TRANSATLANTIC AIRWAY CONFERENCE Oxidative Stress in Airway Diseases Fernando Holguin1 1

Asthma Institute, Division of Pulmonary, Allergy and Critical Care, University of Pittsburgh, Pittsburgh, Pennsylvania

Abstract Airway oxidative stress is broadly defined as an imbalance between prooxidative and antioxidative processes in the airway. Given its direct exposure to the environment, the lung has several mechanisms to prevent an excessive degree of oxidative stress. Both enzymatic and nonenzymatic systems can buffer a wide range of reactive oxidative species and other compounds with oxidative potential. In diseases like asthma and chronic obstructive lung disease, airway oxidative stress can occur from a number of sources, including greater exposure to environmental prooxidants, airway infiltration of inflammatory cells, metabolic deregulation, and reduced levels of antioxidants. Airway oxidative stress has been associated with worse disease severity, reduced lung function, and epigenetic changes that can

diminish response to steroids. Although oxidative stress has been linked to a wide range of adverse biological effects, it has also been associated with adaptive responses and with resolution of inflammation. Therefore, more than being an imbalance with a predictable threshold after which disease or injury ensues, oxidative stress is a dynamic and continuous process. This might explain why supplementing antioxidants has largely failed to improve diseases such as asthma and chronic obstructive pulmonary disease. However, the therapeutic potential of antioxidants could be greatly improved by taking an approach that considers individual and environmental risk factors, instead of treating oxidative airway stress broadly. Keywords: oxidative stress; asthma; chronic obstructive pulmonary disease

(Received in original form May 10, 2013; accepted in final form July 3, 2013 ) Correspondence and requests for reprints should be addressed to Fernando Holguin, M.D., M.P.H., Division of Pediatric Pulmonology, 3705 Fifth Avenue, Pittsburgh, PA 15213. E-mail: [email protected] Ann Am Thorac Soc Vol 10, Supplement, pp S150–S157, Dec 2013 Copyright © 2013 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201305-116AW Internet address: www.atsjournals.org

Oxidative Stress: A Continuum Spectrum Oxidative stress is defined as an imbalance between increased oxidative sources and reduced or defective antioxidant mechanisms. Although conceptually this is adequate, it offers poor insight into how dynamic and highly complicated this process really is. This definition lends itself to the notion that a particular balance does in fact exist and that deviations from this point can affect homeostasis and potentially cause or worsen disease. As such, many research projects have attempted to restore this “balance” by providing antioxidants, which for the most part have ended with disappointing results. This lack of therapeutic effectiveness probably stems from several factors, including our inability to distinguish when, in a given disease process, oxidative stress is a disease driver S150

or merely an epiphenomenon; even worse, treating with antioxidants may prevent some lower degree of oxidative stress from occurring, which could play an important role in controlling inflammation and cellular adaptive responses. Nowhere are these complexities of oxidative stress more evident than in the airways, which are constantly exposed externally to oxidative compounds and internally to recruited and activated inflammatory cells. Given the lack of adequate standardization in the many biomarkers used to define airway oxidative stress, it is difficult to fully gauge their overall contribution to the development or progression of airway diseases. Therefore, instead of presenting a summary of oxidative stress studies, this review proposes a conceptual framework that captures the dynamic interplay among the many oxidative sources and the antioxidant mechanisms; how these balancing forces

exist in a continuum spectrum that can vary in the same individual across time and exposures, between persons, and between different types of airway diseases. This framework, shown in Figure 1, encompasses the major sources of airway oxidative stress and some of the major determinants of antioxidant mechanisms, and Table 1 highlights the heterogeneity in oxidative stress between asthma and chronic obstructive pulmonary disease (COPD). By no means is this intended to be a comprehensive review of the pathophysiology of airway oxidative stress. Sources of Oxidative Stress, Broadly Understood

Perhaps one of the best understood sources for increased airway oxidative stress is the recruitment of inflammatory cells into the airway after exposure to trigger factors; these activated cells can generate anion

