The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease

Infections are thought to be important in the pathogenesis of many heart diseases. Coxsackievirus B3 (CVB3) has been linked to chronic dilated cardiomyopathy, a common cause of progressive heart disease, heart failure and sudden death. We show here that the sarcoma (Src) family kinase Lck (p56lck) is required for efficient CVB3 replication in T-cell lines and for viral replication and persistence in vivo. Whereas infection of wild-type mice with human pathogenic CVB3 caused acute and very severe myocarditis, meningitis, hepatitis, pancreatitis and dilated cardiomyopathy, mice lacking the p56lck gene were completely protected from CVB3-induced acute pathogenicity and chronic heart disease. These data identify a previously unknown function of Src family kinases and indicate that p56lck is the essential host factor that controls the replication and pathogenicity of CVB3.     

In Vivo Delivery of Adenoviral Vector Containing Interleukin-17 Receptor A Reduces Cardiac Remodeling and Improves Myocardial Function in Viral Myocarditis Leading to Dilated Cardiomyopathy

Th17 cells have been implicated in the pathogenesis of myocarditis. Interleukin (IL)-17A produced by Th17 cells is dispensable for viral myocarditis but essential for the progression to dilated cardiomyopathy (DCM). This study investigated whether the adenoviral transfer of the IL-17 receptor A reduces myocardial remodeling and dysfunction in viral myocarditis leading to DCM. In a mouse model of Coxsackievirus B3 (CVB3)-induced chronic myocarditis, the delivery of the adenovirus-containing IL-17 receptor A (Ad-IL17RA:Fc) reduced IL-17A production and decreased the number of Th17 cells in the spleen and heart, leading to the down-regulation of systemic TNF-α and IL-6 production. Cardiac function improved significantly in the Ad-IL17R:Fc- compared with the Ad-null-treated mice 3 months after the first CVB3 infection. Ad-IL17R:Fc reduced the left ventricle dilation and decreased the mortality in viral myocarditis, leading to DCM (56% in the Ad-IL17R:Fc versus 76% in the Ad-null group). The protective effects of Ad-IL17R-Fc on remodeling correlated with the attenuation of myocardial collagen deposition and the reduction of fibroblasts in CVB3-infected hearts, which was accompanied by the down-regulation of A distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1), Matrix metalloproteinase-2(MMP-2), and collagen subtypes I and III in the heart. Moreover, in cultured cardiac fibroblasts, IL-17A induced the expression of ADAMTS-1, MMP-2, and collagen subtypes I and III and increased the proliferation of fibroblasts. We determined that the delivery of IL-17-RA:Fc reduces cardiac remodeling, improves function, and decreases mortality in viral myocarditis leading to DCM, possibly by suppressing fibrosis. Therefore, the adenoviral transfer of the IL-17 receptor A may represent an alternative therapy for chronic viral myocarditis and its progression to DCM.     

Virus-induced dilated cardiomyopathy is characterized by increased levels of fibrotic extracellular matrix proteins and reduced amounts of energy-producing enzymes

The most relevant clinical phenotype resulting from chronic enteroviral myocarditis is dilated cardiomyopathy (DCM). Mice of the susceptible mouse strain A.BY/SnJ mimick well human DCM since they develop as a consequence of persistent infection and chronic inflammation a dilation of the heart ventricle several weeks after coxsackievirus B3 (CVB3) infection. Therefore, this model is well suited for the analysis of changes in the heart proteome associated with DCM. Here, we present a proteomic survey of the dilated hearts based on differential fluorescence gel electrophoresis and liquid chromatography-mass spectrometric centered methods in comparison to age-matched non-infected hearts. In total, 101 distinct proteins, which belong to categories immunity and defense, cell structure and associated proteins, energy metabolism and protein metabolism/modification differed in their levels in both groups. Levels of proteins involved in fatty acid metabolism and electron transport chain were found to be significantly reduced in infected mice suggesting a decrease in energy production in CVB3-induced DCM. Furthermore, proteins associated with muscle contraction (MLRV, MLRc2, MYH6, MyBPC3), were present in significantly altered amounts in infected mice. A significant increase in the level of extracellular matrix proteins in the dilated hearts indicates cardiac remodeling due to fibrosis.     

