Inhibition of influenza infection by glutathione

Free Radical Biology & Medicine, Vol. 34, No. 7, pp. 928 –936, 2003 Copyright © 2003 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter

doi:10.1016/S0891-5849(03)00023-6

Original Contribution INHIBITION OF INFLUENZA INFECTION BY GLUTATHIONE JIYANG CAI,* YAN CHEN,* SHAGUNA SETH,† SATORU FURUKAWA,‡ RICHARD W. COMPANS,† DEAN P. JONES*

and

*Department of Biochemistry, †Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA; and ‡Nutri-Quest, Inc., Chesterfield, MO, USA (Received 27 August 2002; Revised 23 December 2002; Accepted 9 January 2003)

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. © 2003 Elsevier Science Inc. Keywords—Influenza, Glutathione, Antioxidant, Redox, Free radicals

INTRODUCTION

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-conju-

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 symptomfree HIV-infected patients [8]. Viral infection often activates transcription factors, such as AP-1 and NF-␬B, 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 Address correspondence to: Dr. Jiyang Cai, Emory University School of Medicine, Department of Biochemistry, Atlanta, GA 30322, USA; Tel: (404) 727-5865; Fax: (404) 727-3231; E-Mail: [email protected]. 928

Anti-influenza activity of glutathione

gated 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 antiinfluenza activity and suggest that oxidative stress in vivo may enhance susceptibility to infection.

MATERIALS AND METHODS

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 growth medium (Clonetics, San Diego, CA, USA), according to the manufacturer’s instructions. Madin-Darby canine kidney (MDCK) cells and MDBK cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen). Viral infection and GSH treatment When 90% confluent, MDCK cells were washed twice with phosphate-buffered saline (PBS) to remove residual FBS and viral infection was performed as described previously [22]. Briefly, viral stock was inoculated at 0.05– 0.1 plaque-forming units (pfu)/cell in serum-free DMEM medium for 2 h. Cells were then washed with PBS and cultured in DMEM ⫹ 2% FBS, either with or without GSH, for 48 h. Following the addition of GSH to culture medium, pH was adjusted to 7.4 before sterile filtration. For some experiments, GSH was added either before or during viral inoculation. The same infection protocol was used for SAECs cells, except cells at 60% confluence were used because SAECs cells undergo irreversible contact inhibition and differentiation when cultured at high density.

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Hemagglutination (HA) assay Chicken red blood cells (cRBCs) were obtained from Lampire Biological Laboratories (Pipersville, PA, USA). HA assay was carried out in 96-well microtiter plates [22]. Virus-containing cell culture medium was diluted serially by 2-fold to 1:2048 and 0.5% cRBCs (diluted in phosphate-buffered saline) were then added at an equal volume. After 60 min incubation at room temperature, cRBCs in negative wells sedimented and formed red buttons, whereas positive wells had a diffuse appearance with no sedimentation. Plaque assay MDCK cells were cultured in 6-well plates and infected with virus at 10-fold serial dilutions. After removal of unadsorbed virus by washing with PBS, cells were overlaid with DMEM with 1.9% white agar prewarmed to 42°C. After the agar had solidified, the plates were inverted and transferred to a 37°C humidified CO2 incubator. Viral infection resulted in opaque plaques, which were readily visible after 2 to 3 d. In vivo viral infection Female Balb/c mice, 6 to 8 weeks old, were obtained from Harlan (Indianapolis, IN, USA). Preliminary studies with the viral titer of X-31 strain at 109.5/ml was used to establish a suitable dilution and dosing. Mice were lightly anesthetized with CO2 and 50 ␮l diluted virus (1:189,000) was given intranasally. This dose equals 15 mouse infection doses (MID). After 4 d, mice were sacrificed. Virus in lung and trachea homogenates was detected by infecting cultured MDCK cells. GSH measurement Immediately after dissection, lung homogenates were added to an equal volume of 10% perchloric acid with saturated boric acid containing 10 ␮M ␥-glutamyl glutamate as an internal standard. GSH in the acid-soluble fraction was measured by HPLC, as described previously [23]. Apoptosis assay Two days after infection, both attached cells and floating cells were collected and pooled together. Cell viability was determined by staining with LIVE/DEAD viability/cytotoxicity assay kit (Molecular Probes, Eugene, OR, USA) followed by flow cytometry, as described previously [24]. For caspase measurement, cells were stained with 10 ␮M FITC-VAD-FMK (Promega, Madison, WI, USA) for 30 min at 37°C in the dark, washed once with PBS, and analyzed by flow cytometry. For cell surface Fas expression, cells were stained with R-phycoerythrin conjugated anti-Fas antibody (PharMingen, San Diego, CA, USA) at a