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TRANSATLANTIC AIRWAY CONFERENCE increase disease severity or risk of exacerbation. Environmental exposures as contributors to airway oxidative stress. The

Figure 1. Conceptual overview of airway oxidative stress sources and mechanisms in asthma. The figure captures the conceptual framework related to airway oxidative mechanisms throughout the paper. ADMA = asymmetric dimethylarginine; GSH = reduced glutathione; GSSG = oxidized glutathione; HDAC2 = histone deacetylase–2; NADPH = nicotinamide adenine dinucleotide phosphate; NF-kB = nuclear factor kB; Nrf2 = nuclear factor (erythroid-derived 2)-like 2; PM2.5 = particulate matter , 2.5 mm; SOD = superoxide dismutase.

superoxide (O2.2) through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathway. Mitochondrial dysfunction in airway epithelial cells, which occurs in response to mechanical and environmental stimuli, can also contribute to the formation of anion superoxide and airway oxidative stress (1). Anion superoxide is rapidly dismutated to hydrogen peroxide (H2O2) by superoxide dismutase enzymes (SOD). From here, H2O2 can react with transition metals to generate hydroxyl radicals (dOH) or, through the action of eosinophil or neutrophil peroxidases, interact with halides to respectively form hypobromous acid (HOBr) or hypochlorous acid (HOCl) (2, 3). In addition to formation of these hypohalides, increased levels of nitric oxide (NO) are formed by the up-regulation of the epithelial inducible nitric oxide synthase (iNOS). In the presence of reactive oxygen species (ROS), NO rapidly forms reactive nitrogen species (RNS), such as peroxynitrite (4). There has been considerable progress in understanding the downstream effects and clinical correlates related to these compounds. Hypohalides can brominate, chlorinate, or nitrate tyrosine residues affecting protein structure and function. Chloro-, bromo-, and nitrotyrosine compounds have been identified in a number of disease processes.

In asthma, these compounds have been shown to amplify the oxidative and inflammatory airway process and have been associated with poor control (5–8). For example, using proteomic analysis, researchers have experimentally identified and confirmed in humans that oxidative modification by nitro- and chlorotyrosine reduces catalase activity, thereby allowing more H2O2 to accumulate and further propagate oxidative reactions (9). This finding may also partly explain why, when compared with normal subjects, subjects with asthma have greater systemic and airway increased oxidative stress, which is associated with worse asthma severity (10, 11). As with asthma, subjects with COPD have increased airway oxidative stress and nitrosative stress (12). Patients with COPD have a greater degree of immunostaining for nitrotyrosine in the airway epithelium and inflammatory cells in sputum (13). Given the predominant neutrophilic airway inflammation, it is possible that the nitration of tyrosine residues results predominantly from myeloperoxidase (MPO)-induced oxidation of NO22 by H2O2 or through the formation of RNS from the interaction of NO and ROS (13). However, unlike asthma, there are considerably fewer data linking nitrotyrosine measurements with

Holguin: Oxidative Stress in Airway Diseases

lung is exposed to several thousand liters of air per day. Every breath carries with it a very large number of compounds with oxidative potential, including air pollution, pollen, and particulate matter. Although larger particles are efficiently cleared by the nose and upper airways, fine particles can easily access the lower airways and promote increased airway oxidation and inflammation (14, 15). Air pollutants can cause oxidative stress through a variety of mechanisms, such as direct oxidative injury by gas phase pollutants like ozone or nitrogen oxides, or by promoting airway inflammation in the airways (16, 17). For example, fine particulate matter (PM2.5, diameter , 2.5 mm) can react with the respiratory epithelium and promote nuclear translocation of transcription factors with subsequent release of inflammatory cytokines (18). The airway oxidative stress associated with air pollutants is evident even after short-term exposures, particularly among subjects with asthma or other airway diseases. In a study of otherwise healthy subjects with controlled asthma, those taking a 2-hour planned walk through Oxford Street in London (more highly exposed to diesel emissions) had transient decrements in FEV1 and greater levels of MPO, IL-8, and neutrophil count in sputum, when compared with those of similar severity walking through Hyde Park (19). Epidemiological studies have also shown close temporal and exposure associations in children between sulfur dioxide emissions with increased thiobarbituric acid–reactive substances (TBARS) and between fine particulate matter, exhaled NO, and sputum IL-8 levels (20, 21). Other nonpollutant environmental sources can contribute to airway oxidative stress. One interesting example is pollen and subpollen grains, which have been shown to have intrinsic NADPH oxidases and to induce mitochondrial dysfunction in airway epithelial cells; in addition to releasing ROS in the airway epithelium, these particles can activate dendritic cells (DC) (22, 23). Depending on the degree of pollen-induced oxidative stress, the DC response may range from adaptive (at lower concentration) to proinflammatory (at higher concentrations). This dual S151

