Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

ABSTRACT Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a “cytokine storm” attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR‐8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor‐α (TNF‐α) and regulated upon activation normal T‐cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR‐8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF‐α down‐regulated RANTES expression and secretion of RANTES, interleukin (IL)‐8, and monocyte chemotactic protein‐1 (MCP‐1). In addition, siRANTES suppressed interferon (IFN)‐γ expression and secretion of RANTES, IL‐8, and MCP‐1, suggesting that TNF‐α stimulates production of RANTES, IL‐8, MCP‐1, and IFN‐γ, and RANTES also increased IL‐8, MCP‐1, and IFN‐γ. Furthermore, administration of TNF‐α promoted increased secretion of RANTES, IL‐8, and MCP‐1. Administration of RANTES enhanced IL‐6, IL‐8, and MCP‐1 production without PR‐8 infection. These results strongly suggest that, as an initial step, TNF‐α regulates RANTES production, followed by increase of IL‐6, IL‐8, and MCP‐1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL‐8 and MCP‐1 in the presence of the H2O2‐myeloperoxidse (MPO) system, suggesting that NS1 of PR‐8 may induce a “cytokine storm” from epithelial cells in the presence of an H2O2‐MPO system.