Progressive interstitial collagen deposition in Coxsackievirus B3-induced murine myocarditis.

Myocardial fibrosis can be produced in certain inbred strains of mice after coxsackievirus B subtype 3 (CVB3) infection. The mechanism responsible for this interstitial matrix alteration is unknown. The presumption is that fibrosis occurs in areas of myocyte damage and inflammation associated with viral infection, analogous to scar formation after cell injury in other organ systems. To test this hypothesis, we examined the hearts of A/J strain mice infected with three CVB3 variants (Crowell [CVB3-CR], Woodruff [CVB3-WD], and Lerner [CVB3-LR]), each known to cause different degrees of acute myocardial injury. With these three variants, virus was present in the heart until day 28 after inoculation but was absent thereafter. Fourteen days after inoculation, inflammation with myocyte necrosis was seen in discrete foci throughout the myocardium with all three variants. Collapse and disorganization of the usually delicate connective tissue matrix identifiable by silver impregnation was seen in these areas of myocyte injury. Persistent, diffuse lymphocytic infiltration of the myocardium was seen 55 days after inoculation with CVB3-WD and CVB3-LR, but hearts initially infected with CVB3-CR showed only rare interstitial lymphocytes comparable to uninfected control hearts. The focal scars produced by myocyte necrosis 14 days after inoculation were accentuated and heavily collagenized 55 days after inoculation with CVB3-WD and CVB3-LR; however, these foci were indistinct 55 days after inoculation with CVB3-CR. Furthermore, the usually delicate network of interstitial collagen fibers surrounding individual myocytes became thickened throughout the heart 55 days after inoculation with CVB3-WD and CVB3-LR, away from visibly scarred areas produced early after infection with these variants. This diffuse reticulin thickening was not seen after infection with the Crowell variant. Only the virus variants associated with persistent interstitial inflammation at day 55 developed major collagen matrix alterations and interstitial fibrosis. We conclude that this persistent interstitial lymphocytic infiltration reflects altered immune function related to specific virus variants in this animal strain. We postulate that these lymphocytes are part of a delayed immunopathogenic response uncoupled from the original viral injury and inflammatory damage. Potential mechanisms by which this interstitial lymphocytic infiltration results in fibrosis are discussed.     

Coxsackievirus B3-induced myocarditis in MHC class II-deficient mice

OBJECTIVES: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in immunocompetent C57BL/6 and MHC class II knockout mice with identical genetic backgrounds. STUDY DESIGN/METHODS: We analyzed the histology and immunohistology of myocardial injury, the replicating virus titer, and antibody response in the early and late phase of disease. RESULTS: CVB3-infected C57BL/6 mice showed acute myocarditis, with spontaneous healing, virus elimination, anti-CVB3 IgM/IgG production, and neutralizing antibody response. In contrast, MHC class II knockout mice developed less severe acute myocarditis, persistence of infiltrations and strong fibrosis, virus persistence, and weak IgG response, with absence of virus neutralizing antibodies. CONCLUSIONS: Immunodeficient organisms are more susceptible to long-term heart muscle injuries after infection with CVB3. The presence of CD4+ T cells are necessary to prevent the development of chronic disease.     

Coxsackievirus B3-induced chronic myocarditis in outbred NMRI mice

OBJECTIVES: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in adult Han:NMRI mice. The outbred model, in comparison with inbred models, represents better the natural variable susceptibility of the human population. STUDY DESIGN/METHODS: We analyzed the replicating virus titer, the antibody response in the acute and chronic phase of disease, the histology of myocardial injury, and the persistence of viral RNA. RESULTS: NMRI mice infected with 5000 plaque-forming units (PFU) of the CVB3 variant "P"D, a lytic variant to human fibroblast lines, showed a peak of virus replication at day 14 and developed a severe acute myocarditis. The chronic myocarditis was characterized by progressive fibrosis, small foci of infiltrates, persistent viral RNA in the heart, and detectable anti-CVB3 IgG production and neutralizing antibody response up to day 98 postinfection. CONCLUSIONS: CVB3"P"D is able to induce chronic myocarditis in NMRI mice. This model provides a method for examining and proving the mechanisms of myocardial pathogenesis and of developing therapeutic strategies.     

Genetic control of Coxsackievirus B3-induced heart-specific autoantibodies associated with chronic myocarditis.

