Glutathione Peroxidase-1 Reduces Influenza A Virus ... - ATS Journals

Glutathione Peroxidase-1 Reduces Influenza A Virus–Induced Lung Inflammation Selcuk Yatmaz1, Huei Jiunn Seow1, Rosa C. Gualano1, Zi Xin Wong1, John Stambas2, Stavros Selemidis3, Peter J. Crack1, Steven Bozinovski1, Gary P. Anderson1, and Ross Vlahos1 1 Department of Pharmacology, The University of Melbourne, Parkville, Victoria, Australia; 2School of Medicine, Deakin University, Waurn Ponds, Australia; and 3Department of Pharmacology, Monash University, Clayton, Australia

Oxidative stress caused by excessive reactive oxygen species production is implicated in influenza A virus–induced lung disease. Glutathione peroxidase (GPx)-1 is an antioxidant enzyme that may protect lungs from such damage. The objective of this study was to determine if GPx-1 protects the lung against influenza A virus– induced lung inflammation in vivo. Male wild-type (WT) or GPx-12/2 mice were inoculated with HKx31 (H3N2, 1 3 104 plaque-forming units), and bronchoalveolar lavage fluid (BALF)/lung compartments were analyzed on Days 3 and 7 after infection for inflammatory marker expression, histology, and viral titer. WT mice infected with HKx31 had significantly more BALF total cells, macrophages, neutrophils, and lymphocytes at Days 3 and 7 compared with naive WT animals (n ¼ 5–8; P , 0.05). However, infected GPx-12/2 mice had significantly more BALF inflammation, which included more total cells, macrophages, and neutrophils, compared with WT mice, and this was abolished by treatment with the GPx mimetic ebselen. BALF inflammation persisted in GPx-12/2 mice on Day 10 after infection, and GPx-12/2 mice had significantly more influenzaspecific CD81 T cells in spleen compared with WT mice (n ¼ 3–4; P , 0.05). Infected GPx-12/2 mice had greater peribronchial and parenchymal inflammation than WT mice, and viral titer was significantly reduced in GPx-12/2 mice at Day 3 (n ¼ 5; P , 0.05). Gene expression analysis revealed that infected GPx-12/2 mice had higher whole lung TNF-a, macrophage inflammatory protein (MIP)-1a, MIP-2, KC, and matrix metalloproteinase (MMP)-12 mRNA compared with infected WT mice. GPx-12/2 mice had more MIP-2 protein in BALF at Day 3 and more active MMP-9 protease in BALF at Days 3 and 7 than WT mice. These data indicate that GPx-1 reduces influenza A virus–induced lung inflammation. Keywords: oxidative stress; reactive oxygen species; hydrogen peroxide; HKx31; ebselen; antioxidants

Influenza A viruses cause respiratory tract infections and are responsible for significant global morbidity and mortality (1). Current treatments used to combat seasonal and pandemic influenza outbreaks target the key mechanisms of viral infection and replication (2). Antivirals and strain-specific vaccination display clear efficacy for the treatment and prophylaxis of influenza infections (3). However, emerging evidence suggests that

(Received in original form September 29, 2011 and in final form August 23, 2012) This work was supported by the National Health and Medical Research Council of Australia and the Australian Research Council. Author Contributions: R.V. and S.B. were involved in the conception and design of the study. S.Y., H.J.S., R.C.G., Z.X.W., S.S., and R.V. were involved in acquisition of the data. R.V., S.Y., R.C.G., S.B., S.S., J.S., P.J.C., and G.P.A. analyzed and interpreted the data. R.V., S.Y., S.B., R.C.G., S.S., and G.P.A. wrote the article. Correspondence and requests for reprints should be addressed to Ross Vlahos, Ph.D., Department of Pharmacology, The University of Melbourne, Parkville, VIC, 3010 Australia. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 48, Iss. 1, pp 17–26, Jan 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2011-0345OC on September 20, 2012 Internet address: www.atsjournals.org

CLINICAL RELEVANCE Influenza A virus infections claim millions of lives worldwide and continue to impose a major burden on health care systems. Current pharmacological strategies to control influenza A virus–induced lung disease are problematic due to antiviral resistance and the requirement for strain-specific vaccination. There is evidence to suggest that strategies to reduce lung inflammation could improve outcomes in influenza. We have directed our efforts toward targeting host immune responses that underpin lung inflammation, in particular oxidative stress, to modulate influenza A virus– induced lung inflammation. Our study is the first to show that the antioxidant enzyme glutathione peroxidase-1 (GPx1) reduces influenza A virus–induced lung inflammation and that GPx-1, alone or in combination with current antiviral and vaccination strategies, may represent a novel means of controlling influenza infections. certain strains of influenza are developing resistance to these antivirals (4, 5) and that, along with the long lag time for vaccine production and the ongoing threat of new pandemic strains, the global population is at risk. Excessive lung inflammation underlies much of the adverse effects of moderate to severe influenza infections (6). An emerging paradigm regarding the harmful effects of inflammatory cells involves the excessive generation of reactive oxygen species (ROS), in particular superoxide anion (O2 ˉ) and hydrogen peroxide (H2O2). Production of such ROS by leukocytes can be toxic to cells and cause significant injury to surrounding lung tissue when produced in excess (7, 8). Early studies have shown that influenza virus activates macrophages and neutrophils in vitro to produce ROS (8). Mice treated with superoxide dismutase (SOD) survive when subjected to highly virulent influenza (9), and influenza-induced lung injury is prevented in mice overexpressing SOD (10). Mice lacking NADPH oxidase2 (Nox-2), the primary source of superoxide production by inflammatory cells (11, 12), or the key regulator of Nox-2 (i.e., p47phox) (7, 8) show reduced lung oxidative stress, inflammation, and injury in response to influenza and exhibit lung function improvement (13, 14). Glutathione peroxidases (GPxs) are a family of seleniumdependent and -independent antioxidant enzymes that catalyze the reduction of damaging H2O2 into water and oxygen (15). Analysis of the selenoproteome has identified six GPxs in mammals (15). The cytosolic selenium-dependent GPx-1 is the predominant isoform of cellular GPx and is ubiquitously expressed throughout the body (15). Sources in the lung include epithelium, alveolar epithelial lining fluid, and alveolar macrophages (16). A protective role of GPx-1 has been demonstrated in various disease states involving ROS, including ischemia/ reperfusion brain injury (17–19), Parkinson’s and Huntington’s disease (20, 21), cold-induced head trauma (22), cigarette d

