Murine Myocarditis Research Articles

Immunohistochemical characterization of infiltrating mononuclear cells in the rat heart with experimental autoimmune giant cell myocarditis.

The pathogenesis of giant cell myocarditis remains unclear. Subsets of inflammatory infiltrating cells may reflect the pathogenesis and etiology of the disease. Therefore, we examined subsets of infiltrating mononuclear cells in the heart of the rat with experimental giant cell myocarditis. Lewis rats were immunized with cardiac myosin in Freund's complete adjuvant (FCA). Severe myocarditis characterized by congestive heart failure and multinucleated giant cells were elicited. The lesions were composed of predominant mononuclear cells, polymorphonuclear neutrophils and fragments of degenerated myocardial fibres. The subsets of infiltrating mononuclear cells were investigated using MoAbs against rat CD4+ T cell (W3/25), CD8+ T cell (CX8), B cell (OX33) and macrophage (OX42). By serial examination, bound immunoglobulin could only be found on degenerated myocardial fibres. In this model, most infiltrating mononuclear cells were composed of macrophages and CD4+ T cells. The frequencies of macrophages and CD4+ T cells were 73.7% and 13.8%, respectively. CD8+ T cells were scarce and B cells were rare in the lesions. The frequencies of CD8+ T cells and B cells were 4.5% and 0.4%, respectively. The dominance of macrophages and CD4+ T cells was the constant finding among the sites of the lesions and throughout the course of the disease. These characteristic subsets of infiltrating cells were in contrast to those of murine viral myocarditis which were mainly composed of natural killer (NK) cells and CD8+ T cells. Clarifying the subsets of infiltrating cells in myocarditis may contribute to differential diagnosis of myocarditis between viral and autoimmune types. From this study, the pathogenesis of experimental autoimmune giant cell myocarditis seemed to be closely related to CD4+ T cells and macrophages.

Persistence of virus and viral genome in myocardium after coxsackievirus B3-induced murine myocarditis.

Following infection with Coxsackievirus B3 (CVB3), A-strain mice develop ongoing myocarditis that persists after the virus ceases to be cultivatable from heart tissue. We studied the natural history of this virus-induced but apparently autoimmune inflammation by means of in situ hybridization (ISH) and by polymerase chain reaction (PCR). Both ISH and culture allowed detection of virus up to 2 weeks post-infection in virtually all heart tissues. In contrast, PCR revealed the presence of viral genome for a substantially longer period of time, i.e. at least 34 days after CVB3 infection. Similarly, the majority of mice showed myocardial inflammation at this time point. However, the persistence of virus did not correlate with ongoing myocarditis, and vice versa. Most mice with ongoing myocarditis produced heart myosin autoantibodies, most probably as a result of tissue damage. The lack of correlation between presence of ongoing inflammation and persistence of virus supports our previous view that the late phase of CVB3-induced myocarditis is mediated by autoimmunological mechanisms.

Proteasome inhibition attenuates coxsackievirus-induced myocardial damage in mice

Coxsackievirus B3 (CVB3) is one of the most prevalent pathogens of viral myocarditis, which may persist chronically and progress to dilated cardiomyopathy. We previously demonstrated a critical role of the ubiquitin-proteasome system (UPS) in the regulation of coxsackievirus replication in mouse cardiomyocytes. In the present study, we extend our interest to an in vivo animal model to examine the regulation and role of the UPS in CVB3-induced murine myocarditis. Male myocarditis-susceptible A/J mice at age 4-5 wk were randomized to four groups: sham infection + vehicle (n = 10), sham infection + proteasome inhibitor (n = 10), virus + vehicle (n = 20), and virus + proteasome inhibitor (n = 20). Proteasome inhibitor was administered subcutaneously once a day for 3 days. Mice were killed on day 9 after infection, and infected hearts were harvested for Western blot analysis, plaque assay, immunostaining, and histological examination. We showed that CVB3 infection led to an accumulation of ubiquitin conjugates at 9 days after infection. Protein levels of ubiquitin-activating enzyme E1A/E1B, ubiquitin-conjugating enzyme UBCH7, as well as deubiquitinating enzyme UCHL1 were markedly increased in CVB3-infected mice compared with sham infection. However, there was no significant alteration in proteasome activities at 9 days after infection. Immunohistochemical staining revealed that increased expression of E1A/E1B was mainly localized to virus-damaged cells. Finally, we showed that application of a proteasome inhibitor significantly reduced CVB3-induced myocardial damage. This observation reveals a novel mechanism of coxsackieviral pathogenesis, and suggests that the UPS may be an attractive therapeutic target against coxsackievirus-induced myocarditis.