Cardiac-specific autoantibodies to sarcolemmal and cardiac myosin antigens observed during the chronic phase of Coxsackievirus B3-induced myocarditis appear to be under autosomal recessive control. This observation is based on examination of F1 hybrids bred from A/J mice which develop chronic myocarditis and C57BL/6J mice which resolve the virus-induced lesions. Previous mouse studies demonstrated that the prevalence of heart-specific autoantibodies varied with the H-2 complex. However, in 25 H-2 congenic mouse strains the strain background was the predominant determinant of autoantibody presence. Recently, we extended our genetic evaluation of the chromosomal locations governing autoantibody responses by examining 25 AXB and BXA recombinant inbred strains. Two populations of heart-specific autoantibodies were demonstrated against sarcolemmal and cardiac myosin antigens. Analyses of the AXB/BXA strain distribution patterns for these two traits revealed that the anti-sarcolemmal response was controlled by a gene(s) linked to Np-2 and Ter alpha loci on chromosome 14. Linkage could not be assigned for the anti-cardiac myosin response.     

Coxsackievirus B-3 myocarditis. T-cell autoimmunity to heart antigens is resistant to cyclosporin-A treatment.

A cardiotropic variant of coxsackievirus group B, Type 3 (CVB3) induces myocarditis in inbred Balb/c mice. Myocardial injury is predominantly mediated by T lymphocytes recognizing normal myocyte antigens, making this an autoimmune disease. Nonetheless, the autoimmune response cannot be inhibited by cyclosporin A (CSA) treatment of the infected animals. Mortality in treated mice was increased 2-4 times, but neither virus-specific antibody or cytolytic T-lymphocyte responses were affected, and maximal virus concentrations in the hearts of CSA-treated and control animals were similar. Cardiac damage remains T-cell-mediated, because mice given both CSA and rabbit anti-thymocyte serum (ATS) failed to develop significant myocardial inflammation. CSA did suppress immunity in Balb/c mice to an allogeneic C57B1 lymphoma, EL4. Subcutaneous inoculation of mice with 2.5 X 10(6) ascites tumor cells resulted in 100% of the CSA-treated animals having tumors averaging 340 mg 10 days later, compared with less than 10% of control animals having tumors averaging only 30 mg. Humoral immunity to the tumor was absent in CSA-treated mice. Therefore, whether CSA induces immunosuppression depends upon the antigenic stimulus used.     

Coxsackievirus B-3 myocarditis. Acute and chronic forms of the disease caused by different immunopathogenic mechanisms.

Male BALB/c, DBA/2 and A mice inoculated with a myocarditic variant of coxsackievirus group B, Type 3 (CVB3) developed three distinct patterns of myocarditis. Most BALB/c and all DBA/2 mice developed acute cardiac inflammation lasting only 2 weeks, while A animals consistently showed twice as much myocardial damage as the other two strains, with active myocarditis continuing for 56 days after virus inoculation. Although virus elimination from the heart was also delayed in A mice, immunopathogenic, not viral, mechanisms caused cardiac injury in all strains. In vivo depletion of L3T4+ (T helper) and Lyt 2+ (T cytolytic/suppressor) cells using monoclonal antibodies showed that myocarditis in BALB/c mice depended predominantly on Lyt 2+ cells, in DBA/2 mice on L3T4+ cells and in A mice on both Lyt 2+ and L3T4+ cells. IgM heart reactive antibodies (HRAs) occurred in all three strains, while IgG HRAs were detected only in DBA/2 and A mice. Presumably, only the IgG antibody was pathogenic in this system. These data suggest that the host's genetic makeup determines the type of immune response to CVB3 infection of the heart and therefore the pattern of myocarditis which develops.     

Coxsackievirus B-3 myocarditis in Balb/c mice. Evidence for autoimmunity to myocyte antigens.

Male Balb/c mice inoculated with a heart-adapted variant of Coxsackievirus, group B, type 3 (Nancy) (CVB3M), develop extensive myocarditis and cytolytic activity to primary cultures of uninfected and infected myocytes. To elucidate the mechanisms of myocyte injury in myocarditis, two distinct cytolytic T-lymphocyte (CTL) populations were isolated by immunoadsorption of lymph node cells to glutaraldehyde-fixed uninfected and infected myocyte monolayers. One population preferentially adsorbed to and lysed uninfected myocytes (autoreactive CTLs), while the other adsorbed to and lysed CVB3M-infected myocytes (virus-specific CTL). Neither CTL population adsorbed to monolayers of HeLa, L929, or umbilical cord endothelial cells, or to myocytes infected with a related but nonmyocarditic Coxsackievirus B-3 variant ( CVB3o ). While both autoreactive and virus-specific CTLs induced myocarditis in vivo, the lesions caused by autoreactive CTLs were more extensive and necrotizing than those of virus-specific cells. These results support the hypothesis that CVB3 -induced myocarditis results, in part, from autoimmunity to myocyte antigens.     

