Dapagliflozin Alleviates Myocardial Inflammation by Regulating the Macrophage Polarization and Stat3-related Pathways in Coxsackie Virus B3-induced Acute Viral Myocarditis

Viral myocarditis (VMC), which is most prevalently caused by Coxsackievirus B3 (CVB3) infection, is a serious clinical condition characterized by cardiac inflammation. Dapagliflozin, a kind of sodium glucose co-transporters 2(SGLT-2) inhibitor, exhibited protective effects on plenty of inflammatory diseases, while its effect on viral myocarditis has not been studied. Recently we found the protective effect of dapagliflozin on VMC. After CVB3 infection, dapagliflozin were given orally to Balb/c male mice for 8 days and then the severity of myocarditis was assessed. Our results indicated that dapagliflozin significantly alleviated the severity of viral myocarditis, elevated the survival rate, and ameliorated cardiac function. Besides, dapagliflozin can decrease the level of proinflammatory cytokines included IL-1β, IL-6, TNF-α. Furthermore, dapagliflozin can inhibit macrophages differentiate to classically activated macrophages (M1) in cardiac tissue and activate the Stat3 signal pathway which is reported to promote polarization of the alternatively activated macrophage (M2). In conclusion, our study demonstrates that dapagliflozin alleviates myocardial inflammation by regulating the macrophage polarization and Stat3-related pathways in coxsackie virus B3-induced acute viral myocarditis.

Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors. Infection with enteroviruses, including poliovirus and coxsackievirus, such as coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALS due to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathological phenotypes closely resemble ALS. However, a causal relationship has not yet been affirmed. Methods Wild-type C57BL/6J and G85R mutant superoxide dismutase 1 (SOD1G85R) ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin (a broad-spectrum anti-RNA viral drug) was given subcutaneously during the acute or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting at week 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10, week 20 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression. Results We found that sublethal infection (mimicking chronic infection) of SOD1G85R ALS mice with CVB3 resulted in early onset and progressive motor dysfunction, and shortened lifespan, while similar viral infection in C57BL/6J, the background strain of SOD1G85R mice, did not significantly affect motor function and mortality as compared to mock infection within the timeframe of the current study (60 weeks PI). Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies (i.e., TAR DNA-binding protein 43 (TDP-43) pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, we discovered that early (day 1) but not late (day 15) administration of ribavirin could rescue ALS-like neuropathology and symptoms induced by CVB3 infection. Conclusions Our study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.

Identification of Immunogenic Epitopes That Permit the Detection of Antigen-Specific T Cell Reponses to Coxsackievirus B3

Background and Hypothesis:Coxsackievirus B3 (CVB3) is a non-enveloped RNA virus from the Picornaviridae family and is a primary cause of viral myocarditis in the United States. Approximately 5% of all symptomatic CVB3 infections are fatal. Therefore, there is a need to identify the mechanism(s) that regulate a protective immune response to CVB3. However, viral epitopes that stimulate T cell responses to CVB3 remain poorly characterized. To this end, we used a mouse model of CVB3 infection to identify the viral immunogenic CD8 T cell epitopes. We hypothesized that isolated antigen-experienced CD8 T cells from infected mice would be stimulated in the presence of predicted viral epitopes, confirming CVB3-specific T cells. Experimental Design: To identify novel CD8 T cell epitopes, predicted 9-mer MHC binding peptides from the CVB3-Nancy polyprotein were identified using the Immune Epitope Database (IEDB) analysis resource consensus tool. The top ten predicted peptides were synthesized for our assays. Splenocytes from CVB3-infected male and female IFNAR -/- mice were stimulated with each peptide in the presence of brefeldin A for 6 hours at 37˚C. Following stimulation, cells were surfaced stained with antibodies specific for antigen-experienced CD8 T cells. Next, we performed intracellular staining for IFN-gamma. Cells were analyzed using flow cytometry. Candidate epitopes were identified as having results ≥2 standard deviations over the control. Results: Thus far, our analysis has revealed responses to three novel CD8 T cell epitopes within the peptide library, including the viral epitopes within VP1 protein and the RNA-dependent RNA polymerase. Conclusion and Potential ImpactOverall, these data provide an advancement in CVB3 immunology. Further, these data generate new tools like MHC-tetramers to track endogenous T cell responses to CVB3 infection.

