Determinants of SARS-CoV-2 receptor gene expression in upper and lower airways.

The recent outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has led to a worldwide pandemic. One week after initial symptoms develop, a subset of patients progresses to severe disease, with high mortality and limited treatment options. To design novel interventions aimed at preventing spread of the virus and reducing progression to severe disease, detailed knowledge of the cell types and regulating factors driving cellular entry is urgently needed. Here we assess the expression patterns in genes required for COVID-19 entry into cells and replication, and their regulation by genetic, epigenetic and environmental factors, throughout the respiratory tract using samples collected from the upper (nasal) and lower airways (bronchi). Matched samples from the upper and lower airways show a clear increased expression of these genes in the nose compared to the bronchi and parenchyma. Cellular deconvolution indicates a clear association of these genes with the proportion of secretory epithelial cells. Smoking status was found to increase the majority of COVID-19 related genes including ACE2 and TMPRSS2 but only in the lower airways, which was associated with a significant increase in the predicted proportion of goblet cells in bronchial samples of current smokers. Both acute and second hand smoke were found to increase ACE2 expression in the bronchus. Inhaled corticosteroids decrease ACE2 expression in the lower airways. No significant effect of genetics on ACE2 expression was observed, but a strong association of DNA- methylation with ACE2 and TMPRSS2- mRNA expression was identified in the bronchus.

Mitochondrial dysfunction increases pro-inflammatory cytokine production and impairs repair and corticosteroid responsiveness in lung epithelium.

COPD is characterized by chronic lung inflammation and irreversible lung tissue damage. Inhaled noxious gases, including cigarette smoke, are the major risk factor for COPD. Inhaled smoke first encounters the epithelial lining of the lungs, causing oxidative stress and mitochondrial dysfunction. We investigated whether a mitochondrial defect may contribute to increased lung epithelial pro-inflammatory responses, impaired epithelial repair and reduced corticosteroid sensitivity as observed in COPD. We used wild-type alveolar epithelial cells A549 and mitochondrial DNA-depleted A549 cells (A549 Rho-0) and studied pro-inflammatory responses using (multiplex) ELISA as well as epithelial barrier function and repair (real-time impedance measurements), in the presence and absence of the inhaled corticosteroid budesonide. We observed that A549 Rho-0 cells secrete higher levels of pro-inflammatory cytokines than wild-type A549 cells and display impaired repair upon wounding. Budesonide strongly suppressed the production of neutrophil attractant CXCL8, and promoted epithelial integrity in A549 wild-type cells, while A549 Rho-0 cells displayed reduced corticosteroid sensitivity compared to wild-type cells. The reduced corticosteroid responsiveness may be mediated by glycolytic reprogramming, specifically glycolysis-associated PI3K signaling, as PI3K inhibitor LY294002 restored the sensitivity of CXCL8 secretion to corticosteroids in A549 Rho-0 cells. In conclusion, mitochondrial defects may lead to increased lung epithelial pro-inflammatory responses, reduced epithelial repair and reduced corticosteroid responsiveness in lung epithelium, thus potentially contributing to the pathogenesis of COPD.

Impact of acute exposure to cigarette smoke on airway gene expression.

BACKGROUND: Understanding effects of acute smoke exposure (ASE) on airway epithelial gene expression and their relationship with the effects of chronic smoke exposure may provide biological insights into the development of smoking-related respiratory diseases. METHODS: Bronchial airway epithelial cell brushings were collected from 63 individuals without recent cigarette smoke exposure and before and 24 h after smoking three cigarettes. RNA from these samples was profiled on Affymetrix Human Gene 1.0 ST microarrays. RESULTS: We identified 91 genes differentially expressed 24 h after ASE (false discovery rate < 0.25). ASE induced genes involved in xenobiotic metabolism, oxidative stress, and inflammation and repressed genes related to cilium morphogenesis and cell cycle. While many genes altered by ASE are altered similarly in chronic smokers, metallothionein genes are induced by ASE and suppressed in chronic smokers. Metallothioneins are also suppressed in current and former smokers with lung cancer relative to those without lung cancer. CONCLUSIONS: Acute exposure to as little as three cigarettes and chronic smoking induce largely concordant changes in airway epithelial gene expression. Differences in short-term and long-term effects of smoking on metallothionein expression and their relationship to lung cancer requires further study given these enzymes' role in the oxidative stress response.