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Fig. 1. Effect of GSH on influenza viral titer at low MOI in cultured MDCK cells. MDCK cells were infected with influenza A/WSN strain at 0.05– 0.1 pfu/cell for 2 h. After removal of viral inoculation and washing with PBS, cells were maintained in DMEM medium supplemented with 2% serum for 48 h, with the addition of GSH at the indicated concentrations. Viral production in the medium was determined by HA assay. *Significant at p ⬍ .05, n ⫽ 6; one-way ANOVA with linear trend.

concentration of 0.2 ␮g/million cells, according to the manufacturer’s instructions. Measurement of viral matrix protein At 24 h postinfection, cells were collected by gentle scraping and centrifuged at 300 ⫻ g for 3 min. Pellets were resuspended in lysis buffer (50 mM HEPES, pH 7.0, 500 mM NaCl, 1% Nonidet P-40, supplemented with protease inhibitors). After determination of protein concentrations, the viral matrix protein content was determined by SDS-PAGE followed by Western blot analysis with an antibody from ViroStat (Portland, ME, USA). Quantitation of the chemiluminescent signals was performed with a Typhoon 8600 Variable Mode Imager (Amersham Biosciences, Piscataway, NJ, USA).

Fig. 2. Effect of GSH on expression of influenza matrix protein. MDCK cells were infected with influenza A/WSN and cultured for 24 h with the addition of GSH at the indicated concentrations. Viral matrix protein (MP) was measured by Western blot analysis. The relative intensities (RI) of the bands were quantitated and normalized to without GSH. Heat shock protein 70 (HSC70) was used as a loading control. The data presented are representative of three separate experiments.

lowered down to 1:32–1:64. This concentration of GSH is not toxic to MDCK cells, as indicated by their normal morphology and capability of excluding trypan blue (data not shown). Similar results were obtained by measuring viral matrix protein expression at 24 h postinfection (Fig. 2). Therefore, we conclude that GSH showed substantial protection against influenza viral infection in MDCK cells. To determine whether high concentrations of GSH would directly inactivate the virus particle or alter viral binding to MDCK cells, up to 30 mM GSH was added either before or during the viral inoculation period, and viral production was measured by HA assay 48 h later. As shown in Fig. 3, pretreatment of the active virus with 30 mM GSH for 1 h had no effect on infectivity. Similarly, inclusion of 30 mM GSH in the medium only during the viral inocula-

RESULTS

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 significantly inhibited viral production (Fig. 1). At 30 mM, the HA titer was

Fig. 3. Effect of time of addition of GSH on viral production. In addition to treatment of infected cells (■), GSH was either included during the inoculation period (⽧) or was incubated with viral stock prior to inoculation for 1 h at 4°C (●). Viral production was determined by hemagglutinin assay 48 h later. *Significant at p ⬍ .05, n ⫽ 3.

Anti-influenza activity of glutathione

Fig. 4. Effect of influenza infection on cellular GSH. GSH was measured in MDCK cells after infection either with or without the addition of 10 mM GSH. Values are means of two separate experiments with triplicate for each condition.

tion period did not affect the viral production (Fig. 3). GSH inhibited viral production only when it was continuously present in the medium after inoculation. Thus, the observed in vitro antiviral effect of GSH was not due to a direct inactivation of influenza virus and also was not due to interference with the cell adherence and uptake of the virus. Intracellular GSH is not affected by adding GSH to the culture medium Infection by influenza has been reported to deplete GSH and oxidize the cellular glutathione pool [16]. To determine whether exogenous GSH could affect virally induced changes in cellular glutathione, we measured the amount of GSH in control and inoculated cells (Fig. 4A). The results show that the addition of 10 mM GSH did not affect intracellular GSH in control cells, indicating that the MDCK cells did not transport GSH. In cells infected with the influenza virus, cellular GSH dropped by 80% after 24 h and this was not prevented by supplementation of 10 mM GSH in the medium (Fig. 4A). GSH was relatively stable in the cell culture medium after 24 h (Fig. 4B). Thus, in MDCK cells, addition of GSH in the culture medium did not affect the intracellular GSH concentration either before or after viral infection. These results suggest that the effect of GSH occurs extracellularly. Effect of GSH on virally induced apoptosis Influenza viral infection is lethal to host cells and has been shown to trigger apoptotic cell death [25,26]. At low MOI, inhibition of apoptosis could slow viral release and