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 48 2013

smoke–induced lung disease (23), and coxsackie virus–induced cardiac inflammation and damage (24). Because GPx-1 is protective during oxidative stress, we propose that GPx-1 reduces influenza A virus–induced lung inflammation. GPx-12/2 mice inoculated with HKx31 influenza virus had significantly elevated levels of BALF macrophages, neutrophils, proteolytic burden, and whole lung macrophage and neutrophil chemotactic factor gene expression. Histological analysis revealed that infected GPx-12/2 mice had a greater degree of peribronchial and perivascular inflammation and bronchial inflammatory cell exudates. Our data provide new evidence for a protective role of GPx-1 in influenza A virus–induced lung inflammation and suggest the potential therapeutic utility of supplementing GPx-1 activity in vivo. Some of the results of these studies have been previously reported in the form of an abstract (25).

MATERIALS AND METHODS

intranasally with 1 3 104 plaque-forming units (PFU) of the moderately virulent influenza A virus HKx31 (H3N2) in a 30-ml volume diluted in PBS. Further details are provided in the online supplement.

Body Weight, Food Consumption, and Water Intake Mice were weighed daily at approximately the same time each day. Food consumption was calculated by measuring the amount of food remaining in cages every 24 hours and dividing that by the number of mice in the cages. Water intake was calculated by weighing the amount of water remaining in the water bottles every 24 hours.

BAL and Lung Collection Mice were killed by an intraperitoneal injection of sodium pentobarbitone (360 mg/kg) (Sigma Aldrich, St. Louis, MO), and BAL was performed as previously published (13). Further details are provided in the online supplement.

Quantitative Real-Time PCR Details are provided in the online supplement.

Animals Specific pathogen-free male wild-type (WT) (C57BL/6, 8–12 wk old) mice were obtained from the Animal Resource Centre Pty. Ltd. (Perth, Australia), and GPx-12/2 mice (8–12 wk old, C57BL/6 background) were bred at the Department of Pharmacology, The University of Melbourne. Further details are provided in the online supplement.

Lung Homogenization and Virus Titrations Lungs from terminally anesthetized HKx31-infected mice were removed, rinsed in PBS, weighed, and homogenized in 1 ml of DMEM with gentamicin (50 mg/ml) (Invitrogen, Carlsbad, CA). Clarified homogenate was snap frozen and stored at 2808 C until required. Virus was quantitated by plaque assay in MDCK cells as previously published (13, 26).

Influenza A Virus Infection Mice were anesthetized by methoxyflurane (Medical Developments International Ltd., Springvale, Victoria, Australia) inhalation and infected

Protease Expression and Activity in BAL Fluid Details are provided in the online supplement.

Figure 1. Effect of HKx31 virus infection on bronchoalveolar lavage fluid (BALF) cellularity, viral titer, and influenza-specific T-cell numbers in wild-type (WT) and glutathione peroxidase-1 (GPx-1)2/2 mice. Mice were infected with 1 3 104 plaqueforming units (PFU) of HKx31 influenza A virus, and the number of total cells (A), macrophages (B), neutrophils (C), and lymphocytes (D) was determined in the BALF at Days 3, 7, and 10 after infection. Naive represents no virus, open bars represent WT mice, and closed bars represent GPx-12/2 mice. Data are expressed as mean 6 SEM for n ¼ 5 to 11 mice per time point from two independent experiments. *P , 0.05 compared with respective naive (no virus) group (two-way ANOVA and Bonferroni multiple comparison test). #P , 0.05 compared with WT virus group (twoway ANOVA and Bonferroni multiple comparison test). (E) Virus titer determined at Days 3 and 7 after infection. Data in PFU per gram of lung are expressed as mean 6 SEM for n ¼ 5 mice per time point from one experiment. # P , 0.05 compared with WT group (Student’s unpaired t test). (F) Enriched CD81 T cells from spleen were obtained from HKx31-infected WT and GPx-12/2 mice 10 days after infection. Ex vivo intracellular cytokine staining specific for the two immunodominant CD81 T-cell epitopes (DbNP366–372 [open bars] and DbPA224–232 [closed bars]) was assessed, and data were analyzed by flow cytometry. Data are shown as mean 6 SEM for n ¼ 3 or 4 individual mice from one experiment. *P , 0.05 compared with respective WT mice (Student’s unpaired t test). d3 ¼ Day 3; d7 ¼ Day 7; d10 ¼ Day 10.

