Phenotype of asthmatics with increased airway S-nitrosoglutathione reductase activity
- Nadzeya V. Marozkina1,
- Xin-Qun Wang2,
- Vitali Stsiapura3,
- Anne Fitzpatrick4,
- Silvia Carraro5,
- Gregory A. Hawkins6,
- Eugene Bleecker6,
- Deborah Meyers6,
- Nizar Jarjour7,
- Sean B. Fain8,
- Sally Wenzel9,
- William Busse7,
- Mario Castro10,
- Reynold A. Panettieri Jr11,
- Wendy Moore6,
- Stephen J. Lewis1,
- Lisa A. Palmer12,
- Talissa Altes13,
- Eduard E. de Lange13,
- Serpil Erzurum14,15,
- W. Gerald Teague12 and
- Benjamin Gaston1⇑
- 1Dept of Paediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, OH, USA
- 2Dept of Public Health Sciences, University of Virginia, Charlottesville, VA, USA
- 3Dept of Chemistry, University of Virginia, Charlottesville, VA, USA
- 4Dept of Paediatrics, Emory University, Atlanta, GA, USA
- 5Dept of Paediatrics, University of Padua, Padua, Italy
- 6Dept of Medicine, Wake Forest University, Winston-Salem, NC, USA
- 7Dept of Medicine, University of Wisconsin, Madison, WI, USA
- 8Dept of Medical Physics, University of Wisconsin, Madison, WI, USA
- 9University of Pittsburgh, Pittsburgh, PA, USA
- 10Dept of Medicine, Washington University, St. Louis, MO, USA
- 11Pulmonary, Allergy and Critical Care Division, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
- 12Dept of Paediatrics, University of Virginia, Charlottesville, VA, USA
- 13Dept of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA
- 14Dept of Pathobiology, Cleveland Clinic, Cleveland, OH, USA
- 15Dept of Pulmonary, Allergy, and Critical Care Medicine, Cleveland Clinic, Cleveland, OH, USA
- Benjamin Gaston, Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine, Paediatric Pulmonology, BRB Room 727, 10900 Euclid Ave, Cleveland, OH 44106, USA. E-mail: benjamin.gaston{at}case.edu
Abstract
S-Nitrosoglutathione is an endogenous airway smooth muscle relaxant. Increased airway S-nitrosoglutathione breakdown occurs in some asthma patients. We asked whether patients with increased airway catabolism of this molecule had clinical features that distinguished them from other asthma patients.
We measured S-nitrosoglutathione reductase expression and activity in bronchoscopy samples taken from 66 subjects in the Severe Asthma Research Program. We also analysed phenotype and genotype data taken from the program as a whole.
Airway S-nitrosoglutathione reductase activity was increased in asthma patients (p=0.032). However, only a subpopulation was affected and this subpopulation was not defined by a “severe asthma” diagnosis. Subjects with increased activity were younger, had higher IgE and an earlier onset of symptoms. Consistent with a link between S-nitrosoglutathione biochemistry and atopy: 1) interleukin 13 increased S-nitrosoglutathione reductase expression and 2) subjects with an S-nitrosoglutathione reductase single nucleotide polymorphism previously associated with asthma had higher IgE than those without this single nucleotide polymorphism. Expression was higher in airway epithelium than in smooth muscle and was increased in regions of the asthmatic lung with decreased airflow.
An early-onset, allergic phenotype characterises the asthma population with increased S-nitrosoglutathione reductase activity.
Abstract
Asthma patients with increased S-nitrosoglutathione reductase activity have early-onset asthma + are highly allergic http://ow.ly/AuJ9z
Introduction
S-Nitrosoglutathione (GSNO) is one of a class of endogenous sulphur nitric oxide bond-containing species (S-nitrosothiols) that serve as signalling molecules and airway smooth muscle relaxants [1–5]. Nitrosylating and denitrosylating enzymes regulate the formation and breakdown of S-nitrosothiols [4–8]. In the cellular environment, S-nitrosylated proteins are in transnitrosation equilibria with reduced glutathione (GSH) [4–8]. Thus, enzymatic denitrosylation of GSNO depletes S-nitrosylated proteins in a given cell or compartment. The most extensively studied denitrosylating enzyme in the lung is GSNO reductase, also known as alcohol dehydrogenase 5 (ADH5) [4–6, 9, 10].