Interleukin-13 Protects Against Experimental Autoimmune Myocarditis by Regulating Macrophage Differentiation

We report here that interleukin (IL)-13 protects BALB/c mice from myocarditis, whether induced by peptide immunization or by viral infection. In contrast to mild disease in IL-4 knockout (KO) BALB/c mice, IL-13 KO BALB/c mice developed severe coxsackievirus B3 (CVB3)-induced autoimmune myocarditis and myocarditogenic peptide-induced experimental autoimmune myocarditis. Such severe disease was characterized by increased cardiac inflammation, increased total intracardiac CD45(+) leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis. Echocardiography revealed that IL-13 KO mice developed severe dilated cardiomyopathy with impaired cardiac function and heart failure. Hearts of IL-13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL-1beta, IL-18, interferon-gamma, transforming growth factor-beta1, and IL-4 as well as histamine. The hallmark of the disease in IL-13 KO mice was the up-regulation of T-cell responses. CD4(+) T cells were increased in IL-13 KO hearts both proportionally and in absolute number. Splenic T cells from IL-13 KO mice were highly activated, and myosin stimulation additionally increased T-cell proliferation. CD4(+)CD25(+)Foxp3(+) regulatory T-cell numbers were decreased in the spleens of IL-13 KO mice. IL-13 deficiency led to decreased levels of alternatively activated CD206(+) and CD204(+) macrophages and increased levels of classically activated macrophages. IL-13 KO mice had increased caspase-1 activation, leading to increased production of both IL-1beta and IL-18. Therefore, IL-13 protects against myocarditis by modulating monocyte/macrophage populations and by regulating their function.

Protective Effects of Carvedilol in Murine Model With the Coxsackievirus B3-Induced Viral Myocarditis

Carvedilol, a nonselective beta-blocker with additional alpha1-adrenergic blocking and antioxidant properties, has been shown to be cardioprotective in experimental myocarditis. However, the antioxidative effects of carvedilol have not been investigated in the setting of acute viral myocarditis. Therefore, this study investigated whether carvedilol protects against viral myocarditis primarily by its antioxidant and/or antiinflammatory properties. In a coxsackievirus B3 murine myocarditis model (Balb/c), effects of carvedilol and metoprolol on myocardial histopathological changes, cytokine levels, virus titers, malondialdehyde (MDA), and superoxide dismutase (SOD) contents were studied. Carvedilol markedly attenuated myocardial lesions and increased interferon-gamma and interleukin-12 production (cytokines) on day 7 with concomitant reduction of myocardial virus replication. In addition, only carvedilol decreased the content of MDA and increased the content of SOD on day 14. Metoprolol as well as bunazosin (a higly selective alpha1-adrenergic blocking agent) had no significant effects in this model. The results indicate that the superior protection of carvedilol in this model is probably the result of its antioxidative effects and the upregulation of antiinflammatory cytokines.

QiHong Prevents Death in Coxsackievirus B3–Induced Murine Myocarditis Through Inhibition of Virus Attachment and Penetration

