Glutathione modulation during sensitization as well as challenge phase regulates airway reactivity and inflammation in mouse model of allergic asthma.

Biochimie xxx (2014) 1e10

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Research paper

Glutathione modulation during sensitization as well as challenge phase regulates airway reactivity and inflammation in mouse model of allergic asthma Ahmed Nadeem a, *, Nahid Siddiqui b, Naif O. Alharbi a, Mohammad M. Alharbi a, Faisal Imam a, Mohamed M. Sayed-Ahmed a a b

Department of Pharmacology & Toxicology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Amity Institute of Biotechnology, Amity University, Noida, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 December 2013 Accepted 1 April 2014 Available online xxx

Glutathione, being a major intracellular redox regulator has been shown to be implicated in regulation of airway reactivity and inflammation. However, no study so far has investigated the effect of glutathione depletion/repletion during sensitization and challenge phases separately, which could provide an important insight into the pathophysiology of allergic asthma. The aim of the present study was to evaluate the role of glutathione depletion/repletion during sensitization and challenge phases separately in a mouse model of allergic asthma. Buthionine sulphoximine (BSO), an inhibitor of gammaglutamylcysteine synthetase or N-acetyl cysteine (NAC), a thiol donor were used for depletion or repletion of glutathione levels respectively during both sensitization and challenge phases separately followed by assessment of airway reactivity, inflammation and oxidanteantioxidant balance in allergic mice. Depletion of glutathione with BSO during sensitization as well as challenge phase worsened allergen induced airway reactivity/inflammation and caused greater oxidanteantioxidant imbalance as reflected by increased NADPH oxidase expression/reactive oxygen species (ROS) generation/lipid peroxides formation and decreased total antioxidant capacity. On the other hand, repletion of glutathione pool by NAC during sensitization and challenge phases counteracted allergen induced airway reactivity/ inflammation and restored oxidanteantioxidant balance through a decrease in NADPH oxidase expression/ROS generation/lipid peroxides formation and increase in total antioxidant capacity. Taken together, these findings suggest that depletion or repletion of glutathione exacerbates or ameliorates allergic asthma respectively by regulation of airway oxidanteantioxidant balance. This might have implications towards increased predisposition to allergy by glutathione depleting environmental pollutants. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Glutathione NADPH oxidase Reactive oxygen species Antioxidants Asthma

1. Introduction Asthma, a common respiratory disorder affecting millions of people worldwide, is characterized by recurrent episodes of airway hyperreactivity, ongoing airway inflammation and mucus hypersecretion. Many inflammatory and structural cells orchestrate to

Abbreviations: BSO, buthionine sulphoximine; BAL, bronchoalveolar lavage; CHAL, challenged; CON, control; NAC, N-acetyl cysteine; ROS, reactive oxygen species; SEN, sensitized. * Corresponding author. Department of Pharmacology & Toxicology, College of Pharmacy, King Saud University, PO Box 2455, Riyadh, Saudi Arabia. Tel.: þ966 548858471; fax: þ966 4677200. E-mail address: [email protected] (A. Nadeem).

release a variety of mediators such as cytokines, histamine, leukotrienes and reactive oxygen species (ROS) which cause many of the pathophysiological changes found in asthmatic airways [1e3]. Generation of ROS and derangement in antioxidant defenses in asthmatic airways have been shown to be associated with increase in airway reactivity/airway secretions, airway inflammation, and release of chemoattractants [2e5]. Glutathione is ubiquitously found in all cell types as a major non-protein sulfhydryl compound. Glutathione plays a significant role in several respiratory disorders and constitutes the first line of defense against oxidative inflammation along with other enzymatic/non-enzymatic antioxidants. Glutathione can also affect cellular signaling through redox sensitive regulation of kinases, phosphatases and transcription factors [2,6]. Previous studies have

http://dx.doi.org/10.1016/j.biochi.2014.04.001 0300-9084/Ó 2014 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: A. Nadeem, et al., Glutathione modulation during sensitization as well as challenge phase regulates airway reactivity and inflammation in mouse model of allergic asthma, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.001

