The Impact of H5N1 Influenza Virus Replication on Macrophage Replication

Background: Highly pathogenic H5N1 avian influenza viruses pose a public health risk for their pandemic potential and infect humans with a 60% mortality rate. The mechanism of severe disease during H5N1 infection is only partially known. Despite being important for protection against infection with seasonal influenza viruses, macrophages are thought to contribute to severe disease caused by H5N1 viruses. Phagocytosis of influenza virus-infected cells by activated macrophages is an important antiviral defense and inhibition of phagocytosis leads to increased virus replication. Also, H5N1 virus infection is followed by an increase in levels of nitric oxide (NO), a potent inflammatory signaling molecule that contributes to viral pathogenesis. We demonstrated that H5N1 viruses are unique in their ability to replicate in macrophages in a hemagglutinin (HA)-dependent fashion. The objective of this project is to determine the impact of influenza virus replication on macrophage function. We hypothesize that H5N1 influenza virus replication in macrophages alters cellular functions in ways that contribute to increased disease severity. Methods: RAW 264.7 macrophages were infected with live or UV-inactivated H1N1 influenza virus A/California/04/09 virus (CA/09) or CA/09 expressing the HA gene from the H5N1 virus A/Vietnam/1203/04 (H5N1) for 5 hours and the cells were treated with IFN-γ and macrophage function was determined by a phagocytosis and nitric oxide production assays.  NO production in the culture supernatants was quantified by the Griess method. Phagocytic activity of macrophages was measured using the Vybrant Phagocytosis assay kit according to manufacturer’s instructions. Results: Macrophages infected with H5N1 were less efficient at phagocytosis compared to those infected with CA/09. Further, the H5N1 virus inhibited IFN-γ-mediated activation of macrophages in a replication-dependent manner, whereas cells infected with CA/09 were responsive to IFNγ-induced activation. Additionally, H5N1 influenza viruses induced greater NO production in macrophages than CA/09 and this required virus replication.   Conclusion: Our data demonstrate that H5N1 influenza viruses decrease phagocytic capacity and IFN-γ responsiveness of macrophages and increase NO production following virus infection. Importantly, these changes in macrophage function are dependent on H5N1 virus replication, suggesting that macrophage dysfunction during H5 infection is an effect of the ability of these viruses to replicate in macrophages. This study will help to address the potential risk posed by H5N1.