The Influenza Virus H5N1 Infection Can Induce ROS Production for Viral Replication and Host Cell Death in A549 Cells Modulated by Human Cu/Zn Superoxide Dismutase (SOD1) Overexpression

Xian Lin, Ruifang Wang, Wei Zou, Xin Sun, Xiaokun Liu, Lianzhong Zhao, Shengyu Wang, Meilin Jin, Xian Lin, Ruifang Wang, Wei Zou, Xin Sun, Xiaokun Liu, Lianzhong Zhao, Shengyu Wang, Meilin Jin

Abstract

Highly pathogenic H5N1 infections are often accompanied by excessive pro-inflammatory response, high viral titer, and apoptosis; as such, the efficient control of these infections poses a great challenge. The pathogenesis of influenza virus infection is also related to oxidative stress. However, the role of endogenic genes with antioxidant effect in the control of influenza viruses, especially H5N1 viruses, should be further investigated. In this study, the H5N1 infection in lung epithelial cells decreased Cu/Zn superoxide dismutase (SOD1) expression at mRNA and protein levels. Forced SOD1 expression significantly inhibited the H5N1-induced increase in reactive oxygen species, decreased pro-inflammatory response, prevented p65 and p38 phosphorylation, and impeded viral ribonucleoprotein nuclear export and viral replication. The SOD1 overexpression also rescued H5N1-induced cellular apoptosis and alleviated H5N1-caused mitochondrial dysfunction. Therefore, this study described the role of SOD1 in the replication of H5N1 influenza virus and emphasized the relevance of this enzyme in the control of H5N1 replication in epithelial cells. Pharmacological modulation or targeting SOD1 may open a new way to fight H5N1 influenza virus.

Keywords: H5N1; ROS; SOD1; apoptosis; mitochondria; pro-inflamatory response.

Figures

Figure 1
Figure 1
Highly pathogenic H5N1 influenza virus infection induced oxidative stress in A549 cells. (A) A549 cells were infected by H5N1 (DW), H1N1 (PR8), or control. 24 hpi, mean DCF fluorescence was determined via Flow cytometry as described in methods; (B) A549 cells were infected by DW, PR8, or control, 24 hpi, GSH/GSSH of whole cells was tested under manufacturer’s instruction. Data was mean ± SEM. Student’s t-test was used to analyze statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2
H5N1 infection promoted NADPH oxidases transcription, but downregulated key antioxidative genes transcription. A549 cells were infected by DW at 1 MOI, 24 hpi, total RNA was isolated for qRT-PCR. (A) NADPH oxidases transcription upon H5N1 infection. No-significant (NS) indicates p > 0.05; (B) some key antioxidative genes transcription upon H5N1 infection. GAPDH was used for normalization. Data was shown as relative expression to control infection and mean ± SEM of triplicate reactions. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
SOD1 overexpression inhibited H5N1 virus replication in A549 cells. (A) A549 cells were infected by DW or control at 1 MOI, and the mRNA of SOD1 and the protein were detected by RT-PCR (up) and western blot (down) at indicated time points; (B) SOD1 overexpressed A549 cells were infected by DW at 1 MOI, at different time post-infection, RNA and protein was prepared for NP detection by qRT-PCR (up) or western blot (down) respectively; (C) Cells were infected by DW at 2 MOI, at different time post-infecion, the supernatant was collected for virus titer test by plaque assay; (D) Si-RNA targeting human SOD1 was transfected in A549 cells, 36 h later, DW (2 MOI) was infected, at indicated time post-infection, the supernatant of NC or Si-RNA transfected cells upon virus challenge was collected for virus titer test by plaque assay at indicated time post-infection. NC: negative control of Si-SOD1 RNA. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
SOD1 overexpression attenuated intracellular ROS production and decreased pro-inflammatory response induced by H5N1 virus infection. (A) SOD1 or PCAGGS overexpressed A549 cells were infected by DW at 1 MOI, 24 hpi, cellular ROS level was investigated using H2-DCFDA via Flow cytometry; (B) Total RNA of SOD1 or PCAGGS overexpressed A549 cells infected by DW at indicated time post-infection was extracted for qRT-PCR analysis, and (C) IL-6 and IL-8 levels of supernatant were tested by ELISA. Data was mean ± SEM of triplicate reactions. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
SOD1 overexpression inhibited p65 and p38 phosphorylation, and disrupted nuclear export of NP. (A) Control or DW-infected SOD1 overexpressed cells were lysed for indicated protein analysis using corresponding antibodies at 24 hpi; (B) Cells were pretreated with PDTC (NF-κB inhibitor) and SB203580 (p38 kinase inhibitor) with 100 and 5 μM respectively for 1 h followed by DW infection. At 24 hpi, DCF fluorescence was determined by Flow cytometry; (C) PCAGGS or SOD1 overexpressed A549 cells was infected by DW at 5 MOI, 6 hpi, cells were fixed, permeabilized, and reacted with corresponding primary and secondary antibodies. Fluorescence was viewed under a confocal microscope. NP export rate was calculated by counting at least 50 SOD1-positive or negative cells; (D) SOD1 or control transfected A549 cells were infected by DW at 5 MOI, 6hpi, protein of cytoplasm and nucleus were separated for indicated protein detection using corresponding antibodies. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
Forced SOD1 expression inhibited H5N1-induced cellular apoptosis. (A) A549 cells were infected by DW at 1 MOI, at 24 hpi, apoptosis was detected via Flow cytometry; (B) Western blots of cleaved-caspase-3 in SOD1 or control overexpressed A549 upon infection was detected; (C) caspase-3/7 activation was tested using Caspase-Glo 3/7 assay kit. Data was mean ± SEM. ** p < 0.01.
Figure 7
Figure 7
SOD1 overexpression prevented mitochondrial dysfunction caused by H5N1, SOD1, or PCAGGS transfected A549 cells were infected by 1 MOI DW, at 24 hpi, (A) cells were lysed for ATP detection; (B) MMP was investigated using 15 μM JC-10 by Flow cytometry; and (C) Mitochondrial ROS was tested using 5 μM MitoSox as described in methods. MitoSox mean fluorescence was calculated. * p < 0.05, *** p < 0.001.
Figure 7
Figure 7
SOD1 overexpression prevented mitochondrial dysfunction caused by H5N1, SOD1, or PCAGGS transfected A549 cells were infected by 1 MOI DW, at 24 hpi, (A) cells were lysed for ATP detection; (B) MMP was investigated using 15 μM JC-10 by Flow cytometry; and (C) Mitochondrial ROS was tested using 5 μM MitoSox as described in methods. MitoSox mean fluorescence was calculated. * p < 0.05, *** p < 0.001.

