CVB3-infected Mice Research Articles

Interleukin-27 ameliorates coxsackievirus-B3-induced viral myocarditis by inhibiting Th17 cells.

BackgroundInterleukin (IL)-27, which has both pro and anti- inflammatory properties, is a new discovered heterodimeric cytokine that belongs to IL-12 family. However, the expression pattern and functional role of IL-27 in viral myocarditis (VMC) has not been investigated.MethodsBALB/c mice were intraperitoneally (i.p) infected with Coxsackie virus B3 (CVB3) for establishing VMC models. Mice were then injected i.p. with Anti-Mouse IL-27 p28Ab or recombinant IL-27 for neutralization and overexpression of IL-27. The survival rates of mice were recorded and the kinetics of IL-27 expression, the frequencies of Th17 cells and the expression of inflammatory cytokine in CVB3-infected mice were determined by ELISA, real-time PCR and flow cytometry.ResultsThe IL-27 expression in heart tissues and serum in coxsackievirus B3 (CVB3)-induced myocarditis mice peaked on day 4 but then rapidly decreased during the late infectious stage of CVB3, high IL-27 levels were negatively correlated with bodyweight loss (r = −0.71, P = 0.021) and myocardial pathological score (r = −0.85, P = 0.0018). Additionally, neutralization of IL-27 with Anti-IL-27 Ab accelerated, whereas systemic administration of recombinant mouse IL-27 ameliorated CVB3-induced myocarditis. The protective role of IL-27 in VMC was reflected by an improved survival rate, increased bodyweights, and reduced pathological scores in Anti-IL-27 group compared with IgG control group. Mechanistic investigations showed that IL-27 inhibited Th17 cells frequencies and IL-17 production, as well as the Th17-related proinflammatory cytokines in heart tissues.ConclusionsOur results demonstrate that that IL-27 effectively protects the myocardium from the pathogenesis of CVB3 induced myocarditis, which may be attributable to reduced Th17 production. IL-27 might serve as a novel therapeutic treatment for VMC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0418-x) contains supplementary material, which is available to authorized users.

Argonaute proteins in cardiac tissue contribute to the heart injury during viral myocarditis.

MicroRNAs (miRNAs) are a group of short, noncoding, regulatory RNA molecules the dysregulation of which contributes to the pathogenesis of myocarditis. Argonaute proteins are essential components of miRNA-induced silencing complex and play important roles during miRNA biogenesis and function. However, the expression pattern of four AGO family members has not yet been detected in the coxsackievirus B3 (CVB3)-induced myocarditis tissue samples. In this study, we detected the expression of four AGOs in the CVB3-infected mouse heart tissues and found that AGO1 and AGO3 up-regulated significantly at 4 and 8h after CVB3 infection. Further in vitro research indicated that up-regulated AGO1 and AGO3 are related to the down-regulated TNFAIP3, which is a negative regulator of NF-κB pathway. Subsequently, we confirmed that TNFAIP3 is a direct target of miR-19a/b, and during CVB3 infection, the expression of miR-19a/b and miR-125a/b is not significantly changed. TNFAIP3 level is mainly reduced by up-regulated AGO1 and AGO3. This research sheds light on the relationship between overexpressed AGO proteins and CVB3-induced myocarditis, and this provides potential therapeutic target for viral myocarditis.