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A. Nadeem et al. / Biochimie xxx (2014) 1e10

shown that glutathione redox balance can affect airway responses by regulation of immune responses during challenge phase. For instance, glutathione redox status in antigen-presenting cells determines whether immune response would be predominantly Th1 or Th2 type [7,8]. Furthermore, several indoor/outdoor pollutants found in our environment and known to deplete glutathione levels have been linked to increased asthma/allergy prevalence worldwide [9e11]. In support of this, glutathione repletion by thiol donors during diesel exhaust particle exposure has been shown to prevent airway reactivity and inflammation [12,13]. Earlier studies have documented increased ROS generation and airway reactivity when glutathione was depleted during challenge phase in allergic animals [14,15]. On the other hand, repletion of glutathione pool by thiol donors during allergen challenge phase causes reduction in allergic airway inflammation in different animal models [16,17]. However, no study so far has investigated the source of ROS generation during glutathione depletion in mouse model of asthma. Moreover, effect of depletion/repletion during sensitization and challenge phase separately has not been evaluated earlier. ROS and antioxidants have been shown to be involved in bronchoconstriction and inflammatory responses of asthmatic airways [2e4]. It has been shown earlier that depletion of antioxidants such as vitamin E during sensitization and challenge phase leads to increased airway reactivity and inflammation [18]. Conversely, ROS generated by NADPH oxidase or xanthine oxidase have been also shown to cause airway constriction and inflammation [5,19e21]. We hypothesized that glutathione might regulate airway reactivity and inflammation through modulation of NADPH oxidase pathway. No earlier study has looked into the aspect of NADPH oxidase pathway regulation through glutathione redox modulation in mouse model of allergic asthma. Therefore, the present study utilized BSO or NAC during both sensitization and challenge phases separately to evaluate the role of glutathione modulation on NADPH oxidase activity/expression, airway reactivity and inflammation in a mouse model of allergic asthma. Our data show for the first time that depletion or repletion of glutathione exacerbates or ameliorates allergic asthma respectively by regulation of oxidanteantioxidant balance. This might have implications towards increased prevalence of allergy when glutathione is depleted by common environmental pollutants.

For depletion of glutathione levels during sensitization phase, BSO was administered at 2 mmol/kg [15], i.p. 3 h before each sensitization; the same dose and time schedule was used for depletion of glutathione during challenge phase, i.e. days 14, 15 and 16. For repletion of glutathione levels during sensitization phase, NAC was administered at 2 mmol/kg [23], i.p. 3 h before each sensitization; the same dose and time schedule was used for repletion of glutathione during challenge phase, i.e. days 14, 15 and 16. BSO and NAC were dissolved in PBS and administered once only on indicated days. Mice were divided into following groups: Control group (CON): mice received only vehicles for sensitization and challenge; Control group administered BSO or NAC (CON þ BSO or CON þ NAC): mice received only vehicles for sensitization and challenge, and BSO or NAC was administered during challenge phase (days 14, 15 and 16) with the same dosing regimen described above; Sensitized and challenged group (SEN þ CHAL): mice were sensitized and challenged with ovalbumin using the same protocol described above; Sensitized and challenged group administered BSO or NAC during sensitization phase (BSO þ SEN þ CHAL or NAC þ SEN þ CHAL): mice were sensitized and challenged with ovalbumin using the same protocol described above and BSO or NAC was administered on days 1 and 6; Sensitized and challenged group administered BSO during challenge phase (SEN þ BSO þ CHAL or SEN þ NAC þ CHAL): mice were sensitized and challenged with ovalbumin using the same protocol described above and BSO or NAC was administered on days 14, 15 and 16. 2.3. Measurement of airway reactivity in vivo Twenty-four hours after final allergen challenge, airway reactivity to methalcholine in conscious, unrestrained mice were assessed by a whole-body noninvasive plethysmograph (Buxco Electronics Inc.) as described earlier [24,25]. Baseline Penh was determined by exposing mice to nebulized saline. To assess the role of glutathione in NADPH oxidase and xanthine oxidase activity modulation, allergen sensitized and challenged mice administered BSO during sensitization phase (BSO þ SEN þ CHAL) were treated with apocynin (0.3 mmol/kg, i.p.) [26] or allopurinol (10 mmol/kg, i.p.) [27] respectively 12 and 24 h before methacholine reactivity test. The mice were then exposed to increasing concentrations of aerosolized methacholine dissolved in saline (0e32 mg/mL) to obtain a dose response and Penh values were recorded at each dose.