Yatmaz, Jiunn Seow, Gualano, et al.: Gpx-1 and Virus-Induced Lung Inflammation

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Histology

Ebselen Treatment

Details are provided in the online supplement.

Mice were pretreated with ebselen (Sapphire Bioscience, Waterloo, Australia) (10 mg/kg) or vehicle (5% CM-cellulose made up in distilled water) via oral gavage 3 hours before infection with 1 3 104 PFU of HKx31 influenza A virus and then twice daily (morning and evening).

Intracellular Cytokine Staining The number of spleen influenza-specific CD81DbNP3661 and DbPA2241 T cells was assessed using intracellular cytokine staining as previously described (13, 27). Data were acquired on a BD LSRII (Becton Dickinson, Franklin Lakes, NJ) and analyzed using FACS DIVA software.

ELISAs Details are provided in the online supplement.

Figure 2. Histological investigation of lung inflammation in HKx-31 virus–infected WT and GPx-12/2 mice. Hematoxylin and eosin–stained paraffin sections of lungs from naive WT mice (A), naive GPx-12/2 mice (B), and WT and GPx-12/2 mice infected with virus 3 days (C and D) and 7 days (E and F) after infection. Basic lung structures are indicated in A: b ¼ bronchus; v ¼ blood vessel; a ¼ individual alveolus; and p ¼ parenchyma. Lungs from infected GPx-12/2 mice had considerably more peribronchial inflammation and extensive parenchymal inflammation (alveolitis) (D and F) compared with infected WT mice (C and E). Photomicrographs were acquired using a Nikon Eclipse E600 microscope and Image Pro Plus software (Version 5.01). Results shown are typical of the pattern observed in four to eight animals from two independent experiments. Original magnification : 350. (G) Corresponding histopathological scores for peribronchial inflammation and alveolitis. Lung sections were scored blind for alveolitis and peribronchiolar inflammation from 0 to 5 as described in MATERIALS AND METHODS. Data shown represent scores from individual mice (circles) and median values (bar) obtained from one of two independent readers. For each reader, peribronchial inflammation and alveolitis were significantly higher in HKx31-infected GPx-12/2 mice compared with HKx31-infected WT mice (#P , 0.05, two-way ANOVA and Bonferroni multiple comparison test). *P , 0.05 compared with respective naive (no virus) group (two-way ANOVA and Bonferroni multiple comparison test). d3 ¼ Day 3; d7 ¼ Day 7.

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RESULTS GPx-12/2 Mice Show Increased BAL Fluid Inflammation in Response to HKx-31 Virus

Based on previous time-course experiments (13), we chose Day 3 (maximum BAL fluid [BALF] inflammation and peak viral titers) and Day 7 (resolution of infection) to study the BALF cell profile of WT and GPx-12/2 mice. Naive GPx-12/2 mice had similar BALF total cells, macrophages, neutrophils, and lymphocytes to naive WT mice (Figures 1A–1D). However, GPx-12/2 mice infected with HKx31 had significantly more (z 40%) total cells (Figure 1A) and an approximately 2-fold increase in neutrophils (Figure 1C) compared with infected WT mice at Days 3 and 7 (P , 0.05). Infected GPx-12/2 also had a significant increase in macrophage numbers at Days 3 and 7 compared with uninfected mice, and macrophage numbers were significantly higher in GPx-12/2 mice at Day 7 compared with infected WT mice (P , 0.05) (Figure 2B). Although there was an increase in lymphocyte numbers in infected WT and GPx-12/2 mice at Days 3 and 7, there was no difference in lymphocyte numbers between the groups of mice (Figure 1D). To determine if resolution of inflammation is impaired in GPx-12/2 mice, we assessed BALF cellularity in WT and GPx-12/2 mice infected with HKx31 10 days after infection. GPx-12/2 had greater total cells, macrophages, and lymphocytes in BALF compared with WT mice (Figures 1A–1D). GPx-12/2 Mice Show Reduced Viral Load

GPx-12/2 mice infected with HKx31 had significantly lower (z 47%) lung viral titers at Day 3 compared with infected WT mice (P , 0.05) (Figure 1E). However, no significant differences were observed in lung viral titers between GPx-12/2 and WT mice at Day 7 (Figure 1E). Influenza-Specific CD81 T Cells Are Increased in GPx-12/2 Mice Figure 3. Effect of HKx31 virus infection on body weight, food consumption, and water intake in WT and GPx-12/2 mice. (A) Mice were infected with 1 3 104 PFU of HKx31 influenza A virus, and body weight was recorded for up to 7 days after infection. Data are expressed as mean % weight change 6 SEM for n ¼ 6 to 10 mice per time point from two independent experiments. (B) Food consumption was calculated by measuring the amount of food remaining in cages every 24 hours. (C) Water intake was calculated by measuring the amount of water remaining in the water bottle every 24 hours (n ¼ 4, one experiment). Open circles represent naive WT mice, open squares represent naive GPx-12/2 mice, closed circles represent WT mice infected with HKx31, and closed squares represent GPx-12/2 mice infected with HKx31. *P , 0.05 compared with respective naive (no virus) group (two-way ANOVA and Bonferroni multiple comparison test).

Statistical Analyses Because data were normally distributed (assessed using D’Agostino and Pearson Omnibus K2 test where appropriate), they are presented as grouped data expressed as mean 6 SEM; n represents the number of mice. Differences were determined by one- or two-way ANOVA followed by Dunnett’s post hoc test or Bonferroni test for multiple comparisons, where appropriate. In some cases, Student’s unpaired t test was used to determine if there were significant differences between means of pairs. All statistical analyses were performed using GraphPad Prism for Windows (Version 5.02). In all cases, P , 0.05 was used to indicate statistical significance.

