Cytomegalovirus-induced myocarditis - An animal model for understanding pathogenesis and therapy

The inflammatory heart disease myocarditis leads to dilated cardiomyopathy and has been associated with a viral aetiology. The herpesvirus cytomegalovirus induces chronic myocarditis with the development of autoimmunity. Murine models of myocarditis are now well established. Murine cytomegalovirus, a natural pathogen of mice, induces both acute and chronic phases of myocarditis in the susceptible BALB/c mouse strain. Early treatment with the antiviral drugs ganciclovir and cidofovir has been shown to reduce the severity of myocarditis. However, treatment with antivirals in the late stages of infection has limited effectiveness for prevention of the chronic phase of disease. The immunological events in the progression to this chronic cardiac disease have been recently described. New knowledge of the immunopathogenesis of myocarditis can be utilized in the development of strategic antiviral and immunotherapeutic regimes

Immunomodulation of murine cytomegalovirus-induced myocarditis in mice treated with lipopolysaccharide and tumor necrosis factor

Murine cytomegalovirus (MCMV) infection of BALB/c mice produces acute and chronic myocarditis similar to clinical disease in humans. In contrast, MCMV-infected C57BL/6 mice develop only mild acute myocarditis. We have investigated the effect of administration of the immunomodulator lipopolysaccharide (LPS) on the development of postviral myocarditis in mice. LPS exacerbated heart inflammation in both strains of MCMV-infected mice, with normally resistant C57BL/6 mice developing chronic myocarditis. Autoantibodies to cardiac myosin were enhanced with LPS treatment in both MCMV-infected mouse strains. LPS treatment also increased the production of TNF in the sera without affecting virus titers in the spleen, liver, or salivary glands, a target organ most affected during persistent virus infection. In LPS/MCMV-infected BALB/c mice, TNF, IL-6, and IL-10 levels were detected in cultures of heart infiltrating cells but not in splenocytes. Importantly, administration of the bioactive synthetic TNF peptide (amino acids 114-130) increased myocarditis in C57BL/6 mice, similar to that seen with LPS treatment. TNF peptide/MCMV-infected BALB/c and C57BL/6 mice showed distinct differences in the expression pattern of IFN-γ IL-10, and TNF. These data show that the disease may be partly regulated by TNF among other select cytokines and autoantibodies to cardiac myosin. The immunopathological nature of MCMV induced myocarditis is thus highlighted

Synergy of type I interferon-A6 and interferon-B naked DNA immunotherapy for cytomegalovirus infection

Summary: Delivery of type I IFN transgenes by naked DNA immunization can protect against cytomegalovirus infection and myocarditis. Here, we investigate IFN transgene expression, antiviral efficacy, and immunomodulation of myocarditis using various treatment regimes in a mouse CMV model. In vivo expression of the IFN transgene was observed in the sera for 35 days post-DNA inoculation. Prophylactic IFN-A6 and IFN-B DNA treatment for 14 days prior to murine cytomegalovirus (MCMV) infection was more efficacious in significantly reducing viral titres, than 2 days prior to or 2 days post-virus infection. Similarly, IFN-A6 DNA treatment commencing 14 days prior to virus infection was superior in suppressing both acute and chronic myocarditis. Furthermore, reduction of autoantibody titres was more pronounced when IFN was administered 14 days prior to viral infection. Combinational IFN gene therapy was assessed for synergy between IFN subtypes. Combination treatment with either IFN-A6/A9 or IFN-A6/B greatly reduced spleen viral titres while IFN-A6/B and IFN-A9/B reduced virus replication in the liver. Only IFN-A6/A9 and IFN-A9/B reduced acute viral myocarditis, whereas IFNA6/B treatment was most efficacious for autoimmune chronic myocarditis. Finally, treatment with IFN-A6 DNA 2 weeks post-MCMV infection proved effective at inhibiting the development of chronic autoimmune myocarditis. These findings suggest that immunomodulation of both antiviral and autoimmune responses by IFN DNA immunization may be an avenue for improved viral immunotherapy

Characterisation of murine cytomegalovirus myocarditis: Cellular infiltration of the heart and virus persistence

