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Macrophage polarization plays a crucial role in regulating myocardial inflammation and injuries of coxsackievirus B3 (CVB3)-induced viral myocarditis (VM). It has been reported that miR-223 is a potent regulator of inflammatory responses that involved in macrophage polarization. However, the functional roles of miR-223 in CVB3-induced VM still remain unknown. Here, we found that miR-223 expression was significantly down-regulated in heart tissues and heart-infiltrating macrophages of CVB3-infected mice. Up-regulation of miR-223 in vivo protected the mice against CVB3-induced myocardial injuries characterized by the increased body weight and survival, enhanced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), relieved inflammation, depressed creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and aspartate transaminase (AST) levels, reduced production of interferon (IFN)-γ, interleukin (IL)− 6 as well as increased IL-10. We subsequently found that miR-233 up-regulation significantly suppressed the expression of M1 markers (iNOS, TNF-α and CD 86), and promoted the expression of M2 markers (Arginase-1, Fizz-1 and CD 206) in vivo and in vitro. Furthermore, we confirmed that miR-223 directly targeted Pknox1 to inhibit its expression, and the expression of Pknox1 was inversely correlated with miR-223 expression in heart tissues and heart-infiltrating macrophages of CVB3-infected mice. Gain-of-function analyses indicated that Pknox1 overexpression partially reversed the polarization phenotypes regulated by miR-223 overexpression. Taken together, the data suggest that miR-223 protects against CVB3-induced inflammation and myocardial damage, which may partly attribute to the regulation of macrophage polarization via targeting Pknox1. [ABSTRACT FROM AUTHOR]

Semaphorin7A (Sema7A) has been reported to play various roles in nerve axon growth, tumor suppression, and tissue remodeling, as well as regulation of intestinal inflammation diseases. Viral myocarditis (VMC) characterized by viral-myocardial-cell necrosis and inflammatory cell infiltration is a common clinical disease of the cardiovascular system. However, the role of Sema7A in coxsackievirus B3 (CVB3)-induced VMC has not been reported. In this study, we generated an acute VMC mouse model by CVB3 infection, and manipulated Sema7A expression by in vivo polyethyleneimine-mediated Sema7A down-regulation. Our results indicated that Sema7A was up-regulated in cardiomyocytes during VMC, and that Sema7A down-regulation following short hairpin RNA interference or mAb neutralization effectively protected mice from VMC. Additionally, reduced inflammatory responses were observed along with Sema7A down-regulation. Furthermore, adoptive transfer of α1β1-integrin macrophages exacerbated CVB3-induced myocarditis, suggesting the significance of α1β1-integrin macrophages in response to VMC. We observed that co-culture of neonatal myocardiocytes with macrophages increased the percentage of α1β1-integrin macrophages, while Sema7A neutralization reduced α1β1-integrin macrophages in heart tissue of VMC mice. These results demonstrated that Sema7A, as an inflammation regulator in CVB3-induced VMC, might interact with α1β1-integrin in macrophages to enhance the inflammatory response and aggravate disease severity. Our findings provided insight into the potential role of Sema7A as a therapeutic treatment for VMC. [ABSTRACT FROM AUTHOR]

Coxsackieviruses (CVs) are relatively common viruses associated with a number of serious human diseases, including myocarditis and meningo-encephalitis. These viruses are considered cytolytic yet can persist for extended periods of time within certain host tissues requiring evasion from the host immune response and a greatly reduced rate of replication. A member of Picornaviridae family, CVs have been historically considered non-enveloped viruses – although recent evidence suggest that CV and other picornaviruses hijack host membranes and acquire an envelope. Acquisition of an envelope might provide distinct benefits to CV virions, such as resistance to neutralizing antibodies and efficient nonlytic viral spread. CV exhibits a unique tropism for progenitor cells in the host which may help to explain the susceptibility of the young host to infection and the establishment of chronic disease in adults. CVs have also been shown to exploit autophagy to maximize viral replication and assist in unconventional release from target cells. In this article, we review recent progress in clarifying virus replication and dissemination within the host cell, identifying determinants of tropism, and defining strategies utilized by the virus to evade the host immune response. Also, we will highlight unanswered questions and provide future perspectives regarding the potential mechanisms of CV pathogenesis. [ABSTRACT FROM AUTHOR]

