Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible

Role of hemostasis in inflammation induced by influenza A viruses

La grippe est une maladie respiratoire aigüe, due à une infection par des virus influenza et qui représente un problème important de santé publique. Une meilleure compréhension des interactions entre le virus influenza et son hôte nous permettra de mieux comprendre la physiopathologie de l'infection grippale, et donc, à terme, de mieux se protéger contre la maladie. La morbidité et la mortalité, causées par les infections grippales sévères, sont associées à une dérégulation de la réponse immunitaire, au niveau pulmonaire. Cette inflammation délétère serait à l'origine de dommages collatéraux du poumon, entrainant une diminution de la capacité respiratoire du patient. Bien que les mécanismes impliqués ne soient pas totalement élucidés, de récents travaux mettent en évidence un rôle central des cellules endothéliales dans la dérégulation de la réponse de l'hôte face à l'infection grippale. Lors d'une agression de l'endothélium, le processus physiologique de l'hémostase (activation plaquettaire, coagulation et fibrinolyse) s'active afin de permettre la cicatrisation de la plaie et de maintenir l'intégrité des vaisseaux sanguins. Dans de nombreuses maladies inflammatoires, la seule dérégulation de l'hémostase est directement liée à une réponse inflammatoire délétère. Lors de ma thèse, nous avons émis l'hypothèse que l'hémostase pouvait être à l'origine de la dérégulation inflammatoire durant les infections grippales. Nos données montrent le rôle de deux facteurs fortement impliqués dans l'hémostase : le récepteur activé par la thrombine, PAR-1 (Protease Activated Receptor J) ainsi que le plasminogène, dans l'inflammation délétère des poumons et dans la pathogénicité des virus influenza. Outre le rôle de l'hémostase, nous avons également pu mettre en évidence que le virus influenza incorpore des protéines cellulaires dans l'enveloppe virale, lui permettant d'échapper au système immunitaire, ce qui pourrait aussi contribuer à la dérégulation de la réponse de l'hôte. L'ensemble des résultats obtenus ont permis de mieux comprendre les mécanismes à l'origine d'une réponse immunitaire dérégulée dans les infections grippales et de proposer de nouvelles cibles thérapeutiques pour lutter contre la maladieInfluenza is an acute respiratory disease caused by infection with influenza virus and is a major public health problem. A better understanding of the interaction between influenza virus and host allow us to better understand the pathophysiology of influenza infection, and thus, ultimately, to better protect themselves against the disease. Morbidity and mortality caused by severe influenza infections are associated with dysregulation of the immune response in the lung. This deleterious inflammation is the cause of lung collateral damage, causing a decrease in the patient's breathing capacity. Although the mechanisms involved are not fully understood, recent studies point to a central role of endothelial cells in the deregulation of the host response to influenza infection. During endothelium aggression, the physiological process of hemostasis (platelet activation, coagulation and fibrinolysis) is activated in order to allow wound healing and to maintain the integrity of blood vessels. In many inflammatory diseases, the only dysregulation of hemostasis is directly linked to a deleterious inflammatory response. During my thesis, we hypothesized that hemostasis could be the cause of the inflammatory dysregulation during influenza infections. Our data show the role of two factors strongly involved in hemostasis: the thrombin activated receptor, PAR-1 (protease activated receptor 1) and plasminogen, in the deleterious lung inflammation and in the pathogenicity of influenza virus. Besides the role of hemostasis, we have also been able to show that the influenza virus incorporates cellular proteins in the viral envelope, allowing it to evade the immune system, which could also contribute to the deregulation of the host response. All the results obtained allowed to better understand the mechanisms involved in immune response dysregulation during influenza infection and suggest new therapeutic targets to fight against the diseas