IL-12 protects against coxsackievirus B3-induced myocarditis by increasing IFN-gamma and macrophage and neutrophil populations in the heart

Th1-type immune responses, mediated by IL-12-induced IFN-gamma, are believed to exacerbate certain autoimmune diseases. We recently found that signaling via IL-12Rbeta1 increases coxsackievirus B3 (CVB3)-induced myocarditis. In this study, we examined the role of IL-12 on the development of CVB3-induced myocarditis using mice deficient in IL-12p35 that lack IL-12p70. We found that IL-12 deficiency did not prevent myocarditis, but viral replication was significantly increased. Although there were no changes in the total percentage of inflammatory cells in IL-12-deficient hearts compared with wild-type BALB/c controls by FACS analysis, macrophage and neutrophil populations were decreased. This decrease corresponded to reduced TNF-alpha and IFN-gamma levels in the heart, suggesting that macrophage and/or neutrophil populations may be a primary source of TNF-alpha and IFN-gamma during acute CVB3 myocarditis. Increased viral replication in IL-12-deficient mice was not mediated by reduced TNFRp55 signaling, because viral replication was unaltered in TNFRp55-deficient mice. However, STAT4 or IFN-gamma deficiency resulted in significantly increased viral replication and significantly reduced TNF-alpha and IFN-gamma levels in the heart, similar to IL-12 deficiency, indicating that the IL-12/STAT4 pathway of IFN-gamma production is important in limiting CVB3 replication. Furthermore, STAT4 or IFN-gamma deficiency also increased chronic CVB3 myocarditis, indicating that therapeutic strategies aimed at reducing Th1-mediated autoimmune diseases may exacerbate common viral infections such as CVB3 and increase chronic inflammatory heart disease.     

Coxsackievirus B3-Induced Chronic Myocarditis in Mouse: Use of Whole Blood Culture to Study the Activation of TNFα-Producing Cells

The pathogenesis of CVB3-induced chronic myocarditis remains unknown. Activated monocytes and macrophages may maintain ongoing inflammation during a persistent CVB3 infection and possibly represent the major mechanism leading to chronic myocarditis. We decided to study the activation status of cells by studying TNFα secretion in vitro using whole blood culture in CVB3-induced murine chronic myocarditis. Seven DBA/2 +/+ mice and 18 NMRI nu/nu mice were inoculated intraperitoneally with 5 × 10⁵ pfu of CVB3, and mice were mock-infected. Thirty-one days post-infection, all mice were sacrificed, blood samples were obtained from the heart, and the heart was removed. Enteroviral genomic detection by RT-PCR, virus isolation and histological analysis of heart samples were performed. Heparinized whole blood (25 μl) was cultured for 4 hr and 24 hr in sterile 96 well-plate containing 225 μl RPMI in the presence or the absence of activators (LPS + PHA). The TNFα levels in the whole blood from mock-infected DBA/2 (n = 4) and NMRI nu/nu mice (n = 5) were not different. A moderate increase of TNFα was observed in three out of five DBA/2 mice with negative CVB3 that had no histological abnormalities in myocardium. An increased level of TNFα was found in the sole DBA/2 mouse with positive CVB3 detection and chronic myocarditis. An increased level of TNFα was found in one out of nine NMRI nu/nu mice with positive CVB3 detection and chronic myocarditis and in one out of seven mice with positive CVB3 detection exempt of lesions in myocardium. In other infected mice, the level of TNFα was normal. Enteroviral genome was not detected in the blood from infected mice at 31 days post-infection. The increased TNFα level in some mice may be designed for a beneficial inflammatory and immune response, however, an exaggerated release may be associated with an adverse effect. The normal TNFα level in whole blood cultures from mice with chronic myocarditis does not exclude enhanced cytokine production at infected loci such as myocardial tissue. This is the first report to use whole blood cultures to study the production of cytokines in virus-induced disease in a small animal model.     