References

    1. De Jong M.D., Simmons C.P., Thanh T.T., Hien V.M., Smith G.J., Chau T.N., Hoang D.M., Chau N.V., Khanh T.H., Dong V.C., et al. Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat. Med. 2006;12:1203–1207. doi: 10.1038/nm1477.
    1. Perrone L.A., Plowden J.K., Garcia-Sastre A., Katz J.M., Tumpey T.M. H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice. PLoS Pathog. 2008;4:13. doi: 10.1371/journal.ppat.1000115.
    1. De Mochel N.S., Seronello S., Wang S.H., Ito C., Zheng J.X., Liang T.J., Lambeth J.D., Choi J. Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection. Hepatology. 2010;52:47–59. doi: 10.1002/hep.23671.
    1. Pal S., Polyak S.J., Bano N., Qiu W.C., Carithers R.L., Shuhart M., Gretch D.R., Das A. Hepatitis C virus induces oxidative stress, DNA damage and modulates the DNA repair enzyme NEIL1. J. Gastroenterol. Hepatol. 2010;25:627–634. doi: 10.1111/j.1440-1746.2009.06128.x.
    1. Gonzalez-Dosal R., Horan K.A., Rahbek S.H., Ichijo H., Chen Z.J., Mieyal J.J., Hartmann R., Paludan S.R. HSV infection induces production of ROS, which potentiate signaling from pattern recognition receptors: Role for S-glutathionylation of TRAF3 and 6. PLoS Pathog. 2011;7:13. doi: 10.1371/journal.ppat.1002250.
    1. Akaike T., Noguchi Y., Ijiri S., Setoguchi K., Suga M., Zheng Y.M., Dietzschold B., Maeda H. Pathogenesis of influenza virus-induced pneumonia: Involvement of both nitric oxide and oxygen radicals. Proc. Natl. Acad. Sci. USA. 1996;93:2448–2453. doi: 10.1073/pnas.93.6.2448.
    1. Torres M., Forman H.J. Redox signaling and the MAP kinase pathways. BioFactors. 2003;17:287–296. doi: 10.1002/biof.5520170128.
    1. Gloire G., Legrand-Poels S., Piette J. NF-κB activation by reactive oxygen species: Fifteen years later. Biochem. Pharmacol. 2006;72:1493–1505. doi: 10.1016/j.bcp.2006.04.011.
    1. Asehnoune K., Strassheim D., Mitra S., Kim J.Y., Abraham E. Involvement of reactive oxygen species in toll-like receptor 4-dependent activation of NF-κB. J. Immunol. 2004;172:2522–2529. doi: 10.4049/jimmunol.172.4.2522.
    1. Brydon E.W., Morris S.J., Sweet C. Role of apoptosis and cytokines in influenza virus morbidity. FEMS Microbiol. Rev. 2005;29:837–850. doi: 10.1016/j.femsre.2004.12.003.
    1. Finkel T., Holbrook N.J. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408:239–247. doi: 10.1038/35041687.
    1. Oda T., Akaike T., Hamamoto T., Suzuki F., Hirano T., Maeda H. Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer-conjugated SOD. Science. 1989;244:974–976. doi: 10.1126/science.2543070.
    1. Imai Y., Kuba K., Neely G.G., Yaghubian-Malhami R., Perkmann T., van Loo G., Ermolaeva M., Veldhuizen R., Leung Y.H., Wang H., et al. Identification of oxidative stress and Toll-Like receptor 4 signaling as a key pathway of acute lung injury. Cell. 2008;133:235–249. doi: 10.1016/j.cell.2008.02.043.
    1. Bedard K., Krause K.H. The NOX family of ROS-generating nadph oxidases: Physiology and pathophysiology. Physiol. Rev. 2007;87:245–313. doi: 10.1152/physrev.00044.2005.
