Antigen stasis and airway nitrosative stress in human primary ciliary dyskinesia.

Nasal nitric oxide (nNO) is low in most patients with primary ciliary dyskinesia (PCD). Decreased ciliary motion could lead to antigen stasis, increasing oxidant production and NO oxidation in the airways. This could both decrease gas phase NO and increase nitrosative stress. We studied primary airway epithelial cells from healthy controls (HCs) and patients with PCD with several different genotypes. We measured antigen clearance in fenestrated membranes exposed apically to the fluorescently labeled antigen Dermatophagoides pteronyssinus (Derp1-f). We immunoblotted for 3-nitrotyrosine (3-NT) and for oxidative response enzymes. We measured headspace NO above primary airway cells without and with a PCD-causing genotype. We measured nNO and exhaled breath condensate (EBC) H2O2 in vivo. Apical Derp1-f was cleared from HC better than from PCD cells. DUOX1 expression was lower in HC than in PCD cells at baseline and after 24-h Derp1-f exposure. HC cells had less 3-NT and NO3- than PCD cells. However, NO consumption by HC cells was less than that by PCD cells; NO loss was prevented by superoxide dismutase (SOD) and by apocynin. nNO was higher in HCs than in patients with PCD. EBC H2O2 was lower in HC than in patients with PCD. The PCD airway epithelium does not optimally clear antigens and is subject to oxidative and nitrosative stress. Oxidation associated with antigen stasis could represent a therapeutic target in PCD, one with convenient monitoring biomarkers.NEW & NOTEWORTHY The PCD airway epithelium does not optimally clear antigens, and antigen exposure can lead to NO oxidation and nitrosative stress. Oxidation caused by antigen stasis could represent a therapeutic target in PCD, and there are convenient monitoring biomarkers.

Ciliary Function, Antigen Stasis and Asthma.

The prevalence of asthma exceeds 3% of the population. Asthma is observed to be more common in children following severe viral lower respiratory illnesses that affect ciliary function, but mechanisms linking ciliary function to asthma pathogenesis have been obscure. Recent data regarding primary ciliary dyskinesia (PCD) may help us to understand the association. Here, I will review what is known about the relationship between ciliary function and asthma. PCD is caused by pathologic variants in over 50 different genes that affect the structure and function of motile cilia. At the cellular level, a characteristic feature shared by most PCD patients is that antigens and other particles are not cleared from the epithelial surface. Poor antigen clearance results in pro-oxidant pathway activation and airway epithelial damage and may predispose PCD patients to DUOX1- and IL33-mediated asthma. Secondary ciliary dysfunction, such as that caused by viruses or by smoking, can also contribute to asthma development. Moreover, variants in genes that affect the function of cilia can be associated with poor lung function, even in the absence of PCD, and with increased asthma severity. The role of antigen stasis on the surface of dysfunctional airway cilia in the pathophysiology of asthma is a novel area for research, because specific airway clearance techniques and other therapeutic interventions, such as antioxidants, could be of value in preventing the development of asthma.

HSD3B1 genotype identifies glucocorticoid responsiveness in severe asthma.

Asthma resistance to glucocorticoid treatment is a major health problem with unclear etiology. Glucocorticoids inhibit adrenal androgen production. However, androgens have potential benefits in asthma. HSD3B1 encodes for 3ß-hydroxysteroid dehydrogenase-1 (3ß-HSD1), which catalyzes peripheral conversion from adrenal dehydroepiandrosterone (DHEA) to potent androgens and has a germline missense-encoding polymorphism. The adrenal restrictive HSD3B1(1245A) allele limits conversion, whereas the adrenal permissive HSD3B1(1245C) allele increases DHEA metabolism to potent androgens. In the Severe Asthma Research Program (SARP) III cohort, we determined the association between DHEA-sulfate and percentage predicted forced expiratory volume in 1 s (FEV1PP). HSD3B1(1245) genotypes were assessed, and association between adrenal restrictive and adrenal permissive alleles and FEV1PP in patients with (GC) and without (noGC) daily oral glucocorticoid treatment was determined (n = 318). Validation was performed in a second cohort (SARP I&II; n = 184). DHEA-sulfate is associated with FEV1PP and is suppressed with GC treatment. GC patients homozygous for the adrenal restrictive genotype have lower FEV1PP compared with noGC patients (54.3% vs. 75.1%; P < 0.001). In patients with the homozygous adrenal permissive genotype, there was no FEV1PP difference in GC vs. noGC patients (73.4% vs. 78.9%; P = 0.39). Results were independently confirmed: FEV1PP for homozygous adrenal restrictive genotype in GC vs. noGC is 49.8 vs. 63.4 (P < 0.001), and for homozygous adrenal permissive genotype, it is 66.7 vs. 67.7 (P = 0.92). The adrenal restrictive HSD3B1(1245) genotype is associated with GC resistance. This effect appears to be driven by GC suppression of 3ß-HSD1 substrate. Our results suggest opportunities for prediction of GC resistance and pharmacologic intervention.