Treg responses are associated with PM2.5-induced exacerbation of viral myocarditis

The adverse cardiovascular events induced by ambient fine particles (PM2.5) are paid more attention in the world. The current study was conducted to explore the mechanisms of T regulatory cells (Treg) responses in PM2.5-induced exacerbation of viral myocarditis. The male BALB/c mice were administered an intratracheal (i.t.) instillation of 10 mg/kg b.w. PM2.5 suspension. Twenty-four hours later, the mice were injected intraperitoneally (i.p.) with 100 μl of coxsackievirus B3 (CVB3) diluted in Eagle's minimal essential medium (EMEM). Seven days after the treatment, serum, splenetic, and cardiac tissues were examined. The results showed that pre-exposure to PM2.5 aggravated the cardiac inflammation in the CVB3-infected mice along with an increase of Treg cells in the spleen. The mRNA expressions of interleukin-6 (IL-6), TNF-α, transforming growth factor-β (TGF-β), and Foxp3 were up-regulated in the PM2.5-pretreated mice than that in the CVB3-treated mice. Similar results were found in the sera. In addition, compared with the CVB3-treated mice, the cardiac protein expression of TGF-β increased in the PM2.5-pretreated mice. These results demonstrated that preexposure to PM2.5 exacerbated virus-induced myocarditis possibly through the depression of the immune response and increase of inflammation in myocardium through the Treg responses.

Heterogeneous Nuclear Ribonucleoprotein M Facilitates Enterovirus Infection

Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host antiviral defenses. Here, using a proteomics-based approach known as TAILS to identify protease-generated neo-N-terminal peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoproteinM(hnRNP M), a nucleocytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNPMis cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in the hearts of CVB3-infected mice. hnRNPMrelocalizes from the nucleus to the cytoplasm during poliovirus infection. Finally, depletion of hnRNPMusing small interfering RNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus internal ribosome entry site translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNPMis a general strategy utilized by picornaviruses to facilitate viral infection. Enteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases, including respiratory ailments, dilated cardiomyopathy, and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we identify hnRNPMas a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNPMand redirects it to a step in the viral life cycle. We propose that cleavage of hnRNPMis a general strategy that picornaviruses use to facilitate infection.

Coxsackievirus B3 induces viral myocarditis by upregulating toll-like receptor 4 expression.

In the present study, we investigated the potential pathogenesis of coxsackievirus B3 (CVB3)-induced viral myocarditis and the promising protective effect of silencing RNA (small interfering RNA, siRNA). One hundred and twenty mice were included in the study, and 30 mice were intraperitoneally inoculated with CVB3 to establish an acute viral myocarditis model. The survival rate was observed for the CVB3-infected mouse model (MOD), and myocardial injury was examined by HE (hematoxylin and eosin) staining assay. Real-time PCR (RT-PCR) and Western blot assay were selected to detect the toll-like receptor 4 (TLR4) expression in myocardial tissues. The TLR4 gene was silenced for the MOD mice, and the effects of this treatment were observed. The results indicate that the expression of TLR4 mRNA and the protein significantly and persistently increased during the progression of CVB3-induced myocarditis. The activities of cardiac enzymes including CK, LDH, AST, and CK-MB were also enhanced in CVB3-induced myocardial tissues. Interestingly, when the TLR4 gene was silenced, the CVB3-induced TLR4 production was significantly decreased and the severity of myocarditis was significantly lessened. In conclusion, CVB3 may induce viral myocarditis by upregulating toll-like receptor 4 expression. The viral myocarditis can be ameliorated by silencing the TLR4 gene in the CVB3 viral myocarditis model, which may be a feasible therapeutic method for treatment of viral myocarditis.

Platelets interact with Coxsackieviruses B and have a critical role in the pathogenesis of virus‐induced myocarditis