GSH, with its redox cycle partners, serves to maintain the reduced state of protein thiols, which can be achieved by scavenging oxidants or by the covalent reversible binding of GSH to protein thiols. The latter occurs under physiological conditions, is induced upon mild oxidative stress and is known as S-glutathionylation (PSSG) [11]. GSH can be removed from proteins by glutaredoxins (Grx), which restores the function of proteins targeted by PSSG [11]. It has been shown by Kuipers et al. [12] that in the sputum of asthmatics there is increased Grx-1 and decreased protein S-glutathionylation.
Levels of GSNO and other S-nitrosothiols are also decreased in the airway lining fluid of many patients with mild asthma, and intratracheal levels are profoundly low in children with acute asthmatic respiratory failure [10, 13]. The GSNO reductase-deficient mouse is protected from methacholine-induced bronchoconstriction [9] and endogenous S-nitrosothiols prevent tachyphylaxis to β2-adrenergic agonists [3]. Further, inhaled GSNO decreases airway levels of interleukin (IL)-4, -5, -6 and -13, as well as other mediators associated with allergic airway inflammation [5]. Indeed, GSNO reductase gene single nucleotide polymorphisms (SNPs) are associated with increased asthma incidence in specific human asthma subpopulations [14–16]. Thus, it is suggested that GSNO replacement, and/or the inhibition of GSNO reductase, could attenuate asthma symptoms in humans [17].
However, of concern is that long-term treatment, aimed at substantially increasing airway levels of S-nitrosothiols in patients who already have normal levels, could, theoretically, predispose them to adverse consequences [18–20] or could, paradoxically, increase airway hyperresponsiveness at higher doses [5]. Furthermore, most patients with mild asthma have normal airway levels of epithelial GSNO reductase activity [10]. Therefore, not all asthma patients should be treated with S-nitrosothiol repletion or GSNO reductase inhibition. Here, our goal has been to identify the determinants of increased GSNO reductase activity and the phenotypic features of this subpopulation of asthma patients. With this information, personalised therapy might be envisioned for those who are most likely to respond.
Personalised asthma treatment is particularly important for severe asthma. Patient response to standard therapies, including leukotriene receptor antagonists and inhaled corticosteroids, is suboptimal in many patients with severe asthma [21, 22]. Targeting the most probable responders is likely to decrease both the cost and the toxicity of the treatment. Furthermore, severe asthma patients are defined as those who are highly symptomatic despite these treatments; these patients may particularly benefit from a medical approach in which responsiveness is suspected based on a clinical phenotype, diagnostic test and targeted treatment. For example, specific recommendations on the use of sputum eosinophil count and exhaled nitric oxide to guide therapy, as well as treatment with anti-IgE antibody, methotrexate, macrolide antibiotics, antifungal agents and bronchial thermoplasty are provided in the International European Respiratory Society/American Thoracic Society (ATS) guidelines on definition, evaluation and treatment of severe asthma [23]. Here, we have begun to approach this type of paradigm by studying the phenotype of the asthma subpopulation with increased airway GSNO reductase activity.
Methods
Subjects
Non-smoking subjects with severe and nonsevere asthma, as well as healthy controls, who had enrolled in the Severe Asthma Research Program (SARP) underwent previously described characterisation procedures [21] using the ATS consensus pane definition of [22]. A subpopulation underwent hyperpolarised helium (HHe-3) magnetic resonance imaging (MRI) [24]. Not all subjects underwent all tests. All procedures were approved by the Institutional Review Board at the SARP centres, and participants provided informed consent.