    1. Jacoby D.B., Choi A.M. Influenza virus induces expression of antioxidant genes in human epithelial cells. Free Radic. Biol. Med. 1994;16:821–824. doi: 10.1016/0891-5849(94)90198-8.
    1. Knobil K., Choi A.M., Weigand G.W., Jacoby D.B. Role of oxidants in influenza virus-induced gene expression. Am. J. Physiol. 1998;274:L134–L142.
    1. Droge W. Free radicals in the physiological control of cell function. Physiol. Rev. 2002;82:47–95. doi: 10.1152/physrev.00018.2001.
    1. Selemidis S., Sobey C.G., Wingler K., Schmidt H.H., Drummond G.R. NADPH oxidases in the vasculature: molecular features, roles in disease and pharmacological inhibition. Pharmacol. Ther. 2008;120:254–291. doi: 10.1016/j.pharmthera.2008.08.005.
    1. Vlahos R., Stambas J., Bozinovski S., Broughton B.R., Drummond G.R., Selemidis S. Inhibition of NOX 2 oxidase activity ameliorates influenza a virus-induced lung inflammation. PLoS Pathog. 2011;7:13. doi: 10.1371/journal.ppat.1001271.
    1. Amatore D., Sgarbanti R., Aquilano K., Baldelli S., Limongi D., Civitelli L., Nencioni L., Garaci E., Ciriolo M.R., Palamara A.T. Influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by NOX4-derived ROS. Cell Microbial. 2015;17:131–145. doi: 10.1111/cmi.12343.
    1. Thannickal V.J., Fanburg B.L. Reactive oxygen species in cell signaling. Am. J. Physiol. Lung Cell Mol. Physiol. 2000;279:L1005–L1028.
    1. Suliman H.B., Ryan L.K., Bishop L., Folz R.J. Prevention of influenza-induced lung injury in mice overexpressing extracellular superoxide dismutase. Am. J. Physiol. Lung Cell Mol. Physiol. 2001;280:L69–L78.
    1. Rivas-Estilla A.M., Bryan-Marrugo O.L., Trujillo-Murillo K., Perez-Ibave D., Charles-Nino C., Pedroza-Roldan C., Rios-Ibarra C., Ramirez-Valles E., Ortiz-Lopez R., Islas-Carbajal M.C., et al. Cu/Zn superoxide dismutase (SOD1) induction is implicated in the antioxidative and antiviral activity of acetylsalicylic acid in HCV-expressing cells. Am. J. Physiol. Gastrointest. Liver Physiol. 2012;302:G1264–G1273. doi: 10.1152/ajpgi.00237.2011.
    1. Dimayuga F.O., Wang C., Clark J.M., Dimayuga E.R., Dimayuga V.M., Bruce-Keller A.J. SOD1 overexpression alters ROS production and reduces neurotoxic inflammatory signaling in microglial cells. J. Neuroimmunol. 2007;182:89–99. doi: 10.1016/j.jneuroim.2006.10.003.
    1. Pyo C.W., Shin N., Jung K.I., Choi J.H., Choi S.Y. Alteration of Copper-Zinc superoxide dismutase 1 expression by influenza A virus is correlated with virus replication. Biochem. Biophys. Res. Commun. 2014;450:711–716. doi: 10.1016/j.bbrc.2014.06.037.
    1. Pleschka S., Wolff T., Ehrhardt C., Hobom G., Planz O., Rapp U.R., Ludwig S. Influenza virus propagation is impaired by inhibition of the RAF/MEK/ERK signalling cascade. Nat. Cell Biol. 2001;3:301–305. doi: 10.1038/35060098.
    1. Nencioni L., De Chiara G., Sgarbanti R., Amatore D., Aquilano K., Marcocci M.E., Serafino A., Torcia M., Cozzolino F., Ciriolo M.R., et al. Bcl-2 expression and P38MAPK activity in cells infected with influenza A virus: Impact on virally induced apoptosis and viral replication. J. Biol. Chem. 2009;284:16004–16015. doi: 10.1074/jbc.M900146200.