To further understand the role of platelets in the pathogenesis of viral infections we explored platelet interaction with Coxsackieviruses B (CVB) 1 and 3. CVB is a group of viruses that cause the majority of human enterovirus-related viral myocarditis; their receptor (CAR) is expressed on the platelet surface and there is a well-characterized CVB3-induced myocarditis murine model. Human platelets were infected with CVB1 and 3 and viruses were detected in pellets and in supernatants. C57BL/6J mice with or without platelet depletion were inoculated with CVB3 and peripheral blood and heart samples collected at different times post-infection. CVB1 and 3 RNA and a capsid protein were detected in infected platelets. Despite the fact that titration assays in Vero cells showed increasing infectivity titers over time, supernatants and pellets from infected platelets showed similar levels, suggesting that platelets were not susceptible to a replicative infectivity cycle. CVB binding was CAR-independent and resulted in P-selectin and phosphatidylserine (PS) exposure. CVB3-infected mice showed a rapid thrombocytopenia that correlated with an increase in platelet PS exposure and platelet-leukocyte aggregates without modification of platelet P-selectin expression or von Willebrand factor levels. Mortality, viremia, heart viral titers and myocarditis were significantly higher in platelet-depleted than normal animals. Type I IFN levels were not changed but IgG levels were lower in infected and platelet-depleted mice. Our data reveal that platelets play a critical role in host survival and immune response against CVB3 infection.

Coxsackievirus B3 infection reduces female mouse fertility.

Previously we demonstrated coxsackievirus B3 (CVB3) infection during early gestation as a cause of pregnancy loss. Here, we investigated the impacts of CVB3 infection on female mouse fertility. Coxsackievirus-adenovirus receptor (CAR) expression and CVB3 replication in the ovary were evaluated by immunohistochemistry or reverse transcription-polymerase chain reaction (RT-PCR). CAR was highly expressed in granulosa cells (GCs) and CVB3 replicated in the ovary. Histological analysis showed a significant increase in the number of atretic follicles in the ovaries of CVB3-infected mice (CVBM). Estrous cycle evaluation demonstrated that a higher number of CVBM were in proestrus compared to mock mice (CVBM vs. mock; 61.5%, 28.5%, respectively). Estradiol concentration in GC culture supernatant and serum were measured by an enzyme-linked immunosorbent assay. Baseline and stimulated levels of estradiol in GC were decreased in CVBM, consistent with significantly reduced serum levels in these animals. In addition, aromatase transcript levels in GCs from CVBM were also decreased by 40% relative to the mock. Bone mineral density evaluated by micro-computed tomography was significantly decreased in the CVBM. Moreover, the fertility rate was also significantly decreased for the CVBM compared to the mock (CVBM vs. mock; 20%, 94.7%, respectively). This study suggests that CVB3 infection could interfere with reproduction by disturbing ovarian function and cyclic changes of the uterus.

Soluble Coxsackievirus B3 3C Protease Inhibitor Prevents Cardiomyopathy in an Experimental Chronic Myocarditis Murine Model

BackgroundCoxsackievirus B3 (CVB3) is a common cause of myocarditis and dilated cardiomyopathy. CVB3 3C protease (3CP) cleaves the viral polyprotein during replication. We tested whether a water soluble 3CP inhibitor (3CPI) had antiviral effects in a chronic myocarditis model. MethodsChronic myocarditis was established using DBA/2 strain mice. Starting on post-infection (p.i) day 3, CVB3-infected mice (n=41) were treated with 3CPI by daily intraperitoneal (i.p.) injection at a concentration of 50μM (1.7mg/kg/day) per day for 3 consecutive days. Additional mice (n=49) were injected with PBS as a control. ResultsThe 5-week survival rate was significantly higher with 3CPI treatment (82.3% versus 47.9%; P<0.05). Organ virus titers at day 3 and 7 and myocardial damage were significantly lower in 3CPI-treated mice. Echocardiography at day 31 indicated strong protection of heart function by 3CPI (FS, 51.2±1.5 versus 26.1±1.5%; P<0.001). Hemodynamic measurements indicated that 3CPI treatment markedly reduced CVB3-induced LV dysfunction on day 31 (dP/dTmax, 5302±352 versus 4103±408mmHg/s, P<0.05; dP/dTmin, −3798±212 versus −2814±206mmHg/s, P<0.01). ConclusionsWater soluble 3CPI was delivered through i.p. injection after CVB3 infection. This agent preserved heart function and decreased organ viral titers and myocardial damage. Soluble 3CPI may be beneficial in the treatment of cardiomyopathy associated with enterovirus infection.

Ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) in host defense against heart failure in a mouse model of virus-induced cardiomyopathy.

Common causative agents in the development of inflammatory cardiomyopathy include cardiotropic viruses such as coxsackievirus B3 (CVB3). Here, we investigated the role of the ubiquitin-like modifier interferon-stimulated gene of 15 kDa (ISG15) in the pathogenesis of viral cardiomyopathy. In CVB3-infected mice, the absence of protein modification with ISG15 was accompanied by a profound exacerbation of myocarditis and by a significant increase in mortality and heart failure. We found that ISG15 in cardiomyocytes contributed significantly to the suppression of viral replication. In the absence of an intact ISG15 system, virus titers were markedly elevated by postinfection day 8, and viral RNA persisted in ISG15(-/-) mice at postinfection day 28. Ablation of the ISG15 protein modification system in CVB3 infection predisposed mice to long-term disease with deposition of collagen fibers, all leading to inflammatory cardiomyopathy. We found that ISG15 acts as part of the intrinsic immunity in cardiomyocytes and detected no significant effects of ISG15 modification on the cellular immune response. ISG15 modification of CVB3 2A protease counterbalanced CVB3-induced cleavage of the host cell eukaryotic initiation factor of translation eIF4G in cardiomyocytes, thereby counterbalancing the shutoff of host cell translation in CVB3 infection. We demonstrate that ISG15 suppressed infectious virus yield in human cardiac myocytes and the induction of ISG15 in patients with viral cardiomyopathy. The ISG15 conjugation system represents a critical innate response mechanism in cardiomyocytes to fight the battle against invading pathogens, limiting inflammatory cardiomyopathy, heart failure, and death. Interference with the ISG15 system might be a novel therapeutic approach in viral cardiomyopathy.

NK-derived IFN-γ/IL-4 triggers the sexually disparate polarization of macrophages in CVB3-induced myocarditis

Coxsackievirus B3 (CVB3) is a common etiology of myocarditis with an increased morbidity and mortality in males. We previously reported that differential polarization of macrophages contributed to sexually dimorphic susceptibility of mice to CVB3-induced myocarditis. However, the underlying kinetics, impetus as well as the molecular mechanism remain unclear. Here, we demonstrated that myocardial macrophages started to polarize at as early as day 5 post CVB3 infection in both genders of BALB/c mice, with M1 phenotype detected in males and M2a phenotype in females, and this trend was further amplified at day 7 when myocarditis reached peak. In addition, we identified that prevailed IFN-γ in males and dominant IL-4 in females were critical myocardial cytokines for the disparate macrophage polarization, which respectively activated JAK1–STAT1 and JAK3–STAT6 pathways. Strikingly, we found that the main source of IFN-γ and IL-4 cytokines in both genders were myocardial infiltrating NK cells, which differentially secreted cytokines in various microenvironments manifested synergistically by sex hormones and CVB3 infection. Consistently, depletion of NK cells significantly impeded the myocardial macrophage polarization in both genders of CVB3-infected mice. Collectively, these data indicated that myocardial NK-derived IFN-γ/IL-4 was critical for the differential polarization of macrophages in CVB3-induced myocarditis via activating JAK1–STAT1 and JAK3–STAT6 pathways respectively. Our study may help understand the mechanism of sexually differential polarization of macrophages and provide clues for the gender bias in CVB3-induced myocarditis.

Coxsackievirus B3 regulates T-cell infiltration into the heart by lymphocyte function-associated antigen-1 activation via the cAMP/Rap1 axis.