Rôle de l'hémostase dans l'inflammation induite par les virus influenza A

Influenza is an acute respiratory disease caused by infection with influenza virus and is a major public health problem. A better understanding of the interaction between influenza virus and host allow us to better understand the pathophysiology of influenza infection, and thus, ultimately, to better protect themselves against the disease. Morbidity and mortality caused by severe influenza infections are associated with dysregulation of the immune response in the lung. This deleterious inflammation is the cause of lung collateral damage, causing a decrease in the patient's breathing capacity. Although the mechanisms involved are not fully understood, recent studies point to a central role of endothelial cells in the deregulation of the host response to influenza infection. During endothelium aggression, the physiological process of hemostasis (platelet activation, coagulation and fibrinolysis) is activated in order to allow wound healing and to maintain the integrity of blood vessels. In many inflammatory diseases, the only dysregulation of hemostasis is directly linked to a deleterious inflammatory response. During my thesis, we hypothesized that hemostasis could be the cause of the inflammatory dysregulation during influenza infections. Our data show the role of two factors strongly involved in hemostasis: the thrombin activated receptor, PAR-1 (protease activated receptor 1) and plasminogen, in the deleterious lung inflammation and in the pathogenicity of influenza virus. Besides the role of hemostasis, we have also been able to show that the influenza virus incorporates cellular proteins in the viral envelope, allowing it to evade the immune system, which could also contribute to the deregulation of the host response. All the results obtained allowed to better understand the mechanisms involved in immune response dysregulation during influenza infection and suggest new therapeutic targets to fight against the diseaseLa grippe est une maladie respiratoire aigüe, due à une infection par des virus influenza et qui représente un problème important de santé publique. Une meilleure compréhension des interactions entre le virus influenza et son hôte nous permettra de mieux comprendre la physiopathologie de l'infection grippale, et donc, à terme, de mieux se protéger contre la maladie. La morbidité et la mortalité, causées par les infections grippales sévères, sont associées à une dérégulation de la réponse immunitaire, au niveau pulmonaire. Cette inflammation délétère serait à l'origine de dommages collatéraux du poumon, entrainant une diminution de la capacité respiratoire du patient. Bien que les mécanismes impliqués ne soient pas totalement élucidés, de récents travaux mettent en évidence un rôle central des cellules endothéliales dans la dérégulation de la réponse de l'hôte face à l'infection grippale. Lors d'une agression de l'endothélium, le processus physiologique de l'hémostase (activation plaquettaire, coagulation et fibrinolyse) s'active afin de permettre la cicatrisation de la plaie et de maintenir l'intégrité des vaisseaux sanguins. Dans de nombreuses maladies inflammatoires, la seule dérégulation de l'hémostase est directement liée à une réponse inflammatoire délétère. Lors de ma thèse, nous avons émis l'hypothèse que l'hémostase pouvait être à l'origine de la dérégulation inflammatoire durant les infections grippales. Nos données montrent le rôle de deux facteurs fortement impliqués dans l'hémostase : le récepteur activé par la thrombine, PAR-1 (Protease Activated Receptor J) ainsi que le plasminogène, dans l'inflammation délétère des poumons et dans la pathogénicité des virus influenza. Outre le rôle de l'hémostase, nous avons également pu mettre en évidence que le virus influenza incorpore des protéines cellulaires dans l'enveloppe virale, lui permettant d'échapper au système immunitaire, ce qui pourrait aussi contribuer à la dérégulation de la réponse de l'hôte. L'ensemble des résultats obtenus ont permis de mieux comprendre les mécanismes à l'origine d'une réponse immunitaire dérégulée dans les infections grippales et de proposer de nouvelles cibles thérapeutiques pour lutter contre la maladi

[Impact of 5'terminally deleted Enterovirus-B genome on type I IFN response in human cardiomyocytes]

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Coinfection of Parvovirus B19 with Influenza A/H1N1 Causes Fulminant Myocarditis and Pneumonia. An Autopsy Case Report

Parvovirus-B19 (PVB19) is a frequent causative agent of myocarditis. For unclear reasons, viral reactivation can cause acute myocarditis, a leading cause of sudden death in the young. Influenza A/H1N1(2009) virus (IAV/H1N1) is known for causing flu/pneumonia, but the heart is rarely involved. Co-infections of cardiotropic viruses are rarely reported and the mechanisms of viral interactions remain unknown. A 5-year old girl had a flu-like syndrome, when she suddenly presented with a respiratory distress and cardiac arrest. At autopsy, the lungs were found haemorrhagic. Lungs’ histology showed severe bronchiolitis, diffuse haemorrhagic necrosis, and mononuclear inflammation. In the heart, a moderate inflammation was found with no necrosis. IAV/H1N1 was detected in nasal and tracheal swabs, lungs, and the heart. The viral load was high in the lungs, but low in the heart. PVB19 was detected in the heart with a high viral load. Viral co-infection increases the risk of severe outcome but the mechanisms of interaction between viruses are poorly understood. In our case, viral loads suggested a reactivated PVB19-induced acute myocarditis during an IAV/H1N1 pneumonia. Viral interactions may involve an IAV/H1N1-induced cytokine storm, with a fulminant fatal outcome. Clinically, our case shows the importance of investigating inflammatory pathways as therapeutic targets