Emerging understanding of the mechanism of action of Bronchial Thermoplasty in asthma.

Bronchial Thermoplasty (BT) is an endoscopic treatment for moderate-to-severe asthma patients who are uncontrolled despite optimal medical therapy. Effectiveness of BT has been demonstrated in several randomized clinical trials. However, the asthma phenotype that benefits most of this treatment is unclear, partly because the mechanism of action is incompletely understood. BT was designed to reduce the amount of airway smooth muscle (ASM), but additional direct and indirect effects on airway pathophysiology are expected. This review will provide an overview of the different components of airway pathophysiology including remodeling, with the ASM as the key player. Current concepts in the understanding of BT clinical effectiveness with a focus on its impact on airway remodeling will be reviewed.

[Two sisters with lung emphysema]. / Twee zussen met longemfyseem.

BACKGROUND: α1-antitrypsin is an antiprotease that is mainly produced in the liver; it plays a crucial role in the protection of lung parenchyma against the destructive effects of proteases. Mutations in the α1-antitrypsin gene can cause α1-antitrypsin deficiency. Individuals homozygous for the Z-genotype have drastically lowered serum α1-antitrypsine concentrations and often develop lung emphysema at an early age. CASE DESCRIPTION: A 38-year-old woman and her 43-year-old sister both developed lung emphysema at an early age; this could be attributed to severe α1-antitrypsin deficiency. The only treatment for this condition is α1-antitrypsin supplement therapy, but this therapy is not reimbursed by health insurance companies in the Netherlands. CONCLUSION: α1-antitrypsin deficiency is a relatively rare cause of lung emphysema and can be seen as an orphan phenotype of chronic obstructive pulmonary disease (COPD). We appeal for reconsideration of coverage of α1-antitrypsine supplement therapy by basic health insurance in the Netherlands, on the basis of a recent randomised placebo-controlled study in which the protective effect of this therapy on progressive emphysema was demonstrated by CT lung densitometry.

Budesonide and fluticasone propionate differentially affect the airway epithelial barrier.

BACKGROUND: COPD patients have a higher risk of pneumonia when treated with fluticasone propionate (FP) than with placebo, and a lower risk with budesonide (BUD). We hypothesized that BUD and FP differentially affect the mucosal barrier in response to viral infection and/or cigarette smoke. METHODS: We assessed protective effects of equivalent concentrations of BUD and FP on cytokine production and barrier function (electrical resistance) in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) upon exposure to viral mimetic poly-(I:C) and/or cigarette smoke extract (CSE) or epidermal growth factor (EGF). RESULTS: BUD and FP were equally effective in suppressing poly-(I:C)- and/or CSE-induced IL-8 secretion in 16HBE and PBECs. Poly-(I:C) substantially decreased electrical resistance in 16HBE cells and both BUD and FP fully counteracted this effect. However, FP hardly affected 16HBE barrier dysfunction induced by CSE with/without poly-(I:C), whereas BUD (16 nM) provided full protection, an effect likely mediated by affecting EGFR-downstream target GSK-3ß. Similarly, BUD, but not FP, significantly improved CSE-induced barrier dysfunction in PBECs. Finally, BUD, but not FP, exerted a modest but significant protective effect against Streptococcus Pneumoniae-induced barrier dysfunction, and BUD, but not FP, prevented cellular adhesion and/or internalization of these bacteria induced by poly-(I:C) in 16HBE. CONCLUSIONS: Collectively, both BUD and FP efficiently control epithelial pro-inflammatory responses and barrier function upon mimicry of viral infection. Of potential clinical relevance, BUD more effectively counteracted CSE-induced barrier dysfunction, reinforcing the epithelial barrier and potentially limiting access of pathogens upon smoking in vivo.

Frequent sputum production is associated with disturbed night's rest and impaired sleep quality in patients with COPD.