Coxsackievirus B3 (CVB3) is the major pathogen of human viral myocarditis. CVB3 has been found to manipulate and modify the cellular lipid metabolism for viral replication. The cellular AMP-activated protein kinase (AMPK) is a key regulator of multiple metabolic pathways, including lipid metabolism. Here we explore the potential roles AMPK plays in CVB3 infection. We found that AMPK is activated by the viral replication during CVB3 infection in Hela cells and primary myocardial cells. RNA interference mediated inhibition of AMPK could increase the CVB3 replication in cells, indicating that AMPK contributed to restricting the viral replication. Next, we showed that CVB3 replication could be inhibited by several different pharmacological AMPK activators including metformin, A769662 and AICAR. And the constitutively active AMPK mutant (CA-AMPK) could also inhibit the CVB3 replication. Furthermore, we found that CVB3 infection increased the cellular lipid levels and showed that the AMPK agonist AICAR both restricted CVB3 replication and reduced lipid accumulation through inhibiting the lipid synthesis associated gene expression. We further found that CVB3 infection would also induce AMPK activated in vivo . The AMPK agonist metformin, which has been widely used in diabetes therapy, could decrease the viral replication and further protect the mice from myocardial histological and functional changes in CVB3 induced myocarditis, and improve the survival rate of infected mice. Lastly, it was demonstrated that the AICAR-mediated restriction of viral replication could be rescued partially by exogenous palmitate, the first product of fatty acid biosynthesis, demonstrating that AMPK activation restricted CVB3 infection through its inhibition of lipid synthesis. Taken together, these data in the present study suggest a model in which AMPK is activated by CVB3 infection and restricts viral replication by inhibiting the cellular lipid accumulation, and inform a potential novel therapeutic strategy for CVB3-associated diseases. [ABSTRACT FROM AUTHOR]

Abstract: The role of β-adrenergic stimulation on viral myocarditis has been investigated in animal models. The beneficial action of the β-blocker carvedilol in murine viral myocarditis can be explained partly by the resulting heart rate reduction and the inhibition of proinflammatory cytokine production. The modulation of myocardial necrosis and contractile dysfunction by proinflammatory cytokines may be partially mediated by the production of nitric oxide (NO). The selective I f current inhibitor ivabradine reduces the heart rate without affecting cardiac contractility and has been shown to be cardioprotective in failing hearts. However, little is known about the effects of ivabradine in viral myocarditis, and in particular, its effects on inducible NO synthase (iNOS) have not been investigated. This study was therefore designed to examine the effects of ivabradine in murine viral myocarditis. In a coxsackievirus B3 murine myocarditis model, the effects of ivabradine and carvedilol on the myocardial histopathological changes and fibrosis, NO production, iNOS protein and cytokine levels were studied. Both ivabradine and carvedilol similarlyattenuated myocardial lesions and fibrosis, inhibited NO synthesis by iNOS, and decreased the production of TNF-α and IL-6. These results show that ivabradine has a therapeutic benefit in murine CVB3-induced myocarditis. The beneficial effects of ivabradine in viral myocarditis are partially mediated by the inhibition of both the production of proinflammatory cytokines and the synthesis of NO by iNOS. [Copyright &y& Elsevier]

Abstract: Enteroviruses like coxsackievirus B3 (CVB3) are common suspects in myocarditis/dilated cardiomyopathy patients. Autoimmunity has been proposed as an underlying mechanism, but direct evidence of its role is lacking. To delineate autoimmune response in CVB3 myocarditis, we used IAk dextramers for cardiac myosin heavy chain (Myhc)-α 334–352. We have demonstrated that myocarditis-susceptible A/J mice infected with CVB3 generate Myhc-α-reactive CD4 T cells and such a repertoire was absent in naïve mice as measured by proliferative response to Myhc-α 334–352 and IAk dextramer staining. We also detected Myhc-α 334–352 dextramer+ cells in the hearts of CVB3-infected mice. The autoreactive T cell repertoire derived from infected mice contained a high frequency of interleukin-17-producing cells capable of inducing myocarditis in naïve recipients. The data suggest that CVB3, a bona fide pathogen of cardiovascular system that primarily infects the heart can lead to the secondary generation of autoreactive T cells and contribute to cardiac pathology. [Copyright &y& Elsevier]