    1. Shin N., Pyo C.W., Jung K.I., Choi S.Y. Influenza a virus PB1-F2 is involved in regulation of cellular redox state in alveolar epithelial cells. Biochem. Biophys. Res. Commun. 2015;459:699–705. doi: 10.1016/j.bbrc.2015.03.010.
    1. Cheng Z., Ristow M. Mitochondria and metabolic homeostasis. Antioxid. Redox Signal. 2013;19:240–242. doi: 10.1089/ars.2013.5255.
    1. Dada L.A., Sznajder J.I. Mitochondrial Ca2+ and ROS take center stage to orchestrate TNF-α-mediated inflammatory responses. J. Clin. Investig. 2011;121:1683–1685. doi: 10.1172/JCI57748.
    1. Kim R., Emi M., Tanabe K. Role of mitochondria as the gardens of cell death. Cancer Chemother. Pharmacol. 2006;57:545–553. doi: 10.1007/s00280-005-0111-7.
    1. Peterhans E., Grob M., Burge T., Zanoni R. Virus-induced formation of reactive oxygen intermediates in phagocytic cells. Free Radic. Res. Commun. 1987;3:39–46. doi: 10.3109/10715768709069768.
    1. Masella R., di Benedetto R., Vari R., Filesi C., Giovannini C. Novel mechanisms of natural antioxidant compounds in biological systems: involvement of Glutathione and Glutathione-related enzymes. J. Nutr. Biochem. 2005;16:577–586. doi: 10.1016/j.jnutbio.2005.05.013.
    1. Reshi M.L., Su Y.C., Hong J.R. RNA Viruses: ROS-mediated cell death. Int. J. Cell Biol. 2014;2014:467452. doi: 10.1155/2014/467452.
    1. Ciriolo M.R., Palamara A.T., Incerpi S., Lafavia E., Bue M.C., de Vito P., Garaci E., Rotilio G. Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection. J. Biol. Chem. 1997;272:2700–2708. doi: 10.1074/jbc.272.5.2700.
    1. Garaci E., Palamara A.T., di Francesco P., Favalli C., Ciriolo M.R., Rotilio G. Glutathione inhibits replication and expression of viral proteins in cultured cells infected with Sendai virus. Biochem. Biophys. Res. Commun. 1992;188:1090–1096. doi: 10.1016/0006-291X(92)91343-O.
    1. Nucci C., Palamara A.T., Ciriolo M.R., Nencioni L., Savini P., D’Agostini C., Rotilio G., Cerulli L., Garaci E. Imbalance in corneal redox state during Herpes Simplex Virus 1-induced keratitis in rabbits. Effectiveness of exogenous glutathione supply. Exp. Eye Res. 2000;70:215–220. doi: 10.1006/exer.1999.0782.
    1. Nencioni L., Iuvara A., Aquilano K., Ciriolo M.R., Cozzolino F., Rotilio G., Garaci E., Palamara A.T. Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. FASEB J. 2003;17:758–760. doi: 10.1096/fj.02-0508fje.
    1. Braakman I., Helenius J., Helenius A. Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum. Nature. 1992;356:260–262. doi: 10.1038/356260a0.
    1. Sgarbanti R., Nencioni L., Amatore D., Coluccio P., Fraternale A., Sale P., Mammola C.L., Carpino G., Gaudio E., Magnani M., et al. Redox regulation of the influenza hemagglutinin maturation process: A new cell-mediated strategy for anti-influenza therapy. Antioxid. Redox Signal. 2011;15:593–606. doi: 10.1089/ars.2010.3512.
    1. Hosakote Y.M., Liu T., Castro S.M., Garofalo R.P., Casola A. Respiratory syncytial virus induces oxidative stress by modulating antioxidant enzymes. Am. J. Respir. Cell Mol. Biol. 2009;41:348–357. doi: 10.1165/rcmb.2008-0330OC.
    1. Nguyen T., Nioi P., Pickett C.B. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J. Biol. Chem. 2009;284:13291–13295. doi: 10.1074/jbc.R900010200.