Coxsackievirus B3 (CVB3) infection can trigger myocarditis and can ultimately lead to dilated cardiomyopathy. It is known that CVB3-induced T-cell infiltration into cardiac tissues is one of the pathological factors causing cardiomyocyte injury by inflammation. However, the underlying mechanism for this remains unclear. We investigated the mechanism of T-cell infiltration by two types of CVB3: the H3 WT strain and the YYFF attenuated strain. T-cell activation was confirmed by changes in the distribution of lymphocyte function-associated antigen-1 (LFA-1). Finally, we identified which viral gene was responsible for LFA-1 activation. CVB3 could infect and activate T-cells in vivo and in vitro, and activated T-cells were detected in CVB3-infected mouse hearts. LFA-1 expressed on the surface of these T-cells had been activated through the cAMP/Rap1 pathway. Recombinant lentiviruses expressing VP2 of CVB3 could also induce LFA-1 activation via an increase in cAMP, whilst VP2 of YYFF did not. These results indicated that CVB3 infection increased cAMP levels and then activated Rap1 in T-cells. In particular, VP2, among the CVB3 proteins, might be critical for this activation. This VP2-cAMP-Rap1-LFA-1 axis could be a potential therapeutic target for treating CVB3-induced myocarditis.

C-FLIP-Short reduces type I interferon production and increases viremia with coxsackievirus B3.

Cellular FLIP (c-FLIP) is an enzymatically inactive paralogue of caspase-8 and as such can block death receptor-induced apoptosis. However, independent of death receptors, c-FLIP-Long (c-FLIPL) can heterodimerize with and activate caspase-8. This is critical for promoting the growth and survival of T lymphocytes as well as the regulation of the RIG-I helicase pathway for type I interferon production in response to viral infections. Truncated forms of FLIP also exist in mammalian cells (c-FLIPS) and certain viruses (v-FLIP), which lack the C-terminal domain that activates caspase-8. Thus, the ratio of c-FLIPL to these short forms of FLIP may greatly influence the outcome of an immune response. We examined this model in mice transgenically expressing c-FLIPS in T cells during infection with Coxsackievirus B3 (CVB3). In contrast to our earlier findings of reduced myocarditis and mortality with CVB3 infection of c-FLIPL-transgenic mice, c-FLIPS-transgenic mice were highly sensitive to CVB3 infection as manifested by increased cardiac virus titers, myocarditis score, and mortality compared to wild-type C57BL/6 mice. This observation was paralleled by a reduction in serum levels of IL-10 and IFN-α in CVB3-infected c-FLIPS mice. In vitro infection of c-FLIPS T cells with CVB3 confirmed these results. Furthermore, molecular studies revealed that following infection of cells with CVB3, c-FLIPL associates with mitochondrial antiviral signaling protein (MAVS), increases caspase-8 activity and type I IFN production, and reduces viral replication, whereas c-FLIPS promotes the opposite phenotype.

Curcumin Protects Mice From Coxsackievirus B3-Induced Myocarditis by Inhibiting the Phosphatidylinositol 3 kinase/Akt/Nuclear Factor-κB Pathway

Viral myocarditis is an inflammation of the myocardium, and coxsackievirus B3 (CVB3) is one of the most important etiologic agents. Curcumin is an active ingredient of Curcumin longa, which has been used as a traditional Chinese herb for the treatment of various inflammatory diseases. The aim of this study was to explore the therapeutic effect of curcumin on CVB3-induced myocarditis and the underlying mechanism. Our results showed that treatment with curcumin could significantly attenuate CVB3-induced myocarditis, as demonstrated by improved weight loss, increased survival rate, reduced serological level cardiac enzymes, and improved heart histopathology. Of importance, curcumin administration was revealed to significantly reduce the systemic and local myocardial expression of proinflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL) 6, and IL-1β, in the CVB3-infected mice. Further study showed that curcumin treatment significantly inhibited the CVB3-induced activation of nuclear factor-κB (NF-κB), a key transcription factor in the pathogenesis of inflammation, in a phosphatidylinositol 3 kinase (PI3K)/Akt pathway-dependent manner. These data indicate that curcumin has protective effect against CVB3-induced myocarditis by inhibiting PI3K/Akt/NF-κB signaling pathway and thus reducing the inflammatory response.