Abstract: Extracellular cyclophilin A (CyPA) and its receptor Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147) modulate inflammatory processes beyond metalloproteinase (MMP) activity. Recently, we have shown that CyPA and CD147 are upregulated in patients with inflammatory cardiomyopathy. Here we investigate the role of CyPA and CD147 in murine coxsackievirus B3 (CVB3)-induced myocarditis. CVB3-infected CyPA−/− mice (129S6/SvEv) revealed a significantly reduced T-cell and macrophage recruitment at 8days p.i. compared to wild-type mice. In A.BY/SnJ mice, treatment with the cyclophilin-inhibitor NIM811 was associated with a reduction of inflammatory lesions and MMP-9 expression but with enhanced virus replication 8days p.i. At 28days p.i. the extent of lesion areas was not affected bei NIM811, whereas the collagen content was reduced. Initiation of NIM811-treatment on day 12 (after an effective virus defense) resulted in an even more pronounced reduction of myocardial fibrosis. In conclusion, in CVB3-induced myocarditis CyPA is important for macrophage and T cell recruitment and effective virus defense and may represent a pharmacological target to modulate myocardial remodeling in myocarditis. [Copyright &y& Elsevier]

Abstract: Susceptibility to autoimmune myocarditis has been associated with histamine release by mast cells during the innate immune response to coxsackievirus B3 (CVB3) infection. To investigate the contribution of histamine H1 receptor (H1R) signaling to CVB3-induced myocarditis, we assessed susceptibility to the disease in C57BL/6J (B6) H1R−/− mice. No difference was observed in mortality between CVB3-infected B6 and H1R−/− mice. However, analysis of their hearts revealed a significant increase in myocarditis in H1R−/− mice that is not attributed to increased virus replication. Enhanced myocarditis susceptibility correlated with a significant expansion in pathogenic Th1 and Vγ4+ γδ T cells in the periphery of these animals. Furthermore, an increase in regulatory T cells was observed, yet these cells were incapable of controlling myocarditis in H1R−/− mice. These data establish a critical role for histamine and H1R signaling in regulating T cell responses and susceptibility to CVB3-induced myocarditis in B6 mice. [Copyright &y& Elsevier]

Abstract: Coxsackievirus infections are associated with severe diseases such as myocarditis, meningitis and pancreatitis. To study the contribution of the intracellular viral sensor melanoma differentiation-associated protein-5 (MDA-5) in the host immune response to Coxsackievirus B3 (CVB3) we infected C57BL/6 and 129/SvJ mice lacking mda-5. Mice deficient in MDA-5 showed a dramatically increased susceptibility to CVB3 infection. The loss of MDA-5 allowed the virus to replicate faster, resulting in increased liver and pancreas damage and heightened mortality. MDA-5 was not absolutely required for the induction of type 1 interferons (IFNs), but essential for the production of maximal levels of systemic IFN-α early after infection. Taken together, our findings indicate that MDA-5 plays an important role in the host immune response to CVB3 by preventing early virus replication and limiting tissue pathology. [Copyright &y& Elsevier]

