Role Of Proinflammatory Cytokine And P53 Protein In The Pathogenesis Of Avian Influenza In Mammals

Recent outbreaks of H5N1 influenza virus infections had important health and economic consequences and raised concerns that a new influenza pandemic would occur in the near future. In 1997, the H5N1 avian influenza A virus crossed the avian-human species barrier for the first time. Since late 2003, the H5N1 influenza virus has spread across Asia to the Middle East, Europe and Africa, causing outbreaks of disease and death in poultry, mammals, and humans. As of March 2010, approximately 492 laboratory-confirmed human cases of H5N1 virus infection was reported by the WHO, with a human fatality rate above 60%. It is noteworthy that H5N1 influenza virus with the strong pathogenicity, compared with the seasonal human influenza virus infection. In vitro and animal studies suggests that high and disseminated viral replication, and aberrant production of inflammatory cytokines and chemokines,"cytokine storm", may play an important role in the pathogenesis of H5N1 influenza. In support of this hypothesis, the H5N1 virus was found to elicit substantially higher expression of inflammatory cytokines and chemokines, particularly tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), IFN-inducible protein-10 (IP-10), monokine induced by IFN-γ(MIG), monocyte chemoattractant protein-1 (MCP-1), and regulated on activation, normal T cell expressed and secreted protein (RANTES). The current effort was directed toward elucidating whether MIF, HMGB1 and/or MIF, HMGB1-mediated inflammatory response contributes to the pathogenesis of H5N1 virus infection. It has been speculated that inflammatory responses mediated by release of MIF and HMGB1 may contribute to pathogenesis of H5N1 viral infections. MIF is a potent proinflammatory cytokine with broad upstream actions in the inflammatory cascade. Once released, MIF exerts critical autocrine and paracrine activating effects, including the induction of inflammatory cytokines and chemokines. Recent studies have confirmed the involvement of MIF in a number of inflammatory and virus diseases originally studied in animals.The increased levels of MIF in certain pathological conditions may be indicative of its involvement in those diseases. HMGB1 has been established as a critical mediator of lethal systemic inflammation as it plays an important role as a late mediator of lethal endotoxemia and sepsis. During viral infection, excessive production of extracellular HMGB1 causes multiple organ failure and can be more dangerous to the host than the original pathologic manifestation.Firstly, in order to use BALB/c mouse model to study the role of MIF and HMGB1 in the pathogenesis of H5N1 influenza virus, we establish a real-time PCR for H5N1 influenza virus and cytokines(IL-1β, IL-6, TNFα, MIG, IP-10, RANTES, MIF, and HMGB1)mRNA using SYBR GreenⅠ. The sensitivity and specificity of real-time PCR detection method were verified. These results show that real-time PCR for H5N1 influenza virus and cytokines is a specific, accurate and sensitive method and has a potential application in basic and clinical study. In order to investigate whether proinflammatory cytokine macrophage migration inhibitory factor (MIF) and high mobility group box 1 protein (HMGB1) influence H5N1 virus immunopathogenesis in experimental murine model, by assaying MIF and HMGB1 expression in the lungs and serum, and by following the effect of ISO-1 and EP, the inhibitor of MIF and HMGB1, on the virus replication, induction of cytokines and chemokines in the lungs, loss of body weight, and survival rate. Real-time PCR and ELISA assay showed that expression of mRNA encoding MIF and HMGB1, and MIF and HMGB1 protein increased significantly in BALB/c mice infected with Harbin/132 H5N1 virus. The levels of MIF mRNA and protein were elevated in the lungs of BALB/c mice from 3-6 h p.i.. The levels of HMGB1 mRNA and protein were elevated in the lungs of BALB/c mice from 36-72 h p.i.. Immunohistochemistry of the lungs of virus-infected mice showed that bronchiolar epithelial cells, alveolar epithelial cells, and intercellular space contained large amounts of MIF( at 6-48 h p.i.) and HMGB1 (at 48-120 h p.i.), and confirmed its localization in the lung tissue. The lungs of virus-infected ISO-1 and EP -treated mice were assayed at various times p.i. for virus titers and levels for cytokines IL-1β, IL-6 and TNFα, and chemokines IP-10, MIG and RANTES. ISO-1 and EP-untreated mice served as controls. The data showed that virus titers in the lungs had been effected insignificantly by ISO-1 and EP. As for the levels of cytokines and chemokines in the lungs of virus-infected mice, ISO-1 and EP reduced them about 2- to 3-fold as compared with controls.Although ISO-1-and EP treatment did not render the mice more resistant to virus lethality, ISO-1-and EP treatment improved survival and prolonged survival time. The histology of the lungs of ISO-1 and EP-treated virus-infected mice revealed a weaker inflammatory responseas compared with untreated mice. ISO-1-and EP treatment caused a significant reduction in pulmonary inflammatory cytokines and chemokine. These results indicate the involvement of MIF and HMGB1 in inflammatory responses to H5N1 influenza virus infections by induction of pulmonary inflammatory cytokines and chemokines, and suggest that pharmacotherapeutic approaches targeting MIF and HMGB1 may hold promise for the treatment of H5N1 influenza virus. The host anti-viral innate immune against viral infections, played an important role in reduced injury. Cellular control of viral infection is mediated through a variety of processes that affect different stages of the viral life cycle. Infection with H5N1 influenza viruse frequently results in fatal result. Nature of the infection suggests that high and disseminated viral replication,and