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. 2011 Dec;55(12):874-84.
doi: 10.1111/j.1348-0421.2011.00396.x.

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

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Free PMC article

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

Thuy Thi Bich Phung et al. Microbiol Immunol. .
Free PMC article

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 H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.

Figures

Fig. 1
Fig. 1. The survival rate of A549 cells during infection with PR-8
(a) Expression of viral NS1 gene in A549 cells infected with PR-8. (b) Survival rates of infected (black bar) and uninfected (white bar) cells.
Fig. 2
Fig. 2. Protein production and gene expression of cytokines and chemokines in A549 cells after infection
(a) Concentrations of TNF-α and RANTES secreted by infected (black bars) and uninfected (white bars) cells. (b) Quantitative analyses of expression of IL-6 and RANTES genes in infected (black bars) and uninfected (white bars) cells with PR-8 at 2 days post-infection. (c) mRNA expression of cytokine and chemokine genes at 2 days post-infection. Data are shown as mean ± SD of results from three individuals. *P < 0.05 (Student's t-test).
Fig. 3
Fig. 3. Knockdown of expression of TNF-α and RANTES genes
Blockade by siTNF-α and siRANTES of cytokine secretion by A549 cells transfected with si-TNF-α or siRANTES (black bars) or transfected with siGAPDH as controls (white bars). Data are shown as mean ± SD of results from three individuals. *P < 0.05; **P < 0.01 (Student's t-test).
Fig. 4
Fig. 4. Stimulation of uninfected-A549 cells by rTNF-α or rRANTES protein
Addition of rTNF-α or rRANTES protein (black bars), and medium (white bars). Data are shown as mean ± SD of results from three individuals. *P < 0.05; **P < 0.01 (Student's t-test).
Fig. 5
Fig. 5. Explosion of IL-8 and MCP-1 secretion by NS1 of influenza virus collaborating with the H2O2-MPO axis
(a) The influence of H2O2-MPO on the survival rate of A549 cells infected with PR-8 (white bars) or NS1 (black bars). (b) Cytokine production of A549 cells infected with NS1 plasmid (black bars) or PR-8 (white bars) in the presence of H2O2 and MPO. Data are shown as mean ± SD of results from three individuals. *P < 0.05; **P < 0.01 (Student's t-test).
Fig. 6
Fig. 6. A scheme for sequential secretion and explosion of “cytokine storm” in bronchial epithelial cells infected with in fluenza viruses
The first step is TNF-α released from bronchial epithelial cells infected with influenza virus enhances induction of RANTES secretion. In a later phase the epithelial cells associated with viral NS1 protein may enhance production of large amounts of chemokines IL-8 and MCP-1 in the presence of the H2O2-MPO system, suggesting that NS1 of PR-8 may play a key role in “cytokine storm” when the H2O2-MPO system is active.

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