Myocarditis triggered by a viral infection has integral viral and immunological aspects associated with the pathogenesis of disease. The present study was performed to analyse the cellular inflammatory response in the heart and cytomegalovirus replication during the development of myocarditis in vivo. We examined murine cytomegalovirus in an animal model of myocarditis using both susceptible BALB/c and resistant C57BL/6 mice. The heart infiltrating cells of BALB/c mice were found to comprise predominantly CD8+ T cells, with other cells of the CD4+ T cell, macrophage, B cell and neutrophil phenotype. Infectious MCMV titres in the heart were low and replicative virus could not be isolated beyond the first week post-infection (p.i.). Direct viral lysis of myocytes in vitro and apoptosis of cardiac cells in vivo was observed. Furthermore, viral DNA was detected in the heart of both mouse strains throughout the development of chronic disease. Viral gB RNA was detected during the first 35 days p.i. However, viral transcript for ie1 RNA but not gB RNA was found in the heart during the late stage of disease, suggesting latent viral infection of the heart. Our findings suggest that maintenance of the chronic phase of myocarditis involving post-viral immunological responses can occur in the presence of little infectious virus replication in the heart

Cytokine expression in murine cytomegalovirus-induced myocarditis: modulation with interferon-α therapy

Cytomegalovirus-induced myocarditis is largely immune-mediated. BALB/c mice produced higher levels of IL-4 in the heart indicative of a Th2-like response. Although IL-6, IL-10, IL-18, and TNF-α were produced in the heart during acute infection, BALB/c mice lacked a substantial IL-2 and IFN-γ response. Conversely, C57BL/6 mice produced significant levels of IFN-γ in the heart with no significant levels of IL-4 or IL-6, suggestive of a dominant Th1-like response to virus infection. IFN-α/β immunotherapy is known to suppress the development of MCMV-myocarditis. Cytokine secretion in IFN-stimulated MCMV-infected BALB/c myocytes was found to be IFN subtype-dependent with elevation of IL-6 and IL-18 levels. During the chronic phase of disease, IFNA6 DNA treatment in vivo increased IL-18 production in the heart. These results suggest that IFN subtype therapy may have immunomodulating effects in reducing disease severity in BALB/c mice via regulation of cytokine production in the heart

Type I IFN-beta gene therapy suppresses cardiac CD8+ T-cell infiltration during autoimmune myocarditis

Gene therapy using DNA encoding type I IFN subtypes IFNA6, IFNA9 and IFNB suppresses murine cytomegalovirus (MCMV)-myocarditis, a predominantly cell-mediated disease in BALB/c mice. CD8+ T cells are the principal cell type within the inflamed myocardium. As such, we investigated the effects of IFN subtype treatment on this T-cell subset and other cell types in the cardiac infiltrate. In the acute phase of disease, IFNA6 and IFNA9 treatments significantly reduced the number of CD8+ T cells within the foci of cellular infiltration in the heart. During the chronic phase, which is primarily autoimmune in nature, IFNB treatment significantly reduced CD8+ T cells. B-cell and neutrophil numbers in the cardiac infiltrate were also reduced following IFNB immunotherapy. Although early inflammatory responses are important for resolution of virus infection, high numbers of lymphocytes persisting in the myocardium may lead to exacerbation of disease. Our data suggests that type I IFN DNA therapy regulates cardiac cellular infiltration. Thus, treatment with IFN-β administered prophylactically to high-risk patients in acquiring CMV infection may reduce the development of chronic autoimmune myocarditis

Downregulation of IgE antibody and allergic responses in the lung by epidermal biolistic microparticle delivery

Background Biolistic injections provide a needle-free delivery of antigen-laden microparticles to the epithelium. The precision of the injection preferentially targets the Langerhans cell network, which, although ideal for vaccination, might not be suitable for the downregulation of immune responses in immunotherapy. Objective We sought to determine the ability of biolistic injection of antigen into the epithelium of sensitized mice to inhibit IgE antibody and lung inflammatory responses produced by further exposure to antigen. Methods Mice were sensitized by means of a needle injection of ovalbumin (OVA) in alum and given a series of biolistic injections of OVA or vehicle control, followed by a boost of OVA in alum. Serum IgE and IgG antibodies were measured before and after the boost. The mice were then challenged intranasally, and the infiltration of inflammatory cells was measured by means of bronchoalveolar lavage. Airway reactivity of the challenged mice was measured by examining responses to methacholine with forced oscillatory techniques. Results Biolistic injection of OVA into the dorsal skin of sensitized mice markedly inhibited IgE and IgG1 antibody responses induced by boosting. IgG2a antibody responses were reduced rather than stimulated. The eosinophilic inflammation in the bronchoalveolar lavage fluid induced by intranasal challenge was also markedly inhibited. Lung hyperreactivity showed an initial increase and then a decrease of responsiveness to methacholine, with elastance returning to the level of unsensitized mice. Biolistic injection into the buccal epithelium was also inhibitory. Conclusions Biolistic injection of allergen inhibited the boosting of IgE antibody and eosinophilic lung inflammatory responses without inducing TH1 immunity