    1. Minc E., de Coppet P., Masson P., Thiery L., Dutertre S., Amor-Gueret M., Jaulin C. The human Copper-Zinc superoxide dismutase gene (SOD1) proximal promoter is regulated by SP1, EGR-1, and WT1 via non-canonical binding sites. J. Biol. Chem. 1999;274:503–509. doi: 10.1074/jbc.274.1.503.
    1. Afonso V., Santos G., Collin P., Khatib A.M., Mitrovic D.R., Lomri N., Leitman D.C., Lomri A. Tumor necrosis factor-α down-regulates human Cu/Zn superoxide dismutase 1 promoter via JNK/AP-1 signaling pathway. Free Radic. Biol. Med. 2006;41:709–721. doi: 10.1016/j.freeradbiomed.2006.05.014.
    1. Wurzer W.J., Ehrhardt C., Pleschka S., Berberich-Siebelt F., Wolff T., Walczak H., Planz O., Ludwig S. NF-κB-dependent induction of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and FAS/FASl is crucial for efficient influenza virus propagation. J. Biol. Chem. 2004;279:30931–30937. doi: 10.1074/jbc.M403258200.
    1. Cohen G.M. Caspases: The executioners of apoptosis. Biochem. J. 1997;326 Pt 1:1–16. doi: 10.1042/bj3260001.
    1. Sanghavi D.M., Thelen M., Thornberry N.A., Casciola-Rosen L., Rosen A. Caspase-mediated proteolysis during apoptosis: Insights from apoptotic neutrophils. FEBS Lett. 1998;422:179–184. doi: 10.1016/S0014-5793(98)00004-0.
    1. Slee E.A., Adrain C., Martin S.J. Serial killers: Ordering caspase activation events in apoptosis. Cell Death Differ. 1999;6:1067–1074. doi: 10.1038/sj.cdd.4400601.
    1. Wurzer W.J., Planz O., Ehrhardt C., Giner M., Silberzahn T., Pleschka S., Ludwig S. Caspase 3 activation is essential for efficient influenza virus propagation. EMBO J. 2003;22:2717–2728. doi: 10.1093/emboj/cdg279.
    1. Geiler J., Michaelis M., Naczk P., Leutz A., Langer K., Doerr H.W., Cinatl J., Jr. N-acetyl-l-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza a virus. Biochem. Pharmacol. 2010;79:413–420. doi: 10.1016/j.bcp.2009.08.025.
    1. Zamzami N., Susin S.A., Marchetti P., Hirsch T., Gomez-Monterrey I., Castedo M., Kroemer G. Mitochondrial control of nuclear apoptosis. J. Exp. Med. 1996;183:1533–1544. doi: 10.1084/jem.183.4.1533.
    1. Shimizu S., Narita M., Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome C by the mitochondrial channel VDAC. Nature. 1999;399:483–487.
    1. Zamarin D., Garcia-Sastre A., Xiao X., Wang R., Palese P. Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1. PLoS Pathog. 2005;1:13. doi: 10.1371/journal.ppat.0010004.
    1. Ricci J.E., Gottlieb R.A., Green D.R. Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. J. Cell Biol. 2003;160:65–75. doi: 10.1083/jcb.200208089.
    1. Richter C., Schlegel J. Mitochondrial calcium release induced by prooxidants. Toxicol. Lett. 1993;67:119–127. doi: 10.1016/0378-4274(93)90050-8.
    1. Ueda M., Daidoji T., Du A., Yang C.S., Ibrahim M.S., Ikuta K., Nakaya T. Highly pathogenic H5N1 avian influenza virus induces extracellular Ca2+ influx, leading to apoptosis in avian cells. J. Virol. 2010;84:3068–3078. doi: 10.1128/JVI.01923-09.

Source: PubMed

Sponsors en medewerkers

Medische omstandigheden

Geneesmiddelinterventies

3
Abonneren