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.     

A single-amino-acid polymorphism in reovirus protein μ2 determines repression of interferon signaling and modulates myocarditis

Myocarditis is indicated as the second leading cause of sudden death in young adults. Reovirus induces myocarditis in neonatal mice, providing a tractable model system for investigation of this important disease. Alpha/beta-interferon (IFN-α/β) treatment improves cardiac function and inhibits viral replication in patients with chronic myocarditis, and the host IFN-α/β response is a determinant of reovirus strain-specific differences in induction of myocarditis. Virus-induced IFN-β stimulates a signaling cascade that establishes an antiviral state and further induces IFN-α/β through an amplification loop. Reovirus strain-specific differences in induction of and sensitivity to IFN-α/β are associated with the viral M1, L2, and S2 genes. The reovirus M1 gene-encoded μ2 protein is a strain-specific repressor of IFN-β signaling, providing one possible mechanism for the variation in resistance to IFN and induction of myocarditis between different reovirus strains. We report here that μ2 amino acid 208 determines repression of IFN-β signaling and modulates reovirus induction of IFN-β in cardiac myocytes. Moreover, μ2 amino acid 208 determines reovirus replication, both in initially infected cardiac myocytes and after viral spread, by regulating the IFN-β response. Amino acid 208 of μ2 also influences the cytopathic effect in cardiac myocytes after spread. Finally, μ2 amino acid 208 modulates myocarditis in neonatal mice. Thus, repression of IFN-β signaling mediated by reovirus μ2 amino acid 208 is a determinant of the IFN-β response, viral replication and damage in cardiac myocytes, and myocarditis. These results demonstrate that a single amino acid difference between viruses can dictate virus strain-specific differences in suppression of the host IFN-β response and, consequently, damage to the heart.     

Coxsackievirus group B type 3 infection upregulates expression of monocyte chemoattractant protein 1 in cardiac myocytes, which leads to enhanced migration of mononuclear cells in viral myocarditis

Coxsackievirus group B type 3 (CVB3) is an important cause of viral myocarditis. The infiltration of mononuclear cells into the myocardial tissue is one of the key events in viral myocarditis. Immediately after CVB3 infects the heart, the expression of chemokine(s) by infected myocardial cells may be the first trigger for inflammatory infiltration and immune response. However, it is unknown whether CVB3 can induce the chemokine expression in cardiac myocytes. Monocyte chemoattractant protein 1 (MCP-1) is a potent chemokine that stimulates the migration of mononuclear cells. The objective of the present study was to investigate the effect of CVB3 infection on MCP-1 expression in murine cardiac myocytes and the role of MCP-1 in migration of mononuclear cells in viral myocarditis. Our results showed that the expression of MCP-1 was significantly increased in cardiac myocytes after wild-type CVB3 infection in a time- and dose-dependent manner, which resulted in enhanced migration of mononuclear cells in mice with viral myocarditis. The migration of mononuclear cells was partially abolished by antibodies specific for MCP-1 in vivo and in vitro. Administration of anti-MCP-1 antibody prevented infiltration of mononuclear cells bearing the MCP-1 receptor CCR2 in mice with viral myocarditis. Infection by UV-irradiated CVB3 induced rapid and transient expression of MCP-1 in cardiac myocytes. In conclusion, our results indicate that CVB3 infection stimulates the expression of MCP-1 in myocardial cells, which subsequently leads to migration of mononuclear cells in viral myocarditis.     

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.     

柯萨奇病毒B3型诱导的免疫偏离与心肌炎发病关系的研究

目的 研究2种近交系小鼠在柯萨奇病毒B3型(CVB3)感染后辅助性T细胞(Th)免疫偏离对心肌炎发病的影响。方法 用CVB3腹腔感染BALB/c和C57BL/62种近交系小鼠,感染后7d通过检测小鼠血清肌酸激酶(CK)活性,观察心脏外观变化以及心脏石蜡切片H.E染色观察心脏病理改变,比较2种小鼠心肌炎的发病情况;通过体外感染心肌细胞观察病毒复制情况以及体内心脏组织病毒载量的分析,比较2种小鼠对病毒感染和复制的差异;通过检测感染小鼠细胞因子白细胞介素-4(IL-4)、IL-12和γ干扰素(IFN-γ)的表达,抗CVB3VP1抗体的亚型以及T-bet和Gata-3的表达,比较2种小鼠Th免疫偏离的情况。结果 CVB3在体外和体内都可以感染BALB/c和C57BL/6小鼠心肌细胞,但仅BALB/c小鼠感染后可发生明显的病毒性心肌炎,C57BL/6小鼠则不能;BALB/c小鼠感染后表现为Th1型免疫反应而C57BL/6小鼠则偏向于Th2型免疫反应。结论 CVB3感染2种品系小鼠表现为不同的心肌炎发生率,与其诱导了不同类型的免疫偏离密切相关。     

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.     