TRANSATLANTIC AIRWAY CONFERENCE Table 1. Similarities and differences in airway oxidative stress sources and antioxidant mechanisms between asthma and COPD Asthma

COPD

Airway Oxidative Sources Inflammatory airway cells Inflammatory airway cells Predominantly eosinophilic (eosinophil Predominantly neutrophilic peroxidase, bromo- and nitrotyrosine (myeloperoxidase, nitro- and compounds) chlorotyrosine compounds) Mitochondrial dysfunction in airway Mitochondrial dysfunction in airway epithelial cells epithelial cells Air pollution (ozone, PM, NO2) Air pollution (ozone, PM, NO2) Pollen, subpollen particles Pollen, subpollen particles Tobacco (1)* Tobacco (111) Biomass smoke exposure Biomass smoke exposure Airway leptin Metabolic dysregulation in obesity Oxidative stress–mediated dysfunction (higher airway leptin, higher ADMA, in HDAC lower L-arginine), high-fat meals (transient increase in neutrophilic airway inflammation) Antioxidant Airway Mechanisms Higher GSH at baseline; may be lower Higher GSH at baseline; lower GSH levels during exacerbation or with increased with exacerbations; tobacco depletes severity GSH by forming aldehyde derivatives. Reduced SOD and catalase activity Higher levels of extracellular SOD in sputum; greater bronchial epithelium expression of Mn-SOD Lower levels of nonenzymatic antioxidants, Variable results in nonpulmonary, systemic associated with lower lung function and indices of SOD and catalase respiratory symptoms Lipoxin and other electrophilic fatty acids Lower levels of antioxidants associated play a role in the resolution of airway with lower lung function and respiratory inflammation symptoms Definition of abbreviations: ADMA = asymmetric dimethyl arginine; GSH = glutathione; HDAC = histone deacetylase; Mn-SOD = manganese superoxide dismutase; NO2 = nitrogen dioxide; PM = particulate matter; SOD = superoxide dismutase. *Plus signs represent more or less intensity of tobacco exposure playing a role in airway oxidative stress.

response in DC to oxidative stress may lead to different T-cell phenotypes; depending on when the DC-naive T-cell interaction occurs, it may explain why diesel exhaust particle–mediated oxidative stress has been shown to be an adjuvant toward allergic sensitization (24). Thus, air pollution–mediated oxidative stress may not only increase the risk of developing allergic airway diseases (25) but also potentially contribute to other well-known long-term health effects of air pollution, such as reduced lung growth, steeper lung function decline, and reduced response to asthma medications (26, 27). Both active and passive tobacco smoke constitute one of the most significant exposures to airway oxidative stress sources in humans. It is estimated that a single puff contains 1014 oxygen radicals, which exist in gas and tar phases. Gas phase radicals include, among many other ROS, epoxides, S152

peroxides, peroxynitrate, and NO. Tar phase compounds include semiquinone, dOH, and H O radicals. Active smokers 2 2 have greater levels of airway and systemic biomarkers of oxidative stress (28). These biomarkers are increased during COPD exacerbations and are associated with bronchitic symptoms, air trapping, and loss of lung function (29, 30). Biomass exposure is a major public health burden in developing countries and one that is also shown to cause systemic and airway oxidative stress that may play an important role in the development of biomass-related obstructive lung disease and bronchitis (31, 32). Metabolic dysregulation as a source of airway oxidative stress. There is growing