Bronchoscopy
Adult subjects underwent flexible bronchoscopy with BAL and endobronchial biopsies of segmental carinae, using our published methods [25]. Children with severe asthma underwent bronchoscopy for clinical indications according to a shared sample protocol at Emory University (Atlanta, GA, USA) [26]. Biopsies were paraffin embedded and subjected to immunohistochemistry and haematoxylin/eosin staining. At Emory University, the lavage fluid was aspirated in initial and terminal phase fractions [26]. To identify well- and poorly-ventilated lung regions, three subjects scheduled for bronchoscopy underwent HHe-3 MRI; within 12 h, biopsies were performed in airways with and without ventilatory defects.
Biochemical assays
GSNO reductase was measured as NADH-dependent loss of GSNO over time using two separate methods with strong concordance: 1) mass spectrometry and 2) spectrophotometry [18, 28].
Cell culture
Primary human airway smooth muscle (HASM) cells, isolated in the laboratory of Reynold A. Jr Panettieri, were cultured as previously described [29]. Normal human bronchial epithelial cells (NHBE) (Lonza, Walkersville, MD, USA) were grown as monolayers, and primary human bronchial epithelial cells were grown at air-liquid interface as previously described [30]. Cystic fibrosis bronchial epithelial (CFBE41o−) and A549 cell lines were grown as previously described [19, 30].
Immunoblot
GSNO reductase AB (1:1000) (#11051-1-AP; Proteintech Group, Chicago, IL, USA) and second HRP 1:10 000 was used to immunostain for HASM, and CFBE cells. Additionally, airway expression was confirmed using a separate polyclonal rabbit anti-GSNO reductase AB kindly donated by L. Que (Duke University, NC, USA) with goat anti-rabbit secondary AB (#172-1019; Bio-Rad Inc., Hercules, CA, USA).
Immunohistochemistry
Tissue sections were immunoblotted with GSNO reductase AB (1:1000) (#11051-1-AP; Proteintech Group) using secondary goat anti-rabbit (1:10 000) for 30 min at room temperature (#11051-1-AP; Proteintech Group) and ABC reagent (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature, as previously described [19], and scored from 0 (absent) to 4 (intense staining) by two observers who were blinded to the diagnosis.
SNP genotyping and assessment
The ADH5 genotypes were acquired from the Human1M-Duo DNA BeadChip (Illumina, San Diego, CA, USA). The Hardy–Weinberg equilibrium was tested for quality control.
Statistical analysis
By univariate analysis, we studied the relationship between GSNO reductase activity and risk factors. A t-test or nonparametric rank sum test was used for two-sample comparison of continuous variables, a Fisher exact test was used for two-sample comparison of categorical variables, and analysis of variance was used for categorical variables with more than two categories. p<0.05 was considered statistically significant. The statistical analyses were carried out in SAS 9.2 (SAS Institute Inc., Cary, NC, USA).
Results
Subjects
We studied 52 adults (healthy controls n=12, nonsevere asthma n=25, and severe asthma n=15) and 14 children (healthy controls n=2, nonsevere asthma n=6, and severe asthma n=6) (table 1). These subjects were enrolled at five different sites participating in SARP. Through site-specific protocols, subsets of subjects underwent specific procedure (biopsy, bronchoalveolar lavage (BAL) and/or MRI). BALs were performed in the right middle lobe or lingula. Except where directed by MRI, biopsies were performed at a mainstem, third or fourth generation carina contralateral to the BAL site. Genetic analysis was additionally performed on 914 asthma subjects and 344 controls obtained from sites participating in SARP.
GSNO reductase activity
We measured GSNO reductase activity in 54 BAL cell lysates (patients with severe asthma n=16, nonsevere asthma n=25, and controls n=13). Overall the mean values did not differ between the three groups; the p-value was nonsignificant (ns). There was a subpopulation of 44% of the asthma patients with levels >7.5 nmol·min−1·μg−1 protein (more in asthma than controls, p=0.032) (fig. 1a). We performed immunoblot for GSNO reductase on a subpopulation of nine BAL samples (control n=1, severe n=1, and nonsevere n=7). There was a weak concordance between measures of GSNO reductase activity in airway versus alveolar lavage samples (n=11, r2=0.61) (fig. 1b and c) and between BAL GSNO reductase expression and activity (r2=0.54) (fig. 1c), which were consistent with a previous report [10]. Activity did not vary with BAL cell count, differential or other parameters.