Testosterone and interleukin-1β increase cardiac remodeling during coxsackievirus B3 myocarditis via serpin A 3n

Myocarditis and dilated cardiomyopathy (DCM) are often caused by viral infections and occur more frequently in men than in women, but the reasons for the sex difference remain unclear. The aim of this study was to assess whether gene changes in the heart during coxsackievirus B3 (CVB3) myocarditis in male and female BALB/c mice predicted worse DCM in males. Although myocarditis (P = 4.2 × 10(-5)) and cardiac dilation (P = 0.008) were worse in males, there was no difference in viral replication in the heart. Fibrotic remodeling genes, such as tissue inhibitor of metalloproteinase (TIMP)-1 and serpin A 3n, were upregulated in males during myocarditis rather than during DCM. Using gonadectomy and testosterone replacement, we showed that testosterone increased cardiac TIMP-1 (P = 0.04), serpin A 3n (P = 0.007), and matrix metalloproteinase (MMP)-8 (P = 0.04) during myocarditis. Testosterone increased IL-1β levels in the heart (P = 0.02), a cytokine known to regulate cardiovascular remodeling, and IL-1β in turn increased cardiac serpin A 3n mRNA (P = 0.005). We found that 39 of 118 (33%) genes identified in acute DCM patients were significantly altered in the heart during CVB3 myocarditis in mice, including serpin A 3n (3.3-fold change, P = 0.0001). Recombinant serpin A 3n treatment induced cardiac fibrosis during CVB3 myocarditis (P = 0.0008) while decreasing MMP-3 (P = 0.04) and MMP-9 (P = 0.03) levels in the heart. Thus, serpin A 3n was identified as a gene associated with fibrotic cardiac remodeling and progression to DCM in male myocarditis patients and mice.     

Viral myocarditis induced by Coxsackievirus B3 in A.BY/SnJ mice: Analysis of changes in the myocardial proteome

Enteroviral myocarditis displays highly diverse clinical phenotypes ranging from mild dyspnoea or chest pain to cardiogenic shock and death. Despite detailed studies of the virus life cycle in vitro and in vivo, the molecular interplay between host and virus in disease progression is largely unresolved. Murine models of Coxsackievirus B3 (CVB3)‐induced myocarditis well mimic the human disease patterns and can thus be explored to study mechanisms leading from acute to chronic myocarditis. Here, we present a 2‐D gel‐based proteomic survey of the changes in the murine cardiac proteome that occurs following infection with CVB3. In total, 136 distinct proteins were affected. Proteins, which are involved in immunity and defense and protein metabolism/modification displayed pronounced changes in intensity not only during acute but also at later stages of CVB3 myocarditis. Proteins involved in maintenance of cell structure and associated proteins were particularly influenced in the acute phase of myocarditis, whereas reduction of levels of metabolic enzymes was observed in chronic myocarditis. Studies about changes in protein intensities were complemented by an analysis of protein phosphorylation that revealed infection‐associated changes in the phosphorylation of myosin binding protein C, atrial and ventricular isoforms of myosin regulatory light chain 2, desmin, and Rab GDP dissociation inhibitor beta‐2.     

The role of CD8+ T lymphocytes in coxsackievirus B3-induced myocarditis.