Testosterone promotes Coxsackievirus B3 pathogenesis in an oral inoculation mouse model

Enteroviruses initiate infection in the gastrointestinal tract, and sex is often a biological variable that impacts infection. The role of sex hormones on enterovirus pathogenesis, however, is unclear. Previous data indicate that sex hormones can influence intestinal replication of Coxsackievirus B3 (CVB3), an enterovirus in the Picornavirus family. To determine if testosterone promotes CVB3 infection, male mice were castrated and provided placebo or testosterone-filled capsules. We found that testosterone-treated mice shed significantly more CVB3 in the feces and succumbed to CVB3-induced disease at a higher rate than castrated mice given a placebo. Treatment of male mice with an androgen receptor antagonist, flutamide, protected male mice from CVB3-induced lethality, further confirming the role of testosterone in viral pathogenesis. We also observed higher viral loads in peripheral tissues of testosterone-treated mice and an increase in the cytokine and chemokine response. Finally, we found that testosterone treatment in female mice increased fecal CVB3 shedding but had no impact on viral lethality. Overall, these data indicate that testosterone and androgen receptor signaling can promote CVB3 replication in the intestine and enhance CVB3 lethality in a sex-dependent manner.

Polyamines and eIF5A hypusination facilitate SREBP2 translation and cholesterol synthesis to enhance enterovirus attachment and infection

Metabolism is key to cellular processes that ultimately underly the ability of a virus to productively infect a cell. Polyamines are small metabolites that are vital for many host cell processes including cellular proliferation, transcription, and translation, and these molecules are also key in virus infection. Depletion of polyamines inhibits viral infection via diverse mechanisms, including by inhibiting polymerase activity, cellular attachment, and translation of viral proteins. The precise mechanisms underlying many of these phenotypes remain to be understood. We have shown previously that Coxsackievirus B3 requires polyamines for attachment and protease function; however, the mechanism behind this is unknown. Here, we report that polyamines' involvement in translation, through a process called hypusination, globally affects expression of cholesterol synthesis genes by supporting SREBP2 translation, the master transcriptional regulator of cholesterol synthesis genes. By measuring bulk cellular transcription, we found that polyamines enhance the expression of a wide variety of cholesterol synthesis genes, ultimately regulated by SREBP2. The net effect of polyamine depletion on cells negatively impacts CVB3 attachment and replication in polyamine depleted cells by depleting cellular cholesterol. Exogenous cholesterol rescues CVB3 binding and replication, and mutant CVB3 resistant to polyamine depletion exhibits resistance to cholesterol perturbation. This study provides a novel link between polyamine synthesis and cholesterol homeostasis that explains data seen in animals and provides a mechanism through which polyamines impact CVB3 infection.

Interaction between coxsackievirus B3 infection and α-synuclein in models of Parkinson’s disease

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. PD is pathologically characterized by the death of midbrain dopaminergic neurons and the accumulation of intracellular protein inclusions called Lewy bodies or Lewy neurites. The major component of Lewy bodies is α-synuclein (α-syn). Prion-like propagation of α-syn has emerged as a novel mechanism in the progression of PD. This mechanism has been investigated to reveal factors that initiate Lewy pathology with the aim of preventing further progression of PD. Here, we demonstrate that coxsackievirus B3 (CVB3) infection can induce α-syn-associated inclusion body formation in neurons which might act as a trigger for PD. The inclusion bodies contained clustered organelles, including damaged mitochondria with α-syn fibrils. α-Syn overexpression accelerated inclusion body formation and induced more concentric inclusion bodies. In CVB3-infected mice brains, α-syn aggregates were observed in the cell body of midbrain neurons. Additionally, α-syn overexpression favored CVB3 replication and related cytotoxicity. α-Syn transgenic mice had a low survival rate, enhanced CVB3 replication, and exhibited neuronal cell death, including that of dopaminergic neurons in the substantia nigra. These results may be attributed to distinct autophagy-related pathways engaged by CVB3 and α-syn. This study elucidated the mechanism of Lewy body formation and the pathogenesis of PD associated with CVB3 infection.

In Vitro Model Systems of Coxsackievirus B3-Induced Myocarditis: Comparison of Commonly Used Cell Lines and Characterization of CVB3-Infected iCell® Cardiomyocytes

Coxsackievirus B3 (CVB3) belongs to the enteroviruses, which are a well-known cause of acute and chronic myocarditis, primarily infecting cardiac myocytes. As primary human cardiomyocytes are difficult to obtain, viral myocarditis is quite frequently studied in vitro in different non-cardiac and cardiac-like cell lines. Recently, cardiomyocytes that have been differentiated from human-induced pluripotent stem cells have been described as a new model system to study CVB3 infection. Here, we compared iCell® Cardiomyocytes with other cell lines that are commonly used to study CVB3 infection regarding their susceptibility and patterns of infection and the mode of cell death. iCell® Cardiomyocytes, HeLa cells, HL-1 cells and H9c2 cells were infected with CVB3 (Nancy strain). The viral load, CVB3 RNA genome localization, VP1 expression (including the intracellular localization), cellular morphology and the expression of cell death markers were compared. The various cell lines clearly differed in their permissiveness to CVB3 infection, patterns of infection, viral load, and mode of cell death. When studying the mode of cell death of CVB3-infected iCell® Cardiomyocytes in more detail, especially regarding the necroptosis key players RIPK1 and RIPK3, we found that RIPK1 is cleaved during CVB3 infection. iCell® Cardiomyocytes represent well the natural host of CVB3 in the heart and are thus the most appropriate model system to study molecular mechanisms of CVB3-induced myocarditis in vitro. Doubts are raised about the suitability of commonly used cell lines such as HeLa cells, HL-1 cells and H9c2 cells to evaluate molecular pathways and processes occurring in vivo in enteroviral myocarditis.