Characterization of the antibody response in vaccinated mice protected against Coxsackievirus B3-induced myocarditis

Adolescent male CD-1 mice can be rendered resistant to coxsackievirus B3 (CVB3m)-induced myocarditis following inoculation as neonates with a single dose of a temperature-sensitive mutant virus (ts1), derived from the prototype parent virus (CVB3m). Previously, anti-CVB3 neutralizing antibodies were not detected in sera of adolescent ts1 vaccinees by a standard plaque-reduction assay (Gauntt et al 1983. Infect. Immun. 39:851). However, a more sensitive cytopathic effects-reduction assay permitted detection of low titers of anti-CVB3m neutralizing antibodies of the IgG class prior to challenge with CVB3m. Following CVB3m challenge, serum anti-CVB3m neutralizing antibody titers of ts1 vaccines declined on days 1-2 post-inoculation (p.i.) then increased over the next 6 days. The neutralizing antibodies were of both the IgG and IgM classes. Normal mice challenged with CVB3 did not produce detectable serum anti-CVB3m neutralizing antibody until day 4 p.i. and by 8 days p.i. the neutralizing antibody was only of the IgM class. Thus, adolescent murine ts1 vaccines mount a secondary antibody response to CVB3m in both neutralizing IgG and IgM, but resistance to CVB3m-induced myocarditis is due to the presence of low levels of anti-CVB3m IgG neutralizing antibody in serum at the time of challenge with CVB3m.     

The role of Th17 cells and regulatory T cells in Coxsackievirus B3-induced myocarditis

IL-17-producing (Th17) and regulatory T (Treg) cells have been well established in the pathogenesis of many inflammatory diseases. To assess whether Th17 and Treg were altered in acute virus-induced myocarditis (AVMC) mice, we assessed Th17/Treg functions on different levels in AVMC. It was shown that the expression of splenic Th17 cells and Th17-related cytokines (IL-17A, IL-21) markedly increased. Interestingly, the expression of splenic Treg cells and Treg-related cytokines (TGF-β, IL-10) also significantly increased. Using neutralization of IL-17 in the AVMC, we found that Treg cells roughly decreased compared with isotype control mice. However, T cells and perforin dramatically increased, followed by a marked reduction in CVB3 replication. The results suggested that Th17 cells possibly contributed to viral replication through the action of Treg cells in mediating T cells and perforin response in AVMC.     

Susceptibility to Coxsackievirus B3-induced chronic myocarditis maps near the murine Tcr alpha and Myhc alpha loci on chromosome 14.

This study was undertaken to determine the genetic control of host susceptibility to coxsackievirus B3 (CVB3)-induced chronic myocarditis in a mouse model. An autosomal recessive autoimmune myocardial disease (amd) gene (possibly more than one gene), which determined susceptibility to CVB3-induced chronic myocarditis in the A/J and DBA/2J inbred mouse strains, was mapped to a segment of chromosome 14. Data from both the AXB/BXA recombinant inbred (RI) strains and the B10.D2(57N) H-8b congenic mice supported this linkage relationship. Analysis of the AXB/BXA RI strain distribution patterns suggested that amd maps distal to the Np-2, Tcr alpha, and Myhc alpha loci.     

Coxsackievirus B3-induced myocarditis. Autoimmunity is L3T4+ T helper cell and IL-2 independent in BALB/c mice.

Male BALB/c mice inoculated with 6 X 10(4) plaque-forming units (pfu) coxsackievirus, group B, type 3 (CVB3), develop myocarditis within 7 days. Two cytolytic T lymphocyte (CTL) populations arise in infected animals. One population belongs to the Lyt 2+ T (cytolytic/suppressor) lymphocyte subset and reacts specifically with uninfected heart cells (autoreactive CTLs, ACTLs), whereas the other belongs to the L3T4+ T (helper) lymphocyte subset and reacts with infected targets (virus-specific CTLs, VSCTLs). Although both immune T lymphocyte populations can induce cardiac inflammation in vivo, ACTLs predominantly cause tissue injury. VSCTL generation can be inhibited by either anti-Tac (antibody to the interleukin 2 [IL-2] receptor) or anti-Iad but not by anti-IAk, indicating that this response is probably both IL-2-dependent and Class II (major histocompatibility complex [MHC] antigen restricted. ACTL generation is independent of IL-2, because neither anti-Tac or cyclosporin A inhibit this response.     