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provide a mechanism for protection against transmission of the virus to noninfected cells. To determine whether GSH protected against virally induced cell death, we inoculated cells at MOI of 0.05 to 0.1 and measured the viable cells 2 d postinfection. As shown in Fig. 5A, the addition of GSH caused a significant increase of viable cells as compared to infection in the absence of GSH. Apoptotic cell death is a consequence of caspase activation. When stained with a cell permeable fluorogenic substrate, FITC-VAD-FMK, fewer cells showed increased caspase 3-like activity (Fig. 5B). Thus, GSH inhibited influenza-induced apoptosis in MDCK cells. Influenza-induced caspase activation has been found to involve an activation of the cell surface death receptor Fas-mediated pathway [25,26]. The effect of GSH on Fas expression in MDCK cells was measured by flow cytometry 48 h postinfection (Fig. 6). Viral infection upregulated Fas expression, and this effect was inhibited by GSH treatment. Under similar conditions, Fas ligand expression was not affected by GSH (data not shown). Effect of GSH on influenza viral infection in cultured normal epithelial cells We performed experiments with normal human small airway epithelial cells to see whether a similar antiviral effect could be obtained with human cells that are closer to the natural human host cells of influenza. SAECs were inoculated with WSN/A virus at MOI of 0.05 to 0.1 under conditions used for MDCK cells. After 48 h, HA titer in the culture medium of SAECs was 1:128 –1:256, i.e., much lower than that of MDCK cells. The production of active virus was also confirmed by plaque assay (data not shown). Consistent with the data obtained from MDCK cells, the addition of GSH at 10 and 30 mM significantly inhibited viral particle production in SAEC as measured by HA titer (Fig. 7). Effects of GSH on influenza viral infection in mice To determine whether GSH has preventive effects against influenza infection in a mouse model of infection, a mouse-adapted strain, influenza A/X-31, was chosen to infect Balb/c mice [27], based on published protocols. Preliminary experiments showed that the inclusion of 50 mM GSH in the drinking water had no effect on mouse appearance, activity, or weight over a 5 d period. Drinking water containing this concentration of GSH was then administered 1 d before intranasal inoculation with the virus at 15 MID, and the water with GSH was replaced every 12 h. This solution was slightly acidic (pH 5.5– 6.0) and was not extensively oxidized under these conditions (data not shown). A control group treated identically except for the omission of GSH from the drinking water was studied concurrently.

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Fig. 5. Effect of GSH on viral infection-induced apoptosis. (A) Cell viability was determined at 48 h postinfection by ethidium homodimer and calcein staining [24]. Results are from three experiments. **Significantly different from virus alone at p ⬍ .01, one-way ANOVA. (B) Caspase activation was measured by staining with FITC-VAD-FMK. Cells with increased caspase-3-like activity show higher fluorescence. Data presented are representative of four separate experiments.

The infection dose used was not lethal to the mice and no animal died during the experimental period. At 4 d postinfection, mice were sacrificed, weighed, and both trachea and lung homogenates were prepared. No weight loss was observed in any one of the groups (data not shown). Active virus in the homogenates was determined by infecting cultured MDCK cells. The results showed a significant decrease of viral titer in lungs and trachea of mice with GSH in the drinking water (Fig. 8). Measurements of GSH in lung showed no differences due to either virus or GSH under these conditions (Fig. 9). Thus, GSH in the drinking water did not exert its effect on active virus production by preservation of the total lung GSH content. Instead, GSH probably had an effect in the oral, nasal, or upper airway epithelium. Effects of other antioxidants on influenza virus in vitro The in vitro effects of ascorbate phosphate, curcumin, and tetrahydrocurcumin on influenza virus were determined with MDCK cells (Fig. 10). Compared to ascorbic acid, ascorbate phosphate is more stable in aqueous solution [28]. At MOI of 0.05 to 0.1, a dose at which 10

mM GSH effectively inhibited viral production, 10 mM ascorbate phosphate only showed some marginal protection. Ascorbate phosphate was not effective at concentrations lower than 10 mM and was toxic to MDCK cells at higher concentration (data not shown). Curcumin is a naturally occurring compound extracted from turmeric and has been shown to have a broad range of anticancer and anti-inflammatory effects [29]. However, 10 ␮M curcumin and 100 ␮M of the synthetic derivative tetrahydrocurcumin failed to provide any protection in MDCK cells. 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

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Fig. 6. Effect of GSH on viral infection-induced Fas upregulation. Cells were stained with R-phycoerythrin-conjugated anti-Fas antibody 48 h postinfection. On contour plots, cells with increased Fas expression are located at the upper right corner. Data presented are representative of three separate experiments.

