Identification of asthma-associated microRNAs in bronchial biopsies.

BACKGROUND: Changes in microRNA (miRNA) expression can contribute to the pathogenesis of many diseases, including asthma. We aimed to identify miRNAs that are differentially expressed between asthma patients and healthy controls, and explore their association with clinical and inflammatory parameters of asthma. METHODS: Differentially expressed miRNAs were determined by small RNA sequencing on bronchial biopsies of 79 asthma patients and 82 healthy controls using linear regression models. Differentially expressed miRNAs were associated with clinical and inflammatory asthma features. Potential miRNA-mRNA interactions were analysed using mRNA data available from the same bronchial biopsies, and enrichment of pathways was identified with Enrichr and g:Profiler. RESULTS: In total, 78 differentially expressed miRNAs were identified in bronchial biopsies of asthma patients compared with controls, of which 60 remained differentially expressed after controlling for smoking and inhaled corticosteroid treatment. We identified several asthma-associated miRNAs, including miR-125b-5p and miR-223-3p, based on a significant association with multiple clinical and inflammatory asthma features and their negative correlation with genes associated with the presence of asthma. The most enriched biological pathway(s) affected by miR-125b-5p and miR-223-3p were inflammatory response and cilium assembly/organisation. Of interest, we identified that lower expression of miR-26a-5p was linked to more severe eosinophilic inflammation as measured in blood, sputum as well as bronchial biopsies. CONCLUSION: Collectively, we identified miR-125b-5p, miR-223-3p and miR-26a-5p as potential regulators that could contribute to the pathogenesis of asthma.

Respiratory Syncytial Virus Infection Promotes Necroptosis and HMGB1 Release by Airway Epithelial Cells.

Rationale: Respiratory syncytial virus (RSV) bronchiolitis causes significant infant mortality. Bronchiolitis is characterized by airway epithelial cell (AEC) death; however, the mode of death remains unknown.Objectives: To determine whether necroptosis contributes to RSV bronchiolitis pathogenesis via HMGB1 (high mobility group box 1) release.Methods: Nasopharyngeal samples were collected from children presenting to the hospital with acute respiratory infection. Primary human AECs and neonatal mice were inoculated with RSV and murine Pneumovirus, respectively. Necroptosis was determined via viability assays and immunohistochemistry for RIPK1 (receptor-interacting protein kinase-1), MLKL (mixed lineage kinase domain-like pseudokinase) protein, and caspase-3. Necroptosis was blocked using pharmacological inhibitors and RIPK1 kinase-dead knockin mice.Measurements and Main Results: HMGB1 levels were elevated in nasopharyngeal samples of children with acute RSV infection. RSV-induced epithelial cell death was associated with increased phosphorylated RIPK1 and phosphorylated MLKL but not active caspase-3 expression. Inhibition of RIPK1 or MLKL attenuated RSV-induced HMGB1 translocation and release, and lowered viral load. MLKL inhibition increased active caspase-3 expression in a caspase-8/9-dependent manner. In susceptible mice, Pneumovirus infection upregulated RIPK1 and MLKL expression in the airway epithelium at 8 to 10 days after infection, coinciding with AEC sloughing, HMGB1 release, and neutrophilic inflammation. Genetic or pharmacological inhibition of RIPK1 or MLKL attenuated these pathologies, lowered viral load, and prevented type 2 inflammation and airway remodeling. Necroptosis inhibition in early life ameliorated asthma progression induced by viral or allergen challenge in later life.Conclusions: Pneumovirus infection induces AEC necroptosis. Inhibition of necroptosis may be a viable strategy to limit the severity of viral bronchiolitis and break its nexus with asthma.

Differential DNA methylation in bronchial biopsies between persistent asthma and asthma in remission.

