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. 2010 Jul;7(4):263-70.
doi: 10.1038/cmi.2010.25. Epub 2010 May 17.

Cellular response to influenza virus infection: a potential role for autophagy in CXCL10 and interferon-alpha induction

Anna Hing-Yee Law  1 , Davy Chun-Wai Lee, Kwok-Yung Yuen, Malik Peiris, Allan Sik-Yin Lau
Affiliations

Cellular response to influenza virus infection: a potential role for autophagy in CXCL10 and interferon-alpha induction

Anna Hing-Yee Law et al. Cell Mol Immunol. 2010 Jul.

Abstract

Historically, influenza pandemics have arisen from avian influenza viruses. Avian influenza viruses H5N1 and H9N2 are potential pandemic candidates. Infection of humans with the highly pathogenic avian influenza H5N1 virus is associated with a mortality in excess of 60%, which has been attributed to dysregulation of the cytokine system. Human macrophages and epithelial cells infected with some genotypes of H5N1 and H9N2 viruses express markedly elevated cytokine and chemokine levels when compared with seasonal influenza A subtype H1N1 virus. The mechanisms underlying this cytokine and chemokine hyperinduction are not fully elucidated. In the present study, we demonstrate that autophagy, a tightly regulated homeostatic process for self-digestion of unwanted cellular subcomponents, plays a role in cytokine induction. Autophagy is induced to a greater extent by H9N2/G1, in association with cytokine hyperinduction, compared with H1N1 and the novel pandemic swine-origin influenza A/H1N1 viruses. Using 3-methyladenine to inhibit autophagy and small interfering RNA to silence the autophagy gene, Atg5, we further show that autophagic responses play a role in influenza virus-induced CXCL10 and interferon-alpha expression in primary human blood macrophages. Our results provide new insights into the pathogenic mechanisms of avian influenza viruses.

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Figures

Figure 1
Figure 1
Autophagy was induced by H1N1, H9N2/G1 and S-OIV influenza viruses. Human blood macrophages were mock-infected or infected with H1N1, H9N2/G1 or S-OIV. (a, b) Whole-cell lysates were harvested and LC3B expression was examined by western blot analysis. Actin was used as a loading control. (c) Autophagosomes were examined by staining the mock-, H1N1- or H9N2/G1-infected cells with antibodies specific to LC3B. DAPI was used to stain nuclei. Arrows indicate LC3B positive vesicles. Magnification: ×400. (d) p62 expression levels were examined at 19 h.p.i. by western blot analysis. (e) At 2 h.p.i., phosphorylation levels of p70S6K (Thr389) were examined by western blot analysis. Total p70S6K was used as a loading control. Densities of the protein bands were determined with Bio-Rad Quantity One imaging software. The values in parentheses represent LC3B II to LC3B I ratios (a, b). The p62 (d) and phospho-p70S6K (Thr389) (e) intensity values were normalized to the corresponding actin or total p70S6K values. The values in parentheses are the relative normalized intensities compared to those of the mock-treated cells. The results shown are representative figures from three independent experiments. DAPI, 4,6-diamidine-2-phenylindole dihydrochloride; h.p.i., hour post-infection; p70S6K, p70S6 kinase; S-OIV, swine-origin influenza A/H1N1 virus; Thr389, threonine 389.
Figure 2
Figure 2
Knockdown of Atg5 protein by siRNA oligos. Human blood macrophages were transfected with siAtg5 or siCtrl at 100 and 200 nM. At 48 hour post-transfection, whole-cell lysates were harvested and Atg5 expression was measured by western blot with antibodies specific to Atg5. Actin was used as a loading control. Densities of the protein bands were determined with Bio-Rad Quantity One imaging software. The Atg5 intensity values were normalized to the corresponding actin levels. The values in parentheses are the relative normalized intensities compared to those of the siCtrl-transfected cells. The results shown are representative figures from three independent experiments. siAtg5, small interfering RNA oligos specific to Atg5; siCtrl, non-targeting control oligos; siRNA, small interfering RNA.
Figure 3
Figure 3
Involvement of Atg5 in H1N1 and H9N2/G1 virus-induced autophagy. siAtg5- or siCtrl-transfected macrophages were mock-infected or infected with H1N1 or H9N2/G1. Atg5 and LC3B II levels were examined by western blot. Actin was used as a loading control. Densities of the protein bands were determined with Bio-Rad Quantity One imaging software. The values in parentheses are the relative intensities of Atg5 or LC3B II levels compared to those of actin. The results shown are representative figures from three independent experiments. siAtg5, small interfering RNA oligos specific to Atg5; siCtrl, non-targeting control oligos; siRNA, small interfering RNA.
Figure 4
Figure 4
H9N2/G1 induced higher CXCL10 and IFN-α levels than H1N1. Macrophages were either mock-infected or infected with H1N1 or H9N2/G1. (a) At 3 h.p.i., CXCL10 levels were examined by TaqMan gene expression assay; n=6. (b) CXCL10 levels in culture supernatants harvested 8 h.p.i. were measured by ELISA; n=4. (c) IFN-α levels in culture supernatants harvested 8 h.p.i. were measured by ELISA; n=6. *P<0.05. BD, below detection limit; h.p.i., hour post-infection; IFN, interferon.
Figure 5
Figure 5
Inhibition of autophagy suppressed influenza-induced CXCL10 and IFN-α expression. Macrophages were pre-treated with 3-MA (10 mM) before infection with H1N1 (a) or H9N2/G1 (b). CXCL10 mRNA expression at 3 h.p.i. was examined by TaqMan gene expression assay; n=3. Macrophages were transfected with siAtg5 or siCtrl at 200 nM. At 48 hour post-transfection, cells were infected with H1N1 (c) or H9N2/G1 (d). The relative CXCL10 mRNA levels were examined at 3 h.p.i. by TaqMan gene expression assay; n=3. Relative mRNA levels of IFN-α1, -α2 and -α8 in siRNA-transfected cells with H9N2/G1 infections were examined by TaqMan gene expression assay (e); n=3. Data represent the percentages of CXCL10 or IFN-α expression from 3-MA-treated or siAtg5-transfected cells relative to the mock-treated or siCtrl-transfected cells, respectively. *P<0.05, #P<0.01. h.p.i., hour post-infection; IFN, interferon; siAtg5, small interfering RNA oligos specific to Atg5; siCtrl, non-targeting control oligos; siRNA, small interfering RNA; 3-MA, 3-methyladenine.
Figure 6
Figure 6
Atg5 knockdown suppressed H9N2/G1-induced CXCL10 and IFN-α protein expression. Macrophages were transfected with siAtg5 or siCtrl at 200 nM. At 48 hour post-transfection, cells were infected with H9N2/G1. Quantities of CXCL10 (a) (n=4) and IFN-α (b) (n=5) in the culture supernatants at 8 h.p.i. were measured by ELISA. The data represent the percentage of CXCL10 and IFN-α expression from siAtg5-transfected cells relative to siCtrl-transfected cells. *P<0.05, #P<0.01. h.p.i., hour post-infection; IFN, interferon; siAtg5, small interfering RNA oligos specific to Atg5; siCtrl, non-targeting control oligos.
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