2. Materials and methods 2.4. Bronchoalveolar lavage (BAL) 2.1. Animals Male Balb/C mice, 8e10 weeks of age (20e25 g), free of specific pathogens, were used in the experiments. The animals were obtained from Experimental Animal Care Center, College of Pharmacy, KSU. The animals were kept under standard laboratory conditions of 12-h lightedark cycle and 24e26 C ambient temperature. All experimental animals used in this study were under a protocol approved by Animal Care and Research Committee of College of Pharmacy, KSU. 2.2. Mice sensitization and challenge Sensitization was performed according to the protocol described earlier with little modification [22]. Mice were sensitized on days 1 and 6 with i.p. injections of ovalbumin (grade V), 10 mg adsorbed to 4 mg alum. Non-sensitized control animals received only alum with the same volumes. Two weeks after 1st sensitization, the mice were challenged intransally under light anesthesia with 100 mg ovalbumin once daily for three consecutive days (days 14, 15 and 16).

The trachea was cannulated to perform BAL after sacrifice of mice; 0.6 mLl phosphate-buffered saline was introduced into the lungs via the tracheal cannula and the total cells were counted manually in a hemocytometer chamber followed by spinning of cells onto glass slides for differential count. A differential count of at least 300 cells was made according to standard morphologic criteria on cytocentrifuged Diff-Quik stained slides. The number of cells recovered per mouse was calculated and expressed as mean SE per mL for each group. 2.5. Real-time PCR for NADPH oxidase subunits (gp91 phox and p67 phox) Total RNA was isolated from the lung/trachea of different groups using the TRIzol reagent from Life Technologies/Invitrogen followed by DNase treatment to eliminate potential genomic DNA contamination as described earlier [24,25]. This was followed by conversion of 0.5 mg of total RNA into cDNA using High Capacity cDNA archive kit (Applied Biosystems, USA) according to the



manufacturer’s instructions. Real-time PCR was performed on an ABI PRISM 7500 Detection System (Applied Biosystems) using Taqman Universal Mastermix (Applied Biosystems, USA), cDNA, and FAM-labeled Taqman gene expression kit. For the real-time PCR of NADPH oxidase subunits (gp91 phox and p67 phox), the Taqman inventoried assays-on-demand gene expression kits were purchased from Applied Biosystems. 18S rRNA (Ribosomal RNA) was used as an endogenous control. The fold difference in expression of target cDNA was determined using the comparative CT method. The fold difference in gene expression of the target was calculated as described earlier [28]. 2.6. Reactive oxygen species (ROS) assay For reactive oxygen species generation, the tracheal rings/BAL cells (0.25 million) were incubated with 100 mM 6-carboxy-20 ,70 dichlorofluorescin diacetate (DCFH-DA) for 30 min at 37 C. DCFHDA forms a fluorescent product, DCF (dichlorofluorescein) upon oxidation with ROS. Fluorescence caused by DCF in each well was measured and recorded for 30 min at 485 nm (excitation) and 530 nm (emission) by the method of Peshavariya et al. [29] using a multi-mode fluorescent microplate reader (FLUOstar Omega, BMG LabTech, USA) with temperature maintained at 37 C. For in vitro experiments, the compounds [NAC (1 mM), apocyanin (10 uM), and DPI (10 uM)] were incubated with BAL cells/tracheal rings for 30 min followed by measurement of DCF fluorescence as described above. The background fluorescence caused by buffer and DCF were subtracted from the total fluorescence in each well caused by the

cells/tracheal rings in the presence of DCF. Fluorescence intensity was expressed as ROS generation (% control). 2.7. Total antioxidant capacity assay Total antioxidant capacity of low molecular weight nonenzymatic antioxidants in the lung was measured by the method of Benzie and Strain [30] as described earlier by us [4,31]. Briefly, ferric tripyridyltriazine (FeIII-TPTZ) complex reduced by antioxidants present in the lung supernatant is monitored by measuring the change in absorption at 593 nm. The change in absorbance is directly related to the combined or total reducing power of electron donating non-enzymatic antioxidants present in the reaction mixture. The components of plasma that contribute to total antioxidant capacity are ascorbate, alpha-tocopherol, uric acid, bilirubin and remaining antioxidants. Results were expressed in mmol/mg. 2.8. Lipid peroxidation assay Cell membrane damage was monitored through the measurement of malondialdehyde (MDA), a metabolite resulting from lipid peroxidation by the method of Jentzsch et al. [32] as described earlier by us [31]. Briefly, lung supernatant was incubated with butylated hydroxyl toluene, ortho-phosphoric acid and thiobarbituric acid (TBA) in a total volume of 500 mL at 90 C for 45 min, followed by ice-cooling and extraction of MDAeTBA adducts in nbutanol. Absorption was read at 535 and 572 nm for baseline correction in a multititer plate reader. MDAeTBA adducts were