evidence that obesity is a significant comorbidity that can worsen asthma severity and reduce control by mechanisms that are not determined by increased

allergic airway inflammation. In fact, rather paradoxically, among subjects with asthma, increasing body mass index (BMI) has been associated with fewer airway eosinophils and lower exhaled NO levels (33, 34). Some studies have shown that with increasing BMI, there are greater levels of airway oxidative stress biomarkers that are also associated with reduced corticosteroid response in vitro (35, 36). Airway oxidative stress in obese patients with asthma may be explained by the enhanced inflammatory response associated with leptin. Experimentally, leptin increases the number of inflammatory cells and cytokines in the airways (37). In humans, obese subjects with asthma have the greatest levels of airway leptin and have been shown to release proinflammatory cytokines from activated alveolar macrophages (38, 39). Another source of oxidative stress may be the formation of hypochlorous acid from MPO in the setting of airway neutrophilia, which has been described in association with obesity and to occur after a high-fat meal diet (40, 41). Yet another more recently proposed source for airway oxidative stress in obesity is related to the generation of anion superoxide from airway iNOS. During NOS uncoupling, this enzyme produces anion superoxide instead of NO, with the dual effect of contributing to oxidative stress while reducing NO bioavailability (42). Uncoupling has been shown to occur when there is less enzyme substrate (L-arginine) and/or when the levels of endogenous NOS inhibitors are increased. Asymmetric dimethyl arginine (ADMA) is derived from the posttranslational methylation of L-arginine and is one of three NOS inhibitors that can uncouple all NOS isoforms (43). Subjects with asthma, particularly those with more severe disease, have been shown to have lower L-arginine levels (44). In addition, obesity and the metabolic syndrome have been associated with greater ADMA levels (45). Cross-sectionally, plasma L-arginine/ ADMA is inversely related to BMI in subjects with asthma, and lower ratios have been linked with increased frequency of respiratory symptoms (46). Furthermore, the L-arginine/ADMA balance may potentially determine why BMI is inversely associated with exhaled NO. ADMA has been found to be higher in the airways of subjects with asthma and to be inversely related to exhaled NO. In murine models

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TRANSATLANTIC AIRWAY CONFERENCE of airway sensitization, pretreatment of animals with ADMA can augment the airway inflammatory response (42). Therefore, based on these studies, the L-arginine/ADMA balance could be an important determinant of airway oxidative stress and reduced NO bioavailability. The clinical relevance of this pathway could be potentially determined in further studies by supplementing either L-arginine or L-citrulline to obese subjects with asthma, which can block NOS uncoupling and reduce airway oxidative stress (47). This supplementation would be particularly beneficial for those with asthma onset after the age of 12 years, for whom this pathway seems to be more relevant (46). Antioxidants mechanisms in the lung, broadly understood. The lung is equipped

with enzymatic (SOD, catalase, glutathione peroxidase) and nonenzymatic antioxidant systems (ceruloplasmin, ferritin, ascorbic acid, uric acid, thionine, and carotene), which allow the lung to function while constantly buffering a wide range of environmental oxidants. Having inadequate antioxidant levels or mechanisms is a hallmark of airway oxidative stress that is well established and the subject of excellent reviews (48–50). The goal of this section is therefore not to review each pathway but rather to discuss these antioxidants as a dynamic component of oxidative stress with clinical and redox signaling repercussions. Glutathione (GSH) is a tripeptide thiol concentrated in the alveolar epithelial lining fluid that reduces organic hydroperoxides and protects against airway lipid peroxidation. During oxidative reactions, GSH is converted to glutathione disulfide or GSSG, which under normal circumstances is mostly in the form of GSH, and less than 5% is GSSG (51). Compared with healthy control subjects, adults with asthma have higher GSH airway concentrations, which may reflect an adaptive response to increased baseline production of ROS (52). However, children with severe symptomatic asthma have been shown to have significantly lower airway GSH levels and a greater proportion of GSSG, when compared with subjects with mild to moderate asthma or healthy control subjects (53). These children also had the greatest airway levels of H2O2, malondialdehyde, and 8-isoprostanes, which correlated with the degree of oxidized glutathione, suggesting that