failure of the host cellular surveillance are critical to diseases pathogenesis. However, factors determining the host ability to clear the viral infection are incompletely understood. Tumor suppressor p53, the major cellular defense against tumor development, has recently been discovered that p53 also function in the innate antiviral response. Turpin E described that p53 was induced in H1N1 influenza virus-infected cells, and inhibited the viral replication. In this paper, we evaluated the tumor suppressor p53 for its possible effects on the replication of H5N1 influenza virus.Bacuse transgenic mice which was used to research the mechanisms of H5N1 influenza virus pathogenesis almost have the genetic background of C57BL/6 mice. Here we present a C57BL/6 mouse model of H5N1 influenza viral infection. C57BL/6 mice were inoculated intranasally with Harbin/132 H5N1 virus. Three and five days later, three mice of each group were euthanized. Lung injury was assessed by observation of lung histopathology, virus titers. MLD50 were measured. Our data showed that mice infected with H5N1 influenza virus resulted in mainly epithelial injury and interstitial pneumonia, which was characterized by the following features: significant weight loss and dramatically increased lung wet weight:body weight ratios, inflammatory cellular infiltration, alveolar and interstitial edema, and hemorrhage in lungs, and high virus titers in lung; MLD50=106.5/50μL. These results suggested that we successfully established a mouse model of H5N1 influenza virus infection, which may benefit the study of H5N1 influenza virus pathogenesis with p53+/+ and p53-/- mice.In order to explore the dynamic change of p53 protein in the A549 cells and C57BL/6 mice infected with Harbin/132 H5N1 virus, we analyzed the p53 protein in cells and mice collected at different time post infection with western-blot, ELISA, and real-time PCR. We also checked the expression of p53 protein in C57BL/6 mice infected with heat-inactivated virus and UV-inactivated virus to investigate the relationship of the dynamics of p53 protein and the replication of H5N1 influenza virus. The results showed that p53 levels increased in virus-infected C57BL/6 mice at 12h p.i. and remained elevated at 48h p.i. compared to mock-infected mice. p53 levels increased in A549 cells at 9h p.i. and remained elevated at 24h p.i. compared to mock-infected cells. The expression of p53 was stable in C57BL/6 mice infected with heat-inactivated virus, but the increase of the p53 protein was discovered in C57BL/6 mice infected with UN-inactivated H5N1 virus at initial stage. The transcription of p53 gene in H5N1 virus infected cells and mice didn't change. We could draw the conclusion that the up-regulation of p53 protein in H5N1 virus-infected cells and mice was correlative with the viral replication but independent of the transcription of p53 gene.In order to explore the ability of p53 protein to inhibit the replication of H5N1 influenza virus,p53+/+ and p53-/- mice were infected with Harbin/132 H5N1 virus. Virus titers and viral RNA copies in lungs monitored by real-time PCR and TCID50 at 3 and 5d post infection. In addition, p53-/- and p53+/+ MEF, MDCK and MDCK/Mp53(express mouse p53)cells also was infeced with Harbin/132 H5N1 virus. The results showed that virus titers and viral RNA copies in lungs were obviously higher in the p53-/- mice than that of in the p53+/+ mice. There was a a 1-log increase in viral titers in p53-/- MEF compared to those of p53+/+ MEF. These results strongly suggested that mouse p53 protein can inhibit the replication of H5N1 influenza virus.In order to explore the ability of human p53 protein to inhibit the replication of H5N1 influenza virus as same as mouse p53 protein, p53 knock down A549 cells, p53 over expression A549 cells, and A549 cells were infected with Harbin/132 H5N1 virus and viral replication efficiency was compared. The results showed that p53 knock down A549 cells were highly permissive to the replication of H5N1 influenza virus. Within 24 h p.i, there was approximately a 1.5-log increase of virus RNA copies and viral titers in p53 knock down A549 cells compared to the control A549 cells. Conversely, overexpression of p53 significantly suppressed the viral replication. These results strongly suggested that human p53 protein also can inhibit the replication of H5N1 influenza virus. Next, to investigate whether the IFN pathway was relative with the inhibition of p53 protein in the replication of H5N1 influenza virus, we checked the effect of IFN treatment against H5N1 influenza virus in the p53 knock down A549 cells, p53 over expression A549 cells, and A549 cells. These cells infected with H5N1 virus were treated with 1000 U/mL IFNα2a, and the viral replication profile was evaluated by CPE and plaque assay. IFN treatment suppressed the amount of virus titers in both the control A549 cells and the p53 over expression A549 cells. However, the degree of suppression was significantly weaker in A549 cells compared with the p53 over expression A549 cells. IFN treatment didn't influence virus titers in p53 knock down A549 cells. These results indicated the importance of p53 protein for effect of IFN against H5N1 influenza virus.In conclusion, we discovered the role of proinflammatory cytokines MIF and HMGB1 in the pathogenesis of H5N1 influenza virus infection in mammals. The inflammatory injury of MIF and HMGB1 in H5N1 influenza virus infected mammals primarily through the activation and high expression of inflammatory cytokines and chemokines. In addition, we also discovered that p53 protein inhibited the replication of H5N1 influenza viruse. The increased p53 protein may be inhibit the viral replication through the IFN signal pathway. These results could provide important information for the clinical treatment of drug targets against H5N1 influenza virus.