Given the important role of influenza-specific CD81 T cells in virus infection, we examined whether subsets of influenzaspecific CD81 T cells were altered in GPx-12/2 mice. Enriched CD81 T cells from spleen were obtained from HKx31-infected WT and GPx-12/2 mice 10 days after infection. Ex vivo intracellular cytokine staining specific for the two immunodominant CD81 T-cell epitopes (DbNP366–372 and DbPA224–232) revealed that GPx-12/2 mice had more NP- and PA-specific CD81 T cells compared with WT mice (P , 0.05) (Figure 1F). GPx-12/2 Mice Show Increased Lung Inflammation in Response to HKx31 Virus

Lung sections from infected WT mice at Days 3 and 7 were characterized by extensive peribronchial inflammation and cellular inflammation that extended into the parenchyma (Figures 2C and 2E). When observed under higher magnification (data not shown), macrophages and neutrophils were the predominant cell types infiltrating the alveolar spaces of mice. The degree of peribronchial and parenchymal inflammation (alveolitis) was markedly increased in GPx-12/2 mice (Figures 2D, 2F, and 2G). Effect of GPx-1 Deletion on HKx31 Virus–Induced Weight Loss, Food Consumption, and Water Intake

Mouse body weight, food consumption, and water intake were measured each day at approximately the same time. GPx-12/2 mice infected with HKx31 lost similar amounts of body weight


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TABLE 1. EFFECT OF HKx31 VIRUS INFECTION ON WHOLE LUNG CYTOKINE, CHEMOKINE, AND PROTEASE mRNA EXPRESSION IN WILD-TYPE AND GLUTATHIONE PEROXIDASE -12/2 MICE Uninfected Gene Cytokines TNF-a IL-1b GM-CSF IL-6 IFN-g Chemokines MCP-1 MIP-2 KC MIP-1a MIG IP-10 Proteases MMP-9 MMP-12

Day 3 after Infection

GPx-12/2

WT

Day 7 after Infection

GPx-12/2

WT

GPx-12/2

WT

0.97 1.00 1.00 1.00 1.00

6 6 6 6 6

0.04 0.03 0.01 0.02 0.02

1.00 0.99 0.98 1.00 1.05

6 6 6 6 6

0.02 0.03 0.03 0.02 0.12

12.90 17.61 1.25 196 6.66

6 6 6 6 6

0.52 0.18 0.03 1.62 0.42

13.62 15.22 1.46 129 5.92

6 6 6 6 6

0.52 0.23 0.15 2.18 0.68

5.13 1.95 0.41 8.07 12.74

6 6 6 6 6

0.16 0.14 0.02 1.02 0.68

7.54 3.62 0.46 16.58 18.15

6 6 6 6 6

0.23 0.13 0.02 0.77 0.34

0.97 1.00 1.00 1.00 1.01 1.01

6 6 6 6 6 6

0.09 0.11 0.02 0.09 0.10 0.03

1.06 1.01 1.00 1.00 0.99 1.02

6 6 6 6 6 6

0.06 0.18 0.02 0.02 0.03 0.05

198 114 29.28 24.14 67.27 342.2

6 6 6 6 6 6

10.59 5.54 0.80 1.57 3.46 4.62

140.7 146.9 36.22 29.31 40.07 258.1

6 6 6 6 6 6

2.53 1.29 1.64 1.30 0.73 2.44

17.71 2.98 2.39 6.14 82.69 42.54

6 6 6 6 6 6

0.32 0.16 0.25 0.34 3.36 2.26

38.03 12.31 5.44 10.25 123.9 86.2

6 6 6 6 6 6

0.58 1.06 0.27 0.66 1.94 3.54

0.98 6 0.04 1.00 6 0.03

1.00 6 0.01 1.00 6 0.02

2.32 6 0.06 1.75 6 0.07

2.19 6 0.12 2.57 6 0.07

0.44 6 0.02 1.38 6 0.04

0.95 6 0.04 1.63 6 0.08

Definition of abbreviations: GPx-1 ¼ glutathione peroxidase-1; IP-10 ¼ IFN-g–induced protein 10; KC ¼ keratinocyte chemoattractant; MCP ¼ monocyte chemotactic protein; MIG ¼ monokine induced by IFN-g; MIP ¼ macrophage inflammatory protein; WT ¼ wild type. mRNA expression for all genes was measured simultaneously under identical conditions using quantitative real-time PCR. Responses for each time point are shown as fold change relative to uninfected WT mice after normalization to 18S rRNA (housekeeping gene). Data are shown as mean 6 SD for triplicate reactions of five pooled whole lungs as previously published (49, 50).

to infected WT mice over a 7-day period (Figure 3A). In addition, WT and GPx-12/2 mice infected with HKx31 ate less food than uninfected WT and GPx-12/2 mice over the 7-day period (Figure 3B), although GPx-12/2 mice ate less than WT mice regardless of the treatment. When we investigated water intake, naive WT and GPx-12/2 mice drank about the same amount of water over a 7-day period, but, when infected with HKx31, WT and GPx-12/2 mice consumed less water, although water intake was similar for both groups of mice (Figure 3C). Effect of HKx31 Virus Infection on Cytokine, Chemokine, and Protease mRNA Expression in WT and GPx-12/2 Mice