Coxsackievirus infections have previously been shown to cause acute or chronic myocarditis in humans, and several mouse models have been established to study the pathology of this disease. Myocardial injury may result from direct viral effects and/or may be immune mediated. To determine the relative roles of these processes in pathogenesis, we have compared coxsackievirus B3 (CVB3) infections of normal and immuno-compromised transgenic knockout (ko) mice. CVB3 was able to infect all strains used (C57BL/6, CD4ko, and beta-microglobulin ko [beta 2Mko]), and following intraperitoneal injection, two disease processes could be distinguished. First, the virus caused early (3 to 7 days postinfection) death in a viral dose-dependent manner. Immunocompetent C57BL/6 mice were highly susceptible (50% lethal dose = 70 PFU), while immunodeficient transgenic ko mice were less susceptible, showing 10- and 180-fold increases in the 50% lethal dose (for CD4ko and beta 2Mko mice, respectively). Second, a histologic examination of surviving CD4ko mice at 7 days postinfection revealed severe myocarditis; the inflammatory infiltrate comprised 40 to 50% macrophages, 30 to 40% NK cells, and 10 to 20% CD8+ T lymphocytes. The infiltration resolved over the following 2 to 3 weeks, with resultant myocardial fibrosis. In vivo depletion of CD8+ T lymphocytes from these CD4ko mice led to a marked reduction in myocarditis and an increase in myocardial virus titers. beta 2Mko mice, which lack antiviral CD8+ T cells, are much less susceptible to early death and to the development of myocarditis. We conclude that our data support a strong immunopathologic component in CVB3-induced disease and implicate both CD4+ and CD8+ T cells. Compared with immunocompetent animals, (i) mice lacking CD4+ T cells (CD4ko) were more resistant to virus challenge, and (ii) mice lacking CD8+ T cells (beta 2Mko and in vivo-depleted CD4ko) showed enhanced survival and a reduced incidence of the later myocarditis. Nevertheless, the picture is complex, since (iii) removal of the CD4+ component, while protecting against early death, greatly magnified the severity of myocarditis, and (iv) removal of the CD8+ cells from CD4ko mice, although protecting against early death and later myocarditis, led to markedly increased virus titers in the heart. These data underscore the complex balance between the costs and benefits of effective antiviral immune responses.     

Complement receptor 1 and 2 deficiency increases coxsackievirus B3-induced myocarditis, dilated cardiomyopathy, and heart failure by increasing macrophages, IL-1beta, and immune complex deposition in the heart

Complement and complement receptors (CR) play a central role in immune defense by initiating the rapid destruction of invading microorganisms, amplifying the innate and adaptive immune responses, and mediating solubilization and clearance of immune complexes. Defects in the expression of C or CR have been associated with loss of tolerance to self proteins and the development of immune complex-mediated autoimmune diseases such as systemic lupus erythematosus. In this study, we examined the role of CR on coxsackievirus B3 (CVB3)-induced myocarditis using mice deficient in CR1/2. We found that CR1/2 deficiency significantly increased acute CVB3 myocarditis and pericardial fibrosis resulting in early progression to dilated cardiomyopathy and heart failure. The increase in inflammation was not due to increased viral replication, which was not significantly altered in the hearts of CR1/2-deficient mice, but was associated with increased numbers of macrophages, IL-1beta levels, and immune complex deposition in the heart. The complement regulatory protein, CR1-related gene/protein Y (Crry), was increased on cardiac macrophage populations, while immature B220(low) B cells were increased in the spleen of CR1/2-deficient mice during acute CVB3-induced myocarditis. These results show that expression of CR1/2 is not necessary for effective clearance of CVB3 infection, but prevents immune-mediated damage to the heart.     

Impairment of immunoproteasome function by β5i/LMP7 subunit deficiency results in severe enterovirus myocarditis

Proteasomes recognize and degrade poly-ubiquitinylated proteins. In infectious disease, cells activated by interferons (IFNs) express three unique catalytic subunits β1i/LMP2, β2i/MECL-1 and β5i/LMP7 forming an alternative proteasome isoform, the immunoproteasome (IP). The in vivo function of IPs in pathogen-induced inflammation is still a matter of controversy. IPs were mainly associated with MHC class I antigen processing. However, recent findings pointed to a more general function of IPs in response to cytokine stress. Here, we report on the role of IPs in acute coxsackievirus B3 (CVB3) myocarditis reflecting one of the most common viral disease entities among young people. Despite identical viral load in both control and IP-deficient mice, IP-deficiency was associated with severe acute heart muscle injury reflected by large foci of inflammatory lesions and severe myocardial tissue damage. Exacerbation of acute heart muscle injury in this host was ascribed to disequilibrium in protein homeostasis in viral heart disease as indicated by the detection of increased proteotoxic stress in cytokine-challenged cardiomyocytes and inflammatory cells from IP-deficient mice. In fact, due to IP-dependent removal of poly-ubiquitinylated protein aggregates in the injured myocardium IPs protected CVB3-challenged mice from oxidant-protein damage. Impaired NFκB activation in IP-deficient cardiomyocytes and inflammatory cells and proteotoxic stress in combination with severe inflammation in CVB3-challenged hearts from IP-deficient mice potentiated apoptotic cell death in this host, thus exacerbating acute tissue damage. Adoptive T cell transfer studies in IP-deficient mice are in agreement with data pointing towards an effective CD8 T cell immune. This study therefore demonstrates that IP formation primarily protects the target organ of CVB3 infection from excessive inflammatory tissue damage in a virus-induced proinflammatory cytokine milieu.     