Direct gene transfer with IP-10 mutant ameliorates mouse CVB3-induced myocarditis by blunting Th1 immune responses

Background

Myocarditis is an inflammation of the myocardium that often follows the enterovirus infections, with coxsackievirus B3 (CVB3) being the most dominant etiologic agent. We and other groups previously reported that chemokine IP-10 was significantly induced in the heart tissue of CVB3-infected mice and contributed to the migration of massive inflammatory cells into the myocardium, which represents one of the most important mechanisms of viral myocarditis. To evaluate the direct effect of IP-10 on the inflammatory responses in CVB3 myocarditis, herein an IP-10 mutant deprived of chemo-attractant function was introduced into mice to antagonize the endogenous IP-10 activity, and its therapeutic effect on CVB3-induced myocarditis was evaluated.

Methodology/Principal Findings

The depletion mutant pIP-10-AT, with an additional methionine after removal of the 5 N-terminal amino acids, was genetically constructed and intramuscularly injected into BALB/c mice after CVB3 infection. Compared with vector or no treatment, pIP-10-AT treatment had significantly reduced heart/body weight ratio and serum CK-MB level, increased survival rate and improved heart histopathology, suggesting an ameliorated myocarditis. This therapeutic effect was not attributable to an enhanced viral clearance, but to a blunted Th1 immune response, as evidenced by significantly decreased splenic CD4⁺/CD8⁺IFN-γ⁺ T cell percentages and reduced myocardial Th1 cytokine levels.

Conclusion/Significance

Our findings constitute the first preclinical data indicating that interfering in vivo IP-10 activity could ameliorate CVB3 induced myocarditis. This strategy may represent as a new therapeutic approach in treating viral myocarditis.     

Gammadelta T cells promote a Th1 response during coxsackievirus B3 infection in vivo: role of Fas and Fas ligand

Fas/Fas ligand (FasL) interactions regulate disease outcome in coxsackievirus B3 (CVB3)-induced myocarditis. MRL(+/+) mice infected with CVB3 develop severe myocarditis, a dominant CD4(+) Th1 (gamma interferon [IFN-gamma(+)]) response to the virus, and a predominance of gammadelta T cells in the myocardial infiltrates. MRL lpr/lpr and MRL gld/gld mice, which lack normal expression of Fas and express a mutated FasL, respectively, have minimal myocarditis and show a dominant CD4(+) Th2 (interleukin-4 [IL-4(+)]) phenotype to CVB3. Spleen cells from virus-infected wild-type, lpr, and gld animals proliferate equally to virus in vitro. Adoptive transfer of gammadelta T cells from hearts of CVB3-infected MRL(+/+) mice (FasL(+)) into infected MRL gld/gld recipients (FasL(-)/Fas(+)) restores both disease susceptibility and Th1 cell phenotype. However, transfer of these cells into MRL lpr/lpr recipients (FasL(+)/Fas(-)) did not promote myocarditis and the viral response remained Th2 biased. This paralleled the expression of very high surface levels of FasL by myocardial gammadelta T cells, as well as their propensity to selectively lyse Th2 virus-specific CD4(+) T cells. These results demonstrate that Fas/FasL interactions conferred by gammadelta T cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.     

Coxsackievirus B3-induced production of tumor necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes.