Because BALF and lung inflammation were greater in GPx-12/2 mice, we were interested in examining the mediators of this

increased inflammation. HKx31-infected WT and GPx-12/2 mouse lungs were screened for a range of cytokines, chemokines, and proteases by quantitative PCR. Infected WT and GPx-12/2 mice demonstrated increased whole lung mRNA levels of cytokines (TNF-a, IFN-g, IL-1b, IL-6, and GM-CSF), chemokines (monocyte chemotactic protein [MCP]-1, macrophage inflammatory protein [MIP]-1a, MIP-2, KC, monokine induced by IFN-g [MIG], and IFN-g–induced protein 10 [IP10]), and proteases MMP-9 and MMP-12 at both time-points compared with respective naive mice (Table 1). However, we did not detect influenza-induced increases in GM-CSF expression at Day 7. In comparison to WT mice, GPx-12/2 mice had increased levels of the cytokine TNF-a; chemokines MIP1a, MIP-2, KC; and the protease matrix metalloproteinase (MMP)-12 at Day 3 (Table 1). In addition, mRNA levels of

Figure 4. Effect of HKx31 virus infection on protease expression in WT and GPx-12/2 mice. BALF supernatants pooled from HKx31 virus–infected WT and GPx-12/2 were assessed for proteolytic activity using gelatin zymography. (A) Zymography of matrix metalloproteinase (MMP)-9 expression (z 92 kD) for Days 3 and 7 after infection. The BALF from mice in each treatment group (n ¼ 6–8) was pooled. (B) Net free gelatinase activity, as measured by degradation of flurogenic gelatin substrate, using neat BALF assayed from individual mice, indicating the amount of free protease, which is not bound to antiprotease. (C) Net serine protease activity in BALF supernatant from individual mice assayed colorimetrically with substrate N-methoxysuccinyl-ala-ala-proval-4-nitroanilide. Naive represents no virus, open bars represent WT mice, and closed bars represent GPx-12/2 mice. Data for (B) and (C) are shown as mean 6 SEM for n ¼ 5 to 8 mice per treatment group from one experiment. *P , 0.05 compared with naive control group. # P , 0.05 compared with WT virus group (two-way ANOVA and Bonferroni multiple comparison test). d3 ¼ Day 3; d7 ¼ Day 7.

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 48 2013 TABLE 2. EFFECT OF HKx31 VIRUS INFECTION ON CYTOKINE AND CHEMOKINE PROTEIN PRODUCTION IN BRONCHOALVEOLAR LAVAGE FLUID OF WILD-TYPE AND GLUTATHIONE PEROXIDASE-12/2 MICE Uninfected Protein (pg/ml) TNF-a MCP-1 MIP-2

WT 45 6 9 182 6 34 169 6 25

GPx-1

Day 3 after Infection 2/2

45 6 6 213 6 17 129 6 23

WT

GPx-1

50 6 4 612 6 76* 75.22 6 17

Day 7 after Infection 2/2

50 6 8 732 6 187* 348 6 77†

WT

GPx-12/2

26 6 2 214 6 31 15 6 10

38 6 4 405 6 131 19 6 9

Definition of abbreviations: GPx-1 ¼ glutathione peroxidase-1; MCP ¼ monocyte chemotactic protein; MIP ¼ macrophage inflammatory protein; WT ¼ wild type. Data are shown as mean 6 SEM for n ¼ 4–8 individual mice. * P , 0.05 compared with respective uninfected mice (two-way ANOVA and Bonferroni multiple comparison test). y P , 0.05 compared with Day 3 after infection WT mice (two-way ANOVA and Bonferroni multiple comparison test).

the cytokines TNF-a, IFN-g, IL-1b, and IL-6; chemokines MCP-1, MIP-1a, MIP-2, KC, MIG, and IP-10; and the protease MMP-12 were greater in GPx-12/2 mice at Day 7 than in WT mice (Table 1). IL-1b, IL-6, MCP-1, MIG, and IP-10 mRNA expression was lower in infected GPx-12/2 mice than in infected WT mice at Day 3 (Table 1). GPx-12/2 Mice Have Increased Protease Activity in Response to HKx31 Infection

Because MMPs contribute to the recruitment of neutrophils and macrophages into the lung parenchyma, we measured the secretion of MMP-9 in the cell-free BALF of influenza-infected WT and GPx-12/2 mice. There was a marked increase in protease expression in BALF from WT mice infected with HKx31 at Day 3 as assessed by gelatin zymography (Figures 4A and 4B). A major band of protease expression was identified at approximately 92 kD, corresponding to the molecular size of the active form of MMP-9. In addition, a much smaller increase in BALF MMP-9 was observed Day 7 after infection. However, GPx-12/2 mice treated with influenza had significantly more MMP-9 at Days 3 and 7, compared with influenza-infected WT mice (Figures 4A and 4B). In addition, there was a significant increase in net gelatinase (Figure 4C) and net serine protease (Figure 4D) activity in GPx-12/2 mice at Day3 (P , 0.05), whereas there was no difference at Day 7.