Aggravation of coxsackievirus, group B, type 3-induced myocarditis and increase in cellular immunity to myocyte antigens in pregnant Balb/c mice and animals treated with progesterone

Male Balb/c mice inoculated with a heart-adapted variant of coxsackievirus, group B, type 3 (CVB3M) develop severe myocarditis characterized by extensive focal lesions of inflammatory cells and necrosis of the myocardium. Females generally develop minimal myocarditis except when infected during the first and third trimesters of pregnancy. Enhanced myocarditis is usually accompanied by elevations in virus concentrations in the heart, virus-specific antibody titers, and lymphocyte mediated cytolytic activity to both uninfected and CVB3M-infected myocytes in vitro. As previously shown in males, T-lymphocyte-depleted pregnant female mice inoculated with the virus do not develop significant myocarditis indicating that immune rather than virus-mediated myocyte damage is important in myocarditis. Progesterone increases during gestation reaching maximum concentrations during the third week when heart disease is most severe. Administration of progesterone to castrated male and female mice prior to virus inoculation resulted in increased virus concentrations, cellular and humoral CVB3M-specific immunity, and myocarditis. Two hypotheses for exacerbation of the disease with elevated progesterone concentrations have been postulated: the hormone either indirectly increases cellular immune responses by enhancing virus replication, or independently enhances both T-cell responses and virus replication.     

An attenuated variant of Coxsackievirus B3 preferentially induces immunoregulatory T cells in vivo.

BALB/c mice infected with the Woodruff variant of coxsackievirus group B type 3 (CVB3W) develop myocarditis mediated by autoimmune cytolytic T lymphocytes. A variant of CVB3W (designated H3-10A1) which infects the myocardium but induces minimal mortality of myocarditis compared to the parental virus was selected. Although H3-10A1 infections stimulate normal CTL responses to CVB3-infected myocytes, the autoimmune response to myocardial antigens is absent. Treatment of H3-10A1-infected mice with 50 mg of cyclophosphamide per kg of body weight, a treatment which preferentially eliminates suppressor cells, allows both the development of the autoimmune cytotoxic T-lymphocyte response and the expression of myocarditis. Similar treatment of CVB3W-infected mice had no effect on the disease. The presence of the immunoregulatory cells was confirmed by adoptive transfer of T lymphocytes from either H3-10A1 or CVB3W-infected donor mice into syngeneic CVB3W-infected recipients. Animals given H3-10A1-immune cells had minimal myocardial inflammation, while animals given CVB3W-immune lymphocytes developed enhanced cardiac disease. Elimination of the T-lymphocyte population from the donor cells prior to transfer abrogated suppression with the H3-10A1-immune population, showing that immunoregulation depended upon T lymphocytes. Both H3-10A1 and CVB3W have cross-reactive epitopes between the adenine translocator protein and the virion which are indicative of antigenic mimicry and may be the basis for the autoimmunity to cardiac antigens. These results suggest that immunoregulatory T cells may be primarily responsible for the nonpathogenicity of the H3-10A1 variant.     