greater oxidative burden in subjects with severe asthma leads to a more oxidized thiol redox potential. Yet, interestingly, a reduced airway GSH/GSSG ratio is not only a biomarker of oxidative stress but also may propagate inflammation, skew the inflammatory response toward a Th2 phenotype, and impair redox signaling. Indeed, it has been shown that among children with severe asthma with impaired thiol redox balance, there is a corresponding increased expression of Nrf2 (nuclear factor [erythroid-derived 2]like 2), yet this is was highly dysfunctional as it did not increase some of the antioxidant response elements (ARE), including key enzymes involved in glutathione synthesis and conjugation (54). A significant imbalance in the airway thiol metabolism has also been described for patients with COPD, which may be associated with downstream redox transcription changes and proinflammatory events (55). Similar to subjects with asthma, those with COPD have higher airway GSH levels; however, these levels can be reduced by active smoking and during COPD exacerbations (30). Reduced GSH levels in acute exacerbations may reflect an insufficient response to oxidative stress in the form of reduced g-glutamyl cysteine synthetase activity or might result from direct depletion by an overwhelming oxidative burden from ROS released by activated airway neutrophils. In addition, tobacco smoke may deplete GSH by forming glutathione aldehyde derivatives, which cannot be converted to GSSG (56). Regardless of the underlying mechanisms, a thiol airway redox imbalance in COPD causes an impaired antioxidant response, allowing ROS and RNS to produce redox transcription changes that further propagate inflammation and worsen oxidative stress. These changes may partly occur through nuclear factor (NF)-kB activation and subsequent release of proinflammatory cytokines. Also, inhibition of histone deacetylase-2 (HDCA2) can secondarily reduce Nrf2 activity, thereby further impairing the antioxidant response (57). SOD and catalase are also vulnerable targets to the downstream effects of increased airway nitrosative and oxidative stress, which can cause enzymatic modification resulting in loss of activity. Oxidation and nitration of Mn-SOD is present in the asthmatic airway and

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correlates with disease severity (58). Also, in subjects with asthma, lower serum SOD activity is significantly related to the degree of airflow obstruction and is inversely associated with 3-bromotyrosine levels (59). The activity of catalase, a major H2O2 scavenger, is also reduced in asthma. Compared with healthy control subjects, subjects with asthma have reduced catalase activity in the bronchoalveolar lavage. The recovered catalase from the airways of subjects with asthma has increased markers of protein oxidation, including nitrotyrosine and chlorination and oxidation of sulfhydryls, linking oxidative modification to reduced activity in vivo (9). Although oxidative modification of SOD or catalase has not been described to the same extent in COPD, several studies have shown that this condition is associated with changes in the expression and levels of these enzymes. Compared with healthy control subjects, extracellular SOD is increased in the sputum of patients with COPD, with the highest concentrations seen among those who are also active smokers (60). The expression of Mn-SOD has also been shown to be higher in the central bronchial epithelium of smokers with COPD and in the alveolar epithelium of smokers without or with COPD, when compared with nonsmokers (61). Using plasma levels and erythrocyte concentrations as indices of systemic oxidative stress, the results are variable, with some studies showing increased or decreased levels of SOD and catalase (62). These discrepancies may be explained by differences in smoking behavior among subjects. Nonenzymatic antioxidants. In population studies, plasma antioxidant levels have been associated with lung function even after adjusting for smoking and other potential confounders. A change in 1 SD in the serum levels of either b-carotene or vitamin C or vitamin E or selenium is independently associated with changes in FEV1 ranging from 23 to 49 ml, yet when considering the joint effect of these antioxidants, 1 SD is associated with as much as 94 ml; this amount of lung volume is roughly equivalent to the difference in FEV1 between people 4 years apart (63). These epidemiological data provide a convincing argument that some antioxidants are critical to preventing airway obstruction. Compared with healthy control subjects, patients with asthma have S153