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(C) Fig. 1. Effect of glutathione depletion/repletion on allergen induced airway reactivity in mice. BSO or NAC administered i.p. during A) sensitization phase; and B) challenge phase. C) Effect of NADPH oxidase inhibitor, apocynin (APO) and xanthine oxidase inhibitor, allopurinol (ALLO) respectively administered i.p. during challenge phase is also shown in allergen sensitized and challenged mice having depletion of glutathione levels during sensitization phase (BSO þ SEN þ CHAL). Airway reactivity to methacholine was measured as Penh, 24 h after the final allergen challenge using a Buxco system for whole body plethysmography in which mice were exposed to increasing concentrations of methacholine (0e32 mg/ mL). Values are expressed as mean SE, n ¼ 4/group. *P < 0.05, vs. SEN þ CHAL group; #P < 0.05, vs. BSO þ SEN þ CHAL/SEN þ BSO þ CHAL group.


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calculated using the difference in absorption at the two wavelengths compared to the standard curve generated by the use of tetraethoxypropane. Results were expressed in mmol/mg. 2.9. Glutathione assay Lung samples were homogenized in 10% sulfosalicylic acid and centrifuged at 12,000 g for 15 min, followed by measurement of total and oxidized glutathione in the supernatant by the enzymatic recycling method using glutathione reductase [33] as described by us earlier [4]. For measurement of oxidized glutathione, 2vinylpyridine was added to the samples to mask reduced glutathione and prevent its reaction with 5,50 -dithio-bis(2-nitrobenzoic acid; DTNB). Reaction mixture consisted of tissue supernatant, glutathione reductase, DTNB and NADPH. The increase in the absorbance was measured at 412 nm which reflected the rate of 5thio-2-nitrobenzoate formation. Total/oxidized glutathione levels were extrapolated from the standard curves and then normalized to tissue weight. The amount of reduced glutathione was calculated by subtracting the amount of oxidized glutathione from that of total glutathione. 2.10. Chemicals Unless stated otherwise, all chemicals were of the highest grade available and were purchased from Sigma Chemicals (USA). Diphenyliodonium (DPI), was dissolved in dimethyl sulfoxide; while NAC, BSO and apocynin were dissolved in phosphate buffer saline.

2.11. Statistical analysis The data were expressed as mean SEM. Comparisons among different groups were analyzed by ANOVA (analysis of variance) followed by Tukey’s multiple comparison tests. A ‘P’ value of less than 0.05 was considered significant for all statistical tests. All the statistical analyses were performed using Graph Pad Prism statistical package. 3. Results 3.1. Effect of glutathione modulation on airway reactivity in allergic mice As shown in Fig. 1A and B, glutathione depletion by BSO during sensitization as well challenge phase resulted in increase of allergen induced airway reactivity as measured by Penh (Fig. 1A and B). On the other hand, glutathione repletion by NAC during both sensitization and challenge phases attenuated allergen induced airway reactivity (Fig. 1A and B). Control mice administered BSO or NAC during challenge phase had Penh values similar to each other. The Penh values (mean SEM, n ¼ 4) at 0, 2, 8, 16 and 32 mg/L methacholine exposure were 0.77 0.06, 1.15 0.17, 1.79 0.38, 2.48 0.53, and 3.08 0.46 for CON þ BSO group and 0.79 0.04, 1.13 0.13, 1.64 0.10, 2.68 0.13, and 3.12 0.30 for CON þ NAC group respectively. To know the role of NADPH oxidase in airway reactivity during glutathione depletion, allergen sensitized and challenged mice having glutathione depletion during sensitization