Tissue GSNO reductase expression
Next, we studied GSNO reductase tissue expression in random endobronchial biopsies (i.e. not directed by HHe3 imaging; n=18, which included: severe asthma n=6, nonsevere asthma n=4 and controls n=8). There was less GSNO reductase expression in smooth muscle cells than in epithelial cells (p<0.05) (fig. 2). However, GSNO reductase immunoreactivity did not differ between asthma and control patients in epithelial, smooth muscle or inflammatory cells. Immunoreactivity localised primarily to the apical regions of airway epithelial cells of healthy lungs. This was not the case in epithelium from asthmatic subjects, where immunoreactivity localised to basal cells (fig. 2). Consistent with these data, HASM cells expressed less GSNO reductase, relative to protein load control, than NHBE cells in cultured monolayers, NHBE cells grown as pseudostratified epithelium at the air–fluid interface and the established airway cell lines CFBE (fig. 3a). Of note, interleukin (IL) 13 increased epithelial GSNO reductase expression in severe asthma native BAL cell pellets ex vivo, and in cultured epithelial cells in vitro, whereas IL-4 was not effective (fig. 3b, c and d).
Consistent with previous reports [24, 27], HHe-3 imaging showed regions focal ventilatory defects in the lungs of subjects with severe asthma (n=3) (fig. 4). In figure 4a (coronal image) and figure 4b (transverse image) ventilation defects are shown at baseline in stable, severe asthma patient. In figure 4c a second image acquired after albuterol administration shows only moderate improvement in the left upper lobe ventilation defect. Image-guided endobronchial biopsy done in the segmental airways supporting well-ventilated lung region (green arrows, fig. 4a) showed relatively preserved epithelial integrity, less GSNOR immunoreactivity and reduced inflammation (fig. 4 g–i) than biopsy taken from airways supporting region with focal ventilatory defects (red arrows, fig. 4a and d–f). Overall, when epithelium was present, it was immunostained more for GSNOR (3-4/4 in obstructed airways versus 2-3/4 in nonobstructed airways).
Phenotype analysis of patients with increased GSNO reductase activity
We next analysed the clinical phenotype of the well characterised SARP patients with evidence for increased airway GSNO reductase activity. Of the asthmatic subjects with BAL GSNO reductase activity measures, 31 were fully characterised in the SARP database (table 2). Those with GSNO reductase activity >7.5 nmol·min−1·μg−1 protein had higher IgE (p=0.01) and were diagnosed with asthma and allergies at a younger age (p=0.02). Also, they were younger, had a lower body mass index (BMI), had a lower daytime sleepiness snores on obstructive sleep apnoea (OSA) screening and were less likely to have exercise-induced symptoms (each p<0.05). Asthmatic subjects with increased GSNO reductase activity did not have a significantly lower methacholine PC20 (provocative concentration causing a 20% fall in forced expiratory volume in 1 s (FEV1))±sd (1.02±0.92) than those with low GSNO reductase activity (4.32±6.79, p=0.22).