Myosin-induced acute myocarditis is a T cell-mediated disease

The heart is a target organ in several autoimmune diseases, and therefore it is important to understand more about the effector cells involved in immune-mediated mechanisms of myocardial cell death. Because immune T lymphocytes are central to many immune responses, we wanted to study the role of T cells in causing cardiac specific inflammation. We used purified mouse cardiac myosin to cause acute myocarditis in mice. The adoptive transfer of purified T cells from C.B-17 mice with active myocarditis to SCID recipients successfully transferred the disease into SCID hosts. In contrast, transfer of serum with high-titer antimyosin antibodies to SCID hosts did not cause myocarditis. Using mAb to deplete A/J mice of CD4+ T cells, we showed that these mice were protected against the induction of myocarditis. Depletion of CD8+ T cells reduced the severity of inflammation but did not prevent induction of myocarditis. We were also able to prevent the induction of myocarditis using major histocompatibility class II protein-binding, nonimmunogenic, competitor peptides. These blocking studies also indicated that in H-2k mice, myocarditis is an I-Ak-restricted disease, and provided further evidence that CD4+ T cells are critical to the induction of disease. Together, these studies provide direct evidence that myosin-induced myocarditis is a T cell-mediated disease.     

Activation of apoptotic signalling events in human embryonic stem cells upon Coxsackievirus B3 infection

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate to a wide range of specialized cells and hold great promise as models for human development and disease, as well as for drug discovery and cell-replacement therapies. Group B Coxsackie viruses (CVBs) produce acute myocarditis, pancreatitis, non-septic meningitis and encephalitis in neonates, children and young adults. Moreover, CVBs can produce spontaneous miscarriage after early embryo infection. It was reported that hESCs express CVBs receptors and are susceptible to CVB3 infection. Apoptosis is one of the hallmarks of CVBs infection although details regarding CVB3 involvement in the apoptotic processes remain elusive. In order to evaluate the mechanisms of cell death induced by CVB3 in these pluripotent cells, we infected HUES-5 (H5) and WA01 (H1) hESC lines with CVB3. After validating the maintenance of stemness in these hESC lines when grown as confluent monolayers in feeder-free conditions, we analysed several aspects of programmed cell death triggered by CVB3. In all cases, we detected chromatin condensation, DNA fragmentation and caspase-9 and 3 cleavages. Moreover, we observed the presence of cleaved PARP product which was preceded by the appearance of p17, the catalytically active fragment of caspase-3. Mitochondrial function assays revealed a MOI dependent decrease in cell viability at 24 h post-infection (pi). No appreciable modifications in Bcl-2, Bcl-X_L and Bax protein levels were observed upon CVB3 infection during 5–24 h observation period. However, a marked decrease in pro-apoptotic Bad abundance was detected without changes in its mRNA levels. In this study we found that the hESCs are highly susceptible to CVB3 infection and display elevated apoptosis rates, thus emerging as suitable human non-transformed in vitro models to study CVB3-induced apoptosis and resulting relevant to understand CVBs pathogenesis.     

Demonstration of suppressor cells in coxsackievirus group B, type 3 infected female Balb/c mice which prevent myocarditis

Coxsackievirus group B type 3 (CVB3) induces myocarditis in male Balb/c mice but produces little cardiac injury in females. Males develop cytolytic T lymphocytes (CTL) reactive to heart antigens which primarily cause the inflammation and cardiac injury observed in the disease. Infected female mice lack this CTL response because they rapidly produce suppressor cells inhibiting both cellular immunity and cardiac inflammation. Four lines of evidence demonstrate suppressor cells in females. First, females develop myocarditis when treated with low-dose cyclophosphamide under conditions known to preferentially eliminate suppressor cells but not other immune cells. Second, lymphocytes obtained from females at various times after infection prevent myocarditis when adoptively transferred into CVB3-infected males. Virus concentrations in the hearts of males receiving immune female cells and control males were equivalent. Thus protection did not result from accelerated virus elimination in recipient males. Third, CTL from CVB3 infected male mice could induce myocarditis in infected T-lymphocyte depleted but not in intact females suggesting the presence of an inhibitory T cell in the intact animals. Finally, male lymphocytes cultured on heart cell monolayers for 5 days generate significant cytolytic activity to myocyte targets. CTL generation could be inhibited by co-culture of the male cells with immune female lymphocytes. Nonimmune female cells were not inhibitory.     