SNAP47 Interacts with ATG14 to Promote VP1 Conjugation and CVB3 Propagation

Coxsackievirus B3 (CVB3), an enterovirus (EV) in the family of Picornaviridae, is a global human pathogen for which effective antiviral treatments and vaccines are lacking. Previous research demonstrated that EV-D68 downregulated the membrane fusion protein SNAP47 (synaptosome associated protein 47) and SNAP47 promoted EV-D68 replication via regulating autophagy. In the current study, we investigated the interplay between CVB3 and cellular SNAP47 using HEK293T/HeLa cell models. We showed that, upon CVB3 infection, protein levels of SNAP47 decreased independent of the activity of virus-encoded proteinase 3C. We further demonstrated that the depletion of SNAP47 inhibited CVB3 infection, indicating a pro-viral function of SNAP47. Moreover, we found that SNAP47 co-localizes with the autophagy-related protein ATG14 on the cellular membrane fractions together with viral capsid protein VP1, and expression of SNAP47 or ATG14 enhanced VP1 conjugation. Finally, we revealed that disulfide interactions had an important role in strengthening VP1 conjugation. Collectively, our study elucidated a mechanism by which SNAP47 and ATG14 promoted CVB3 propagation through facilitating viral capsid assembly.

Inhibition of calpain reduces cell apoptosis by suppressing mitochondrial fission in acute viral myocarditis

Cardiomyocyte apoptosis is critical for the development of viral myocarditis (VMC), which is one of the leading causes of cardiac sudden death in young adults. Our previous studies have demonstrated that elevated calpain activity is involved in the pathogenesis of VMC. This study aimed to further explore the underlying mechanisms. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin were infected with coxsackievirus B3 (CVB3) to establish a VMC model. Apoptosis was detected with flow cytometry, TUNEL staining, and western blotting. Cardiac function was measured using echocardiography. Mitochondrial function was measured using ATP assays, JC-1, and MitoSOX. Mitochondrial morphology was observed using MitoTracker staining and transmission electron microscopy. Colocalization of dynamin-related protein 1 (Drp-1) in mitochondria was examined using immunofluorescence. Phosphorylation levels of Drp-1 at Ser637 site were determined using western blotting analysis. We found that CVB3 infection impaired mitochondrial function as evidenced by increased mitochondrial ROS production, decreased ATP production and mitochondrial membrane potential, induced myocardial apoptosis and damage, and decreased myocardial function. These effects of CVB3 infection were attenuated by inhibition of calpain both by PD150606 treatment and calpastatin overexpression. Furthermore, CVB3-induced mitochondrial dysfunction was associated with the accumulation of Drp-1 in the outer membrane of mitochondria and subsequent increase in mitochondrial fission. Mechanistically, calpain cleaved and activated calcineurin A, which dephosphorylated Drp-1 at Ser637 site and promoted its accumulation in the mitochondria, leading to mitochondrial fission and dysfunction. In summary, calpain inhibition attenuated CVB3-induced myocarditis by reducing mitochondrial fission, thereby inhibiting cardiomyocyte apoptosis.

Sublethal Enteroviral Infection Exacerbates Disease Progression in an ALS Mouse Model

Background: Amyotrophic lateral sclerosis (ALS) is a fatalneurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors.Infection with enteroviruses, including poliovirus and coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALSdue to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathologicalphenotypes closely resembleALS. However, a causal relationship has not yet been affirmed.Methods:Wild-type C57BL/6J and SOD1G85R ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin (a broad-spectrum anti-RNA viral drug) was given subcutaneously during the acute and/or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting atweek 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression.Results: We found that sublethal infection (mimicking chronic infection) of SOD1G85R ALS mice with CVB3 resulted inearly onset and progressive motor dysfunction, andshortened lifespan, while similar viral infection in C57BL/6J, the background strain of SOD1G85R mice, did not significantly affect motor function and mortality as compared to mock infection within the timeframe of the current study (60 weeksPI).Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies (i.e., TDP-43 pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, wediscovered that early (day 1) but not late (day 15) administration of ribavirincould rescue ALS-like neuropathology and symptoms induced by CVB3 infection.Conclusions: Our study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.