The mitochondrial respiratory chain has a critical role in the antiviral process in Coxsackievirus B3-induced myocarditis

Well-established differences in Coxsackievirus B3 (CVB3) elimination in resistant C57BL/6 and permissive A.SW/SnJ mice provide suitable models for studying the significance of the link between mitochondrial respiratory chain (RC), antioxidative stress components and mitochondrion-related apoptosis in the context of myocardial virus elimination. Distinct myocardial CVB3 titer in C57BL/6 (2.5 ± 1.4 × 10(4) plaque-forming units (p.f.u.)/g tissue) and A.SW/SnJ mice (1.4 ± 0.8 × 10(7) p.f.u./g) were associated with differences in the cardiac mitochondrial function 8 days post infection (p.i.). Infected C57BL/6 mouse hearts disclosed increased complex I (CI) and CIII activity, but restricted CII and normal CIV activity of RC. Reduced expression of the antioxidative catalase was accompanied by elevated lipid peroxidation (LPO), indicating oxidative stress. Intrinsic apoptosis was activated demonstrated by elevated levels of Bax, Bcl-2, caspase 3 and DNA degradation. In contrast, all myocardial RC complex activities were restricted in CVB3-infected A.SW/SnJ mice. The antioxidative system provided sufficient protection against oxidative stress shown by an elevated catalase expression and unaltered LPO. Bax and Bcl-2 levels were unchanged in CVB3-infected A.SW/SnJ mice, while caspase 3 was moderately increased but no DNA degradation was detectable. Correlation analyses including data from the two mouse strains revealed that reduced CVB3 titer correlated with increased CI and CIII activity, oxidative stress as well as active apoptosis during acute myocarditis (MC). C57BL/6 mice completely eliminated CVB3 and inflammation and normalized all intracellular parameters, while A.SW/SnJ mice showed permanently restricted CI activity in chronic MC 90 days p.i., at which time the replicating virus was no longer detectable but immunological processes were still active. Consequently, the regulation of energy metabolism appears crucial for an effective virus elimination and may be of prognostic and therapeutic significance for patients with virus-induced MC.     

Murine acinar pancreatitis preceding necrotizing myocarditis after Coxsackievirus B3 inoculation.

Balb/C weanling mice were inoculated intraperitoneally with a myocarditic variant of coxsackie-virus B3, with the aim of characterizing more fully the cell damage induced in the heart as well as in other organs. During the first week postinfection (pi), all animals developed acinar pancreatitis, followed by focal myocarditis. In accordance with the increasing infectivity titers, such progressive histopathological changes correlated with local viral replication. From day 4 pi, acinar degeneration accompanied by diffuse inflammatory exudate was observed in the pancreas, followed by fatty tissue replacement by day 8. In the heart, focal necrosis rather than inflammatory reaction first appeared at 4 days pi and became widespread by 6-8 days pi. Necrotic foci usually presented calcium deposits, with absence of myofibrils in the affected fibers. The fact that both periodic acid Schiff (PAS) and Best carmine staining remained positive even after diastase treatment ruled out basophilic necrosis. In summary, the pancreas appeared to be the site of primary viral replication leading to viremia.     

Levamisole exacerbates coxsackievirus B3-induced murine myocarditis.

Levamisole administration to several strains of adolescent mice at the time of or up to 4 days post-inoculation (p.i.) with a myocarditic variant of coxsackievirus B3 (CVB3m) increased the number of myocarditic lesions above that found in CVB3m-inoculated mice. Virus replication in heart tissues in vivo was not affected by levamisole administration to the mice, nor was production of neutralizing antibody to CVB3m. Lymphocytes from nodes of virus-inoculated mice treated with levamisole at 2 days p.i. exhibited an increased reactivity to phytohemagglutinin on days 6 and 8 p.i., compared with respective responses by nodal T lymphocytes from CVB3m-inoculated mice. Levamisole treatment of CVB3m-inoculated mice also increased the reactivity of splenic and peripheral blood T lymphocytes to phytohemagglutinin on day 8 p.i., but not day 6 p.i., compared with the respective responses by lymphocytes from CVB3m-inoculated mice. The proportion of theta antigen-bearing lymphocytes in the total lymphocyte population in peripheral blood of CVB3m-inoculated mice was not altered by levamisole treatment. However, CVB3m-induced reduction in this subpopulation of lymphocytes in the nodes was restored to control levels by levamisole treatment. Reactivities of cytotoxic T lymphocytes from CVB3m-inoculated mice were increased against both normal and CVB3m-inoculated target cells after levamisole treatment of these mice. The results suggest that levamisole may contribute to CVB3m induction of myocarditis by several mechanisms, such as increasing the blastogenic activity of the phytohemagglutinin-responding subset of T lymphocytes, by possibly altering T-lymphocyte distribution in the body and by nonspecifically increasing reactivities of cytotoxic T lymphocytes.     