Viral myocarditis affects about 5% to 20% of the population. So far, there are not many effective antiviral treatments available. QiHong, the combination of the extracts from Astragali (Huangqi), Rhadiola rosea (Hongjingtian), and Sophora flavescens (Kushen), was developed based on laboratory research. The aim of this study was to investigate the effect and mechanism of QiHong on coxsackievirus B3 (CVB3)-induced myocarditis. The antiviral activity of QiHong in vitro was evaluated on HeLa and Vero cells infected by CVB3. Ribavirin was chosen as positive control. Our results showed that QiHong possessed potent antiviral effects on CVB3 by sodium 3'-[1-(phenylamino-carbonyl)-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid and plaque-forming assay (50% inhibitory concentrations [IC50] were 7.16 +/- 0.8 microg/ml and 2.63 +/- 0.5 microg/ml, respectively). The 50% cytotoxicity concentration (CC50) was 16-fold higher in QiHong-treated cells than in ribavirin-treated cells. Time course studies demonstrated that the antiviral effect of QiHong was mainly found during 0-4 hrs of infection, and it blocked the attachment and penetration of CVB3 into cells. In vivo 4-week-old male Balb/C mice were used and inoculated intraperitoneally with CVB3 suspension or normal saline. At 48 hrs after inoculation, the infected mice were gavaged with QiHong or ribavirin. On Day 6, myocardial virus titers were significantly lower in the QiHong-treated group than in the viral-infected groups. On Day 14, QiHong significantly ameliorated CVB3-induced myocardium necrosis; on Day 28, QiHong treatment increased survival rate 4-fold compared with CVB3-infected controls (64% vs. 16%; P < 0.05). The results showed that QiHong is a very promising potent antiviral agent with a highly significant favorable effect on survival and pathologic changes in CVB3-induced myocarditis with less toxicity than ribavirin. The antiviral activity of QiHong is at least partially due to an inhibitory effect on virus attachment and penetration.

Amelioration of Coxsackievirus B3-Mediated Myocarditis by Inhibition of Tissue Inhibitors of Matrix Metalloproteinase-1

Coxsackievirus B3 (CVB3) is a major cause of acute myocarditis, a serious condition that is refractory to treatment. Myocardial damage results in tissue remodeling that, if too extensive, may contribute to disease. Remodeling is achieved by extracellular proteolysis mediated by the matrix metalloproteinases (MMPs), and MMP activity is counterbalanced by tissue inhibitors of MMPs (TIMPs). We show herein that TIMP-1 expression is induced in the myocardium by CVB3 infection. Surprisingly, TIMP-1 knockout mice exhibited a profound attenuation of myocarditis, with increased survival. The amelioration of disease in TIMP-1 knockout mice was not attributable to either an altered T-cell response to the virus or to reduced viral replication. These data led us to propose a novel function for TIMP-1: its highly localized up-regulation might arrest the MMP-dependent migration of inflammatory cells at sites of infection, thereby anatomically focusing the adaptive immune response. The benefits of TIMP-1 blockade in treating viral myocarditis were confirmed by administering, to wild-type animals, TIMP-1-specific siRNA or polyclonal antisera, both of which diminished CVB3-induced myocarditis. These unexpected findings indicate that increased TIMP-1 expression exacerbates, rather than ameliorates, CVB3-induced myocarditis and, thus, that TIMP-1 may represent a target for the treatment of virus-induced heart disease.

Basal Expression Levels of IFNAR and Jak-STAT Components Are Determinants of Cell-Type-Specific Differences in Cardiac Antiviral Responses

Viral myocarditis is an important human disease, and reovirus-induced murine myocarditis provides an excellent model system for study. Cardiac myocytes, like neurons in the central nervous system, are not replenished, yet there is no cardiac protective equivalent to the blood-brain barrier. Thus, cardiac myocytes may have evolved a unique antiviral response relative to readily replenished cell types, such as cardiac fibroblasts. Our previous comparisons of these two cell types revealed a conundrum: reovirus T3D induces more beta-interferon (IFN-beta) mRNA in cardiac myocytes, yet there is a greater induction of IFN-stimulated genes (ISGs) in cardiac fibroblasts. Here, we investigated possible underlying molecular determinants. We found that greater basal expression of IFN-beta in cardiac myocytes results in greater basal activated nuclear STAT1 and STAT2 and greater basal ISG mRNA expression and provides greater basal antiviral protection relative to cardiac fibroblasts. Conversely, cardiac fibroblasts express greater basal IFN-alpha/beta receptor 1 (IFNAR1) and greater basal cytoplasmic Jak1, Tyk2, STAT2, and IRF9, leading to a greater increase in reovirus T3D- or IFN-induced nuclear activated STAT1 and STAT2 and greater induction of ISGs for a greater IFN-induced antiviral protection relative to cardiac myocytes. Our results suggest that high basal IFN-beta expression in cardiac myocytes prearms this vulnerable, nonreplenishable cell type, while high basal expression of IFNAR1 and latent Jak-STAT components in adjacent cardiac fibroblasts renders these cells more responsive to IFN and prevents them from inadvertently serving as a reservoir for viral replication and spread to cardiac myocytes. These studies provide the first indication of an integrated network of cell-type-specific innate immune components for organ protection.