Effect of HKx31 Virus Infection on Cytokine and Chemokine Protein Production in BALF from WT and GPx-12/2 Mice

Because BALF and lung inflammation were greater in GPx-12/2 mice, we performed ELISAs to measure a selected number of key cytokines and chemokines that regulate macrophage/monocyte and neutrophil influx. Infected WT and GPx-12/2 mice had increased BALF MCP-1 at Day 3 after infection, but the levels were similar for genotypes (Table 2). However, the levels of MIP-2 were significantly greater in GPx-12/2 mice at Day 3 after infection compared with WT mice (P , 0.05). Levels of TNF-a in infected WT and GPx-12/2 mice were similar to uninfected mice (Table 2). Effect of Ebselen on BALF Cellularity, Body Weight, Food Consumption, and Virus Titer in HKx31 Virus–Infected Mice

Compared with naive (no virus) WT mice, vehicle-treated WT mice infected with HKx31 had significantly increased BALF total cells, macrophages, neutrophils, and lymphocytes at Days 3 and 7 (P , 0.05) (Figures 5A–5D). However, treatment of infected WT mice with the GPx mimetic ebselen (10 mg/kg, based on previous studies from our laboratory [23, 28] and others [29, 30]) before infection had no effect on BALF total cells, macrophages, neutrophils, and lymphocytes at Days 3 and 7 after infection (Figures 5A–5D).

Figure 5. Effect of ebselen on BALF cellularity in HKx31 virus–infected WT mice. WT mice were pretreated with ebselen (10 mg/kg) or vehicle (5% CM-cellulose) 3 hours before infection with 1 3 104 PFU of HKx31 influenza A virus, and the number of total cells (A), macrophages (B), neutrophils (C), and lymphocytes (D) was determined in BALF at Days 3 and 7 after infection. Hatched bars represent naive mice, open bars represent vehicle-treated mice, and closed bars represent ebselen-treated mice. Data are expressed as mean 6 SEM for n ¼ 5 to 8 mice per time point from one experiment. *P , 0.05 compared with respective naive (no virus) group (two-way ANOVA and Bonferroni multiple comparison test). d3 ¼ Day 3; d7 ¼ Day 7.


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Effect of Ebselen on Cytokine, Chemokine, and Protease mRNA Expression and Protein Production in HKx31 Virus–Infected WT Mice

Vehicle-treated mice infected with HKx31 had increased mRNA levels of cytokines (TNF-a, IL-1b, GM-CSF, IL-6, and INF-g), chemokines (MCP-1, MIP-2, KC, MIP-1a, MIG, and IP-10), and proteases MMP-9 and MMP-12 at Days 3 and 7 compared with naive mice (see Table E1 in the online supplement). Ebselen treatment reduced TNF-a, MIG, and MMP-12 at Day 3 and TNF-a and IP-10 mRNA expression at Day 7. In contrast to whole lung mRNA expression, BALF levels of TNF-a, MCP-1, and MIP-2 protein in ebselen-treated mice were similar to those of vehicle-treated mice at Days 3 and 7 after infection (Table E2). Vehicle- and ebselen-treated mice infected with HKx31 had lower levels of MIP-2 than naive uninfected mice. Ebselen Abolished the Increased BALF Inflammation Observed in HKx31 Virus–Infected GPx-12/2 Mice

As shown in Figure 1, GPx-12/2 mice had enhanced BALF inflammation (total cells, macrophages and neutrophils) in response to HKx31 compared with WT mice (Figures 7A–7D). However, administration of ebselen (10 mg/kg) to HKx31infected GPx-12/2 mice abolished the enhanced virus-induced BALF total cell numbers, macrophages, and neutrophils (P , 0.05) (Figures 7A–7D). Ebselen did not reduce the increased expression (mRNA) and production (protein) of key inflammatory mediators observed in infected GPx-12/2 mice (Tables E3 and E4). In fact, the mRNA levels of IL-1b, IL-6, MCP-1, KC, and MMP-12 were greater in ebselen-treated virus-infected GPx-12/2 mice compared with vehicle-treated virus-infected GPx-12/2 mice.

DISCUSSION Figure 6. Effect of ebselen on body weight, food intake, and viral titer in HKx31 virus–infected WT mice. (A) Mice were pretreated with vehicle (5% CM-cellulose) or ebselen (10 mg/kg) 3 hours before infection with 1 3 104 PFU of HKx31 influenza A virus, and body weight was recorded for up to 7 days after infection (A). Data are expressed as mean % weight change 6 SEM for n ¼ 6 to 8 mice per time point for one experiment. (B) Food consumption was calculated by measuring amount of food remaining in cages every 24 hours. Open circles represent naive WT mice, open squares represent WT mice infected with HKx31 and treated with vehicle, and closed squares represent WT mice infected with HKx31 and treated with ebselen (10 mg/kg). (C) Vehicle (5% CM-cellulose)- and ebselen -treated mice (10 mg/kg) were infected with 1 3 104 PFU of HKx31 influenza A virus, and viral titer was determined at Days 3 and 7 after infection. Open bars represent vehicle-treated mice, and closed bars represent ebselen-treated mice. Data in PFU per gram of lung are expressed as mean 6 SEM for n ¼ 5 mice per time point. *P , 0.05 compared with respective naive (no virus) group (two-way ANOVA and Bonferroni multiple comparison test). d3 ¼ Day 3; d7 ¼ Day 7.

Vehicle-treated WT mice infected with HKx31 lost significant weight compared with vehicle-treated WT naive mice (P , 0.05) (Figure 6A). In addition, vehicle-treated WT mice infected with HKx31 ate less food than vehicle-treated WT naive mice (Figure 6B). However, ebselen treatment did not affect virus-induced weight loss and food intake over the 7-day period (Figures 6A and 6B). Similarly, ebselen treatment had no effect on viral titer at Days 3 and 7 after infection (Figure 6C).