Benefits of Airway Androgen Receptor Expression in Human Asthma.

Rationale: Androgens are potentially beneficial in asthma, but AR (androgen receptor) has not been studied in human airways.Objectives: To measure whether AR and its ligands are associated with human asthma outcomes.Methods: We compared the effects of AR expression on lung function, symptom scores, and fractional exhaled nitric oxide (FeNO) in adults enrolled in SARP (Severe Asthma Research Program). The impact of sex and of androgens on asthma outcomes was also evaluated in the SARP with validation studies in the Cleveland Clinic Health System and the NHANES (U.S. National Health and Nutrition Examination Survey).Measurements and Main Results: In SARP (n = 128), AR gene expression from bronchoscopic epithelial brushings was positively associated with both FEV1/FVC ratio (R2 = 0.135, P = 0.0002) and the total Asthma Quality of Life Questionnaire score (R2 = 0.056, P = 0.016) and was negatively associated with FeNO (R2 = 0.178, P = 9.8 × 10-6) and NOS2 (nitric oxide synthase gene) expression (R2 = 0.281, P = 1.2 × 10-10). In SARP (n = 1,659), the Cleveland Clinic Health System (n = 32,527), and the NHANES (n = 2,629), women had more asthma exacerbations and emergency department visits than men. The levels of the AR ligand precursor dehydroepiandrosterone sulfate correlated positively with the FEV1 in both women and men.Conclusions: Higher bronchial AR expression and higher androgen levels are associated with better lung function, fewer symptoms, and a lower FeNO in human asthma. The role of androgens should be considered in asthma management.

Photolytic Measurement of Tissue S-Nitrosothiols in Rats and Humans In Vivo.

S-nitrosothiols are labile thiol-NO adducts formed in vivo primarily by metalloproteins such as NO synthase, ceruloplasmin, and hemoglobin. Abnormal S-nitrosothiol synthesis and catabolism contribute to many diseases, ranging from asthma to septic shock. Current methods for quantifying S-nitrosothiols in vivo are suboptimal. Samples need to be removed from the body for analysis, and the S-nitrosothiols can be broken down during ex vivo processing. Here, we have developed a noninvasive device to measure mammalian tissue S-nitrosothiols in situ non-invasively using ultraviolet (UV) light, which causes NO release in proportion to the S-nitrosothiol concentration. We validated the assay in vitro; then, we applied it to measure S-nitrosothiols in vivo in rats and in humans. The method was sensitive to 0.5 µM, specific (did not detect other nitrogen oxides), and was reproducible in rats and in humans. This noninvasive approach to S-nitrosothiol measurements may be applicable for use in human diseases.

Cyclic compression increases F508 Del CFTR expression in ciliated human airway epithelium.

The mechanisms by which transepithelial pressure changes observed during exercise and airway clearance can benefit lung health are challenging to study. Here, we have studied 117 mature, fully ciliated airway epithelial cell filters grown at air-liquid interface grown from 10 cystic fibrosis (CF) and 19 control subjects. These were exposed to cyclic increases in apical air pressure of 15 cmH2O for varying times. We measured the effect on proteins relevant to lung health, with a focus on the CF transmembrane regulator (CFTR). Immunoflourescence and immunoblot data were concordant in demonstrating that air pressure increased F508Del CFTR expression and maturation. This effect was in part dependent on the presence of cilia, on Ca2+ influx, and on formation of nitrogen oxides. These data provide a mechanosensory mechanism by which changes in luminal air pressure, like those observed during exercise and airway clearance, can affect epithelial protein expression and benefit patients with diseases of the airways.

Nitrogen chemistry and lung physiology.

The versatile chemistry of nitrogen is important to pulmonary physiology. Indeed, almost all redox forms of nitrogen are relevant to pulmonary physiology and to pathophysiology. Here we review the relevance to pulmonary biology of (a) elemental nitrogen; (b) reduced forms of nitrogen such as amines, ammonia, and hydroxylamine; and (c) oxidized forms of nitrogen such as the nitroxyl anion, the nitric oxide free radical, and S-nitrosothiols. Our focus is on oxidized nitrogen in the form of S-nitrosothiol bond-containing species, which are now appreciated to be important to every type of cell-signaling process in the lung. We also review potential clinical applications of nitrogen oxide biochemistry. These principles are being translated into clinical practice as diagnostic techniques and therapies for a range of pulmonary diseases including asthma, cystic fibrosis, adult respiratory distress syndrome, primary ciliary dyskinesia, and pulmonary hypertension.