GWAS data from the SARP
One well-studied SNP in the GSNO reductase promoter associated with good responsiveness to β2 adrenergic treatment (rs1154400) [15] was available on the Human1M-Duo DNA BeadChip used in the SARP. This SNP is in a region associated with protection from asthma in Caucasian populations [15–17] and the C/C genotype was associated with improved asthma outcomes and β2 responsiveness in an African-American population [15]; however, the authors did not measure airway GSNOR activity. The rs1154400 genotypes were in the Hardy Weinberg equilibrium in the SARP population. Of the subjects who had both BAL GSNO reductase analysis and GWAS, none with high GSNO reductase activity in BAL had the C/C genotype, but the differences did not achieve significance because of sample size. In the SARP population as a whole, those asthmatics with C/T and T/T genotypes were more allergic than those with the C/C genotype (IgE 362 ± 846 IU·mL−1 versus 245 ± 374 IU·mL−1, n=838 and n=76, respectively, p=0.028); and, of those with a positive skin-prick test, wheal diameters were greater to Dermatophagoyetes farinae (10.8±6.0 mm (n=449) versus 8.8±4.5 mm (n=42), respectively, p=0.009). Note that the C/T and C/T genotype patients had lower exhaled breath condensate (EBC) pH than C/C (7.7±0.8 (n=449) versus 8.0±0.5 (n=45), respectively, p=0.0004), consistent with data that high EBC formic acid levels may be associated with increased airway GSNO reductase activity [31]. Consistent with the data by Moore et al. [25], though C/C did not differ from C/T-T/T with regard to baseline FEV1 (p=ns), FEV1 after four and six puffs of albuterol was higher in the C/C group than in the C/T-T/T group (four puffs 91.7±17.3% pred (n=81) versus 86.0±20.0% pred (n=697), respectively, p=0.013; and six puffs 92.7±17.1% pred (n=72) versus 87.4±20.2% pred (n=697), respectively, p=0.034). The genotype groups did not differ in terms of age, sex or BMI.
Discussion
GSNO and other S-nitrosothiols cause airway smooth muscle relaxation in humans and other species [2, 4, 32–34]. Levels of S-nitrosothiols are low, and activity of GSNO reductase is high, in the airways of children with severe asthma who are experiencing an acute exacerbation associated with respiratory failure [13]. Here, we studied airway GSNO reductase expression and activity in stable outpatients with severe and non-severe asthma.
Activity of GSNO reductase was increased in the BAL cells of some stable outpatients with asthma. However, it was normal in many severe asthma patients; increased activity is not a defining pathophysiological feature of severe asthma. Consistent with the work of Que et al. [10], activity was also normal in the BAL of a large proportion of stable, nonsevere asthmatics. Our data extends the observation to cases of severe asthma. In part, this is likely because there is population heterogeneity in GSNO reductase expression that is genetically determined. Previous studies have shown that increased airway GSNO reductase activity, and associated SNPs, are present only in a minority of patients with asthma [10, 14–16].
GSNO reductase has been of growing interest in the targeted asthma treatment [23]. It has been shown that that sputum glutathione disulfide levels were significantly decreased in patients with asthma, especially in the two inflammatory phenotypes. However, no correlation with the percentage of eosinophils or neutrophils was observed [12].
There is interest in treating asthma patients with GSNO reductase inhibition. Because a substantial chronic excess of S-nitrosothiols in the context of nitrosative stress has the potential to be associated with adverse effects [5, 18–20], it is important to target nitrosothiol repletion only to those patients who have an airway S-nitrosothiol deficiency and/or excessive GSNO catabolism associated with asthma. The SARP data suggest a specific phenotype. Patients with increased expression were more allergic, with earlier onset of asthma and allergies. They were younger and smaller, with reduced OSA. However, they were not more “severe” [22]. This proposed phenotype will require validation.
In a mouse model, GSNO inhalation modestly suppresses both T-helper cell type (Th)1 and Th2 cytokine responses, including IL-13 production, to antigen [5]. We also found that SARP subjects with a GSNO reductase-associated SNP, and associated with impaired β2 responsiveness in Moore et al. [15] study, were more allergic. As predicted by Whalen et al. [3], data from Choudhry et al. [16] show that asthma and impaired β2 responsiveness are associated with genotypes causing GSNO reductase gain-of-function. Here, we have confirmed the finding that the bronchodilator responsiveness of subjects with C/C was great than that of subjects with CT/and T/T [14]. This process may feed forward in asthma. Increased Th2 activation and IL-13 production would further increase GSNO reductase expression and activity (current study data) [5], particularly favouring increased GSNO reductase expression in allergic patients (fig. 5). We have shown that it is IL-13, not IL-4 that increases GSNOR expression. One of the possible mechanisms is that IL-13 can stimulate endothelial nitric oxide synthase activity, even in inflammatory cells. This effect can involve a feed-forward mechanism involving transforming growth factor β1. In turn, these effects may stimulate GSNOR in a counter-regulatory fashion. However, more detailed cellular analysis will be required to confirm this hypothesis.
