mousesignificantly raised the levels of serum IFNγ in CVB3-infected mice.In summary, the results suggested that about the patient infected with CVB3,treatment with TLR9agonists including CpG ODN should be careful. But properamount of TLR9agonists including CpG ODN might play the prophylactic effect toCVB3infection.