S-Nitrosoglutathione formation at gastric pH is augmented by ascorbic acid and by the antioxidant vitamin complex, Resiston.

CONTEXT: Exogenous nitrogen oxides must be made bioavailable to sustain normal physiology because nitric oxide synthase (NOS) deficient mice are viable. In the stomach, S-nitrosoglutathione (GSNO) is formed from ingested nitrite and high levels of airway glutathione (GSH) that are cleared and swallowed. However, gastric GSNO may be broken down by nutrients like ascorbic acid (AA) before it is absorbed. OBJECTIVE: To study the effect of AA on GSNO formation and stability. MATERIALS AND METHODS: GSH and nitrite were reacted with or without 5 mM AA or Resiston (5 mM AA with retinoic acid and α-tocopherol). GSNO was measured by reduction/chemiluminescence and HPLC. AA and reduced thiols were measured colorimetrically. O-Nitrosoascorbate and AA were measured by gas chromatography-mass spectrometry (GC-MS). RESULTS: GSNO was formed in saline and gastric samples (pH ∼4.5) from physiological levels of GSH and nitrite. Neither AA nor Resiston decreased [GSNO] at pH >3; rather, they increased [GSNO] (0.12 ± 0.19 µM without AA; 0.42 ± 0.35 µM with AA; and 0.43 ± 0.23 µM with Resiston; n = 4 each; p ≤ 0.05). However, AA compounds decreased [GSNO] at lower pH and with incubation >1 h. Mechanistically, AA, but not dehydroascorbate, increased GSNO formation; and the O-nitrosoascorbate intermediate was formed. CONCLUSIONS: AA, with or without other antioxidants, did not deplete GSNO formed from physiological levels of GSH and nitrite at pH >3. In fact, it favoured GSNO formation, likely through O-nitrosoascorbate. Gastric GSNO could be a NOS-independent source of bioavailable nitrogen oxides.

Phenotype of asthmatics with increased airway S-nitrosoglutathione reductase activity.

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.

In Vivo Analysis of Tissue S-Nitrosothiols in Pediatric Sepsis.

S-nitrosothiols are endogenous, bioactive molecules. S-nitrosothiols are implicated in many diseases, including sepsis. It is currently cumbersome to measure S-nitrosothiols clinically. We have previously developed an instrument to measure tissue S-nitrosothiols non-invasively using ultraviolet light. We have performed a prospective case control study of controls and children with sepsis admitted to the PICU. We hypothesized that tissue S-nitrosothiols would be higher in septic patients than controls. Controls were patients with no cardiopulmonary instability. Cases were patients with septic shock. We measured S-nitrosothiols, both at diagnosis and after resolution of shock. A total of 44 patients were enrolled: 21 controls and 23 with sepsis. At baseline, the controls were younger [median age 5 years (IQR 0, 9) versus 11 years (IQR: 6, 16), p-value = 0.012], had fewer comorbidities [7 (33.3%) vs. 20 (87.0%), p-value < 0.001], and had lower PELOD scores [0 (IQR: 0, 0) vs. 12 (IQR: 11, 21), p-value < 0.001]. S-nitrosothiol levels were higher in sepsis cohort (1.1 ppb vs. 0.8 ppb, p = 0.004). Five patients with sepsis had longitudinal measures and had a downtrend after resolution of shock (1.3 ppb vs. 0.9 ppb, p = 0.04). We dichotomized patients based on S-nitrosothiol levels and found an association with worse clinical outcomes, but further work will be needed to validate these findings.

S-Nitrosylation signaling regulates cellular protein interactions.

BACKGROUND: S-Nitrosothiols are made by nitric oxide synthases and other metalloproteins. Unlike nitric oxide, S-nitrosothiols are involved in localized, covalent signaling reactions in specific cellular compartments. These reactions are enzymatically regulated. SCOPE: S-Nitrosylation affects interactions involved in virtually every aspect of normal cell biology. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation. MAJOR CONCLUSIONS AND SIGNIFICANCE: S-Nitrosylation is a regulated signaling reaction.

Hsp 70/Hsp 90 organizing protein as a nitrosylation target in cystic fibrosis therapy.