Approximately 40% of asthmatics experience remission of asthma symptoms. A better understanding of biological pathways leading to asthma remission may provide insight into new therapeutic targets for asthma. As an important mechanism of gene regulation, investigation of DNA methylation provides a promising approach. Our objective was to identify differences in epigenome wide DNA methylation levels in bronchial biopsies between subjects with asthma remission and subjects with persistent asthma or healthy controls.We analysed differential DNA methylation in bronchial biopsies from 26 subjects with persistent asthma, 39 remission subjects and 70 healthy controls, using the limma package. The comb-p tool was used to identify differentially methylated regions. DNA methylation of CpG-sites was associated to expression of nearby genes from the same biopsies to understand function.Four CpG-sites and 42 regions were differentially methylated between persistent asthma and remission. DNA methylation at two sites was correlated i n cis with gene expression at ACKR2 and DGKQ Between remission subjects and healthy controls 1163 CpG-sites and 328 regions were differentially methylated. DNA methylation was associated with expression of a set of genes expressed in ciliated epithelium.CpGs differentially methylated between remission and persistent asthma identify genetic loci associated with resolution of inflammation and airway responsiveness. Despite the absence of symptoms, remission subjects have a DNA methylation profile that is distinct from that of healthy controls, partly due to changes in cellular composition, with a higher gene expression signal related to ciliated epithelium in remission versus healthy controls.

Genetic variance is associated with susceptibility for cigarette smoke-induced DAMP release in mice.

Chronic obstructive pulmonary disease (COPD) is characterized by unresolved neutrophilic airway inflammation and is caused by chronic exposure to toxic gases, such as cigarette smoke (CS), in genetically susceptible individuals. Recent data indicate a role for damage-associated molecular patterns (DAMPs) in COPD. Here, we investigated the genetics of CS-induced DAMP release in 28 inbred mouse strains. Subsequently, in lung tissue from a subset of strains, the expression of the identified candidate genes was analyzed. We tested whether small interfering RNA-dependent knockdown of candidate genes altered the susceptibility of the human A549 cell line to CS-induced cell death and DAMP release. Furthermore, we tested whether these genes were differentially regulated by CS exposure in bronchial brushings obtained from individuals with a family history indicative of either the presence or absence of susceptibility for COPD. We observed that, of the four DAMPs tested, double-stranded DNA (dsDNA) showed the highest correlation with neutrophilic airway inflammation. Genetic analyses identified 11 candidate genes governing either CS-induced or basal dsDNA release in mice. Two candidate genes (Elac2 and Ppt1) showed differential expression in lung tissue on CS exposure between susceptible and nonsusceptible mouse strains. Knockdown of ELAC2 and PPT1 in A549 cells altered susceptibility to CS extract-induced cell death and DAMP release. In bronchial brushings, CS-induced expression of ENOX1 and ARGHGEF11 was significantly different between individuals susceptible or nonsusceptible for COPD. Our study shows that genetic variance in a mouse model is associated with CS-induced DAMP release, and that this might contribute to susceptibility for COPD.

Cigarette smoke-induced necroptosis and DAMP release trigger neutrophilic airway inflammation in mice.

Recent data indicate a role for airway epithelial necroptosis, a regulated form of necrosis, and the associated release of damage-associated molecular patterns (DAMPs) in the development of chronic obstructive pulmonary disease (COPD). DAMPs can activate pattern recognition receptors (PRRs), triggering innate immune responses. We hypothesized that cigarette smoke (CS)-induced epithelial necroptosis and DAMP release initiate airway inflammation in COPD. Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE), and necrotic cell death (membrane integrity by propidium iodide staining) and DAMP release (i.e., double-stranded DNA, high-mobility group box 1, heat shock protein 70, mitochondrial DNA, ATP) were analyzed. Subsequently, BEAS-2B cells were exposed to DAMP-containing supernatant of CS-induced necrotic cells, and the release of proinflammatory mediators [C-X-C motif ligand 8 (CXCL-8), IL-6] was evaluated. Furthermore, mice were exposed to CS in the presence and absence of the necroptosis inhibitor necrostatin-1, and levels of DAMPs and inflammatory cell numbers were determined in bronchoalveolar lavage fluid. CSE induced a significant increase in the percentage of necrotic cells and DAMP release in BEAS-2B cells. Stimulation of BEAS-2B cells with supernatant of CS-induced necrotic cells induced a significant increase in the release of CXCL8 and IL-6, in a myeloid differentiation primary response gene 88-dependent fashion. In mice, exposure of CS increased the levels of DAMPs and numbers of neutrophils in bronchoalveolar lavage fluid, which was statistically reduced upon treatment with necrostatin-1. Together, we showed that CS exposure induces necrosis of bronchial epithelial cells and subsequent DAMP release in vitro, inducing the production of proinflammatory cytokines. In vivo, CS exposure induces neutrophilic airway inflammation that is sensitive to necroptosis inhibition.