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(C) Fig. 2. Effect of glutathione depletion/repletion on allergen induced airway inflammation in mice. BSO or NAC administered i.p. during A) sensitization phase; and B) challenge phase. C) Effect of NADPH oxidase inhibitor, apocynin (APO) and xanthine oxidase inhibitor, allopurinol (ALLO) respectively administered i.p. during challenge phase is also shown in allergen sensitized and challenged mice having depletion of glutathione levels during sensitization phase (BSO þ SEN þ CHAL). Airway inflammation in BAL was assessed 48 h after the final allergen challenge through total cell and eosinophil cell counts. Values are expressed as mean SE, n ¼ 5e6/group. *P < 0.05, vs. SEN þ CHAL group; #P < 0.05, vs. BSO þ SEN þ CHAL/SEN þ BSO þ CHAL group.



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Fig. 3. Effect of glutathione depletion/repletion on the levels of reduced and oxidized glutathione in the lung of allergic mice. BSO or NAC administered i.p. during A) sensitization phase; and B) challenge phase. Reduced and oxidized glutathione levels were measured in all the groups biochemically after the last allergen challenge. Values are expressed as mean SE, n ¼ 5e6/group. *P < 0.05, vs. SEN þ CHAL group; #P < 0.05, vs. BSO þ SEN þ CHAL/SEN þ BSO þ CHAL group.

phase were treated in the challenge phase with NADPH oxidase inhibitor, apocynin followed by dose response with methacholine. Treatment with NADPH oxidase inhibitor, apocyanin significantly decreased airway reactivity in this group of mice (Fig. 1C). On the

other hand, allopurinol, a xanthine oxidase inhibitor did not show any significant effect on airway reactivity in allergen sensitized and challenged mice having glutathione depletion during sensitization phase (Fig. 1C). These data show that glutathione depletion

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Fig. 4. Effect of glutathione depletion/repletion on allergen induced NADPH oxidase expression in lung and trachea of mice. BSO or NAC administered i.p. during A & C) sensitization phase; and B & D) challenge phase. Expression of gp91phox and p67 phox subunits in all the groups was assessed by real time PCR. For mRNA expression by comparative CT method using real time PCR, first column was made as the calibrator against which the other groups were compared. Values are expressed as mean SE, n ¼ 5e6/group. *P < 0.05, vs. SEN þ CHAL group; #P < 0.05, vs. BSO þ SEN þ CHAL/SEN þ BSO þ CHAL group.


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increases whereas glutathione repletion decreases allergen induced airway reactivity. Further, the increase/decrease in airway reactivity is likely to be through modulation of NADPH oxidase activity. 3.2. Effect of glutathione modulation on airway inflammation in allergic mice As shown in Fig. 2A and B, glutathione depletion by BSO during sensitization as well challenge phase resulted in increase of allergen induced airway inflammation as reflected by an increase in total cell and eosinophil counts (Fig. 2A and B). On the contrary, glutathione repletion by NAC during sensitization as well challenge phase attenuated allergen induced airway inflammation (Fig. 2A and B). Control mice administered BSO or NAC during challenge phase did not differ in inflammatory BAL cell counts. Total cell and eosinophil cell counts (104/mL; mean SEM, n ¼ 6) were 19 1.15 and 0.57 0.26 for CON þ BSO group and 17.17 1.53 and 0.50 0.23 for CON þ NAC group respectively. When allergen sensitized and challenged mice having glutathione depletion during sensitization phase were treated with NADPH oxidase inhibitor, apocynin in the challenge phase, it resulted in significant reduction of airway inflammation, whereas xanthine oxidase inhibitor, allopurinol caused no significant reduction in inflammation (Fig. 2C); thus showing that glutathione probably regulates allergic inflammation through NADPH oxidase pathway.

3.4. Effect of glutathione modulation on expression of NADPH oxidase subunits in allergic mice To confirm the role of glutathione in modulation of NADPH oxidase activity during allergic airway responses; effect of glutathione depletion/repletion during both sensitization and challenge phases was assessed on NADPH oxidase subunits expression.