Abstract 1407: Interferon Regulatory Factor-9 (irf-9) Mediates Short Term Host Protection, But Promotes Long Term Immune Injury In Evolution Of Myocarditis Leading To Dilated Cardiomyopathy

INTRODUCTION: Evolution of viral myocarditis to dilated cardiomyopathy (DCM) represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. However, the influence of innate immunity in general and IRF-9 in particular on acquired immunity and DCM is unknown. HYPOTHESIS: IRF-9 signaling provides immediate host protection through interferon production, but stimulates acquired immunity leading to DCM in a coxsackievirus murine myocarditis model. METHODS: Interferon-regulatory factor 9 (IRF-9) homozygous knockout mice were generated, and show impaired type I interferon production. Wild-type (WT, n=51) and IRF-9 −/− (n=124) littermates were inoculated with 10^2 p.f.u. of coxsackievirus B3 as previously described. Survival, viral titers, cardiac hypertrophy, inflammation and fibrosis were evaluated on days 0, 4, 7, 10, 14, 28 and 42. Splenocyte subpopulations were cell sorted and quantitated by FACS. RESULTS: IRF9−/− mice showed dramatically increased mortality compared to the wild-type littermates (0% WT vs 72% IRF-9 −/− on day 14, P&lt;0.0001). On day 42, there was less cardiac hypertrophy and inflammation in IRF-9 −/− mice compared to WT controls (p&lt;0.05). There was no difference in fibrosis. The mature T-lymphocyte population, defined as CD4 or CD8 single positive, was statistically identical in the two populations up until and including day 28 post-infection. However on day 42 there was a dramatic increase in the number of cytotoxic and helper T-Cells in the wild-type mice that was not observed in the IRF-9 −/− spleens (p&lt;0.05). CONCLUSIONS: These data suggest a novel dual role of IRF-9 in not only regulating interferon in acute stage of viral infection in myocarditis, but also late acquired immunity activation, including CD4/8 populations, contributing to the development of chronic cardiomyopathy.

Recombinant coxsackievirus vectors for prevention and therapy of virus-induced heart disease

Cardiovascular diseases are the major cause of human death and have been linked to many different risk factors. Among them, coxsackievirus B3 (CVB3), as a member of the enterovirus group, is one of the most important infectious agents of virus-induced myocarditis. Despite the fact that the molecular structure of this pathogen has been characterized very precisely, there is no virus-specific preventive or therapeutic procedure against CVB3-induced heart disease in clinical use today. A promising approach to prevent CVB3-caused myocarditis represents the mutation of the viral genome in a way that coding sequences of cytokines are integrated into the viral RNA. Recombinant cytokine-expressing CVB3 variants were established to increase the local cytokine concentration and to modulate TH1-/TH2-specific immune responses. Especially protective against CVB3-induced murine myocarditis is the application of an interferon- γ (IFN- γ)-expressing recombinant coxsackievirus variant. The local and simultaneous expression of an immuno-relevant cytokine by the virus itself induces a strong and long-lasting immune response which protects laboratory animals against lethal infections.

Interferon-γ Induces Chronic Active Myocarditis and Cardiomyopathy in Transgenic Mice

Chronic heart failure is associated with an activation of the immune system characterized among other factors by the cardiac synthesis and serum expression of proinflammatory cytokines. There is unequivocal clinical and experimental evidence that the cytokine tumor necrosis factor-alpha is involved in the development of chronic heart failure, but a putative cardiotoxic potential of the proinflammatory cytokine interferon (IFN)-gamma remains primarily unknown. To investigate this issue we analyzed the cardiac phenotype of SAP-IFN-gamma transgenic mice, which constitutively express IFN-gamma in their livers and hence exhibit high circulating serum levels of this cytokine. SAP-IFN-gamma mice spontaneously developed chronic active myocarditis, characterized by the infiltration of not only CD4(+) and CD8(+) T cells but also Mac2(+) (galectin 3(+)) macrophages and CD11c(+) dendritic cells, eventually culminating in cardiomyopathy. Echocardiographic analyses exhibited a left ventricular dilation and impaired systolic function induced by IFN-gamma overexpression. IFN-gamma-mediated cardiotoxicity was associated with high-level cardiac transcription of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-12 and the macrophage-attracting chemokines MCP1 and MIP1-alpha. Myotoxic IFN-gamma effects could not be detected in smooth or striated muscle tissue, suggesting cardiomyocellular specificity of the toxic IFN-gamma effect. The precise mechanism of IFN-gamma cardiotoxicity remains to be elucidated.