Abstract: Coxsackievirus B3 (CVB3) is a gastrointestinal virus causing myocarditis in human and mice. An ideal vaccine for CVB3-myocarditis requires both humoral and cellular immunity at systemic and mucosal compartments. We described here an enhancing strategy for chitosan-pVP1 vaccine by co-immunizing with lymphotactin (LTN) gene, a T cell-attractive-chemokine, encapsulated in chitosan particle to provide more protection against CVB3. Mice were intranasally co-immunized with 4 doses of chitosan–DNA vaccines separately encapsulating VP1 and LTN plasmids by 2 week-intervals and challenged with CVB3 4 weeks after the last immunization. Compared with chitosan-pVP1 alone, co-immunization with chitosan-pLTN significantly increased high-avidity-neutralizing antibody levels in serum and in intestinal mucosa, and promoted systemic and mucosal Th1 and CD8+CTL immune responses. Accordingly, enhanced resistance to CVB3-myocarditis was evidenced by reduced myocardial viral load, profound subsidence of myocarditis and increased survival rate. This strategy represents a promising platform for Th1 polarization and protection against mucosal infectious pathogens. [Copyright &y& Elsevier]

Abstract: Ultraviolet (u.v.) inactivated coxsackievirus B3 (CVB3) induces rapid calcium flux in naïve BALB/c CD4+ T cells. CD4+ cells lacking decay accelerating factor (DAF−/−) show little calcium flux indicating that virus cross-linking of this virus receptor protein is necessary for calcium signaling in CVB3 infection. Interaction of CVB3 with CD4+ cells also activates NFAT DNA binding. To show that NFAT activation is crucial to CVB3 induced disease, wild-type mice and transgenic mice expressing dominant-negative NFAT (dnNFAT) mutant in T cells were infected and evaluated for myocarditis and pancreatitis 7 days later. Inhibition of NFAT in T cells prevented myocarditis but had no effect on pancreatitis. Virus titers in pancreas were equivalent in wild-type and dnNFAT animals but cardiac virus titers were increased in dnNFAT mice. Interferon-gamma (IFNγ) expression was reduced in both CD4+ and Vγ4+ T cells from dnNFAT mice compared to controls. FasL expression by Vγ4+ cells was also suppressed. Inhibition of FasL expression by Vγ4+ cells is consistent with myocarditis protection in dnNFAT mice. [Copyright &y& Elsevier]

Myocarditis is a disease with a variable clinical presentation, ranging from asymptomatic to a fatal outcome. Among the recognized causes of myocarditis are mutations in multiple genes; infection by bacterial, rickettsial, mycotic, protozoan, and viral agents; and exposure to drugs, toxins, and alcohol. Some subtypes of myocarditis, such as giant cell myocarditis or eosinophilic necrotizing myocarditis, are suspected to be caused by an autoimmune inflammation. Several lines of evidence support the involvement of autoimmunity in myocarditis. These include the production of antibodies against relevant self-antigens, the fact that myocarditis symptoms can be relieved by immunosuppressive therapy in some patients, and a co-occurrence of myocarditis with other autoimmune diseases. Most of the evidence that myocarditis is an autoimmune disease comes from animal models. In this chapter, we discuss coxsackievirus B3-induced myocarditis and myosin-induced myocarditis as models of both viral and autoimmune inflammation in the heart. The latest advances in the study of autoimmunity have been concentrated on T helper cells, particularly the newly discovered subset, Th17 cells. Experimental autoimmune myocarditis (EAM), a mouse model of myocarditis induced by cardiac myosin, is partly an IL-17-driven disease. However, we have shown recently in IL-13 knockout mice that the disease can be driven through other pathways, and that the Thl helper cells also lead to severe heart inflammation. Most importantly, IL-17A knockout mice are not fully protected against EAM and still develop mild myocarditis. The most abundant cells in heart infiltrate in human giant cell myocarditis or EAM are monocyte/macrophages, and there is now evidence that macrophages play a decisive role in the course of EAM. [ABSTRACT FROM AUTHOR]