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Glutathione ratio was measured to determine its effects on airway responses and NADPH oxidase signaling in the lungs of allergic mice. As shown in Fig. 3A and B, glutathione depletion by BSO during sensitization as well challenge phase resulted in decreased ratio of reduced to oxidized glutathione in allergic mice (Fig. 3A and B). The changes in glutathione redox caused by allergen challenge alone were less in magnitude than caused in combination with BSO in allergic lungs. On the other hand, glutathione repletion by NAC during sensitization as well challenge phase restored cellular ratio of glutathione redox in allergic mice (Fig. 3A and B). These data suggest that BSO or NAC cause effects opposite to each other on airway responses in allergic mice through increase or decrease of glutathione redox ratio respectively.

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Fig. 5. Effect of glutathione depletion/repletion on allergen induced ROS generation in trachea and BAL cells of mice. BSO or NAC administered i.p. during A) sensitization phase; and B) challenge phase. C) Effect of NADPH oxidase inhibitor, apocynin (APO) and xanthine oxidase inhibitor, allopurinol (ALLO) respectively administered i.p. during challenge phase is also shown in allergen sensitized and challenged mice having depletion of glutathione levels during sensitization phase (BSO þ SEN þ CHAL). D) Effect of NADPH oxidase inhibitors, apocynin (10 mM)/diphenyliodonium (DPI, 10 mM) and thiol donor, NAC (1 mM) is also shown in allergen sensitized and challenged mice administered BSO during sensitization phase (BSO þ SEN þ CHAL) in vitro. ROS were measured as end products of NADPH oxidase activation in all the groups biochemically. Values are expressed as mean SE, n ¼ 4e5/ group. *P < 0.05, vs. SEN þ CHAL group; #P < 0.05, vs. BSO þ SEN þ CHAL/SEN þ BSO þ CHAL group.



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Depletion of glutathione by BSO during sensitization as well challenge phase led to increased mRNA transcripts for NADPH oxidase subunits, p67 phox and gp91 phox (Fig. 4AeD) in trachea and lung of allergic mice. On the other hand, repletion of glutathione by NAC during sensitization as well challenge phase resulted in attenuation of NADPH oxidase subunits expression (Fig. 4AeD). These data show that glutathione is associated with modulation of NADPH oxidase subunits expression in allergic mice.

inhibited ROS generation in allergen sensitization and challenged mice having glutathione depletion during sensitization phase (Fig. 5D). These data show that glutathione regulates airway reactivity and inflammation through NADPH oxidase mediated ROS production in murine allergic airways.

3.5. Effect of glutathione modulation on reactive oxygen species (ROS) in allergic mice

Since ROS generation is associated with alteration in oxidante antioxidant balance, we measured, total antioxidant capacity (combined non-enzymatic antioxidant potential) and lipid peroxides formation in the lung. Glutathione depletion with BSO during both sensitization as well as challenge phase worsened whereas glutathione repletion with NAC ameliorated allergen induced changes in total antioxidant capacity (Fig. 6A and B) and lipid peroxides (Fig. 6C and D). These data suggest that glutathione regulates airway oxidanteantioxidant balance through ROS generated by NADPH oxidase in the airways of allergic mice.

Modulation of NADPH oxidase activity due to depletion/repletion of glutathione in allergic mice was also confirmed by measuring ROS production in trachea and BAL cells. Glutathione depletion during sensitization as well as challenge phases led to increase in allergen induced ROS generation in trachea and BAL cells (Fig. 5A). Conversely, glutathione repletion in both phases resulted in attenuation of allergen induced ROS generation in trachea and BAL cells (Fig. 5B). NADPH oxidase inhibitor, apocynin administered in vivo (Fig. 5C) and in vitro (Fig. 5D) reduced allergen induced ROS production. Xanthine oxidase inhibitor, allopurinol administered in vivo did not have any significant effect on allergen induced ROS production (Fig. 5C). Effect of glutathione repletion was also assessed on ROS generation in vitro. Treatment of trachea and BAL cells with NAC

3.6. Effect of glutathione modulation on antioxidants and lipid peroxides in allergic mice

4. Discussion Our study shows for the first time that glutathione is not only important during the sensitization but also during challenge phase in regulation of airway reactivity and inflammation which is

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Fig. 6. Effect of glutathione depletion/repletion on allergen induced changes in total antioxidant capacity and lipid peroxides in the lung of mice. BSO or NAC administered i.p. during A & C) sensitization phase; and B & D) challenge phase. Total antioxidant capacity and lipid peroxides were measured in all the groups biochemically. Values are expressed as mean SE, n ¼ 5e6/group. *P < 0.05, vs. SEN þ CHAL group; #P < 0.05, vs. BSO þ SEN þ CHAL/SEN þ BSO þ CHAL group.


