The Protective Effect Of Interfering TLR9-IRF5 Signaling Pathway On The Development Of CVB3-induced Myocarditis

Viral myocarditis(VMC)is a complication of inflict significant damage on cardiomyocytes caused by virus infection through direct injury and secondary immune reactions,and is also the leading cause of sudden death and heart failure.However,there is still no sufficient treatment for VMC nowadays.Since toll-like receptor 9(TLR9)or interferon regulatory factor 5(IRF5)was reported to be associated with the development of myocarditis,we wondered if the TLR9-IRF5 pathway could contribute to the development of coxsackievirus B3(CVB3)-induced myocarditis.We detected the signaling molecules of TLR9-IRF5 pathway in CVB3-infected patients and mice,and compared them with the severity of the hearts injury during CVB3 infection.In addition,we interfered the TLR9-IRF5 signaling pathway in the VMC mice by an AAAG ODN with IRF5 interfering activities,and found that the AAAG ODN could significantly alleviate the myocarditis,and decrease the levels of the TLR9-IRF5 pathway molecules in hearts,spleens as well as white blood cells in the CVB3-infected mice.The data suggest that interfering TLR9-IRF5 pathway could be an approach to treat CVB3-induced myocarditis.The results were as followed:1.The expression of TLR9,IRF5,TNF-? and IL-6 in patients with CVB3-induced myocarditisTo explore whether the TLR9-IRF5 pathway is activated during the development of CVB3-induced myocarditis,we tested the expressions of IRF5-related cytokines in the serum and the TLR9,IRF5,TNF-? and IL-6 in the pericardial fluids from the VMC patients.The results showed that:1.1 The expression of IRF5-related cytokines in the serum from the VMC patients(1)The levels of TNF-?,IL–6,IL-12 and IFN-? were increased in the serum from the VMC patients.(2)The levels of IL-10 and IL-10 were not significantly elevated in the VMC patients.The results indicated that the IRF5 might be involved in the development of CVB3-induced myocarditis.1.2 The expression of TLR9,IRF5,TNF-? and IL-6 in the pericardial fluids from the VMC patients(1)The levels of TLR9,IRF5 were increased in the cells of pericardial fluids from the VMC patients.(2)The levels of TNF-? and IL-6 were increased in the pericardial fluids from the VMC patients.These observations support that the TLR9-IRF5 pathway could be activated in hearts of the VMC patients with CVB3 infection.2.The effect of TLR9-IRF5 signaling on the VMC miceUpon the findings that activation of the TLR9-IRF5 pathway is related to the VMC development in human,we infected mice with CVB3 to induce myocarditis.We detected the pathological changes of the heart at different times after CVB3 infection,and the expression levels of TLR9-IRF5 signaling pathway molecules in the hearts,and explored the correlation between them;Also,we used CVB3 to infect H9C2 cells,treated the cells with HMGB1 neutralizing antibody and the TLR9-IRF5 signal interfering agents,respectively,and then detected the expressions of HMGB1,TLR9,IRF5,TNF-?,IL-6 and the cell viability after CVB3 infection.2.1 The relationship between the levels of the molecules in TLR9-IRF5 pathway and the extent of cardiac injury in the mice after CVB3 infection(1)The lymphocytes minimally infiltrated in the hearts on day 1,became apparently on day 4,peaked on day 7,and gradually reduced until day 14.(2)The CVB3 induced obviously elevated expressions of TLR9,IRF5,and IL-6 on day 4post-infection and peak expression of IRF5 on day 7,the steadily increased TLR9 and decreased IL-6 up to the day 14,and also induced the increased expression of TNF-?on day 7.(3)The levels of the molecules in TLR9-IRF5 pathway is positively correlated to the extent of cardiac injury in the mice after CVB3 infection.The resultsindicated that vigorous activation of the TLR9-IRF5 signaling pathway could be involved in the cardiac injury induced by CVB3 infection.2.2 The TLR9-IRF5 signaling activation induced by CVB3 infection(1)The expression of HMGB1 increased in CVB3 infected mice and H9C2 cells.(2)The expressions of TLR9,IRF5,TNF-?,IL-6 in H9C2 cells infected with CVB3 increased.