PURPOSE: In this study, we measured night's rest parameters measured with an accelerometer and sleep quality in mild to very severe patients with COPD. Furthermore, our aim was to investigate the association between night's rest parameters and clinical variables and the association between sleep quality and quality of life or health status. METHODS: Mild to very severe COPD patients were recruited from general practitioners and outpatient clinics of general hospitals to participate in a cross-sectional study on physical activity in patients with COPD. A total of 103 patients (mean age 65 years, 67 % male) wore the accelerometer during night's rest for at least four nights and were included in the analyses. RESULTS: No significant associations were found between objectively measured body movements during night's rest or subjective sleep quality and lung function, dyspnoea severity, body composition and physical activity during the day. Patients with frequent sputum production during the day had a higher number of sitting transitions during the night (5.3 vs 4.3 sitting transitions) and more frequently got out of bed compared to patients who hardly ever produced sputum during the day (1.0 vs 0.8 times per night). Furthermore, these patients also reported worse sleep quality (Pittsburgh sleep quality index (PSQI) score 4 vs 3). CONCLUSIONS: Our results indicate that objectively measured body movements during night's rest like body postures and transitions are not related to sleep quality in patients with COPD. We did find an association between frequent sputum production and disturbances during night's rest and sleep quality. Future studies should investigate whether the treatment of mucus hypersecretion leads to improved night's rest.

Effects of small airway dysfunction on the clinical expression of asthma: a focus on asthma symptoms and bronchial hyper-responsiveness.

BACKGROUND: The small airways are an important site of inflammation in asthma. However, the relation between small airway dysfunction and clinical expression of asthma has hardly been studied. AIM: To investigate the association of small and large airway dysfunction with asthma symptoms and bronchial hyper-responsiveness (BHR). METHODS: Fifty-eight patients with asthma were characterized with spirometry, body plethysmography, impulse oscillometry, alveolar and bronchial exhaled nitric oxide, and a methacholine provocation. Symptoms of nocturnal asthma, exercise-related symptoms, BHR symptoms, and respiratory symptoms were assessed with the Asthma Control Questionnaire and Bronchial Hyper-responsiveness Questionnaire. Perception of dyspnea was rated with the Borg score during the provocation test. RESULTS: Small and large airway dysfunction did not associate with higher scores for nocturnal, exercise-related, or BHR symptoms. Only higher scores on wheezing were significantly associated with higher values of difference between R5 and R20 (R5-R20) (r = 0.367, P < 0.01) and AX (r = 0.354, P < 0.01). Lower FEF25-75% (P = 0.024) and higher R5-R20 (P = 0.003) values were independently associated with more severe BHR to methacholine, but not FEV1 or R20 values. The increase in dyspnea during the methacholine provocation was strongly and independently correlated with the decrease in FEV1 and reactance of the respiratory system at 5 Hertz. CONCLUSION: Small and large airway dysfunction poorly associate with asthma symptoms in our patients. However, deteriorations in small airway dysfunction are strongly related to an increase in dyspnea during bronchial provocation with methacholine. Small airway dysfunction contributes also independently to the clinical expression of asthma, as reflected by the severity of BHR.

DAMPs activating innate and adaptive immune responses in COPD.

Chronic obstructive pulmonary disease (COPD), a progressive lung disease characterized by sustained neutrophilic airway inflammation, is caused by chronic exposure to noxious stimuli, e.g., cigarette smoke. This chronic exposure can induce immunogenic cell death of structural airway cells, inducing the release of damage-associated molecular patterns (DAMPs). Levels of several DAMPs, including S100 proteins, defensins, and high-mobility group box-1 (HMGB1), are increased in extracellular lung fluids of COPD patients. As DAMPs can attract and activate immune cells upon binding to pattern recognition receptors, we propose that their release may contribute to neutrophilic airway inflammation. In this review, we discuss the novel role of DAMPs in COPD pathogenesis. Relevant DAMPs are categorized based on their subcellular origin, i.e. cytoplasm, endoplasmic reticulum, nucleus, and mitochondria. Furthermore, their potential role in the pathophysiology of COPD will be discussed.

Less small airway dysfunction in asymptomatic bronchial hyperresponsiveness than in asthma.