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through modulation of NADPH oxidase activity/expression in murine model of allergic asthma. Glutathione depletion during sensitization as well as challenge phase worsened allergen induced increase in airway reactivity and inflammation and also led to increased oxidant antioxidant imbalance as reflected by increased NADPH oxidase activity and decreased antioxidants. On the other hand, glutathione repletion during sensitization as well as challenge phase attenuated allergen induced increase in airway reactivity and inflammation and led to inhibition of NADPH oxidase activity and an increase in antioxidants. These data suggest that glutathione deficiency in allergic mice is linked to NADPH oxidase activation/ROS generation and altered antioxidant defenses. Glutathione mediated effects on airway reactivity and inflammation could be due to several factors: a) Th1/Th2 balance regulation as shown earlier by several studies [7,10]; b) alteration in nitric oxide metabolism through formation of S-Nitrosoglutathione, which has been shown to be associated with modulation of airway responses [34]; and c) ROS generation and alteration in antioxidant defenses [35]. Our study supports the last notion and highlights one more dimension of glutathione mediated signaling during allergic responses. Glutathione-mediated effects on ROS production and oxidative stress in allergic animals have been shown in earlier studies but the source of ROS was not indicated [14,15]. Moreover, effect of glutathione alteration during sensitization and challenge phases separately has not been investigated previously. Our study showed that decrease in ratio of reduced to oxidized glutathione had worsening effect on airway responses through ROS generation in allergic mice. This was confirmed by Penh, biochemical and real-time PCR data. Real-time PCR data from this study showed an increase in expression of p67 phox and gp91 phox, subunits of NADPH oxidase in allergen sensitized and challenged trachea and lung after glutathione depletion as compared to allergen sensitized and challenged mice. ROS, which are end products of NADPH oxidase activation, were higher in trachea and BAL cells of allergen sensitized and challenge mice having glutathione depletion as compared to allergen sensitized and challenged mice. Treatment with NADPH oxidase inhibitor, apocyanin led to decreased airway reactivity, inflammation and ROS in allergen sensitized and challenged mice having glutathione depletion. Treatment with NAC, a thiol donor during sensitization or challenge phase also led to decreased airway reactivity, inflammation and ROS generation. NAC has been shown to reduce expression of NADPH oxidase subunits as well as ROS generation previously in other disease models [36,37]. It has also been shown earlier in alveolar epithelial cells in vitro that shift of redox equilibrium towards an oxidizing microenvironment through the use of BSO leads to inflammation via upregulation of ROS whereas repletion of cellular glutathione pool by NAC leads to attenuation of inflammation through reduction of ROS [38]. Our study confirms these observations in airways of allergic mice. ROS generated by NADPH oxidase or xanthine/xanthine oxidase systems have been shown to cause airway reactivity and inflammation [19e21]. Apocynin treatment in human asthmatics and allergic mice has been shown to decrease airway reactivity/ inflammation [26,39]. Moreover, gp91 phox as well as p47 phox deficient allergic mice have decreased airway reactivity and inflammation, which further supports the role of NADPH oxidase generated ROS in asthma [20,21]. Lack of effect on any of the parameters, i.e. airway reactivity, inflammation and ROS generation by xanthine oxidase inhibitor, allopurinol suggests that ROS generation in allergen sensitized and challenged mice having glutathione depletion is via NADPH oxidase pathway. Glutathione being a major intracellular thiol may affect ROS generation through different mechanisms. Earlier studies carried