only marginal and not statistically significant [16]. Injection of pyran polymer-conjugated SOD previously has been reported to prevent lethality caused by influenza virus [19]. In a clinical trial with a total of 262 subjects, continuous supplementation of NAC at the dose of 600 mg twice daily for 6 months showed significant protection against flu-like symptoms, although NAC may not protect the rate of infection per se [30]. GSH is a major water-soluble antioxidant that is normally present in epithelial cells at millimolar concentrations [31] and regulates a variety of cellular functions by redox-dependent mechanisms [32]. The results of the present study show that, when added extracellularly, GSH had a dose-dependent anti-influenza effect in cultured cells. This was confirmed with two different types of cell lines by hemagglutanin assay, plaque assay, and Western blot analysis. Protection was also observed with an in vivo mouse model with a nonlethal dose for infection, which reflects infection at low MOI. Because GSH is an abundant natural antioxidant that is broken down to

Fig. 7. Effect of GSH on viral infection in normal human small airway epithelial cells (SAEC). After removing viral inoculation, cells were cultured for up to 72 h in medium with or without GSH. Viral production was determined by HA assay and normalized to the titer in the absence of GSH.

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Fig. 10. Effect of other antioxidant compounds on influenza viral production in vitro. After viral inoculation, MDCK cells were cultured for 2 d in the presence of different compounds. Active virus in the medium was measured with plaque assay and normalized to virus alone. GSH (10) ⫽ 10 mM GSH; Asc-P (1) and (10) ⫽ ascorbate phosphate at 1 mM and 10 mM; TH-curcumin ⫽ tetrahydrocurcumin. Data presented are representative of two separate experiments. *Significant at p ⬍ .05, one-way ANOVA.

Fig. 8. Effect of GSH on low-dose viral infection in mice. Balb/c mice were infected with influenza A/X-31 strain at 15 mouse infection dose (MID); 50 mM GSH was added to the drinking water. Viral production in both trachea and lung homogenates were assayed 4 d after infection. GSH showed statistically significant protection both in lung and trachea. *p ⬍ .05, Student’s t-test; n ⫽ 8 –10 per group for each of two experiments.

normal amino acids and has no known toxicity, it may be useful in the prevention of influenza infection. GSH may protect against influenza infection by multiple mechanisms. As measured by both viability assay and caspase activity assay, GSH inhibited apoptosis of infected MDCK cells (Fig. 5). Viral infection often results in apoptosis in host cells, and the release of active virus from dead cells can initiate subsequent rounds of infection. Therefore, an inhibition of apoptosis by anti-

Fig. 9. Effect of GSH supplement on lung GSH. Mice were fed with 50 mM GSH in their drinking water. GSH concentration of lung homogenates was measured by HPLC. No significant differences were detected; n ⫽ 8 –10 per group for each of two experiments.

oxidants, as seen in many other systems [33], can reduce infection at low MOI. Caspase activation is a tightly controlled process that is subjected to redox regulation at different stages [33]. Consistent with our previous findings on human retinal pigmet epithelial cells [34], extracellular GSH inhibited the upregulation of death receptor Fas and thereby inhibited the initiation of caspase activation. Thus, a central component of the mechanism for inhibition of influenza infection appears to be GSH-dependent inhibition of apoptosis. An additional mechanism for protection by extracellular GSH could also occur if post-transcriptional processing of viral peptides is redox dependent. The viral hemagglutanin antigen must be proteolytically cleaved for the virus to bind to cell surface glycoproteins and be internalized. This processing occurs for some strains in the endoplasmic reticulum but can also occur extracellularly. Hennet et al. examined whether proteolytic activation in the lung lining fluid could be potentiated by oxidants [17]. Their results showed that hypochlorite treatment resulted in a 10,000-fold activation of the virus due to oxidant inactivation of the protease inhibitor that prevented cleavage of HA. In principle, this mechanism could have a critical role in determining susceptibility to influenza infection, since the capacity to maintain antiproteases in their reduced and functional form may be limited in the fluids lining the oral, nasal, and upper airway epithelia. Such a mechanism could be especially relevant to transmission at low MOI. The present study only measured viral titer and lung GSH. Additional studies are needed to examine the effects of GSH on lung pathology after influenza infection. GSH supplementation has been shown to enhance T cell-mediated immune response in aging mice [35].

Anti-influenza activity of glutathione

Whether such a mechanism contributes to GSH’s protection against influenza infection remains to be determined. Annual vaccination with inactivated virus remains the major preventive strategy in controlling flu epidemic [36]. People at high risk can also be treated with antiviral drugs such as amantadine and rimantadine [35]. Our results suggest that GSH provides an alternate strategy to limiting influenza infection. Such a strategy may be especially useful given the recent finding that selenium deficiency increases influenza virus mutations and pathology [20,21]. Furthermore, the reports that extracellular thiol/disulfide redox in humans is oxidized with increasing age [37] and chronic ethanol consumption [38] suggest that GSH may be particularly useful for prevention of influenza infection in some at-risk populations. 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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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