TRANSATLANTIC AIRWAY CONFERENCE lower plasma levels of carotenes, ascorbic acid, and vitamin E (64). Reduced ascorbic acid levels have been associated with increased respiratory symptoms, higher odds of being diagnosed with asthma, and diminished lung function, whereas lower plasma carotene levels have also been associated with higher odds for asthma diagnosis (65, 66). Reduced plasma antioxidant levels in subjects with asthma may be secondary to greater airway and systemic oxidative burden known to occur in these patients. Similarly, COPD is characterized by low plasma antioxidant levels. Having reduced levels of vitamin C and b-carotene is associated with bronchitic symptoms and lower FEV1 and FVC in subjects with chronic airflow obstruction, with steeper associations noted among active smokers (66, 67).

Oxidative Stress: A Role for Resolution of Inflammation It is commonly misunderstood that in oxidative stress, oxidation reactions are “the bad element” or the injurious processes that propagate inflammation and promote injury, whereas lack of adequate antioxidants is the loss of the “good element” that needs to be restored to return things to a certain physiologic homeostasis. Yet, failure of antioxidant supplementation to successfully improve many diseases provides convincing evidence that the oxidative stress imbalance is a complex phenomenon. Oxidative stress, as mentioned earlier, is rather a continuous process in which oxidative reactions play a role in mitigating inflammation and in adaptive cellular responses. One of these oxidative reactions is the lipid products generated by the interaction of RNS/ROS with unsaturated fatty acids to form nitrated fatty acids or lipid hydroperoxides. Nitrated fatty acids are nonenzymatically produced by the interaction of unsaturated fatty acids with secondary nitrogen oxidation products, such as nitrogen dioxide, nitrite, and peroxynitrate (68). The addition of a nitro group to a double bond at the carbon chain of an unsaturated fatty acid leads to an alkenyl nitro-configuration providing electrophilic reactivity. During conditions of higher oxygen levels, nonenzymatic oxidation reactions predominate, forming lipid hydroperoxides that are also potent S154

electrophiles. Both nitroalkenes and hydroperoxides can react with other nucleophiles, such as Cys or His residues, by forming Michael adducts (68). These reactions produce reversible posttranslational modification in signaling proteins, such as the peroxisome proliferated activated receptors (PPAR), and the keap1/Nrf2 pathways (68, 69). Both nitro oleic and nitro linolenic acids are examples of these nitrated fatty acids that have been shown to be endogenous PPARg ligands and to inhibit proinflammatory cytokine production (69). These nitroalkenes have also been shown to induce expression of the hemeoxygenase-1 (HO-1) gene, which is part of the antioxidant response elements targeted by Nrf2 and is known to provide cytoprotection against oxidative lung injury (70, 71). In addition to induction of ARE, nitrated fatty acids interact with the p65 subunit if NF-kB, preventing its nuclear translocation and release of proinflammatory cytokines (72). Other forms of electrophilic fatty acids that share similar pharmacological properties are formed enzymatically. Lipoxins are formed in mucosal surfaces by the interaction of leukocyte 5-lypoxygenase and epithelial 15-lipoxygenease (73). Unlike other arachidonic acid derivatives, lipoxins are protective against bronchial constriction and reduce airway inflammation (73). Cycloxygenase-2 (Cox-2) mediates the formation of electrophilic fatty acid oxoderivatives, from omega-3 fatty acids (74). These compounds, like nitrated fatty acids, are known to be endogenous PPAR ligands and to induce the ARE via Nrf2 activation (74). However, whether oxidative stress plays a role in determining the formation of enzymatically derived electrophilic fatty acids is unclear at this time. Autophagy is a homeostatic process involved in adaptive responses and cell survival, in which ROS can play an important role. Autophagy provides a pathway for the turnover of cytoplasmic organelles and proteins through a lysosomedependent degradation process and can be triggered by oxidative stress, among other factors. In the lung, HO-1 has been shown to modulate autophagic activation, resulting in less cell death (71). Thus, HO-1 could be a link between the formation of electrophilic fatty acids and autophagy, as part of an adaptive response to oxidative stress (75).