BACKGROUND: Bronchial hyperresponsiveness (BHR) can be present in subjects without any respiratory symptoms. Little is known about the role of the small airways in asymptomatic subjects with BHR. METHODS: We investigated small airway function assessed by spirometry and impulse oscillometry, as well as Borg dyspnea scores at baseline and during a methacholine provocation test in 15 subjects with asymptomatic BHR, 15 asthma patients, and 15 healthy controls. RESULTS: At baseline, small airway function (R5 -R20 and X5 ) was comparable between subjects with asymptomatic BHR and healthy controls, whereas asthma patients showed small airway dysfunction as reflected by higher R5 -R20 and lower X5 values. During methacholine provocation, small airway dysfunction was more severe in asthma patients than in subjects with asymptomatic BHR. Interestingly, a higher increase in small airway dysfunction during methacholine provocation was associated with a higher increase in Borg dyspnea scores in subjects with asymptomatic BHR, but not in asthma patients. CONCLUSION: Subjects with asymptomatic BHR may experience fewer symptoms in daily life because they have less small airway dysfunction.

Treatment of the bronchial tree from beginning to end: targeting small airway inflammation in asthma.

Asthma is a chronic respiratory disease, characterized by airway obstruction and inflammation. Increasing evidence shows that the small airways contribute significantly to the clinical expression and severity of asthma. Traditionally, high levels of disease activity are thought to be necessary before symptoms occur in the small airways because of their large reserve capacity. However, this concept is being challenged and increasing evidence shows small airway disease to be associated with symptoms, disease severity, and bronchial hyper-responsiveness. Particle size and distribution are of key importance when developing inhaled treatments for small airway disease. The availability of small-particle aerosols such as HFA-ciclesonide and HFA-beclomethasone dipropionate (HFA-BDP) enables a higher drug deposition into the peripheral lung and potentially provides additional clinical benefits compared with large-particle treatment. However, improved methods are needed to monitor and assess small airway disease and its response to treatment because conventional spirometry mainly reflects large airway function. This remains a challenging area requiring further research. The aim of the current manuscript is to review the clinical relevance of small airway disease and the implications for the treatment of asthma.

Interleukin-17A induces glucocorticoid insensitivity in human bronchial epithelial cells.

A subset of asthma patients suffer from glucocorticoid (GC) insensitivity. T-helper cell type 17 cells have an emerging role in GC insensitivity, although the mechanisms are still poorly understood. We investigated whether interleukin (IL)-17A induces GC insensitivity in airway epithelium by studying its effects on responsiveness of tumour necrosis factor (TNF)-α-induced IL-8 production to budesonide in human bronchial epithelial 16HBE cells. We unravelled the underlying mechanism by the use of specific pathway inhibitors, reporter and overexpression constructs and a histone deacetylase (HDAC) activity assay. We demonstrated that IL-17A-induced IL-8 production is normally sensitive to GCs, while IL-17A pre-treatment significantly reduced the sensitivity of TNF-α-induced IL-8 production to budesonide. IL-17A activated the p38, extracellular signal-related kinase (ERK) and phosphoinositide-3-kinase (PI3K) pathways, and the latter appeared to be involved in IL-17A-induced GC insensitivity. Furthermore, IL-17A reduced HDAC activity, and overexpression of HDAC2 reversed IL-17A-induced GC insensitivity. In contrast, IL-17A did not affect budesonide-induced transcriptional activity of the GC receptor, suggesting that IL-17A does not impair the actions of the ligated GC receptor. In conclusion, we have shown for the first time that IL-17A induces GC insensitivity in airway epithelium, which is probably mediated by PI3K activation and subsequent reduction of HDAC2 activity. Thus, blockade of IL-17A or downstream signalling molecule PI3K may offer new strategies for therapeutic intervention in GC-insensitive asthma.

Particle size matters: diagnostics and treatment of small airways involvement in asthma.

Small airways are an important site of inflammation and obstruction in asthma, which contributes to the severity of airway hyperresponsiveness (AHR) that is usually measured by nebulisation of large-particle stimuli. We investigated whether small and large particle sizes of aerosolised adenosine monophospate (AMP) provoke similar severity of AHR. Additionally, effects of the small-particle inhaled corticosteroid (ICS) ciclesonide and large-particle ICS fluticasone on AHR to large- and small-particle size AMP were assessed. After a 4-week run-in period using open-label fluticasone (100 µg b.i.d.), 37 mild-to-moderate asthmatics underwent provocations with standard-size (3.7 µm), large-particle (9.9 µm) and small-particle (1.06 µm) AMP. Subjects received 4-week ciclesonide (160 µg s.i.d.) or fluticasone (100 µg b.i.d.) treatment (double-blind and double-dummy) followed by large- and small-particle AMP provocation. Small-particle AMP induced a 20% decrease in forced expiratory volume in 1 s (FEV(1)) at a significantly higher dose than large-particle AMP. Ciclesonide and fluticasone had comparable effects on AMP provocations. Not all subjects reached the provocative concentration causing a 20% fall in FEV(1) (PC(20)) at the highest AMP dose. In those who did, ciclesonide improved small-particle AMP PC(20) by 1.74 doubling doses (DD) (p = 0.03), whereas fluticasone did not. Conversely, fluticasone improved large-particle AMP PC(20) significantly (1.32 DD; p = 0.03), whereas ciclesonide did not. Small-particle AMP provocation appears to be a promising tool to assess changes in small airway inflammation. Future adjustments are necessary taking into account the very small particle size used, with large exhaled fractions. In asthmatics reaching a PC(20) with small- and large-particle AMP provocations, ciclesonide improves hyperresponsiveness to small particle size AMP, and fluticasone to large particle size. This warrants further research to target provocations and treatment to specific airway sizes.

Airway eosinophilia in remission and progression of asthma: accumulation with a fast decline of FEV(1).

BACKGROUND: As it is unknown whether complete asthma remission or progression of asthma is associated with airway inflammation and remodeling, we assessed these characteristics in bronchial biopsies of relevant subsets of asthma patients. METHODS: Sputum and bronchial biopsies were obtained from asthma patients in remission (PC(20) histamine> 32 mg/ml, PC(20) AMP> 320 mg/ml) and from those with either a slow FEV(1) decline (< 30 ml/year) or fast decline (> 30 ml/year). Inflammatory cells and mediators were determined in sputum, inflammatory cells and aspects of airway remodeling in bronchial biopsies. RESULTS: Asthmatics in remission and asthma patients with a slow FEV(1) decline had a similar extent of airway inflammation and remodeling in sputum and bronchial biopsies. Asthma patients with a fast FEV(1) decline had high sputum eosinophil numbers. Moreover, FEV(1) decline (ml/year) correlated with sputum eosinophil numbers (Rs=0.51, p=0.003) and ECP levels (Rs=0.57, p=0.001). Airway remodeling, i.e. basement membrane thickness, correlated with sputum eosinophils (Rs=0.69, p<0.001), sputum ECP (Rs=0.46, p=0.018) and airway wall eosinophil numbers (Rs=0.49, p=0.002). CONCLUSIONS: Asthma, even when in remission, is accompanied by airway inflammation and remodeling. Data suggest that eosinophils are important in a subset of asthma patients by association to accelerated FEV(1) decline and change of basement membrane thickness.

Physiology of the small airways: A gender difference?

Small airways are affected in asthma, but gender differences have not been investigated. This study aims to assess whether gender differences exist in the extent of small airway involvement in asthma. Sixteen patients with mild-to-moderate asthma (seven males, median (range) age 39 (19-56) years, FEV(1)%predicted 89% (62-120), PC(20) Methacholine (MCh) 0.4 mg/mL (0.1-4.2)) participated in the study. MCh-induced air trapping at end-expiratory CT scans, alveolar and bronchial exhaled Nitric Oxide (eNO), Single-Breath N(2) closing volume, FEF(25-75%), FEF(50%) and % change in FVC at PC(20) adenosine-5'-monophosphate (AMP) and at PC(20)MCh were evaluated. Multiple regression analyses evaluated whether gender, body mass index and age were explanatory variables for the severity of air trapping. Males had significantly larger MCh-induced increases in low attenuation areas on CT than females, 6% (1-9) versus 2% (-1-7), p=0.012. Males had significantly more signs of MCh-induced air trapping in the non-dependent lung lobes than females. Male gender was the sole factor explaining higher values of MCh-induced signs of air trapping. Females had higher bronchial eNO values than males, 4 nL/s (2-7) versus 2 nL/s (1-4), p=0.04. Small airway involvement in asthma as reflected by MCh-induced air trapping at CT was significantly larger in males than females even in this relatively small group. Bronchial eNO, reflecting partially small airway inflammation, was higher in females than males. These data suggest that the small airways are affected differently in males and females with asthma. Males exhibit small airway involvement by attenuated small airway patency and females by small airway inflammation.

[Pedometers for monitoring and improvement of the level of physical activity]. / Stappentellers voor het monitoren en bevorderen van het lichamelijke activiteitenniveau.

Pedometers are cheap and easy to handle devices that have proven their accuracy to measure the number of steps taken during a time period (day or week). However, at lower walking speeds and if used by obese persons the accuracy of several pedometers decreases significantly. Pedometers are not able to record the intensity of daily physical activity. In addition, the number of steps depends on age, gender, body weight and season. Nevertheless, monitoring of daily physical activity with a pedometer may be an effective method to enhance the daily physical activity of selected subgroups. For instance, subjects with an increased risk of obesity or hypertension, sedentary patients with diabetes mellitus type 2, patients with coronary heart disease and cancer patients can more easily be stimulated to become physically active and meet healthy living standards. Pedometers combined with existing effective counseling methods may be an effective strategy for many health care providers for adoption and enhancement of healthy lifestyles by sedentary patients.

Ciclesonide improves measures of small airway involvement in asthma.

Ciclesonide is delivered as a small-particle inhaled corticosteroid and improves lung function and airway hyperresponsiveness. The objective of the present study was to assess whether ciclesonide can specifically improve small airway function in asthma. A total of 16 mild-to-moderate asthma patients (seven males; median (range) age 39 (19-56) yrs and forced expiratory volume in one second (FEV(1)) 89 (62-120)% predicted) were randomised to 5 weeks' treatment with placebo or 320 mug ciclesonide once daily. The following small airway parameters were assessed: mean forced expiratory flow between 25 and 75% of forced vital capacity (FVC), percentage fall in FVC at provocative dose of adenosine-5'-monophosphate and of methacholine (MCh) causing a 20% fall in FEV(1), expiratory lung volume on computed tomography (CT) scan after MCh challenge, single-breath nitrogen closing volume and alveolar exhaled nitric oxide (eNO). Seven subjects received placebo and nine received ciclesonide. Both alveolar eNO and CT measurements of expiratory lung volume after MCh challenge decreased significantly with ciclesonide (median (range) decrease 4.4 (54.8-1.4) ppb and 59 (1,569- -117) mL, respectively), and compared with placebo (-0.4 (7.3- -3.4) ppb and -121 (20- -236) mL respectively). Ciclesonide did not significantly improve other small airways parameters. Inflammation and patency of small airways, reflected by alveolar exhaled nitric oxide and air trapping on computed tomography scan, both improve with ciclesonide even in this small number of patients. This indicates that ciclesonide exerts anti-inflammatory effects on small airways.

Chronic bronchitis sub-phenotype within COPD: inflammation in sputum and biopsies.

The presence of chronic bronchitis predicts a more rapid decline of forced expiratory volume in one second (FEV(1)) in patients with chronic obstructive pulmonary disease (COPD). The hallmark of COPD is airway inflammation. It was hypothesised that COPD patients with chronic bronchitis are characterised by a distinct inflammatory cell profile, as measured in bronchial biopsies and sputum. From 114 COPD patients (male/female ratio 99/15, mean+/-sd age 62+/-8 yrs, current smoking 63%, post-bronchodilator FEV(1) 63+/-9% predicted, no steroids), with and without chronic bronchitis, inflammatory cell counts in bronchial biopsies and induced sputum were measured. Analysis was carried out by logistic regression. COPD patients with chronic bronchitis had lower eosinophil counts in biopsies and higher percentages of sputum eosinophils than patients without those symptoms, which remained after adjustment for smoking and sex. Patients with chronic bronchitis also showed higher percentages of macrophages and lower percentages of neutrophils in sputum, which could be explained by differences in smoking and sex. It was concluded that chronic bronchitis reflects an inflammatory sub-phenotype among patients with chronic obstructive pulmonary disease. The present results indicate a preferential distribution of eosinophils towards the airway lumen in patients with chronic bronchitis. This may have implications for anti-inflammatory treatment of chronic obstructive pulmonary disease patients with chronic bronchitis.