Interferon Regulatory Factor-9 (IRF-9) Mediates Short Term Host Protection, but Promotes Long Term Immune Injury in Evolution of Myocarditis Leading to Dilated Cardiomyopathy

Introduction: Evolution of viral myocarditis to dilated cardiomyopathy (DCM) represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. However, the influence of innate immunity in general and IRF-9 in particular on acquired immunity and DCM is unknown. Hypothesis: IRF-9 signaling provides immediate host protection through interferon production, but stimulates acquired immunity leading to DCM in a coxsackievirus murine myocarditis model.

Roles of programmed death-1 (PD-1)/PD-1 ligands pathway in the development of murine acute myocarditis caused by coxsackievirus B3

This study was designed to investigate the roles of programmed death-1 (PD-1) and PD-1 ligands (PD-L) in the development of murine acute myocarditis caused by Coxsackievirus B3. PD-1/PD-L belong to the CD28/B7 superfamily, and the PD-1/PD-L pathway is known to transduce a negative immunoregulatory signal that antagonizes the T-cell receptor-CD28 signal and inhibits T-cell activation. We first analyzed the expression of PD-L1/PD-L2 on cardiac myocytes in vivo and in vitro. Second, we examined the effects of in vivo treatment with an anti-PD-1, PD-L1, or PD-L2 monoclonal antibodies on the development of myocardial inflammation in C3H/He mice infected with Coxsackievirus B3. Third, to investigate the effects of anti-PD-1 monoclonal antibody treatment on the activation of the infiltrating cells, we examined the expression of interleukin (IL)-2, interferon (IFN)-gamma, CD40 ligand (CD40L), Fas ligand (FasL), and perforin as activation markers in mouse hearts by a semiquantitative PCR method. PD-L1 was markedly induced on cardiac myocytes with acute myocarditis. In vivo anti-PD-1 or -PD-L1 blocking monoclonal antibody treatment increased the myocardial inflammation whereas anti-PD-1 stimulating monoclonal antibody treatment decreased the myocardial inflammation, and anti-PD-L2 monoclonal antibody treatment had no effect. Anti-PD-1 monoclonal antibody treatment significantly increased the expression of IFN-gamma, FasL, CD40L, perforin, and Coxsackievirus B3 genomes in myocardial tissue. Our findings strongly suggest that the PD-1/PD-L1 pathway played a pivotal role in suppressing myocardial inflammation and raise the possibility of immunotherapy by stimulating the PD-1/PD-L1 pathway to prevent myocardial damage in viral myocarditis.

Effect of astragaloside on cardiomyocyte apoptosis in murine coxsackievirus B3 myocarditis

Astragaloside is the major component of Astragalus membranaceus, one of the Chinese medical herbs, and has several pharmacological actions on cardiovascular system, including positive inotropic, anti-arrhythmia and anti-oxidant activities. We have investigated the effect of astragaloside on cardiomyocyte apoptosis and expression of apoptosis-associated genes in mice with coxsackievirus B3 (CVB3)-induced myocarditis, the former of which has been detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay and the latter determined by using a focused microarray. Results showed that cardiomyocyte apoptosis and expression of various apoptosis associated genes are inhibited after treatment with astragaloside. The anti-apoptotic activity of astragaloside may contribute to the improvement of clinical outcomes in treating myocarditis with pharmaceutics of Astragalus membranaceus.

