Inactivated Influenza A H1N1pdm (Canada/6294/09)

Neutrophils are the most abundant cells in the human immune system. Here, the researchers explored the role of neutrophils in host defense during influenza A virus infection, specifically evaluating whether they are involved in the pathogenesis of H5N1 influenza virus. The results showed that influenza virus-infected alveolar epithelial cells allowed neutrophil migration. Compared with seasonal influenza H1N1 virus-infected alveolar epithelial cells, the number of migrating neutrophils was significantly increased in H5N1 influenza virus-infected alveolar epithelial cells. Neutrophils were equally sensitive to H5N1 and H1N1 virus infection and had similar levels of viral gene transcription. Efficient replication was observed in H5N1 virus-infected neutrophils. H5N1 virus-induced cytokine and chemokine gene transcription levels were higher than those in H1N1 virus-infected neutrophils, including TNF-α, IFN-β, CXCL10, MIP-1α, and IL-8. This infers that the inflammatory response caused by H5N1 virus is more intense than that of H1N1 virus. Notably, NADPH oxidase-dependent NET formation was observed in neutrophils only at 6 h after infection with H1N1 virus, whereas NET formation was not observed after infection with H5N1 virus. These findings demonstrate that NET formation is abrogated after H5N1 influenza virus infection and may exacerbate the severity of H5N1 disease.

Here, the researchers performed an infection experiment to examine whether influenza viruses are able to infect and replicate in neutrophils. Influenza M genes were readily detected at 1 hpi, with a copy number of 106 for both influenza virus subtypes, H5N1 and H1N1. There was no trend toward an increase in viral gene copy number in cells at subsequent time points, 3, 6, to 16 hpi (Figure 1a). De novo viral proteins were investigated by immunofluorescence assay at 16 hpi, revealing evidence of viral antigens in the multilobed nuclei of neutrophils infected with the four viruses, while mock-infected cells remained unstained. This finding suggests that viral protein translation has occurred in H5N1- and H1N1-infected neutrophils (Figure 1b). To examine whether infectious virions were formed, culture supernatants collected at different time points post-infection were titrated (Figure 1c). For better comparison, the researchers set up heat-killed controls of H5N1, seasonal, and pandemic H1N1 (H1N1pdm). After 16 hpi, the infectious virus titers in the culture supernatants of cells infected with H5N1 virus were significantly higher than those of the heat-inactivated control, while the infectious virus titers in the culture supernatants of cells infected with seasonal and pandemic H1N1 virus were significantly lower than those of the heat-inactivated control. This suggests that H5N1 virus replicates efficiently in neutrophils infected with H5N1 virus, while H1N1 virus is actively inactivated by neutrophils (Figure 1c).

Figure 1. Influenza virus infection and replication competence in naïve neutrophils. (Chan L L Y, et al., 2020)