Original contributionInhibition of influenza infection by glutathione
Introduction
Viral infection is often associated with redox changes characteristic of oxidative stress 1, 2. Cultured cells infected with herpes simplex virus type 1 [3], Sendai virus [4], and human immunodeficiency virus (HIV) [5] have decreased intracellular GSH, increased generation of reactive oxygen species (ROS), and oxidation of the cellular GSH pool. T cells isolated from HIV-infected patients have lower cysteine and glutathione contents 6, 7. Plasma GSH drops significantly even in symptom-free HIV-infected patients [8]. Viral infection often activates transcription factors, such as AP-1 and NF-κB, by redox-dependent mechanisms [9] and leads to increased production of various cytokines that contribute to most of the symptoms and tissue damage [10].
A more oxidized environment can favor viral infection. Coxsackie virus infection is greatly potentiated by selenium deficiency [1], which can result in decreased activities of selenoproteins with antioxidant function such as glutathione peroxidase and thioredoxin reductase [11]. Patients with low selenium intake have higher risk of developing virally induced cardiomyopathy and Keshan disease [12]. Infection of Se-deficient mice with an amyocarditic strain of Coxsackie virus converted the virus to a much more virulent form [13]. Extensive studies have demonstrated that HIV replication and viral protein synthesis are stimulated by oxidants 14, 15.
Influenza viral infection induces oxidative stress in mice 16, 17. The tissue concentrations of the antioxidants glutathione and ascorbic acid decreased in lung after infection [17]. The bronchoalveolar lavage fluid from infected mice showed an increased rate of superoxide production [18], increased activity of the O2−-generating enzyme xanthine oxidase 16, 19, decreased total glutathione, increased GSSG, and an increased level of malondialdehyde, which is an indicator of lipid peroxidation [16]. Transgenic mice carrying overexpressed extracellular superoxide dismutase had less severe lung injury after influenza infection [16]. In contrast, selenium-deficient mice had more severe inflammatory response in the lung [20], and the infected virus became more virulent with increased mutations in viral M1 gene [21]. Intravenous injection of a pyran copolymer-conjugated Cu,ZnSOD protected mice from death induced by influenza viral infection [19].
The purpose of this study was to determine whether supplemental GSH, a naturally occuring thiol antioxidant that is also present intracellularly at a high concentration, has an anti-influenza effect. Results show that GSH significantly inhibited production of active influenza virus both in cultured MDCK cells and a normal human small airway epithelial cell line. Consistent with these in vitro data, the inclusion of GSH in the drinking water of mice inoculated with influenza inhibited viral titer in trachea and lung. These results indicate that supplemental GSH has an anti-influenza activity and suggest that oxidative stress in vivo may enhance susceptibility to infection.
Section snippets
Viral stock and cell culture
Viral stock of influenza A/WSN strain was prepared by infecting cultured Madin-Darby bovine kidney (MDBK) cells. Mouse-adapted influenza A/X-31 strain was provided by Dr. Jacqueline Katz (Centers for Disease Control and Prevention, Atlanta, GA, USA). This strain of virus was grown in chicken eggs and purified from allantoic fluid. Normal human small airway epithelial cells (SAECs) were obtained from Clonetics (San Diego, CA, USA) and cultured in serum-free bronchial/tracheal epithelial cell
Effect of GSH on influenza virus infection of cultured MDCK cells
To determine whether GSH has an antiviral effect in in vitro cultured cells, we infected MDCK cells with influenza A/WSN strain at 0.05–0.1 pfu/cell, and subsequently we cultured the cells for 2 to 3 d in the presence of different concentrations of GSH. Viral particle production was detectable as early as 12 h postinfection, as measured by HA assay (data not shown). At 48 h postinfection, the viral HA titer was 1:512–1:1024. At concentrations of 5 mM or higher, GSH in the culture medium
Discussion
Oxidative stress, either systemically or localized within the infected tissues and cells, might be a common consequence of RNA virus infection [1]. Therefore, antioxidants are potentially useful strategies against either viral infection or infection-associated symptoms. Overexpressing extracellular superoxide dismutase in transgenic mice attenuated tissue damage and inflammatory response, although its inhibition of viral production was only marginal and not statistically significant [16].
Abbreviations
GSH—glutathione
HA assay—hemagglutination
MDCK cells—Madin-Darby canine kidney cells
MOI—multiplicity of infection
pfu—plaque-forming unit
SAEC—small airway epithelial cell
Acknowledgements
The authors thank Dr. Jacqueline Katz (Centers for Disease Control and Prevention, Atlanta, GA, USA) for her generous support with experimental materials as well as helping us to set up the in vivo and in vitro models of influenza infection. This research is partly supported by NIH grant ES 09047 and by Kyowa Hakko Co., Ltd.
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