Infections by coxsackievirus B3 (CVB3) have previously been shown to cause acute and chronic myocarditis characterized by a heavy mononuclear leukocyte infiltration and myocyte necrosis. Because clinical and experimental evidence suggested that cardiac damage may result from immunologic rather than viral mechanisms, we examined in this study the in vitro interaction of CVB3 with human monocytes. CVB3 was capable of infecting freshly harvested monocytes as revealed by immunofluorescence and release of infectious virus particles. Virus infection did not reduce monocyte viability but, on the contrary, enhanced spreading and adherence. In a dose-dependent manner, CVB3 stimulated the release of cytokines from monocytes. Whereas a potent production of TNF-alpha, IL-1 beta, and IL-6 was dependent on exposure to infectious CVB3, IFN release was also induced by UV-inactivated virus. On a molecular level, CVB3 stimulated cytokine gene expression as shown by a marked TNF-alpha, IL-1 beta, and IL-6 mRNA accumulation. Supernatants of CVB3-infected monocytes displayed cytotoxic activity against Girardi heart cells which could be abrogated by an anti-TNF-alpha antiserum. These data suggest that CVB3-induced cytokine release from monocytes may participate in virus-induced organ damage such as myocarditis, which may either occur by a direct cytotoxicity of cytokines or by activation of cytotoxic lymphocytes.     

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.     

Cardiac injury in myocarditis induced by Coxsackievirus group B, type 3 in Balb/c mice is mediated by Lyt 2 + cytolytic lymphocytes

Male Balb/c mice inoculated with a heart-adapted variant of Coxsackievirus, group B, type 3 (CVB3) develop severe myocarditis 7 days later. The lesions are characterized by mononuclear cell inflammation and myocyte necrosis. Infected T-lymphocyte-deficient mice show either minimal or no cardiac injury, although virus concentrations in the hearts of T-cell-deficient and -sufficient animals are similar. Adoptive transfer of 2 X 10(6) CVB3 immune Thy 1+ cells into CVB3-infected T-cell-deficient mice effectively restored myocarditis to levels observed in intact animals. Similar reconstitution with immune Ig+ cells or serum resulted in only a minimal increase in cardiac injury. To determine whether T-lymphocyte-dependent humoral or cellular immunity was responsible for myocarditis. T lymphocytes were obtained from Balb/c mice 6 days after infection with CVB3, separated into Lyt 1+2- (helper) and Lyt 1-2+ (cytolytic/suppressor) cell populations, and 2 X 10(6) of the enriched helper and cytolytic cells were adoptively transfused into infected T-cell-deficient recipients. Animals receiving the immune Lyt2+ cells developed severe myocarditis, had cytolytic T lymphocytes to both CVB3-infected and uninfected myocytes, but lacked a detectable IgG antibody response. Recipients of the Lyt 1+ cells failed to develop either myocarditis or cytolytic T cells but had normal serum IgG antibody titers to the virus. These results demonstrate that cardiac myocarditis is the product of cellular immune 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.     

Truncated monocyte chemoattractant protein‐1 can alleviate cardiac injury in mice with viral myocarditis via infiltration of mononuclear cells

BALB/c mice inoculated intraperitoneally with coxsackievirus group B type 3 (CVB3) were allocated to five groups; namely, a viral myocarditis group infected with CVB3 alone (control group), an antibody intervention group that received intracardiac anti‐MCP‐1, an antibody intervention control group that received goat IgG, a tMCP‐1 intervention group that received plasmid pVMt expressing tMCP‐1, and a tMCP‐1 intervention control group that received plasmid pVAX1. There was also a normal control group. The ratio of murine heart weight to body weight, pathological score of myocardial tissue, serum creatine kinase‐MB titers and CVB3 loading of myocardial tissue were assessed. The cardiac lesions in mice that received 20, 40 or 60 µg pVMt (P < 0.05) were less severe than those in control mice with untreated viral myocarditis. In addition, fewer mononuclear cells had infiltrated the myocardium of mice who received 40 or 60 µg pVMt intramyocardially (P < 0.01), whereas there was no difference in mononuclear cell infiltration between mice with viral myocarditis and those that received 20 µg pVMt (P > 0.05). There was also no difference between mice that received anti‐MCP‐1 antibody and those that received 40 µg pVMt in ratio of HW/BW, serum CK‐MB titers and pathological score (P > 0.05). This study showed that tMCP‐1 can alleviate cardiac lesions and cardiac injury in mice with viral myocarditis via infiltration of mononuclear cells. Thus, tMCP‐1 may be an alternative to anti‐MCP‐1 antibody treatment of viral myocarditis. Further research is required.     

