Host immune and apoptotic responses to avian influenza virus H9N2 in human tracheobronchial epithelial cells.

The avian influenza virus H9N2 subtype has circulated in wild birds, is prevalent in domestic poultry, and has successfully crossed the species boundary to infect humans. Phylogenetic analyses showed that viruses of this subtype appear to have contributed to the generation of highly pathogenic H5N1 viruses. Little is known about the host responses to H9N2 viruses in human airway respiratory epithelium, the primary portal for viral infection. Using an apically differentiated primary human tracheobronchial epithelial (TBE) culture, we examined host immune responses to infection by an avian H9N2 virus, in comparison with a human H9N2 isolate. We found that IFN-ß was the prominent antiviral component, whereas interferon gamma-induced protein 10 kDa (IP-10), chemokine (C-C motif) ligand (CCL)-5 and TNF-α may be critical in proinflammatory responses to H9N2 viruses. In contrast, proinflammatory IL-1ß, IL-8, and even IL-6 may only play a minor role in pathogenicity. Apparently Toll-like receptor (TLR)-3, TLR-7, and melanoma differentiation-associated gene 5 (MDA-5) contributed to the innate immunity against the H9N2 viruses, and MDA-5 was important in the induction of IFN-ß. We showed that the avian H9N2 virus induced apoptosis through the mitochondria/cytochrome c-mediated intrinsic pathway, in addition to the caspase 8-mediated extrinsic pathway, as evidenced by the cytosolic presence of active caspase 9 and cytochrome c, independent of truncated BH3 interacting domain death agonist (Bid) activation. Further, we demonstrated that FLICE-like inhibitory protein (FLIP), an apoptotic dual regulator, and the p53-dependent Bcl-2 family members, Bax and Bcl-x(s), appeared to be involved in the regulation of extrinsic and intrinsic apoptotic pathways, respectively. The findings in this study will further our understanding of host defense mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium.

Roles of the ERK MAPK in the regulation of proinflammatory and apoptotic responses in chicken macrophages infected with H9N2 avian influenza virus.

The mitogen-activated protein kinase (MAPK) family is responsible for important signalling pathways which regulate cell activation, differentiation, apoptosis and immune responses. Studies have shown that influenza virus infection activates MAPK family members in mammals. While the extracellular signal-regulated kinase (ERK)1/2 is important for virus replication, activation of p38 controls the expression of RANTES, interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha. In this study, we report that avian influenza virus (AIV) activates ERK, p38 and Jun-N-terminal kinases in avian species. In chicken macrophages, while ERK was required for H9N2 AIV replication, ERK regulated proinflammatory cytokines IL-1beta, IL-6 and IL-8, which is distinct from what has been previously reported in mammalian cells. Moreover, ERK alone suppressed TNF-alpha and FasL and inhibited TNF-family-mediated extrinsic apoptosis in H9N2-infected chicken macrophages. Taken together, these findings suggest that ERK signalling may uniquely play important roles in avian host responses to AIV infection.