Coxsackievirus B3 induction of NFAT: requirement for myocarditis susceptibility

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.     

Coxsackievirus B3-induced myocarditis: differences in the immune response of C57BL/6 and Balb/c mice

Coxsackievirus B3 (CVB3) infections are the most frequent causes of human myocarditis, often resulting in chronic stages characterized by fibrosis and loss of function. This disease is called dilated cardiomyopathy (DCM). Persistent virus in the myocardium may lead to chronic activation of fibroblasts, and subsequently, to fibrosis of the myocardium. Studies with immunodeficient mice have shown that certain defects of the immune system retard the rate at which virus is eliminated from the heart, thus leading to viral persistence. Therefore, we followed the immune response of two immunocompetent mouse strains (C57BL/6 and Balb/c) to CVB3 infection. These two strains have been reported to develop different immune responses to infections and we expected a similar reaction to viral infections as well. The two mouse strains recovered completely from CVB3 infection and expressed identical levels of cytokine mRNA in the heart. However, the virus in heart tissue decreased more slowly in Balb/c than in C57BL/6 mice. This was accompanied by a strong virus-specific IgG and weak IgM response in the C57BL/6 mice, in comparison to the Balb/c mice. We conclude, therefore, that viral-specific IgG is of importance for CVB3 elimination from infected hearts.     

Extracellular matrix remodelling after coxsackievirus B3-induced murine myocarditis.

Weanling inbred Balb/c mice were intraperitoneally inoculated with a myocarditic variant of coxsackievirus B3. At days 1, 2, 4, 6, 8, 10, 14, 24 and 30 post-infection (p.i.), myocardial tissue was harvested for viral infectivity titrations and histological studies, including routine techniques (haematoxylin-eosin, Masson trichrome and von Kossa) and specialized procedures (silver impregnation for reticulin, picrosirius red stain for collagen and immunoperoxidase labelling for laminin). Virus was isolated as from day 2, reached maximal infectivity at days 6-8 and decreased gradually to become undetectable by day 14. Early histological findings during the 1st week consisted mainly of scattered foci of necrotic myocytes showing calcium deposits; slight mononuclear cell infiltration and fragmentation of both reticulin fibres and pericellular laminin were also present. From the 2nd up to 4th week p.i., inflammatory reaction abated concomitantly with the gradual development of fibrosis, as evidenced by reticulin fibre thickening, irregular laminin distribution and collagen fibre increase. Our results suggest that viral-induced necrosis is able to trigger marked extracellular matrix remodelling even in the case of minimal inflammation.     

Coxsackievirus B3-induced myocarditis: infection of females during the estrus phase of the ovarian cycle leads to activation of T regulatory cells

Transgenic female mice expressing the TNFα gene under the cardiac myosin promoter (TNF1.6) develop substantially increased myocarditis and increased numbers of CD4+Th1 (interferon gamma+) cells when infected with coxsackievirus B3 (CVB3) during the diestrus and proestrus phases of the estrus cycle compared to females infected during the estrus and metestrus phases. Cardiac virus titers were increased in females infected in estrus compared to females infected during the other phases. T regulatory cells (CD4+CD25+FoxP3+) were increased in both peripheral blood and inflammatory cells in the heart in females infected during estrus. Exogenous administration of 200 ng/mouse 17-β-estradiol to females protected against CVB3 induced myocarditis and increased CD4+CD25+FoxP3+ cells. These results demonstrate that hormonal fluctuations occurring in normally cycling females can determine T regulatory cell response and control virus-induced pathogenesis.     

Coxsackievirus B3 persistence and myocarditis in NFR nu/nu and +/nu mice

NFR nude (nu/nu) and euthymic (+/nu) littermates were infected with coxsackievirus B3 (CBV-3) and assayed for virus persistence in the heart, pancreas and spleen, for development of myocarditis and for antibody production. The virus grew to higher titer and persisted longer in hearts of nu/nu mice. In both types of mice there was comparable myocarditis with a mononuclear cell inflammatory response, and there was some evidence of chronic lesions for up to 21 days in +/nu and 28 days in nu/nu mice. Antibody of the IgM class was present at 7 days in both strains of mice. Thereafter, +/nu mice produced high titers of virus-specific IgG antibody, while nu/nu mice produced little or no viral IgG antibody. The persistence of virus for up to 28 days in NFR nude mice is longer than has been reported previously, and offers an opportunity for further study of the role of T-cells in virus persistence and myocarditis.