A20 (TNFAIP3) Alleviates CVB3-Induced Myocarditis via Inhibiting NF-κB Signaling

Background

Viral myocarditis, which is most prevalently caused by Coxsackievirus B3 (CVB3) infection, is a serious clinical condition characterized by cardiac inflammation. However, efficient therapies targeting inflammation are still lacking and much needed. A20, also known as tumor necrosis factor alpha induced protein 3 (TNFAIP3) is a key negative regulator of inflammation. But whether A20 may affect cardiac inflammation during acute viral myocarditis remains to be elucidated. The aim of this study was to investigate the potential protective effect of A20 on CVB3-induced myocarditis.

Methodology/Principal Findings

Mice were intraperitoneally inoculated with CVB3 to establish acute viral myocarditis model. We found that the expression of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and monocyte chemotactic protein-1 (MCP-1) were markedly and persistently increased during the progression of CVB3-induced myocarditis, and positively correlated with the disease severity. Notably, intravenous injection in vivo with adenovirus expressed A20 (Ad-A20) remarkably reduced CVB3-induced pro-inflammatory cytokines production and alleviated the severity of myocarditis. Further, we observed that nuclear factor-kappaB (NF-κB) signaling which mediates inflammatory response was significantly inhibited in CVB3-infected mice with Ad-A20 treatment. Finally, we revealed that A20 was required to inhibit CVB3-induced NF-κB signaling by restricting TNF receptor associated factor 6 (TRAF6) ubiquitylation.

Conclusion/Significance

This study demonstrates the protective role of A20 against CVB3-induced myocarditis, which may provide a new therapeutic strategy for the treatment of viral myocarditis.     

The importance of cardiac MRI as a diagnostic tool in viral myocarditis-induced cardiomyopathy

Myocarditis is an acute or chronic inflammatory disease of the myocardium which can be viral, postinfectious immune or primarily organ-specific autoimmune. Clinical manifestations of acute and chronic myocarditis are extremely varied, ranging from mild to severe. Affected patients may recover or develop (dilated) cardiomyopathy (DCM) with life-threatening symptoms including heart failure, conduction disturbances, arrhythmias, cardiogenic shock or sudden cardiac death. The diagnosis of myocarditis is a challenging process and not only because of a diverse presentation; other problems are limited sensitivity of endomyocardial biopsies (EMB) and overlapping symptoms. Furthermore, the diagnosis is not well defined. However, early diagnosis is mandatory to address specific aetiology-directed therapeutic management in myocarditis that influences patient morbidity and mortality. Currently, EMB remains the only way to confirm the presence of a viral genome and other histopathological findings allowing proper treatment to be implemented in cases of myocarditis. Increased recognition of the role of myocardial inflammatory changes has given rise to interest in noninvasive imaging as a diagnostic tool, especially cardiovascular magnetic resonance imaging (CMR). In this review we discuss the current role of CMR in the evaluation of myocarditis-induced inflammatory cardiomyopathies. (Neth Heart J 2009;17:481–6.)     

IL-22-producing Th22 cells play a protective role in CVB3-induced chronic myocarditis and dilated cardiomyopathy by inhibiting myocardial fibrosis

Background

A new subset of T helper (Th) cells, named IL-22-producing Th22 cells, was identified recently. Th22 cells have been implicated in immunity and inflammation. However, the role of these cells in the progression from acute viral myocarditis (AVMC) to dilated cardiomyopathy (DCM) and myocardial fibrosis remains unknown.

Methods

BALB/c mice were repeatedly i.p. infected with Coxsackie virus B3 (CVB3) to establish models of AVMC, chronic myocarditis and DCM. On week 2, 12 and 24 post initial injection, the percentage of splenic Th22 cells, the levels of plasma IL-22, cardiac IL-22 receptor (IL-22R) expression, and indicators of myocardial fibrosis were measured. Further, mice with AVMC and chronic myocarditis were treated with an anti-IL-22 neutralizing antibody (Ab). The collagen volume fraction (CVF), the percentage of splenic Th22 cells, plasma IL-22 levels, cardiac IL-22R expression and indicators of myocardial fibrosis were then monitored.