Can Airway Oxidative Stress Be Treated? Should It Be Treated? In asthma, treatment with cysteine-donor antioxidants has been problematic due to reduced bioavailability, potential side effects including increased airway resistance and enhanced bronchial hyperresponsiveness, and the inability to improve gas exchange or lung volumes. For COPD, treatment with cysteine-donor antioxidants such as N-acetylcysteine (NAC) has led to more encouraging results. In the BRONCUS study, patients with COPD were randomized to 600 mg/d of NAC versus placebo for more than 3 years. Both groups showed similar rates of lung function decline, yet in post hoc analysis, patients on the NAC arm showed less hyperinflation. In this study, NAC did not influence yearly exacerbation rate, but the HR for a COPD exacerbation was reduced by 22% among those not taking inhaled steroids (76). In a recent metaanalysis, among 723 patients from nine studies, 48.5 versus 31.2% of subjects treated with NAC had no COPD exacerbations (relative benefit, 1.56; 95% confidence interval, 1.37–1.77); in five studies, patients receiving NAC reported symptom improvement (relative benefit, 1.78; 95% confidence interval, 1.5–2.0) (77). A recent study of patients with COPD with a median Global Initiative for Chronic Obstructive Lung Disease score of II evaluated the use of a high dose NAC of 1,200 mg/d versus placebo for 1 year. NAC was associated with a lower rate of yearly COPD exacerbations (1.71 vs. 0.9; P = 0.01) and improvements in forced expiratory flow in the midexpiratory phase; in addition, 53.8% in the NAC arm versus 37.5% in the placebo remained exacerbation-free at 1 year (78). Based on these results, it would appear that patients with moderate COPD might benefit from a high dose of NAC, whereas no clinical benefit from this type of therapy has been demonstrated for patients with asthma. There are several potential factors that might explain the lack of anticipated antioxidant therapy in airway diseases, including (1) insufficient knowledge of the clinical pharmacology of antioxidants and the role and timing of antioxidants in the pathophysiology of airway diseases (i.e., knowing when antioxidants are needed to reduce further inflammation or oxidative stress vs. when would antioxidants, by

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TRANSATLANTIC AIRWAY CONFERENCE blocking oxidative reactions, mostly impair mechanisms involved in resolution of inflammation), (2) insufficient dosing or duration due to limited dose–response studies, (3) method of antioxidant delivery, (4) selection of unstandardized unvalidated primary outcome indicators, and (5) lack of surrogate biomarkers of oxidative stress to accompany functional outcomes (51).

Should We Treat Oxidative Stress Routinely? Although there are no indications for the use of antioxidant supplementation in the treatment of airway disorders for the aforementioned reasons, there are situations in which, given the nature of environmental exposures or individual factors, supplementation of specific antioxidants could be warranted. For example, among children with asthma living in highly polluted environments, supplementation with antioxidant vitamins prevented lung function changes associated with ambient ozone exposure, particularly among those with prevalent glutathione

s-transferase polymorphisms, like GSTM1 null (79, 80). Heavy smokers with low vitamin C, and particularly those with functional polymorphisms in the glutamate-cysteine ligase, are at highest risk from accelerated lung function decline; it is therefore possible that vitamin C supplementation in this population could be protective (81), whereas it might not have the same degree of efficacy in other populations without these risks factors. Although we are clearly far from determining the individual characteristics (genetic or habit-related) or the interaction with disease and environment that would make a person a candidate for antioxidant supplementation, it is only through an individualized phenotypic (including environment) and genomic approach that we can learn the real therapeutic potential of antioxidant supplementation.