(3)HMGB1 neutralizing antibody and chloroquine could decrease the expressions of TLR9,IRF5,TNF-? and IL-6 in H9C2 cells,while the AAAG ODN reduced the expressions of IRF5,TNF-? and IL-6.The results showed that the TLR9-IRF5 signaling activation could be induced by CVB3 infection.2.3 The effect of TLR9-IRF5 signaling intervention on the injury of CVB3-infected cardiomyocytes(1)The Cp G 1826,as a TLR9 activator,could reduce the activity of H9C2 cells infected with CVB3 and increase the content of CKMB in cell supernatant.(2)There was no significant change in the activity and the content of CKMB in the supernatant of H9C2 cells cultured with chloroquine.(3)AAAG ODN could enhance the activity of H9C2 cells infected after CVB3 infection and reduce the content of CKMB in the supernatant.The results showed that the interventions of TLR9-IRF5 signaling,such as AAAG ODN,may alleviate the heart injury induced by CVB3 infection.3.The effect of interfering TLR9-IRF5 signaling pathway on the development of CVB3-induced myocarditisSince the AAAG ODN was demonstrated to interfere the TLR9-IRF5 signaling pathway,we were curious to find whether the AAAG ODN could also alleviate the CVB3-induced inflammatory damage in hearts.The CVB3-infected mice were treated with AAAG ODN once on day-1,0,1,or 4 prior or post-infection,respectively,and pathologically analyzed the the hearts of the mice.Also,we detected the influence of the expressions of TLR9-IRF5 pathway molecules,including TLR9,IRF5,TNF-? and IL-6,in the hearts and the in peripheral immune cells,and also we detected the effect of AAAG ODN on the CVB3 load in hearts of the VMC mice.3.1 The roles of AAAG ODN on CVB3-induced myocarditis in miceThe AAAG ODN treatment once on day 4 post-infection obviously improved the general status,and dramatically lessened the heart damage of the VMC mice,while the AAAG ODN treatment on-1,0 and 1 day failed to.The results showed that the AAAG ODN could alleviate the myocardial injury induced by CVB3 infection,and the day 4 post-infection should be an appropriate time point for treating the CVB3-induced myocarditis.3.2 The influence of AAAG ODN on TLR9-IRF5 pathway in heart of mice with CVB3-induced myocarditis(1)The AAAG ODN treatment on day 4 post-infection significantly decreased cardiac expressions of TLR9,IRF5,TNF-? and IL-6 on day 7.In contrast,the AAAG ODN treatment on day-1 increased the expressions of TLR9,IRF5,TNF-? and decreased the IL-6.Whereas the AAAG ODN treatment on day 0 only increased the TLR9,and the treatment on day 1 only decreased the IL-6.(2)The AAAG ODN treatment on day 4 post-infection decreased the cardiac expressions of TLR9,IRF5 and TNF-? on day 14 post-CVB3 infection.These results suggested that the AAAG ODN treatment on day 4 could alleviate the CVB3-induced myocarditis by suppressing the expression of the TLR9-IRF5 pathway molecules in hearts.3.3 The effect of AAAG ODN on expression of TLR9-IRF5 pathway molecules in peripheral immune cells of the mice with CVB3 infection(1)In the spleen cells,the AAAG ODN treatment on day 4 post-infection decreased the expressions of TLR9,IRF5 and TNF-?,and the increases couldn't be detected on day 14 in the VMC mice.(2)In the WBCs,the AAAG ODN treatment on day 4 post-infection decreased the expressions of TLR9,IRF5 and TNF-?,and the increases couldn't be detected on day 14 in the VMC mice.The results indicate that the AAAD OND treatment on day 4 participates in the myocardial inflammatory responses in the mice by regulating the immune cells in periphery.3.4 The effect of AAAG ODN on the CVB3 load in hearts of the mice with CVB3 infectionThe cardiac CVB3 titers of the mice treated with the AAAG ODN were within the same range as that of the mice treated with the PBS,suggesting that the AAAGODN couldn't induce the inhibition on CVB3.As above,we found that the increased expression of cytokines regulated by IRF5 in the sera and the TLR9-IRF5 pathway molecules in the pericardial fluid from the VMC patients,and the activated TLR9-IRF5 pathway during the development of CVB3-induced myocarditis,and alsofound that interfering the signaling activation with AAAG ODN could lessen the cardiac detrimental inflammation induced by the CVB3 infection.The study may provide valuable experimental data for clinical treatment of the VMC.