Results

Compared to control mice at the same time points, AVMC, chronic myocarditis and DCM mice have higher percentage of splenic Th22 cells, higher plasma IL-22 levels, increased cardiac IL-22R, as well as increased collagen typeI-A1 (COL1-A1), collagen type III-A1 (COL3-A1) and matrix metalloproteinase-9 (MMP9) expression. However, the expression of tissue inhibitor of metalloproteinase-1(TIMP-1) was decreased. Treatment of AVMC and chronic myocarditis mice with an anti-IL-22 Ab decreased the survival rate and exacerbated myocardial fibrosis. The percentage of splenic Th22 cells, plasma IL-22 levels and cardiac IL-22R expression also decreased in anti-IL-22 Ab treatment group as compared to IgG and PBS treated groups of AVMC and chronic myocarditis mice. Moreover, increased expression of COL1-A1, COL3-A1, MMP9 but decreased expression of TIMP-1 were observed in anti-IL-22 Ab mouse group.

Conclusions

Th22 cells play an important role in the pathogenesis of CVB3-induced mouse chronic myocarditis and DCM. IL-22 is a myocardium-protective cytokine by inhibiting myocardial fibrosis. Therefore, Th 22 cells may be considered as potential therapeutic targets for DCM.

     

Myosin autoimmunity is not essential for cardiac inflammation in acute Chagas' disease

Infection with the protozoan parasite Trypanosoma cruzi leads to acute myocarditis that is accompanied by autoimmunity to cardiac myosin in susceptible strains of mice. It has been difficult to determine the contribution of autoimmunity to tissue inflammation, because other inflammatory mechanisms, such as parasite-mediated myocytolysis and parasite-specific immunity, are coincident during active infection. To begin to investigate the contribution of myosin autoimmunity to myocarditis, we selectively inhibited myosin autoimmunity by restoring myosin tolerance via injection of myosin-coupled splenocytes. This tolerization regimen suppressed the strong myosin-specific delayed-type hypersensitivity (DTH) that normally develops in infected mice, although it did not affect myosin-specific Ab production. Suppression of myosin autoimmunity had no effect on myocarditis or cardiac parasitosis. In contrast, myosin tolerization completely abrogated myocarditis in mice immunized with purified myosin, which normally causes severe autoimmune myocarditis. In this case, myosin-specific DTH and Ab production were significantly reduced. We also examined the contribution of T. cruzi-specific immunity to inflammation by injection of T. cruzi-coupled splenocytes before infection. This treatment reduced T. cruzi DTH, although there was no effect on parasite-specific Ab production. Interestingly, cardiac inflammation was decreased, cardiac parasitosis was significantly increased, and mortality occurred earlier in the parasite-tolerized animals. These results indicate that myosin-specific autoimmunity, while a potentially important inflammatory mechanism in acute and chronic T. cruzi infection, is not essential for inflammation in acute disease. They also confirm previous studies showing that parasite-specific cell-mediated immunity is important for myocarditis and survival of T. cruzi infection.     

Variation in susceptibility of Balb/c mice to coxsackievirus group B type 3-induced myocarditis with age

The severity of cardiac lesions in coxsackievirus group B, type 3 (CVB3)-infected Balb/c mice depends upon both the age and the sex of the animal at the time of viral inoculation. Suckling animals (1-3 weeks old) of either sex develop few cardiac lesions. Thereafter, males rapidly demonstrate increasing disease susceptibility peaking at 16-18 weeks old and then decreasing susceptibility from 20 to 40 weeks of age. Female susceptibility increases much more gradually and myocarditis in this sex never reaches maximal levels as seen in males. Increased susceptibility correlates with virus concentrations in the heart and anti-CVB3 titers in the serum. Cardiac injury is dependent on functional T lymphocytes since treatment of the animals with rabbit anti-mouse thymocyte serum abrogates inflammation and myocyte necrosis. Sex-associated steroid hormones influence both virus concentrations and immune responses in mice and are probably responsible for variations in disease susceptibility throughout the animal's life.