Summary Airway oxidative stress is a complex phenomenon with important physiological and pathophysiological implications in

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many airway disorders. We conceptually understand that airway oxidative stress occurs from an imbalance between oxidative and antioxidative processes, yet we lack basic understanding of when oxidative stress is largely an epiphenomenon or when it is a major driver of disease. Given this lack of knowledge, coupled with an absence of standardized biomarkers that link to clinical or functional outcomes, it is not surprising that antioxidant therapy for airway diseases has largely been unsuccessful. Airway oxidative stress should be framed in the context of individual (phenotypical and genetic) risk factors and environmental exposures, to determine which specific cases could benefit from antioxidant therapy. Recommending treatment widely to restore some measure of airway oxidative stress is not recommended; not only will it not be not beneficial for many patients but also it has the potential to harm, if it blocks mechanisms aimed at resolving inflammation that depend on some low degree of oxidative stress. n Author disclosures are available with the text of this article at www.atsjournals.org.

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TRANSATLANTIC AIRWAY CONFERENCE 57 Malhotra D, Thimmulappa RK, Mercado N, Ito K, Kombairaju P, Kumar S, Ma J, Feller-Kopman D, Wise R, Barnes P, et al. Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients. J Clin Invest 2011;121:4289–4302. 58 Demicheli V, Quijano C, Alvarez B, Radi R. Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide. Free Radic Biol Med 2007;42:1359–1368. 59 Comhair SA, Ricci KS, Arroliga M, Lara AR, Dweik RA, Song W, Hazen SL, Bleecker ER, Busse WW, Chung KF, et al. Correlation of systemic superoxide dismutase deficiency to airflow obstruction in asthma. Am J Respir Crit Care Med 2005;172:306–313. 60 Regan EA, Mazur W, Meoni E, Toljamo T, Millar J, Vuopala K, Bowler RP, Rahman I, Nicks ME, Crapo JD, et al. Smoking and COPD increase sputum levels of extracellular superoxide dismutase. Free Radic Biol Med 2011;51:726–732. 61 Harju T, Kaarteenaho-Wiik R, Sirvio¨ R, Pa¨ akk ¨ o¨ P, Crapo JD, Oury TD, Soini Y, Kinnula VL. Manganese superoxide dismutase is increased in the airways of smokers’ lungs. Eur Respir J 2004;24:765–771. 62 Nadeem A, Raj HG, Chhabra SK. Increased oxidative stress and altered levels of antioxidants in chronic obstructive pulmonary disease. Inflammation 2005;29:23–32. 63 Hu G, Cassano PA. Antioxidant nutrients and pulmonary function: the Third National Health and Nutrition Examination Survey (NHANES III). Am J Epidemiol 2000;151:975–981. 64 Misso NL, Brooks-Wildhaber J, Ray S, Vally H, Thompson PJ. Plasma concentrations of dietary and nondietary antioxidants are low in severe asthma. Eur Respir J 2005;26:257–264. 65 Allen S, Britton JR, Leonardi-Bee JA. Association between antioxidant vitamins and asthma outcome measures: systematic review and meta-analysis. Thorax 2009;64:610–619. 66 Romieu I, Trenga C. Diet and obstructive lung diseases. Epidemiol Rev 2001;23:268–287. 67 Rautalahti M, Virtamo J, Haukka J, Heinonen OP, Sundvall J, Albanes D, Huttunen JK. The effect of alpha-tocopherol and beta-carotene supplementation on COPD symptoms. Am J Respir Crit Care Med 1997;156:1447–1452. 68 Trostchansky A, Bonilla L, Gonzalez-Perilli ´ L, Rubbo H. Nitro-fatty acids: formation, redox signaling, and therapeutic potential. Antioxid Redox Signal (In press) 69 Ferreira AM, Minarrieta L, Lamas Bervejillo M, Rubbo H. Nitro-fatty acids as novel electrophilic ligands for peroxisome proliferatoractivated receptors. Free Radic Biol Med 2012;53:1654–1663. 70 Khoo NK, Rudolph V, Cole MP, Golin-Bisello F, Schopfer FJ, Woodcock SR, Batthyany C, Freeman BA. Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression

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