The endogenous signaling molecule S-nitrosoglutathione (GSNO) and other S-nitrosylating agents can cause full maturation of the abnormal gene product DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR). However, the molecular mechanism of action is not known. Here we show that Hsp70/Hsp90 organizing protein (Hop) is a critical target of GSNO, and its S-nitrosylation results in DeltaF508 CFTR maturation and cell surface expression. S-nitrosylation by GSNO inhibited the association of Hop with CFTR in the endoplasmic reticulum. This effect was necessary and sufficient to mediate GSNO-induced cell-surface expression of DeltaF508 CFTR. Hop knockdown using siRNA recapitulated the effect of GSNO on DeltaF508 CFTR maturation and expression. Moreover, GSNO acted additively with decreased temperature, which promoted mutant CFTR maturation through a Hop-independent mechanism. We conclude that GSNO corrects DeltaF508 CFTR trafficking by inhibiting Hop expression, and that combination therapies--using differing mechanisms of action--may have additive benefits in treating CF.

Asthma innovations from the first International Collaborative Asthma Network forum.

Background: Many patients have uncontrolled asthma despite available treatments. Most of the new asthma therapies have focused on type 2 (T2) inflammation, leaving an unmet need for innovative research into mechanisms of asthma beyond T2 and immunity. An international group of investigators developed the International Collaborative Asthma Network (ICAN) with the goal of sharing innovative research on disease mechanisms, developing new technologies and therapies, organising pilot studies and engaging early-stage career investigators from across the world. This report describes the purpose, development and outcomes of the first ICAN forum. Methods: Abstracts were solicited from interdisciplinary early-stage career investigators with innovative ideas beyond T2 inflammation for asthma and were selected for presentation at the forum. Breakout sessions were conducted to discuss innovation, collaboration and research translation. Results: The abstracts were categorised into: 1) general omics and big data analysis; 2) lung-brain axis and airway neurology; 3) sex differences; 4) paediatric asthma; 5) new therapeutic targets inspired by airway epithelial biology; 6) new therapeutics targeting airway and circulating immune mediators; and 7) lung anatomy, physiology and imaging. Discussions revealed that research groups are looking for opportunities to further their findings using larger scale collaboration and the ability to translate their in vitro findings into clinical treatment. Conclusions: Through ICAN, teams that included interdisciplinary early-stage career investigators discussed innovation, collaboration and translation in asthma and severe asthma research. With a combination of fresh ideas and energetic, collaborative, global participation, ICAN has laid a firm foundation and model for future collaborative global asthma research.

S-nitrosoglutathione reductase in human lung cancer.

S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.

Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium.

Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air-liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbß expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, ß, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbß gene expression were associated with lower FEV1 in asthma. Both Hbß knockdown and overexpression affected cell morphology. Hbß and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbß gene expression were associated with airflow obstruction. Hbß and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

An Update on Thiol Signaling: S-Nitrosothiols, Hydrogen Sulfide and a Putative Role for Thionitrous Acid.

Long considered vital to antioxidant defenses, thiol chemistry has more recently been recognized to be of fundamental importance to cell signaling. S-nitrosothiols-such as S-nitrosoglutathione (GSNO)-and hydrogen sulfide (H2S) are physiologic signaling thiols that are regulated enzymatically. Current evidence suggests that they modify target protein function primarily through post-translational modifications. GSNO is made by NOS and other metalloproteins; H2S by metabolism of cysteine, homocysteine and cystathionine precursors. GSNO generally acts independently of NO generation and has a variety of gene regulatory, immune modulator, vascular, respiratory and neuronal effects. Some of this physiology is shared with H2S, though the mechanisms differ. Recent evidence also suggests that molecules resulting from reactions between GSNO and H2S, such as thionitrous acid (HSNO), could also have a role in physiology. Taken together, these data suggest important new potential targets for thiol-based drug development.

Nasal Nitric Oxide Measurement in Primary Ciliary Dyskinesia. A Technical Paper on Standardized Testing Protocols.

Nasal nitric oxide concentrations are extremely low in primary ciliary dyskinesia (PCD), and measurement of this nasal gas is recommended as a PCD diagnostic test in cooperative patients aged 5 years and older. However, nasal nitric oxide measurements must be performed with chemiluminescence analyzers using a standardized protocol to ensure proper results, because nasal nitric oxide values can be influenced by various internal and external factors. Repeat nasal nitric oxide testing on separate visits is required to ensure that low diagnostic values are persistent and consistent with PCD. This technical paper presents the standard operating procedures for nasal nitric oxide measurement used by the PCD Foundation Clinical and Research Centers Network at various specialty centers across North America. Adherence to this document ensures reliable nasal nitric oxide testing and high diagnostic accuracy when employed in a population with appropriate clinical phenotypes for PCD.

Voltage-gated potassium channel proteins and stereoselective S-nitroso-l-cysteine signaling.

S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase-independent (sGC-independent) effects are stereoselective - that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) - and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays - followed by unbiased proteomic analysis - to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO-Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvß2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.