BMP7 counteracts TGF beta1 induced endothelial-to-mesenchymal transition in viral cardiomyopathy and its potential mechanism

Purpose: Cardiac fibrosis is the most important histological characteristics of viral cardiomyopathy and is associated with poor prognosis and therapeutic interventions. BMP7 is a member of TGF-β superfamily and was reported to counteract the effects of TGF-β1. This study was designed to evaluate whether BMP7 administration could reduce fibrosis induced by CVB3 infection and its potential mechanism. Methods: Viral myocarditis mice model was made and BMP7 was administrated to infected mice. Fourteen days after CVB3 infection, echocardiography, Sirius Red staining and hematoxylin-eosin (HE) were performed to describe the cardiac function and histological characteristics. Colocation of endothelial markers and mesenchymal markers were identified using confocal immunofluorescence staining. Western blot was performed to evaluate the TGF-β1/smad and Wnt/β-catenin signaling pathway. Results: A mice model of CVB3 myocarditis was made and BMP7 was administrated to CVB3-infected mice. Histological data demonstrated that BMP7 administration reduced inflammatory cells accumulation and cardiac fibrosis in response to CVB3 challenge. Echo data described cardiac dysfunction was recovered after BMP7 intervention. Double labeling of endothelial and mesenchymal markers showed BMP7-treated mice had significantly reduced the double-positive cells. Western blot described that TGF-β1/smad and Wnt/β-catenin signaling pathway was involved in this pathogenesis. Conclusions: BMP7 counteracts TGF-β1 induced endothelial-to-mesenchymal transition in viral cardiomyopathy through both TGF-β1/smad and Wnt/β-catenin signaling pathway. The research is supported by grant from the Health Joint-research Program of the National Natural Science Foundation of China and Canadian Institutes of Health Research (81010007) and National Natural Science Foundation of China (31070786)

Preexposure to PM2.5 exacerbates acute viral myocarditis associated with Th17 cell

It is increasingly recognized that exposure to ambient fine particles (PM(2.5)) is a risk factor for the development of cardiovascular events. This study was to explore the link between PM(2.5) exposure and viral myocarditis in the functional mechanism of Th17 cells. Male BALB/c mice were administered an intratracheal (i.t.) instillation of 10 mg/kg b.w. PM(2.5) particles. Twenty-four hours later, the mice were injected intraperitoneally (i.p.) with 100 μl of coxsackievirus B3 (CVB3) diluted in Eagle's minimal essential medium (EMEM). Seven days after the treatment, pulmonary and cardiac tissues were examined. The results showed that preexposure to PM(2.5) increased the cardiac and pulmonary injuries and viral replication in the heart of CVB3-infected mice along with an increase in CD4(+) IL-17(+) cells in the spleen and heart. The mRNA expressions of interleukin-17A (IL-17A), perforin, transforming growth factor-β (TGF-β) and RORγt were up-regulated in PM(2.5)-pretreated mice than that in the virus-treated mice. Additionally, compared to virus-treated mice, the cardiac protein expressions of IL-17A and matrix metalloproteinases-2 (MMP-2) were increased, but interferon-γ (IFN-γ) and metalloproteinases-1 (TIMP-1) were decreased in PM(2.5)-pretreated mice. Interestingly, PM(2.5) caused IFN-γ decreased, whereas CVB3 caused a dramatic increase in IFN-γ. Subsequently, preexposure to PM(2.5) induced a slight increase of IFN-γ in the sera of CVB3-infected mice. These results demonstrated that PM(2.5) exposure exacerbated virus-induced myocarditis possibly through the increase in Th17-mediated viral replication, perforin response and imbalance of MMP-2/TIMP-1. These findings provided supportive evidence for the epidemiological research that ambient particles could increase the occurrence and development of cardiovascular diseases.