Analysis of transcription and translation viral activities of wild type (WT) and 5’ terminally deleted enteroviruses in a model of cultured primary human cardiomyocytes

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Early Emergence of 5′ Terminally Deleted Coxsackievirus-B3 RNA Forms Is Associated with Acute and Persistent Infections in Mouse Target Tissues

Major EV-B populations characterized by 5′ terminal deletions (5′TD) have been shown to be associated with the development of myocarditis and type 1 diabetes in mice or humans. To date, the dynamics of EV-B 5′TD-RNA forms’ emergence during the course of infection and their impact on cellular functions remain unclear. Using a RACE-PCR approach in CVB3/28-infected mouse organs, we showed an early (3 days post infection, DPI) emergence of major 5′TD populations associated with minor full-length RNA forms. Viral replication activities with infectious particle production were associated with heart, liver, and pancreas acute inflammatory lesions, whereas clearance of viral RNA without organ lesions was observed in the brain, lung, intestines, and muscles from 3 to 7 DPI. At 28 DPI, low viral RNA levels, +/-RNA ratios < 5 associated with viral protein 1 expression revealed a persistent infection in the heart and pancreas. This persistent infection was characterized by molecular detection of only 5′TD RNA forms that were associated with dystrophin cleavage in the heart and insulin production impairment in beta-pancreatic cells. These results demonstrated that major EV-B 5′TD RNA forms can be early selected during systemic infection and that their maintenance may drive EV-induced acute and persistent infections with target cell dysfunctions

Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication

International audienceDuring the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-gamma)-induced Stat phosphorylation and IFN-gamma-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-gamma signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-gamma antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes

Replication Activities of Major 5′ Terminally Deleted Group-B Coxsackievirus RNA Forms Decrease PCSK2 mRNA Expression Impairing Insulin Maturation in Pancreatic Beta Cells

Emergence of 5′ terminally deleted coxsackievirus-B RNA forms (CVB-TD) have been associated with the development of human diseases. These CVB-TD RNA forms have been detected in mouse pancreas during acute or persistent experimental infections. To date, the impact of the replication activities of CVB-TD RNA forms on insulin metabolism remains unexplored. Using an immunocompetent mouse model of CVB3/28 infection, acute and persistent infections of major CVB-TD populations were evidenced in the pancreas. The inoculation of mice with homogenized pancreases containing major CVB-TD populations induced acute and chronic pancreatic infections with pancreatitis. In the mouse pancreas, viral capsid protein 1 (VP1) expression colocalized with a decrease in beta cells insulin content. Moreover, in infected mouse pancreases, we showed a decrease in pro-hormone convertase 2 (PCSK2) mRNA, associated with a decrease in insulin plasmatic concentration. Finally, transfection of synthetic CVB-TD50 RNA forms into cultured rodent pancreatic beta cells demonstrated that viral replication with protein synthesis activities decreased the PCSK2 mRNA expression levels, impairing insulin secretion. In conclusion, our results show that the emergence and maintenance of major CVB-TD RNA replicative forms in pancreatic beta cells can play a direct, key role in the pathophysiological mechanisms leading to the development of type 1 diabetes

Major Group-B Enterovirus populations deleted in the noncoding 5' region of genomic RNA modulate activation of the type I interferon pathway in cardiomyocytes and induce myocarditis.

Major 5'-terminally deleted (5'TD) RNA forms of group-B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-β (IFN-β) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5'TD RNA have been characterized as replicative viral populations that impair IFN-β production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5'-terminal CVB3 RNA domain I, modifying stem-loops "b", "c" or "d", were found to impair IFN-β production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-β production and to reduce the burden of CVB-5'TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5' noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5'TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis