Updated reference values for static lung volumes from a healthy population in Austria.

BACKGROUND: Reference values for lung volumes are necessary to identify and diagnose restrictive lung diseases and hyperinflation, but the values have to be validated in the relevant population. Our aim was to investigate the Global Lung Function Initiative (GLI) reference equations in a representative healthy Austrian population and create population-derived reference equations if poor fit was observed. METHODS: We analysed spirometry and body plethysmography data from 5371 respiratory healthy subjects (6-80 years) from the Austrian LEAD Study. Fit with the GLI equations was examined using z-scores and distributions within the limits of normality. LEAD reference equations were then created using the LMS method and the generalized additive model of location shape and scale package according to GLI models. RESULTS: Good fit, defined as mean z-scores between + 0.5 and -0.5,was not observed for the GLI static lung volume equations, with mean z-scores > 0.5 for residual volume (RV), RV/TLC (total lung capacity) and TLC in both sexes, and for expiratory reserve volume (ERV) and inspiratory capacity in females. Distribution within the limits of normality were shifted to the upper limit except for ERV. Population-derived reference equations from the LEAD cohort showed superior fit for lung volumes and provided reproducible results. CONCLUSION: GLI lung volume reference equations demonstrated a poor fit for our cohort, especially in females. Therefore a new set of Austrian reference equations for static lung volumes was developed, that can be applied to both children and adults (6-80 years of age).

Alterations in the molecular control of mitochondrial turnover in COPD lung and airway epithelial cells.

Abnormal mitochondria have been observed in bronchial- and alveolar epithelial cells of patients with chronic obstructive pulmonary disease (COPD). However, it is unknown if alterations in the molecular pathways regulating mitochondrial turnover (mitochondrial biogenesis vs mitophagy) are involved. Therefore, in this study, the abundance of key molecules controlling mitochondrial turnover were assessed in peripheral lung tissue from non-COPD patients (n = 6) and COPD patients (n = 11; GOLDII n = 4/11; GOLDIV n = 7/11) and in both undifferentiated and differentiated human primary bronchial epithelial cells (PBEC) from non-COPD patients and COPD patients (n = 4-7 patients/group). We observed significantly decreased transcript levels of key molecules controlling mitochondrial biogenesis (PPARGC1B, PPRC1, PPARD) in peripheral lung tissue from severe COPD patients. Interestingly, mRNA levels of the transcription factor TFAM (mitochondrial biogenesis) and BNIP3L (mitophagy) were increased in these patients. In general, these alterations were not recapitulated in undifferentiated and differentiated PBECs with the exception of decreased PPARGC1B expression in both PBEC models. Although these findings provide valuable insight in these pathways in bronchial epithelial cells and peripheral lung tissue of COPD patients, whether or not these alterations contribute to COPD pathogenesis, underlie changes in mitochondrial function or may represent compensatory mechanisms remains to be established.

The effect of body compartments on lung function in childhood and adolescence.

BACKGROUND: There is an association between body composition and lung function, assessed by spirometry, but the effects of body compartments on static lung volumes and its changes during lung growth remain to be explored. We aimed to investigate the association of appendicular lean mass, reflecting skeletal muscle mass, and fat mass on forced and static lung function measures in childhood and adolescence. METHODS: In total, 1489 children and adolescents (6-18 years) of the observational, longitudinal (first and second visit within 4 years), general population-based LEAD study have been investigated. The association of appendicular lean mass and fat mass indices (ALMI and FMI; assessed by dual-energy X-ray absorptiometry) on lung function by spirometry (FEV1, FVC) and body plethysmography (TLC, RV, FRC) was investigated cross-sectionally. Longitudinal associations between lung function and body compartment changes between the two visits were analyzed. FINDINGS: The ALMI is positively associated with FEV1, FVC, and TLC. Contrary, FMI is inversely associated with lung function measures including FRC and RV. During the phase of lung growth, higher gain in muscle mass is associated with higher increases of FVC and TLC. INTERPRETATION: This study demonstrates the different effects of muscle and fat mass on forced expiratory and static lung volumes. Achieving and maintaining muscle mass in childhood and adolescence might become an important preventive strategy for lung health in adulthood.

Diagnostic Potential of Oscillometry: A Population-based Approach.

Rationale: Respiratory resistance (Rrs) and reactance (Xrs) as measured by oscillometry and their intrabreath changes have emerged as sensitive parameters for detecting early pathological impairments during tidal breathing. Objectives: This study evaluates the prevalence and association of abnormal oscillometry parameters with respiratory symptoms and respiratory diseases in a general adult population. Methods: A total of 7,560 subjects in the Austrian LEAD (Lung, hEart, sociAl, boDy) Study with oscillometry measurements (computed with the Resmon Pro FULL; Restech Srl) were included in this study. The presence of respiratory symptoms and doctor-diagnosed respiratory diseases was assessed using an interview-based questionnaire. Rrs and Xrs at 5 Hz, their inspiratory and expiratory components, the area above the Xrs curve, and the presence of tidal expiratory flow limitation were analyzed. Normality ranges for oscillometry parameters were defined. Measurements and Main Results: The overall prevalence of abnormal oscillometry parameters was 20%. The incidence of abnormal oscillometry increased in the presence of symptoms or diagnoses: 17% (16-18%) versus 27% (25-29%), P < 0.0001. All abnormal oscillometry parameters except Rrs at 5 Hz were significantly associated with respiratory symptoms/diseases. Significant associations were found, even in subjects with normal spirometry, with abnormal oscillometry incidence rates increasing by 6% (4-8%; P < 0.0001) in subjects with symptoms or diagnoses. Conclusions: Abnormal oscillometry parameters are present in one-fifth of this adult population and are significantly associated with respiratory symptoms and disease. Our findings underscore the potential of oscillometry as a tool for detecting and evaluating respiratory impairments, even in individuals with normal spirometry.

Supranormal lung function: Prevalence, associated factors and clinical manifestations across the lifespan.

BACKGROUND AND OBJECTIVE: It is now well established that there are different life-long lung function trajectories in the general population, and that some are associated with better or worse health outcomes. Yet, the prevalence, clinical characteristics and risk factors of individuals with supranormal FEV1 or FVC values (above the upper-limit of normal [ULN]) in different age-bins through the lifetime in the general population are poorly understood. METHOD: To address these questions, we investigated the prevalence of supranormal FEV1 and FVC values in the LEAD (Lung, hEart, sociAl and boDy) study, a general population cohort in Austria that includes participants from 6 to 82 years of age. RESULTS: We found that: (1) the prevalence of supranormal pre-bronchodilator FEV1 and FVC values was 3.4% and 3.1%, respectively, and that these figures remained relatively stable through different age-bins except for participants >60 years., in whom they increased (5.0% and 4.2%, respectively). Approximately 50% of supranormal individuals had both increased FEV1 and FVC values; (2) supranormal spirometric values were consistently accompanied by higher static lung volumes and lower specific airway resistance through the lifespan, indicating better overall lung function; and (3) multivariate regression analysis identified that female sex, higher muscle mass (FFMI), less diabetes and fewer respiratory symptoms were consistently associated with supranormal FEV1 and FVC values. CONCLUSION: Supranormal FEV1 and/or FVC values occur in about 3% of the general population in different age bins and are associated with better health markers.

Asthma Prevalence and Phenotyping in the General Population: The LEAD (Lung, hEart, sociAl, boDy) Study.

Background: Asthma is a chronic heterogeneous respiratory disease involving differential pathophysiological pathways and consequently distinct asthma phenotypes. Objective and Methods: In the LEAD Study, a general population cohort (n=11.423) in Vienna ranging from 6-82 years of age, we addressed the prevalence of asthma and explored inflammatory asthma phenotypes that included allergic and non-allergic asthma, and within these phenotypes, an eosinophilic (eosinophils ≥300 cells/µL, or ≥150 cells/µL in the presence of ICS medication) or non-eosinophilic (eosinophils <300 cells/µL, or <150 cells/µL in the presence of ICS) phenotype. In addition, we compared various factors related to biomarkers, body composition, lung function, and symptoms in control subjects versus subjects with current asthma (current doctor's diagnosis of asthma). Results: An overall prevalence of 4.6% was observed for current asthma. Furthermore, an age-dependent shift from allergic to non-allergic asthma was found. The non-eosinophilic phenotype was more prominent. Obesity was a prevalent condition, and body composition including visceral adipose tissue (VAT), is affected in current asthma versus controls. Conclusion: This broad-aged and large general population cohort identified differential patterns of inflammatory asthma phenotypes that were age-dependent. The presence of eosinophilia was associated with worse asthma control, increased asthma medication, increased VAT, and lower lung function, the opposite was found for the presence of an allergic asthma.

Prevalence of restrictive lung function in children and adults in the general population.

BACKGROUND: Restrictive lung function (RLF) is characterized by a reduced lung expansion and size. In the absence of lung volume measurements, restriction can be indirectly assessed with restrictive spirometric patterns (RSP) by spirometry. Prevalence data on RLF by the golden standard body plethysmography in the general population are scarce. Therefore, we aimed to evaluate the prevalence of RLF and RSP in the general population by body plethysmography and to determine factors influencing RLF and RSP. METHODS: Pre-bronchodilation lung function data of 8891 subjects (48.0% male, age 6-82 years) have been collected in the LEAD Study, a single-centered, longitudinal, population-based study from Vienna, Austria. The cohort was categorized in the following groups based on the Global Lung Initiative reference equations: normal subjects, RLF (TLC < lower limit of normal (LLN)), RSP (FEV1/FVC ≥ LLN and a FVC < LLN), RSP only (RSP with TLC ≥ LLN). Normal subjects were considered those with FEV1, FVC, FEV1/FVC and TLC between LLN and ULN (upper limit of normal). RESULTS: The prevalence of RLF and RSP in the Austrian general population is 1.1% and 4.4%. Spirometry has a positive and negative predictive value of 18.0% and 99.6% to predict a restrictive lung function. Central obesity was associated with RLF. RSP was related to smoking and underweight. CONCLUSIONS: The prevalence of true restrictive lung function and RSP in the Austrian general population is lower than previously estimated. Our data confirm the need for direct lung volume measurement to diagnose true restrictive lung function.

Macrophage-intrinsic DUOX1 contributes to type 2 inflammation and mucus metaplasia during allergic airway disease.

The NADPH oxidase DUOX1 contributes to epithelial production of alarmins, including interleukin (IL)-33, in response to injurious triggers such as airborne protease allergens, and mediates development of mucus metaplasia and airway remodeling in chronic allergic airways diseases. DUOX1 is also expressed in non-epithelial lung cell types, including macrophages that play an important role in airway remodeling during chronic lung disease. We therefore conditionally deleted DUOX1 in either lung epithelial or monocyte/macrophage lineages to address its cell-specific actions in innate airway responses to acute airway challenge with house dust mite (HDM) allergen, and in chronic HDM-driven allergic airway inflammation. As expected, acute responses to airway challenge with HDM, as well as type 2 inflammation and related features of airway remodeling during chronic HDM-induced allergic inflammation, were largely driven by DUOX1 with the respiratory epithelium. However, in the context of chronic HDM-driven inflammation, DUOX1 deletion in macrophages also significantly impaired type 2 cytokine production and indices of mucus metaplasia. Further studies revealed a contribution of macrophage-intrinsic DUOX1 in macrophage recruitment upon chronic HDM challenge, as well as features of macrophage activation that impact on type 2 inflammation and remodeling.

Redox Dysregulation in Aging and COPD: Role of NOX Enzymes and Implications for Antioxidant Strategies.

With a rapidly growing elderly human population, the incidence of age-related lung diseases such as chronic obstructive pulmonary disease (COPD) continues to rise. It is widely believed that reactive oxygen species (ROS) play an important role in ageing and in age-related disease, and approaches of antioxidant supplementation have been touted as useful strategies to mitigate age-related disease progression, although success of such strategies has been very limited to date. Involvement of ROS in ageing is largely attributed to mitochondrial dysfunction and impaired adaptive antioxidant responses. NADPH oxidase (NOX) enzymes represent an important enzyme family that generates ROS in a regulated fashion for purposes of oxidative host defense and redox-based signalling, however, the associations of NOX enzymes with lung ageing or age-related lung disease have to date only been minimally addressed. The present review will focus on our current understanding of the impact of ageing on NOX biology and its consequences for age-related lung disease, particularly COPD, and will also discuss the implications of altered NOX biology for current and future antioxidant-based strategies aimed at treating these diseases.

Oxidation-Dependent Activation of Src Kinase Mediates Epithelial IL-33 Production and Signaling during Acute Airway Allergen Challenge.

The respiratory epithelium forms the first line of defense against inhaled pathogens and acts as an important source of innate cytokine responses to environmental insults. One critical mediator of these responses is the IL-1 family cytokine IL-33, which is rapidly secreted upon acute epithelial injury as an alarmin and induces type 2 immune responses. Our recent work highlighted the importance of the NADPH oxidase dual oxidase 1 (DUOX1) in acute airway epithelial IL-33 secretion by various airborne allergens associated with H2O2 production and reduction-oxidation-dependent activation of Src kinases and epidermal growth factor receptor (EGFR) signaling. In this study, we show that IL-33 secretion in response to acute airway challenge with house dust mite (HDM) allergen critically depends on the activation of Src by a DUOX1-dependent oxidative mechanism. Intriguingly, HDM-induced epithelial IL-33 secretion was dramatically attenuated by small interfering RNA- or Ab-based approaches to block IL-33 signaling through its receptor IL1RL1 (ST2), indicating that HDM-induced IL-33 secretion includes a positive feed-forward mechanism involving ST2-dependent IL-33 signaling. Moreover, activation of type 2 cytokine responses by direct airway IL-33 administration was associated with ST2-dependent activation of DUOX1-mediated H2O2 production and reduction-oxidation-based activation of Src and EGFR and was attenuated in Duox1 -/- and Src +/- mice, indicating that IL-33-induced epithelial signaling and subsequent airway responses involve DUOX1/Src-dependent pathways. Collectively, our findings suggest an intricate relationship between DUOX1, Src, and IL-33 signaling in the activation of innate type 2 immune responses to allergens, involving DUOX1-dependent epithelial Src/EGFR activation in initial IL-33 secretion and in subsequent IL-33 signaling through ST2 activation.

Downregulation of DUOX1 function contributes to aging-related impairment of innate airway injury responses and accelerated senile emphysema.

Aging is associated with a gradual loss of lung function due to increased cellular senescence, decreased regenerative capacity, and impaired innate host defense. One important aspect of innate airway epithelial host defense to nonmicrobial triggers is the secretion of alarmins such as IL-33 and activation of type 2 inflammation, which were previously found to depend on activation of the NADPH oxidase (NOX) homolog DUOX1, and redox-dependent signaling pathways that promote alarmin secretion. Here, we demonstrate that normal aging of C57BL/6J mice resulted in markedly decreased lung innate epithelial type 2 responses to exogenous triggers such as the airborne allergen Dermatophagoides pteronyssinus, which was associated with marked downregulation of DUOX1, as well as DUOX1-mediated redox-dependent signaling. DUOX1 deficiency was also found to accelerate age-related airspace enlargement and decline in lung function but did not consistently affect other features of lung aging such as senescence-associated inflammation. Intriguingly, observations of age-related DUOX1 downregulation and enhanced airspace enlargement due to DUOX1 deficiency in C57BL/6J mice, which lack a functional mitochondrial nicotinamide nucleotide transhydrogenase (NNT), were much less dramatic in C57BL/6NJ mice with normal NNT function, although the latter mice also displayed impaired innate epithelial injury responses with advancing age. Overall, our findings indicate a marked aging-dependent decline in (DUOX1-dependent) innate airway injury responses to external nonmicrobial triggers, but the impact of aging on DUOX1 downregulation and its significance for age-related senile emphysema development was variable between different C57BL6 substrains, possibly related to metabolic alterations due to differences in NNT function.

Glutathione S-transferases and their implications in the lung diseases asthma and chronic obstructive pulmonary disease: Early life susceptibility?

Our lungs are exposed daily to airborne pollutants, particulate matter, pathogens as well as lung allergens and irritants. Exposure to these substances can lead to inflammatory responses and may induce endogenous oxidant production, which can cause chronic inflammation, tissue damage and remodeling. Notably, the development of asthma and Chronic Obstructive Pulmonary Disease (COPD) is linked to the aforementioned irritants. Some inhaled foreign chemical compounds are rapidly absorbed and processed by phase I and II enzyme systems critical in the detoxification of xenobiotics including the glutathione-conjugating enzymes Glutathione S-transferases (GSTs). GSTs, and in particular genetic variants of GSTs that alter their activities, have been found to be implicated in the susceptibility to and progression of these lung diseases. Beyond their roles in phase II metabolism, evidence suggests that GSTs are also important mediators of normal lung growth. Therefore, the contribution of GSTs to the development of lung diseases in adults may already start in utero, and continues through infancy, childhood, and adult life. GSTs are also known to scavenge oxidants and affect signaling pathways by protein-protein interaction. Moreover, GSTs regulate reversible oxidative post-translational modifications of proteins, known as protein S-glutathionylation. Therefore, GSTs display an array of functions that impact the pathogenesis of asthma and COPD. In this review we will provide an overview of the specific functions of each class of mammalian cytosolic GSTs. This is followed by a comprehensive analysis of their expression profiles in the lung in healthy subjects, as well as alterations that have been described in (epithelial cells of) asthmatics and COPD patients. Particular emphasis is placed on the emerging evidence of the regulatory properties of GSTs beyond detoxification and their contribution to (un)healthy lungs throughout life. By providing a more thorough understanding, tailored therapeutic strategies can be designed to affect specific functions of particular GSTs.

Downregulation of epithelial DUOX1 in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by small airway remodeling and alveolar emphysema due to environmental stresses such as cigarette smoking (CS). Oxidative stress is commonly implicated in COPD pathology, but recent findings suggest that one oxidant-producing NADPH oxidase homolog, dual oxidase 1 (DUOX1), is downregulated in the airways of patients with COPD. We evaluated lung tissue sections from patients with COPD for small airway epithelial DUOX1 protein expression, in association with measures of lung function and small airway and alveolar remodeling. We also addressed the impact of DUOX1 for lung tissue remodeling in mouse models of COPD. Small airway DUOX1 levels were decreased in advanced COPD and correlated with loss of lung function and markers of emphysema and remodeling. Similarly, DUOX1 downregulation in correlation with extracellular matrix remodeling was observed in a genetic model of COPD, transgenic SPC-TNF-α mice. Finally, development of subepithelial airway fibrosis in mice due to exposure to the CS-component acrolein, or alveolar emphysema induced by administration of elastase, were in both cases exacerbated in Duox1-deficient mice. Collectively, our studies highlight that downregulation of DUOX1 may be a contributing feature of COPD pathogenesis, likely related to impaired DUOX1-mediated innate injury responses involved in epithelial homeostasis.

The Transient Receptor Potential Channel Vanilloid 1 Is Critical in Innate Airway Epithelial Responses to Protease Allergens.

The airway epithelium plays a critical role in innate responses to airborne allergens by secreting IL-1 family cytokines such as IL-1α and IL-33 as alarmins that subsequently orchestrate appropriate immune responses. Previous studies revealed that epithelial IL-33 secretion by allergens such as Alternaria alternata or house dust mite involves Ca2+-dependent signaling, via initial activation of ATP-stimulated P2YR2 (type 2 purinoceptor) and subsequent activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase DUOX1. We sought to identify proximal mechanisms by which epithelial cells sense these allergens and here highlight the importance of PAR2 (protease-activated receptor 2) and TRP (transient receptor potential) Ca2+ channels such as TRPV1 (TRP vanilloid 1) in these responses. Combined studies of primary human nasal and mouse tracheal epithelial cells, as well as immortalized human bronchial epithelial cells, indicated the importance of both PAR2 and TRPV1 in IL-33 secretion by both Alternaria alternata and house dust mite, based on both pharmacological and genetic approaches. TRPV1 was also critically involved in allergen-induced ATP release, activation of DUOX1, and redox-dependent activation of EGFR (epidermal growth factor receptor). Moreover, genetic deletion of TRPV1 dramatically attenuated allergen-induced IL-33 secretion and subsequent type 2 responses in mice in vivo. TRPV1 not only contributed to ATP release and P2YR2 signaling but also was critical in downstream innate responses to ATP, indicating potentiating effects of P2YR2 on TRPV1 activation. In aggregate, our studies illustrate a complex relationship between various receptor types, including PAR2 and P2YR2, in epithelial responses to asthma-relevant airborne allergens and highlight the central importance of TRPV1 in such responses.

Proteomic Methods to Evaluate NOX-Mediated Redox Signaling.

The NADPH oxidase (NOX) family of proteins is involved in regulating many diverse cellular processes, which is largely mediated by NOX-mediated reversible oxidation of target proteins in a process known as redox signaling. Protein cysteine residues are the most prominent targets in redox signaling, and to understand the mechanisms by which NOX affect cellular pathways, specific methodology is required to detect specific oxidative cysteine modifications and to identify targeted proteins. Among the many potential redox modifications involving cysteine residues, reversible modifications most relevant to NOX are sulfenylation (P-SOH) and S-glutathionylation (P-SSG), as both can induce structural or functional alterations. Various experimental approaches have been developed to detect these specific modifications, and this chapter will detail state-of-the-art methodology to selectively evaluate these modifications in specific target proteins in relation to NOX activation. We also discuss some of the limitations of these procedures and potential complementary approaches.

Dysregulated Redox Regulation Contributes to Nuclear EGFR Localization and Pathogenicity in Lung Cancer.

Lung cancers are frequently characterized by inappropriate activation of epidermal growth factor receptor (EGFR)-dependent signaling and epigenetic silencing of the NADPH oxidase (NOX) enzyme DUOX1, both potentially contributing to worse prognosis. Based on previous findings linking DUOX1 with redox-dependent EGFR activation, the present studies were designed to evaluate whether DUOX1 silencing in lung cancers may be responsible for altered EGFR regulation. In contrast to normal epithelial cells, EGF stimulation of lung cancer cell lines that lack DUOX1 promotes EGF-induced EGFR internalization and nuclear localization, associated with induction of EGFR-regulated genes and related tumorigenic outcomes. Each of these outcomes could be reversed by overexpression of DUOX1 or enhanced by shRNA-dependent DUOX1 silencing. EGF-induced nuclear EGFR localization in DUOX1-deficient lung cancer cells was associated with altered dynamics of cysteine oxidation of EGFR, and an overall reduction of EGFR cysteines. These various outcomes could also be attenuated by silencing of glutathione S-transferase P1 (GSTP1), a mediator of metabolic alterations and drug resistance in various cancers, and a regulator of cysteine oxidation. Collectively, our findings indicate DUOX1 deficiency in lung cancers promotes dysregulated EGFR signaling and enhanced GSTP1-mediated turnover of EGFR cysteine oxidation, which result in enhanced nuclear EGFR localization and tumorigenic properties.