Hepatic damage caused by coxsackievirus B3 is dependent on age-related tissue tropisms associated with the coxsackievirus-adenovirus receptor

Coxsackievirus B (CVB) and enterovirus 71 (EV71) are important causes of severe enteroviral diseases in neonates or young children in Taiwan. CVB can cause fulminant hepatitis, myocarditis or meningoencephalitis. This study was designed to explore the role of coxsackievirus-adenovirus receptor (CAR) in the pathogenesis of CVB3-infected hepatocytes via in vitro and mice studies. CVB3 (CVB3/2630) was isolated from liver tissue of a neonate with fulminant hepatitis. Cell lines A549, HeLa, HEp2 and Huh-7 were maintained in Dulbecco's modified Eagle's medium. Mice progeny 1 or 7 days old were used in the experiments. Viremia was noted in 7-day-old ICR mice 2 h after intraperitoneal injection. The highest viral titers were detected in blood, liver and spleen. Histopathological studies of the liver demonstrated polymorphonuclear cell infiltration, massive hepatic cell necrosis and apoptosis. CAR was expressed more in liver than in other tissues. Expression of CAR decreased with mouse age. Anti-CAR monoclonal antibody prevented infection of Huh-7 cells from CVB3. Furthermore, anti-CAR monoclonal antibody pretreatment can reduce mortality and decrease the level of liver enzymes in CVB3-infected mice. These findings indicate that CAR plays an important role in the initiation of CVB infections and is closely associated with hepatotropism and age-specific susceptibility.

Coxsackievirus B3 regulates T cell infiltration by LFA-1 modification through Rap1 activation (P5075)

Abstract Coxsackievirus B3 (CVB3) infection has the potential to induce myocarditis. The following may be the mechanisms of CVB3-induced pathogenesis: viral infection directly causes injuries to cardiomyocytes through viral cytopathic effect and virus-activated T cell infiltration indirectly causes injuries to cardiomyocytes through inflammation. However, the detailed mechanism of T cell infiltration remains unknown. The present study observed that infiltrated T cells could be detected in the hearts of CVB3-infected mice. Meanwhile, attenuated CVB3 (YYFF) did not elicit T cell infiltration in infected mouse hearts. Rap-1, Ras family of small GTPases, is a critical regulator of T cell activation. CVB3 infection was found to activate Rap1 through cAMP induction. The downstream molecules of Rap1, ERK and p-CREB, which are involved in T cell activation and differentiation, were also activated after CVB3 infection. However, this result did not occur in the case of YYFF. This Rap-1 activation leads to an active form of LFA-1 on T cells that can be bound to intracellular adhesion molecule-1 (ICAM-1) on myocytes for T cell infiltration after CVB3 infection. This mechanism of CVB3-induced T cell infiltration may contribute to be a potential therapeutic agent for virus-induced myocarditis. Keywords: Coxsackievirus B3, YYFF, LFA-1

MiR-21 confers resistance against CVB3-induced myocarditis by inhibiting PDCD4-mediated apoptosis

The participation of microRNAs (miRNAs) in cardiovascular diseases suggests them as potential targets for novel preventive and therapeutic strategies. In this study, the key myocardial miRNA, miR-21, was identified in the murine coxsackievirus B3 (CVB3)-induced myocarditis model and its contribution to disease progression was explored. Myocardial microRNA expression changes in CVB3-infected mice were analyzed by real-time PCR and miR-21 was found to be the miRNA whose expression was significantly reduced. Mice were injected with plasmid encoding miR-21 (pMDH-miR-21) at day 1 post CVB3 infection and myocarditis severity was evaluated 7 days post-infection. The underlying mechanism of miR-21 in viral myocarditis was also investigated. Myocardial miR-21 expression was negatively related to viral myocarditis severity. Recovery of miR-21 expression, by injecting with pMDH-miR-21, significantly relieved CVB3-induced myocarditis as shown by increased body weight, reduced myocardial injury, lowered myocarditis score and increased survival rate. Further study showed that miR-21 could protect myocardial apoptosis by specifically inhibiting its target programmed cell death 4 (PDCD4) expression. miR-21 administration efficiently alleviated CVB3-induced myocarditis by repressing PDCD4-mediated apoptosis. Our study not only helps to better understand the pathogenesis of viral myocarditis, but also proves the potential of miR-21 as a novel therapeutic target for treatment of CVB3-induced myocarditis and other apoptosis-mediated cardiovascular diseases.