Immunological Relationships between Heart Tissue and Non-Cardiac Organs in Murine Eosinophilc and Lymphocytic Myocarditis

The Interleukin-4 signal transducer and activator of transcription 6 (IL-4-STAT6) signaling pathway plays a pivotal role in regulation of gene transcription. We have previously identified a defective STAT6 activational phenotype in response to IL-4 in patients from our familial Inflammatory Bowel Disease registry. This has been termed Stat6null and Stat6high is the normal phenotype. The purpose of this study was to investigate the defect in Stat6 activation in Stat6null cells.Stat6null and Stat6high Epstein Barr virus transformed cell lines were stimulated with 10 ng/mL of IL-4 for 0, 10, 30, or 60 min and cytoplasmic and nuclear proteins harvested. Western blot for STAT6, phosphorylated STAT6 (pSTAT6), Janus Kinase (Jak)1 and Jak3 was performed. Cells were also cultured for 48 h and interferon gamma (IFNγ) measured in the supernatant. Additional cells were cultured with 20 ng/mL of IFNγ for 90 min or 5 ug of antibody to IFNγ for 48 h, and then stimulated with IL-4.There were no differences in cytoplasmic STAT6 in Stat6null versus Stat6high cells. In Stat6high cells, STAT6 was rapidly phosphorylated and translocated to the nucleus. In Stat6null cells there was minimal phosphorylation and translocation of pSTAT6 to the nucleus. Spontaneous secretion of IFNγ by Stat6null cells was significantly higher than Stat6null cells. Addition of IFNγ decreased pSTAT6 in Stat6high cells to Stat6null levels while blocking IFNγ increased baseline pSTAT6 in Stat6null cells to levels similar to Stat6high cells.This suggests that the spontaneously produced IFNγ in the Stat6null cell lines suppresses STAT6 function and creates the Stat6null phenotype.

Combination Therapy of MRL-1237 and PCI-186 in Murine Coxsackievirus B3 Myocarditis

Objective: MRL-1237, a new anti-enterovirus drug, showed beneficial effects to acute viral myocarditis. MCI-186 (edaravone), a novel free radical scavenger, protected against acute experimental autoimmune myocarditis in rats. We evaluated combination therapy of MRL-1237 and MCI-186 in murine coxsackievirus B3 myocarditis.

Mechanism of Inflammation in Murine Eosinophilic Myocarditis Produced by Adoptive Transfer with Ovalbumin Challenge

Background: Interleukin (IL)-5, RANTES and CC chemokine receptor 3 (CCR3) are essential for induction of eosinophil recruitment in organs, but the precise pathogenesis of eosinophilic myocarditis is still unclear. We investigated the relationships between these cytokines and receptors in the development of inflammation in murine myocarditis produced by adoptive transfer, with reference to eosinophil infiltration and signal transduction. Methods: The splenocytes from male donor DBA/2 mice were separated after ovalbumin (OVA) sensitization. These cells had a CD4/CD8 ratio of approximately 3.0. Cells (2.0 × 10⁷) were individually transfused to recipient adoptive male DBA/2 mice, and OVA challenge was performed serially. The heart and spleen of the recipient were analyzed to determine the kinetics of IL-5, RANTES, CCR3 and eosinophil production with simultaneous determination of Janus kinase 3 (JAK3) mRNA. Results: Approximately 85% of recipient mice developed myocarditis; 35% had recognizable cell infiltration in the left ventricular endocardium, an effect which was absent in control mice. Eosinophilic myocarditis was usually associated with animals having several degenerative changes in myocardial cells, and IL-5, RANTES and CCR3 expressions were usually present in these eosinophils (p < 0.05). CCR3 and JAK3 mRNAs were detected in the spleens and hearts of recipient animals providing histological evidence for kinetics related to eosinophil infiltration. Conclusion: The murine model of adoptive transferred myocarditis is suitable for studying the mechanism of eosinophilic myocarditis. A unique pathogenesis of this disorder may be controlled by the synergism of CD4 dominancy in the donor and JAK-STAT signaling in the recipient, which may cause recruitment of eosinophils into heart lesions.

Immunomodulation by interleukin-4 suppresses matrix metalloproteinases and improves cardiac function in murine myocarditis