Bronchoscopic results can be flawed in asthma patients. Here, we have shown that GSNO reductase activity levels in patients may have reflected the lung region sampled, not simply overall phenotype. In our preliminary studies, expression was not uniform throughout the airway. One possible explanation is that the regional variations in expression result from treatment: inhaled corticosteroids may access the well-ventilated units, inhibiting inflammation and GSNO reductase expression. A previous HHe-3 study has shown that roughly 50% of poorly ventilated lung units have persistent defects [24], perhaps reflecting chronic lack of therapy. It is also possible that some airways, like the skin, have inherently different patterns of inflammation. Either way, it is important to recognise that there may be substantial intra-subject airway heterogeneity. Ongoing studies in the SARP and other programmes are investigating this regional variability more prospectively.
Further, bronchoscopy is not without expense and potential risk. Therefore, noninvasive biomarkers may prove useful for diagnosing of the severe asthma. Possibly consistent with low pH in the non-C/C genotype population, our previous data suggest that increased EBC formic acid levels might be a noninvasive marker for increased GSNO reductase activity [31]. We have previously shown that inhaled GSNO increases exhaled nitric oxide, followed by time-dependent decay [35]. Therefore, an inhalational challenge with GSNO may test for the presence and/or the disinhibition of GSNO reductase activity in human asthmatics: the greater the activity of GSNO reductase, the higher the fractional exhaled nitric oxide (FeNO) after challenge. Airway homogeneity, in turn, could be assayed using different FeNO flow rates. We believe that this kind of challenge test, and/or EBC formic acid test, may prove clinically useful to identify severe asthma patients who could be candidates for S-nitrosothiol replacement and/or inhibition of GSNO reductase.
Of note that is that we found increased GSNO reductase activity in asthmatics was not significantly associated with methacholine PC20, though there was a trend in this direction. This could be explained by corticosteroid treatment. In our study 83% of patients with nonsevere and 100% of patients with severe asthma received inhaled steroids. In the study by Que et al. [10], only subjects with mild asthma were included, and they did not receive either a high-dose inhaled or oral corticosteroids.
In summary, we found marked heterogeneity in GSNO reductase activity in subjects with both severe and nonsevere asthma. Increased GSNO reductase activity is not, in general, the cause of severe asthma. We also present evidence that patients with increased airway GSNO reductase activity may have a more allergic phenotype. If validated, this high GSNO reductase phenotype could be suspected clinically based on history and physical examination and confirmed with genetic analysis and/or with biomarker testing in the lung function laboratory. This information, in turn, can be used to personalise treatment. We believe that this paradigm will be of increasing value in subspecialty asthma management.
Acknowledgements
We would like to thank all the Severe Asthma Research Program coordinators.
Footnotes
This version of this article has been amended. The amendment is outlined in the erratum published in the June 2015 issue of the European Respiratory Journal [DOI: 10.1183/09031936.50042414].
Support statement: B. Gaston received funding for this study from: NIH Severe Asthma Research Program Grant #5U10HL109250-02; NIH Program Project Grant #5P01HL101871-02; and NIH R01 Grant #5RO1-HL059337-12. R.A. Panettieri has received funding for this study from NIH R01 Grant #HL097796 and NIEHS P30 ES013508. E.E. de Lang has received funding for this study from NIH RO1 HL66479.
Conflict of interest: Disclosures can be found alongside the online version of this article at erj.ersjournals.com
- Received March 4, 2014.
- Accepted August 12, 2014.
- Copyright ©ERS 2015