The flame-retardant BDE-99 dose-dependently affects viral replication in CVB3-infected mice

The flame retardant component 2,2′,4,4′,5-penta-BDE (BDE-99) is found in the environment and in human tissues and fluids. In mice the common human coxsackievirus B3 (CVB3) infection has been shown to change the tissue distribution of BDE-99. We now investigate how CVB3 infection in mice affects liver uptake of 14C at two doses of radiolabelled BDE-99, and whether increased tissue levels are related to changed virus replication and gene expression of the proinflammatory chemokine monocyte chemoattractant protein-1 (MCP-1). Mice were infected on day 0, orally treated either with 200μg or 20mg 14C-BDE-99/kgbw on day 1, and euthanised on day 3. Serum and liver levels of 14C-BDE-99, as well as virus levels and gene expressions of MCP-1 in the liver, were measured. In non-infected mice, there was a dose-dependent uptake of BDE-99 in both liver and serum, and in infected animals the liver BDE-99 levels was further increased. When comparing infected mice exposed to the two BDE-99 doses, the higher BDE dose resulted in increased virus amounts in the liver, and decreased infection-induced expression of MCP-1. Consequently, a high enough dose/tissue concentration of BDE-99 may result in a disturbed mobilisation of immune cells into infected tissues that could explain higher virus titres and a possibly altered clinical course of the disease. Moreover, the fact that CVB3 infection increased the BDE-99 levels in liver but not in serum may impair the risk assessment of polybrominated diphenyl ethers (PBDEs) in subclinical and clinically infected individuals, as serum levels is the common marker of exposure.

Targeting of the Osteoclastogenic RANKL–RANK Axis Prevents Osteoporotic Bone Loss and Soft Tissue Calcification in Coxsackievirus B3–Infected Mice

Bone mineralization is a normal physiological process, whereas ectopic calcification of soft tissues is a pathological process that leads to irreversible tissue damage. We have established a coxsackievirus B3 (CVB3)-infected mouse model that manifests both osteoporosis and ectopic calcification specifically in heart, pancreas, and lung. The CVB3-infected mice showed increased serum concentrations of both cytokines including IL-1β, TNF-α, and the receptor activator of NF-κB ligand (RANKL) that stimulate osteoclast formation and of the osteoclast-derived protein tartrate-resistant acid phosphatase 5b. They exhibited more osteoclasts in bone, with no change in the number of osteoblasts, and a decrease in bone formation and the serum concentration of osteoblast-produced osteocalcin. These results indicate that CVB3-induced osteoporosis is likely due to upregulation of osteoclast formation and function, in addition to decreased osteoblast activity. In addition, the serum in the CVB3-infected mice contained a high inorganic phosphate content, which causes ectopic calcification. RANKL treatment induced an increase in the in vitro cardiac fibroblast calcification by inorganic phosphate via the upregulation of osteogenic BMP2, SPARC, Runx2, Fra-1, and NF-κB signaling. We finally observed that i.p. administration of RANK-Fc, a recombinant antagonist of RANKL, prevented bone loss as well as ectopic calcification in CVB3-infected mice. Thus, our results indicate that RANKL may contribute to both abnormal calcium deposition in soft tissues and calcium depletion in bone. In addition, our animal model should provide a tool for the development of new therapeutic agents for calcium disturbance in soft and hard tissues.