Coxsackievirus B3, a cytopathic virus in the family Picornaviridae, induces degenerative changes in host cell morphology. Here we demonstrate cytochrome c release and caspases-2, -3, -6, -7, -8, and -9 processing. Enforced Bcl-2 and Bcl-xL expression markedly reduced release of cytochrome c, presentation of the mitochondrial epitope 7A6, and depressed caspase activation following infection. In comparison, cell death using TRAIL ligand caused caspase-8 processing prior to cytochrome c release and executioner caspases and cell death was only partially rescued by Bcl-2 and Bcl-xL overexpression. Disruption of the mitochondrial inner membrane potential following CVB3 infection was not inhibited by zVAD.fmk treatment. Bcl-2 or Bcl-xL overexpression or zVAD.fmk treatment delayed the loss of host cell viability and decreased progeny virus release following infection. Our data suggest that mitochondrial release of cytochrome c may be an important early event in caspase activation in CVB3 infection, and, as such, may contribute to the loss of host-cell viability and progeny virus release. [Copyright &y& Elsevier]

Viral myocarditis (VMC) is a type of inflammation affecting myocardial cells caused by viral infection and has been an important cause of dilated cardiomyopathy (DCM) worldwide. Type B3 coxsackievirus (CVB3), a non-enveloped positive-strand RNA virus of the Enterovirus genus, is one of most common agent of viral myocarditis. Till now, effective treatments for VMC are lacking due to lack of drugs or vaccine. Lithium chloride (LiCl) is applied in the clinical management of manic depressive disorders. Accumulating evidence have demonstrated that LiCl, also as an effective antiviral drug, exhibited antiviral effects for specific viruses. However, there are few reports of evaluating LiCl's antiviral effect in mice model. Here, we investigated the inhibitory influence of LiCl on the CVB3 replication in vitro and in vivo and the development of CVB3-induced VMC. We found that LiCl significantly suppressed CVB3 replication in HeLa via inhibiting virus-induced cell apoptosis. Moreover, LiCl treatment in vivo obviously inhibited virus replication within the myocardium and alleviated CVB3-induced acute myocarditis. Collectively, our data demonstrated that LiCl inhibited CVB3 replication and negatively regulated virus-triggered inflammatory responses. Our finding further expands the antiviral targets of LiCl and provides an alternative agent for viral myocarditis. • Lithium chloride inhibits coxsackievirus B3 virus replication in vitro and in vivo. • Lithium chloride inhibits CVB3 replication via inhibiting virus-induced cell apoptosis. • LiCl treatment in vivo obviously alleviated CVB3-induced acute myocarditis. [ABSTRACT FROM AUTHOR]

• Protease-activated receptor 4 (PAR4) reduces Coxsackievirus B3 (CVB3) myocarditis and H1N1 influenza A virus (IAV) infection. • PAR4−/− mice exhibited increased virus load and reduced innate immune response in the heart after CVB3 infection. • PAR4−/− mice exhibit more severe myocarditis and heart function impairment. • PAR4−/− mice exhibited more pronounced cellular and cytokine immune responses in the lung after IAV infection. • More severe H1N1 IAV infection in PAR4−/− mice led to more lung injury and death compared to WT mice. PAR4 is expressed by a variety of cells, including platelets, cardiac, lung and immune cells. We investigated the contribution of PAR4 to viral infections of the heart and lung. Toll-like receptor (TLR) 3-dependent immune responses were analyzed after co-stimulation of PAR4 in murine bone-marrow derived macrophages, embryonic fibroblasts and embryonic cardiomyocytes. In addition, we analyzed Coxsackievirus B3 (CVB3) or H1N1 influenza A virus (H1N1 IAV) infection of PAR4−/− (ΔPAR4) and wild-type (WT) mice. Lastly, we investigated the effect of platelet inhibition on H1N1 IAV infection. In vitro experiments revealed that PAR4 stimulation enhances the expression of TLR3-dependent CXCL10 expression and decreases TLR3-dependent NFκB-mediated proinflammatory gene expression. Furthermore, CVB3-infected ΔPAR4 mice exhibited a decreased anti-viral response and increased viral genomes in the heart leading to more pronounced CVB3 myocarditis compared to WT mice. Similarly, H1N1 IAV-infected ΔPAR4 mice had increased immune cell numbers and inflammatory mediators in the lung, and increased mortality compared with infected WT controls. The study showed that PAR4 protects mice from viral infections of the heart and lung. [ABSTRACT FROM AUTHOR]