Immune response is critically involved in determining the course of viral myocarditis and immunomodulation. Different cytokines may have either deleterious or protective effects. Following acute Coxsackievirus B3 infection, intramyocardial inflammation is associated with altered myocardial matrix metalloproteinase (MMP) expression and left ventricular dysfunction. In this study, we evaluated the effect of exogenous interleukin-4 treatment on myocardial inflammation, MMPs and left ventricular function in Coxsackievirus B3-induced acute murine myocarditis. Eight-week-old inbred male BALB/c (H-2 d) mice (The Jackson Laboratory, Bar Harbor, Maine, USA) were used. Myocardial inflammation was measured by immunohistochemical detection of CD3 +-, CD8a +-T-lymphocytes, and CD11b + macrophages. In situ hybridization was used to detect enteroviral genome in the myocardium. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was employed to detect cytokine and MMP mRNA. MMP activity was quantified by zymography analysis. Detection of myocytolysis was performed by Luxol fast blue staining. In the early acute phase, in comparison to infected mice without treatment, interleukin-4 administration (200 ng daily) reduced intramyocardial inflammation (CD3 + lymphocytes: 55.3 ± 7.0 vs. 72.1 ± 13.7 cells/mm 2, P < 0.05; CD8a + lymphocytes: 31.7 ± 3.6 vs. 64.2 ±7.7 cells/mm 2, P < 0.05; CD11b + macrophages: 5.1 ± 2.3 vs. 13.2 ± 2.5 cells/mm 2, P < 0.05). It also down-regulated interleukin-2 (IL) (1.7-fold, P < 0.001) but increased transforming growth factor-β 1 (TGF) (1.5-fold, P < 0.001) and IL-4 (1.4-fold, P < 0.001). IL-4 suppressed MMP-2/-3/-9 transcription and activity. These biochemical alterations were accompanied by a significant improvement of left ventricular function as assessed by Milar tip catheter (left ventricular endsystolic pressure, 1.3-fold, P < 0.01; d P / d t max, 1.5-fold, P < 0.01). Immunomodulation by exogenous IL-4 treatment may lead to an anti-inflammatory effect with the inhibition of Th 1 cell phenotypic response, which may further mediate the down-regulation of MMPs. A significant suppression of MMPs may mainly contribute to an improvement of left ventricular dysfunction in acute murine CVB3-induced myocarditis.

CD1d expression on hemopoietic cells promotes CD4+ Th1 response in coxsackievirus B3 induced myocarditis

Coxsackievirus B3 induced murine myocarditis depends upon CD1d expression and upon a population of CD1d-restricted Vγ4+ T cells. Infection upregulates CD1d expression in CD4+ T cells. Bone marrow chimeras were made between BALB/c and BALB/c CD1d−/− mice and showed that CD1d expression in either hemopoietic and non-hemopoietic cells induces myocarditis, although CD1d expression on hemopoietic cells was more effective in increasing Vγ4+ cell numbers and activation, and CD4+ IFNγ+ cell response than CD1d expression on non-hemopoietic cells. Co-culture of enriched CD4+ cells from infected CD1d−/− and BALB/c mice with Vγ4+ T cells demonstrated that the Vγ4+ cells bias the CD4+ cell response to the Th1 phenotype through CD1d. Anti-CD1d antibody effectively blocked promotion of IFNγ expression by the CD4+ cell population. These results show that Vγ4+ cells modulate developing adaptive immunity through recognition of CD1d on CD4+ T cells, and that this interaction, more than Vγ4+ cell interaction with infected cardiocytes, determines pathogenicity.

TNFR-Fc fusion protein expressed by in vivo electroporation improves survival rates and myocardial injury in coxsackievirus induced murine myocarditis

Tumor necrosis factor-α (TNF-α) is one of the major cytokines that modulate the immune response in viral myocarditis, but its role has not yet been thoroughly evaluated. We antagonized TNF-α using the expressed soluble p75 TNF receptor linked to the Fc portion of the human IgG1 gene (sTNFR:Fc) by in vivo electroporation, and evaluated its effects on experimental coxsackieviral B3 (CVB3) myocarditis. A plasmid DNA encoding sTNFR:Fc (15 μg/mouse) was injected into the gastrocnemius muscles of Balb/C male mice followed by electroporation (day −1). Control mice were injected with an empty vector. One day after electroporation, mice were infected with CVB3 (day 0). Serum levels of sTNFR:Fc increased from day 2 and peaked at day 5 following electroporation. The heart virus titers of sTNFR:Fc mice were higher than those of controls at day 3. However, subsequent to day 12, the survival rates of the sTNFR:Fc mice were significantly higher than those of the controls (36% versus 0% at day 27, P < 0.01). Histopathological examination indicated that inflammation and myocardial fibrosis were significantly decreased in sTNFR:Fc mice at day 12. The expressed sTNFR:Fc could modulate the inflammatory process during the post-viremic phase of viral myocarditis.