out with thiol donors in vitro/in vivo have shown that glutathione repletion suppresses transcription factors such as NF-kB and kinases such as p38MAPK/PI3K which are thought to be involved in allergic airway reactivity/inflammation [38,40e42]. It has been reported that increased ROS generation during a state of glutathione depletion may result from activation of kinases such as PKC and MAPK [2,38,43,44]. Increased NADPH oxidase expression/activity observed in our study after glutathione depletion in allergic mice may be due to activation of upstream kinases sensitive to redox regulation by glutathione. In support of this observation, it has been reported previously that NAC may inhibit activation of kinases which are thought to be involved in regulation of NADPH oxidase activity [45]. Therefore, it is reasonable to infer that glutathione modulates NADPH oxidase pathway through redox sensitive mechanisms in allergic airways. The possibility of feedback regulation of NADPH oxidase by ROS generated by itself cannot be ruled out; however, this needs further investigation with specific pathway inhibitors through in vitro cell culture and this is a limitation of our study. ROS generation can lead to formation of lipid peroxides through attack on cellular membranes as well as decrease in nonenzymatic antioxidants, which are considered to be first line of defense against them [2,3]. Total antioxidant capacity (combined non-enzymatic antioxidant potential) shows overall picture of antioxidant status of the organ and obviates the need to measure each non-enzymatic antioxidant separately. Total antioxidant capacity has also been shown to be associated negatively with airway obstruction in asthmatic human subjects [4]. Therefore, we chose total antioxidant capacity and lipid peroxides along with ROS to represent overall oxidanteantioxidant balance in the airways. Our study shows decreased airway total antioxidant capacity along with increased lipid peroxides in sensitized and challenged mice having glutathione depletion. Decrease in total antioxidant capacity observed in our study is likely due to scavenging of NADPH oxidase-mediated ROS by non-enzymatic antioxidants thus leading to diminished protection of cellular structures from oxidative inflammation caused by glutathione depletion. Treatment with NAC either during sensitization or challenge phase led to normalization of oxidanteantioxidant balance along with concomitant improvement in allergic airway reactivity and inflammation. Earlier studies in human and animals have shown that thiol antioxidants play an important role in airway reactivity and inflammation [4,14e16,21,40,41,46,47]. For example, L-2oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine reduced airway hyperresponsiveness and inflammation by increasing glutathione levels in allergic mice [46]. Another study showed that administration of a more hydrophobic and membrane permeable form of NAC known as N-acetyl cysteine amide (NACA) led to attenuation of airway hyper responsiveness and inflammation in allergen challenged mice [40]. A recent study with another novel compound having two thiol groups, i.e. CB3 also resulted in significant reduction of allergen induced airway reactivity and inflammation [41]. Supplementation with NAC has also been reported to attenuate BAL ROS generation and airway reactivity/ inflammation in occupational mouse model of asthma [21]. Recently, OTC and a-lipoic acid (capable of regenerating reduced glutathione from its oxidized form intracellularly) have been shown to inhibit ROS mediated airway remodeling in mouse model of asthma [47]. All of these studies show that restoration of glutathione redox is crucial in normalization of airway responses. Our study supports these earlier observations. Most importantly, our study highlights for the first time that NADPH oxidase pathway and airway reactivity/inflammation are linked to each other through modulation of glutathione redox in airways of allergic mice.



BAL is a mixture of different inflammatory cells such as macrophages, neutrophils, lymphocytes and eosinophils, all of which contribute significantly to the pathogenesis of asthma. ROS in asthmatic lungs have been shown to be generated from almost all inflammatory cell types, therefore ROS generation in BAL cells in our study most likely shows combined ROS generation from all the cells present in BAL. Glutathione and other antioxidants have been reported to be decreased during exposure to common indoor and outdoor environmental pollutants [10,17]. It has also been shown earlier that environmental pollutants such as diesel exhaust particles and ultrafine particles are associated with increased airway responses to allergen [10,11]. Combination of these factors is likely to predispose the individuals to increased allergy leading to enhanced airway reactivity and inflammation. To support this view, administration of NAC and NACA during challenge phase has been shown to attenuate allergen as well as diesel exhaust/ultrafine particle-mediated airway inflammation through restoration of cellular glutathione pool [10,12,13,17]. In conclusion, our data suggests that glutathione redox status is critically involved in determination of allergic airway responses during sensitization phase as well as phase through modulation of oxidant antioxidant balance. Therefore, strategies aimed at augmentation of glutathione through thiol donors should be considered to blunt increased predisposition to allergy by indoor/ outdoor pollutants.

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

Conflict of interest [22]

The authors declare that there is no conflict of interest. [23]

Acknowledgements This study was funded (Project No. RGP-VPP-305) by Deanship of Scientific Research, College of Pharmacy, KSU.

[24]

[25]

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