GPx-1 is a ubiquitous antioxidant enzyme that plays a critical role in maintaining the local and systemic redox status and its function in protecting against various disease states involving ROS is well established (17–19, 23). Given that no definitive role for GPx-1 during influenza infection has been described, the primary aim of this study was to investigate the role of GPx1 in influenza A virus–induced lung inflammation. This study showed that GPx-12/2 mice infected with influenza A virus have enhanced BALF inflammation, suggesting that GPx-1 is required to control influenza-induced lung inflammation. The similarity in BALF cell count numbers between naive (no virus) WT and GPx-12/2 mice can help explain the absence of any overt phenotype in GPx-12/2 mice and strengthens the hypothesis that GPx-1 may be protective in influenzainduced lung inflammation where there is an enhanced oxidant burden and is entirely consistent with the known biology of GPx-1, which appears to be limited to protection during oxidative stress (17–19, 23). Influenza-infected GPx-12/2 mice had increased expression of lung mRNA for cytokines (TNF-a and GM-CSF) and chemokines (MIP-1a, MIP-2, and KC) at Day 3. Consistent with the fact that, after an influenza infection, alveolar macrophages and respiratory epithelial cells trigger the production and release of cytokines and chemokines that have proinflammatory and chemotactic properties (31), we observed a concurrent increase of total cells in the BALF at Day 3, manifested mainly by macrophages and neutrophils. The increase in mRNA for macrophage chemoattractant MIP-1a and the macrophage growth factor GM-CSF in GPx-12/2 mice should reflect an increase in macrophage numbers at Day 3; however,

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Figure 7. Effect of ebselen on BALF cellularity in HKx31 virus–infected WT and GPx-12/2 mice. WT and GPx-12/2 mice were pretreated with ebselen (10 mg/kg) or vehicle (5% CM-cellulose) 3 hours before infection with 1 3 104 PFU of HKx31 influenza A virus, and the number of total cells (A), macrophages (B), neutrophils (C), and lymphocytes (D) was determined in BALF 3 days after infection. Open bars represent vehicle-treated mice, and closed bars represent ebselen-treated mice. Data are expressed as mean 6 SEM for n ¼ 3 to 6 mice per time point for one experiment. *P , 0.05 compared with respective WT group (twoway ANOVA and Bonferroni multiple comparison test). #P , 0.05 compared with GPx-12/2/vehicle group (two-way ANOVA and Bonferroni multiple comparison test).

we observed no difference in macrophage numbers between GPx-12/2 and WT mice. A decrease in MCP-1 mRNA (also a macrophage chemoattractant) and no further increase in MCP-1 protein levels in GPx-12/2 mice could be responsible for maintaining a balanced macrophage chemotactic gradient at this time point. Furthermore, the increase in MIP-2 (mRNA and protein) and KC (mRNA), both possessing potent chemotactic activity for neutrophils, could explain the increase in BALF neutrophils at Day 3 in GPx-12/2 mice. The increase in neutrophil number was associated with increased clearance of virus at Day 3 in GPx-12/2 mice, which is reflected by the decrease in viral titer. As shown by Tate and colleagues (32), neutrophils play a major role in clearance of virus and virus replication in the lungs of mice. Although our data agree with this, the clearance of virus does not necessarily associate with decreased lung pathology. In mouse models of airway inflammation, the absence of neutrophils ameliorated lung injury (33, 34), reiterating the connection between neutrophils and lung injury, especially in greater numbers than necessary, as seen in GPx-12/2 mice. In fact, neutrophils are long known to be an important source of serine proteases, such as elastase and proteinase-3, as well as MMP-9 (gelatinase B) (35, 36). Although these proteases play an important role in host defense, extensive amounts lead to degradation of extracellular matrix and basement membrane in the lungs (37). Infected GPx-12/2 mice had increased net serine protease activity as well as increased activity of MMP-9 revealed by zymography. Moreover, MMP-12 expression was increased in infected GPx-12/2 mice, reinforcing the fact that proteases like MMP-9 and MMP12 can contribute to lung inflammation and damage (37). At this early stage of infection (Day 3), GPx-1 plays a role in regulating pathology associated with inflammation via altering the response of mRNA and protein levels for key cytokines and chemokines involved in the recruitment of cells and in the expression and activity of proteases. This is supported by Beck and colleagues, who showed that selenium-deficient mice display greater lung inflammation and pathology in response to influenza A/Bangkok/1/79 (H3N2) infection and that this increase in pathology was associated with significant alterations in mRNA levels for cytokines and chemokines involved in proinflammatory responses (38). At a later stage of infection (Day 7), BALF inflammation persists in GPx-12/2 mice. There is an overexpression of mRNA

