Original contribution
Inhibition of influenza infection by glutathione

https://doi.org/10.1016/S0891-5849(03)00023-6Get rights and content

Abstract

Infection by RNA virus induces oxidative stress in host cells. Accumulating evidence suggests that cellular redox status plays an important role in regulating viral replication and infectivity. In this study, experiments were performed to determine whether the thiol antioxidant glutathione (GSH) blocked influenza viral infection in cultures of Madin-Darby canine kidney cells or human small airway epithelial cells. Protection against production of active virus particles was observed at a low (0.05–0.1) multiplicity of infection (MOI). GSH inhibited expression of viral matrix protein and inhibited virally induced caspase activation and Fas upregulation. In BALB/c mice, inclusion of GSH in the drinking water decreased viral titer in both lung and trachea homogenates 4 d after intranasal inoculation with a mouse-adapted influenza strain A/X-31. Together, the data suggest that the thiol antioxidant GSH has an anti-influenza activity in vitro and in vivo. Oxidative stress or other conditions that deplete GSH in the epithelium of the oral, nasal, and upper airway may, therefore, enhance susceptibility to influenza infection.

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.

References (38)

  • D.P. Jones

    Redox potential of GSH/GSSG coupleassay and biological significance

    Methods Enzymol

    (2002)
  • M.A. Beck

    The influence of antioxidant nutrients on viral infection

    Nutr. Rev

    (1998)
  • E. Peterhans

    Oxidants and antioxidants in viral diseasesdisease mechanisms and metabolic regulation

    J. Nutr

    (1997)
  • W. Droge et al.

    Functions of glutathione and glutathione disulfide in immunology and immunopathology

    FASEB J

    (1994)
  • Staal, F. J.; Roederer, M.; Israelski, D. M.; Bubp, J.; Mole, L. A.; McShane, D.; Deresinski, S. C.; Ross, W.; Sussman,...
  • B. de Quay et al.

    Glutathione depletion in HIV-infected patientsrole of cysteine deficiency and effect of oral N-acetylcytseine

    AIDS

    (1992)
  • F. Jahoor et al.

    Erythrocyte glutathione deficiency in symptom-free HIV infection is associated with decreased synthesis rate

    Am. J. Physiol

    (1999)
  • A.M.K. Choi et al.

    Oxidant stress responses in influenza virus pneumonia gene expression and transcription factor activation

    Am. J. Physiol

    (1996)
  • Observations on effect of sodium selenite in prevention of Keshan disease

    Chin. Med. J

    (1979)
  • Cited by (173)

    • Tackle the free radicals damage in COVID-19

      2020, Nitric Oxide - Biology and Chemistry
      Citation Excerpt :

      Similarly, erythromycin may be effective for COVID-19 as it can also suppress the generation of NO and superoxide [29]. Other less toxic antioxidant medicines such as glutathione [30] and N-acetylcysteine [31] should also be considered for COVID-19 treatment. The Koch's postulates are the dogma of infectious disease, hence people focus on targeting SARS-CoV-2 in COVID-19.

    View all citing articles on Scopus
    View full text