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. 2015 Mar;89(5):2672-83.
doi: 10.1128/JVI.03529-14. Epub 2014 Dec 17.

Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via upregulation of SOCS1 and SOCS3

Siying Ye  1 , Sue Lowther  2 , John Stambas  3
Affiliations

Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via upregulation of SOCS1 and SOCS3

Siying Ye et al. J Virol. 2015 Mar.

Abstract

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species.

Importance: Highly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Wide-spread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses after infection. Disease severity following avian influenza virus infection can be attributed in part to hyperinduction of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low-pathogenicity avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and downregulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combination with antiviral therapy may have important therapeutic benefits.

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Figures

FIG 1
FIG 1
Immunofluorescence staining of influenza-infected cells. The influenza viral NP protein (green) was detected by immunofluorescence using a mouse anti-NP monoclonal antibody in A549 (A), DF-1 (B), and HD-11 (C) cells infected with PR8, H5N3, or H5N1 in the presence of 1% DMSO vehicle control or 1 mM apocynin at 24 hpi. Uninfected cells treated with 1% DMSO for 24 h were used as negative controls. DAPI staining (blue) shows the total nuclei.
FIG 2
FIG 2
Cytokine and chemokine gene expression profiles in influenza virus-infected human and chicken cell lines. (A to C) A549 cells (A), DF-1 cells (B), and HD-11 (C) cells were infected with influenza viruses as indicated. Differences were expressed as the fold change compared to uninfected cells calculated using the 2–ΔΔCT method. The data shown are the means ± the SD (*, P < 0.05; ***, P < 0.001 [PR8 versus H5N1]; #, P < 0.05; ###, P < 0.001 [H5N1 versus H5N3]; †††, P < 0.001 [H5N1 versus H7N9]).
FIG 3
FIG 3
Anti-inflammatory effects of apocynin in human and chicken cell lines infected with influenza viruses. (A to C) A549 cells (A), DF-1 cells (B), and HD-11 (C) cells were infected with influenza viruses as indicated in the presence of 1% DMSO control or 1 mM apocynin for 24 h. Negative controls included uninfected cells cultured in medium supplemented with 1% DMSO or 1 mM apocynin. The level of influenza M gene was expressed as the log of its cDNA copy number relative to 106 cells. The data shown are the means ± the SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001 [PR8 versus H5N1]; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 [H5N1 versus H5N3]; †, P < 0.05; ††, P < 0.01; †††, P < 0.001 [H5N1 versus H7N9]).
FIG 4
FIG 4
Apocynin reduced ROS production in influenza-infected human and chicken cell lines. (A to C) A549 cells (A), DF-1 cells (B), and HD-11 cells (C) were infected with influenza viruses as indicated in the presence of 1% DMSO control or 1 mM apocynin for 24 h. Uninfected cells cultured in medium containing 1% DMSO were used as negative controls. ROS production (red) was detected by immunofluorescence with CellROX Deep Red reagent, and the total nuclei were shown with DAPI staining (blue). Fluorescence was analyzed and quantified using a CellInsight system. ROS fluorescence was normalized against DAPI staining. The data represent the mean ROS fluorescence ± the SD from three experiments (**, P < 0.01; ***, P < 0.001 [compared to H5N1-infected cells with the same DMSO or apocynin treatment]; ##, P < 0.01; ###, P < 0.001 [compared to H5N1-infected cells treated with DMSO control]).
FIG 5
FIG 5
Expression of SOCS1, SOCS3, and SOCS2 in A549 cells infected with PR8, H5N3, H5N1, or H7N9. SOCS1 (A) and SOCS3 (B) gene expression levels in A549 cells after influenza virus infection were measured by qRT-PCR over a 24-h time course. The effects of apocynin on SOCS1 (C) and SOCS3 (D) gene expression in uninfected and infected A549 cells were also determined at 24 hpi. The data shown are the means ± the SD. *, P < 0.05; ***P < 0.001 (compared to uninfected cells in medium with 1% DMSO); ###, P < 0.001 (compared to H5N1); †††, P < 0.001 (significantly different between infected cells treated with DMSO control and apocynin). (E) Protein expression of SOCS1 and SOCS3 in A549 cells infected with H5N1 in the absence or presence of 0.1, 0.5, or 1 mM apocynin at 6 or 24 hpi. Representative Western blots and quantification of protein band intensity from three individual experiments are shown. The protein band intensity data represent the means ± the SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001). SOCS2 gene expression levels were also analyzed during a 24-h time course (F) or at 24 hpi in the presence of 1% DMSO or 1 mM apocynin (G). The “0” time point represents uninfected cells cultured in medium only.
FIG 6
FIG 6
Effects of apocynin on the expression of phospho-FoxO3-Ser253 and TAM receptors in influenza virus-infected A549 cells. (A) The presence of phospho-FoxO3-Ser253 protein was determined in A549 cells infected with H5N1 and H7N9 in the presence of 1% DMSO or 1 mM apocynin at 24 hpi. Gene expression levels of the TAM receptors Axl (B), Mer (C), and Tyro3 (D) were analyzed by qRT-PCR. *, P < 0.05, ***, P < 0.001 (compared to uninfected controls); #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (compared to H5N1 virus infection group); †††, P < 0.001 (significantly different between infected cells treated with DMSO control and apocynin). (E) Total endogenous Tyro3 protein expression was analyzed in A549 cells infected with H5N1 and H7N9 in the presence of 1% DMSO or 1 mM apocynin at 24 hpi. Representative Western blots and quantification of protein band intensity from three individual experiments are shown. The protein band intensity data represent mean ± the SD (*, P < 0.05; **, P < 0.01).
FIG 7
FIG 7
Schematic diagram showing the proposed mechanism for influenza virus-induced cytokine dysregulation and the anti-inflammatory effects of apocynin. SOCS1 and SOCS3 expression are positively regulated by FoxO3 and the TAM receptor, Tyro3. The infection of influenza virus, especially HPAI H5N1 and LPAI H7N9 influenza viruses, causes increased ROS production and hypercytokinemia. Influenza virus-mediated cytokine dysregulation can be induced by enhancing the accumulation of the inactivated phospho-FoxO3-Ser253 (Fig. 6A). This in turn reduces SOCS3 expression and contributes to the overproduction of cytokines and chemokines following influenza virus infection. H5N1 and H7N9 infection also inhibit Tyro3 expression in infected cells (Fig. 6E), interrupting the transcription of SOCS1 and SOCS3, resulting in cytokine dysregulation. In addition, yet to be identified host factors may also influence gene expression in an attempt to restore cytokine homeostasis, resulting in an upregulation of SOCS1 and SOCS3 (Fig. 5E, 24 hpi). Treatment of influenza virus-infected cells with apocynin significantly reduces influenza virus-stimulated cytokine/chemokine overproduction (Fig. 3) via the upregulation of SOCS1 and SOCS3 (Fig. 5E). One of the underlying mechanisms suggests that apocynin influences the host immune response by reducing influenza virus-induced ROS production, which in turn reduces phospho-FoxO3-Ser253 accumulation (Fig. 6A) and upregulation of SOCS3 (Fig. 5E). Apocynin also elevates SOCS1 expression through a yet-to-be-determined mechanism.
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