for cytokines (TNF-a, INF-g, IL-1b, and IL-6) and chemokines (MCP-1, MIP-1a, MIP-2, KC, MIG, and IP-10) and a slight increase in protein levels of MCP-1, but protein levels of TNF-a and MIP-2 were similar to virus-infected WT mice. The number of total macrophages was increased and the neutrophil response remained high in GPx-12/2 mice. In addition, we found that BALF inflammation in GPx-12/2 mice had not resolved by Day 10 after infection because these mice displayed increased levels of macrophages and lymphocytes. Although viral titer and protease activity at Day 7 remained similar between GPx-12/2 and WT mice, the aberrant, persistent trafficking of cells into the lung, reflected in BALF, may culminate in potentially deleterious lung inflammation. This was the case in animals used to model highly virulent strains of influenza, such as the 1918 Spanish flu (39, 40). It is believed that one of the main culprits of the lung injury seen in these highly virulent strains is ROS (41). We also addressed whether deletion of GPx-1 influences important processes of the adaptive immune system. For example, does GPx-1 play a role in antigen processing and presentation to CD81 T lymphocytes? This process is crucial for effective activation of the adaptive immune response because it stimulates proliferation of CD81 T lymphocytes that recognize viral antigens presented by infected cells. We have previously shown that in the absence of the antioxidant enzyme NADPH oxidase-2 (Nox-2), antigen-specific T-cell–mediated immunity is preserved (13). However, in the present study we found that GPx-12/2 mice had increased numbers of NP- and PA-specific CD81 T cells, suggesting that antigen-specific T-cell–mediated immunity is enhanced. Snelgrove and colleagues showed a modest increase in CD81 T-cell numbers in the BALF of Nox-22/2 mice compared with WT mice and suggested this is likely to explain the enhanced viral clearance in this strain of mice. This mechanism may also contribute to the reduced viral titers in GPx-12/2 mice. The absence of GPx-1 leads to a dysregulation of the immune response to influenza. Because GPx-1 catalyzes the reduction of toxic H2O2 into water and oxygen, this altered immune response may be due to an oxidant/antioxidant imbalance, resulting in oxidative stress due to an accumulation of H2O2. This buildup of H2O2 can activate intracellular signaling pathways. Several studies have implicated a role of H2O2 in activation of the redox-sensitive transcription factor NF-kB during inflammation, but the exact mechanism has not been determined. Ultimately,


H2O2–dependent activation of NF-kB results in the up-regulation of a number of proinflammatory genes, including IL-6, GMCSF, MCP-1, and TNF-a (42, 43). The increase in protease expression and activity in GPx-12/2 mice further supports this proposed mechanism. ROS not only activate proteases such as MMP-9 (44) but also can inactivate protease inhibitors such as tissue inhibitor of metalloproteinase-1 (45). Cytokine-mediated weight loss has long been established as a hallmark of influenza infection. Proinflammatory cytokines, including TNF-a and IL-1b, have been shown to increase the production and release of leptin from adipose tissues in mice, which in turn acts on the hypothalamus to repress appetite, leading to the weight loss observed during influenza infection (46, 47). However, it appears from our study that influenza-induced weight loss is attributed to reduced food and water intake. To further investigate the protective role of GPx-1 against influenza-induced lung inflammation, we used a pharmacological approach and tested the GPx mimetic ebselen in virus-infected mice. Ebselen has been shown to have therapeutic benefit in various disease states involving ROS, including lung inflammation (23, 28). In addition, various studies have shown reduced lung inflammation and injury in the absence of oxidative stress during an influenza infection (9, 13, 14). Therefore, we expected that mice treated with ebselen would show a reduction in influenza-mediated lung inflammation. However, ebselen did not inhibit virus-induced increases in BALF total cells, macrophages, neutrophils, and T lymphocytes. Furthermore, no differences were observed for body weight, food intake, and viral titers at any time point after infection. However, a paradoxical variation in cytokine (e.g., TNF-a and IL-6) and chemokine (e.g., MCP-1 and MIP-1a) expression and production was seen at Days 3 and 7 in the ebselen-treated group in comparison to the vehicle-treated group, which makes it difficult to account for the lack of effect of ebselen on BALF inflammation. This is in contrast to studies showing that ebselen is effective against LPS-, sephadex-, and cigarette smoke–induced lung inflammation (23, 29, 30). An explanation for this could be that under conditions of existing GPx-1 activity, or where GPx-1 activity is not completely overwhelmed, supplementation of GPx-1 has no further beneficial effect. Our observation that the enhanced BALF inflammation observed in HKx31-infected GPx-12/2 was abolished by ebselen administration supports this idea. This is in accord with our previous study showing that pretreatment of GPx-12/2 mice with ebselen restored microvascular perfusion, limited the induction and activation of MMP-9, and attenuated the increases in infarct size and vascular permeability (28). From our studies, it appears that the mechanism behind this antiinflammatory response does not involve reductions in the key neutrophil and macrophage chemotactic factors MIP-1a, MCP-1, MIP-2, and KC, the neutrophil and macrophage survival factor GM-CSF, and the proteases MMP-9 and MMP-12. It could be possible that ebselen’s antioxidant properties of removing H2O2, scavenging ONOOˉ, and enhancing pulmonary expression of copper/zinc and manganese SODs (which can contribute to a decrease in the formation of ONOOˉ by lowering the concentration of available O2 ˉ) may have contributed to reduced inflammation, although this was not measured in our study. It could also be possible that ebselen reduced influenzainduced BALF inflammation by inducing cell death as previously described by Guerin and Gauthier (48), although this was not measured in our study. Thus, targeting GPx-1 with mimetics such as ebselen might exert antiinflammatory and antioxidant effects in vivo. However, one should carefully monitor treatment when supplementing with GPx-1 mimetics because the reduction in inflammation may lead to uncontrolled infection. d

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In conclusion, current strategies for the treatment of influenza A virus–induced lung disease are focused primarily on halting mechanisms of viral infection and replication. However, far less attention has been directed to investigating mechanisms that modulate host responses, which lead to lung inflammation and pathology. Oxidative stress and ROS are key mechanisms of the host immune response and are implicated in influenza A virus– induced lung inflammation and damage. We are the first to show that the antioxidant enzyme GPx-1 reduces influenza A virus–induced lung inflammation and that GPx-1, alone or in combination with antiviral and vaccination strategies, may represent a novel means of controlling influenza infections. Author disclosures are available with the text of this article at www.atsjournals.org.

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