大黄在体内抗柯萨奇病毒B3的实验研究

柯萨奇病毒 (Ｃｏｘｓａｃｋｉｅｖｉｒｕｓ ,ＣＶ)属小核糖核酸病毒科肠道病毒属 ,根据其对乳鼠的致病能力不同分为Ａ、Ｂ两组 ,Ａ组病毒能够引起乳鼠广泛性肌炎及坏死 ,Ｂ组病毒可致局灶性肌炎。研究表明 ,ＣＶＢ是病毒性心肌炎的主要病因[1,2 ] 。为寻找一种有效的抗ＣＶＢ3 治疗药物 ,笔者通过体外实验发现大黄注射液有抗柯萨奇病毒作用[3] ,并根据中药大黄五脏皆治的理论 ,本研究进一步利用ＣＶＢ3 病毒性心肌炎小鼠模型观察了该药的抗病毒作用。现报告如下 :1　材料与方法1.1　细胞Ｈｅｐ 2细胞由武汉大学典型培养物保藏中心提供。细…     

Antiviral and myocyte protective effects of murine interferon-beta and -{alpha}2 in coxsackievirus B3-induced myocarditis and epicarditis in Balb/c mice

The present study tested the hypothesis that murine (m)IFN-beta or mIFN-alpha(2) can eliminate cardiac viral load and protect cardiomyocytes from injury in animals infected with coxsackievirus B3 (CVB3). CVB3-inoculated male Balb/c mice exhibited signs of illness, including lethargy, progressive weight loss, and death (10% on day 3 and 100% on day 8). Cardiac viral load was high [4,277 +/- 1,009 plaque-forming units and 25 +/- 5 copies CVB3/hypoxanthine guanine phosphoribosyl transferase 1 mRNA] on day 4. The cardiac tissue exhibited severe inflammatory infiltration and myocyte damage with an average myocarditis integrated pathology score of 2.1 +/- 0.2 on day 7. Most of the mice infected with CVB3 also developed epicarditis, and 55% had intraventricular thrombi present. Treatment with mIFN-beta [2.5 to 10 million international units (MIU)/kg] dose-dependently improved the general health status in CVB3-inoculated mice, as evidenced by reduction in weight loss, prevention of death, elimination of cardiac viral load, protection of myocytes from injury, decrease in inflammatory cell infiltration, and attenuation of intraventricular thrombus formation. Treatment with 10 MIU/kg mIFN-alpha(2) resulted in a similar level of efficacy as that induced by 5 MIU/kg mIFN-beta, with the exception that mIFN-alpha(2) did not reduce cardiac CVB3 mRNA. However, mIFN-alpha(2) , but not any dose group of mIFN-beta, significantly attenuated CVB3-induced epicarditis. These data demonstrate antiviral effects for both mIFN-beta and mIFN-alpha(2), which lead to protection of the mice from CVB3-induced myocarditis. However, the potential mechanisms leading to a differential host response for the two isoforms of mIFN remain to be elucidated.     

Blockade of interleukin-17A protects against coxsackievirus B3-induced myocarditis by increasing COX-2/PGE2 production in the heart

The Th17/interleukin (IL)-17 axis controls inflammation and might be important in the pathogenesis of experimental autoimmune myocarditis (EAM) and other autoimmune diseases. However, the mechanism underlying the increased Th17 cell response in coxsackievirus-induced myocarditis remains unclear. This study aimed to elucidate the regulatory mechanisms affected by blocking IL-17A responses in acute virus-induced myocarditis (AVMC) mice. The results showed that IL-17A and COX-2 proteins were significantly increased in the cardiac tissue of acute myocarditis, as were Th17 cells in the spleen. Using anti-mouse IL-17Ab to block IL-17A on day 7 of the viral myocarditis led to decreased expressions of cardiac tumor-necrosis factor alpha, IL-17A and transforming growth factor beta in AVMC mice compared to isotype control mice. COX-2 and prostaglandin E2 proteins were dramatically elevated, followed by marked reductions in CVB3 replication and myocardial injury. These results hint that the Th17/IL-17 axis is intimately associated with viral replication in acute myocarditis via induction of COX-2 and prostaglandin E2.     

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.     

黄芪总黄酮对病毒性心肌炎小鼠心律失常与内质网应激因子的影响

目的：研究黄芪总黄酮（TFA）对病毒性心肌炎小鼠心律失常与内质网应激及缝隙连接蛋白作用,明确TFA抗病毒性心肌炎合并心律失常作用机制。方法：36只雄性Balb/c小鼠分为正常对照组、病毒性心肌炎组和TFA组（n=12）,病毒性心肌炎组腹腔内无菌注射含0.1 ml/d 10-950 TCID柯萨奇B3病毒（CVB3）,注射3 d制备Balb/c小鼠病毒性心肌炎模型,TFA组给予CVB3同时尾静脉注射0.1 ml TFA （20 mg/L）,共7 d。实验结束后心电图检测心律失常发生率后处死小鼠,取心脏行HE染色,观察心肌病理改变,Western blot检测各组小鼠心肌细胞葡萄糖调节蛋白78（GRP78）、内质网应激信号通路因子激活转灵因子4（ATF4）及缝隙连接蛋白（Cx43）表达。结果：与正常组比较,病毒性心肌炎组GRP78与ATF4的表达显著升高（P<0.01）,Cx43表达明显下降（P<0.01）;与病毒性心肌炎组比较,TFA组小鼠心肌细胞GRP78与内质网应激信号通路因子ATF4表达明显减少（P<0.01）,Cx43表达明显增多（P<0.01）。结论：TFA抗心律失常作用可能与缓解内质网应激及增加Cx43表达有关。     

A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3.

Coxsackievirus B3 (CVB3) infections induce myocarditis in humans and mice. Little is known about the molecular characteristics of CVB3 that activate the cellular immunity responsible for cardiac inflammation. Previous experiments have identified an antibody escape mutant (H310A1) of a myocarditic variant of CVB3 (H3) that attenuates the myocarditic potential of the virus in mice in spite of ongoing viral replication in the heart. We have cloned full-length infectious cDNA copies of the viral genome of both the wild-type myocarditic H3 variant of CVB3 and the antibody escape mutant H310A1. Progeny viruses maintained the myocarditic and attenuated myocarditic potential of the parent viruses, H3 and H310A1. The full sequence of the H3 viral cDNA is reported and compared with those of previously published CVB3 variants. Comparison of the full sequences of H3 and H310A1 viruses identified a single nonconserved mutation (A to G) in the P1 polyprotein region at nucleotide 1442 resulting in an asparagine-to-aspartate mutation in amino acid 165 of VP2. This mutation is in a region that corresponds to the puff region of VP2. Nucleotide 1442 of the H3 and H310A1 cDNA copies of the viral genome was mutated to change amino acid 165 of VP2 to aspartate and asparagine, respectively. The presence of asparagine at amino acid 165 of VP2 is associated with the myocarditic phenotype, while an aspartate at the same site reduces the myocarditic potential of the virus. In addition, high-level production of tumor necrosis factor alpha by infected BALB/c monocytes is associated with asparagine at amino acid 165 of VP2 as has been previously demonstrated for the H3 virus. These findings identify potentially important differences between the H3 variant of CVB3 and other previously published CVB3 variants. In addition, the data demonstrate that a point mutation in the puff region of VP2 can markedly alter the ability of CVB3 to induce myocarditis in mice and tumor necrosis factor alpha secretion from infected BALB/c monocytes.     

Antiviral effects of sophoridine against coxsackievirus B3 and its pharmacokinetics in rats

Coxsackievirus B3 (CVB3) is a major pathogen for acute and chronic viral myocarditis. The aim of this study was to investigate the antiviral effects of sophoridine, an alkaloid extracted from Chinese medicinal herb, Sophora flavescens, against CVB3, and the underlying pharmacokinetics. First, we determined the antiviral effects of sophoridine against CVB3 in in vitro (primarily cultured myocardial cells), in vivo (BALB/c mice) and serum pharmacological experiments. Then, we determined the pharmacokinetic behavior in serum samples of SD rats after oral administration by HPLC. Finally, we determined the effects of sophoridine on the production of cytokines in a murine viral myocarditis model by measuring mRNA expression of some important cytokines in hearts of infected BALB/c mice by RT-PCR. We found that sophoridine exhibited obvious antiviral effects both in vitro and in vivo, and serum samples obtained from rats with oral administration of sophoridine reduced the virus titers in infected myocardial cells. The serum concentration profile correlated closely with antiviral activity profile. Moreover, sophoridine significantly enhanced mRNA expression of IL-10 and IFN-gamma, but decreased TNF-alpha mRNA expression. In conclusion, sophoridine possesses antiviral activities against CVB3, by regulating cytokine expression, and it is likely that sophoridine itself, not its metabolites, is mainly responsible for the antiviral activities. Therefore, sophoridine may represent a potential therapeutic agent for viral myocarditis.