Susceptibility for cigarette smoke-induced DAMP release and DAMP-induced inflammation in COPD.

Cigarette smoke (CS) exposure is a major risk factor for chronic obstructive pulmonary disease (COPD). We investigated whether CS-induced damage-associated molecular pattern (DAMP) release or DAMP-mediated inflammation contributes to susceptibility for COPD. Samples, including bronchial brushings, were collected from young and old individuals, susceptible and nonsusceptible for the development of COPD, before and after smoking, and used for gene profiling and airway epithelial cell (AEC) culture. AECs were exposed to CS extract (CSE) or specific DAMPs. BALB/cByJ and DBA/2J mice were intranasally exposed to LL-37 and mitochondrial (mt)DAMPs. Functional gene-set enrichment analysis showed that CS significantly increases the airway epithelial gene expression of DAMPs and DAMP receptors in COPD patients. In cultured AECs, we observed that CSE induces necrosis and DAMP release, with specifically higher galectin-3 release from COPD-derived compared with control-derived cells. Galectin-3, LL-37, and mtDAMPs increased CXCL8 secretion in AECs. LL-37 and mtDAMPs induced neutrophilic airway inflammation, exclusively in mice susceptible for CS-induced airway inflammation. Collectively, we show that in airway epithelium from COPD patients, the CS-induced expression of DAMPs and DAMP receptors in vivo and the release of galectin-3 in vitro is exaggerated. Furthermore, our studies indicate that a predisposition to release DAMPs and subsequent induction of inflammation may contribute to the development of COPD.

Advanced glycation endproducts and their receptor in different body compartments in COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by chronic airway inflammation and emphysema, and is caused by exposure to noxious particles or gases, e.g. cigarette smoke. Smoking and oxidative stress lead to accelerated formation and accumulation of advanced glycation end products (AGEs), causing local tissue damage either directly or by binding the receptor for AGEs (RAGE). This study assessed the association of AGEs or RAGE in plasma, sputum, bronchial biopsies and skin with COPD and lung function, and their variance between these body compartments. METHODS: Healthy smoking and never-smoking controls (n = 191) and COPD patients (n = 97, GOLD stage I-IV) were included. Autofluorescence (SAF) was measured in the skin, AGEs (pentosidine, CML and CEL) and sRAGE in blood and sputum by ELISA, and in bronchial biopsies by immunohistochemistry. eQTL analysis was performed in bronchial biopsies. RESULTS: COPD patients showed higher SAF values and lower plasma sRAGE levels compared to controls and these values associated with decreased lung function (p <0.001; adjusting for relevant covariates). Lower plasma sRAGE levels significantly and independently predicted higher SAF values (p < 0.001). One SNP (rs2071278) was identified within a region of 50 kB flanking the AGER gene, which was associated with the gene and protein expression levels of AGER and another SNP (rs2071278) which was associated with the accumulation of AGEs in the skin. CONCLUSION: In COPD, AGEs accumulate differentially in body compartments, i.e. they accumulate in the skin, but not in plasma, sputum and bronchial biopsies. The association between lower sRAGE and higher SAF levels supports the hypothesis that the protective mechanism of sRAGE as a decoy-receptor is impaired in COPD.

Cigarette smoke-induced damage-associated molecular pattern release from necrotic neutrophils triggers proinflammatory mediator release.

Cigarette smoking, the major causative factor for the development of chronic obstructive pulmonary disease, is associated with neutrophilic airway inflammation. Cigarette smoke (CS) exposure can induce a switch from apoptotic to necrotic cell death in airway epithelium. Therefore, we hypothesized that CS promotes neutrophil necrosis with subsequent release of damage-associated molecular patterns (DAMPs), including high mobility group box 1 (HMGB1), alarming the innate immune system. We studied the effect of smoking two cigarettes on sputum neutrophils in healthy individuals and of 5-day CS or air exposure on neutrophil counts, myeloperoxidase, and HMGB1 levels in bronchoalveolar lavage fluid of BALB/c mice. In human peripheral blood neutrophils, mitochondrial membrane potential, apoptosis/necrosis markers, caspase activity, and DAMP release were studied after CS exposure. Finally, we assessed the effect of neutrophil-derived supernatants on the release of chemoattractant CXCL8 in normal human bronchial epithelial cells. Cigarette smoking caused a significant decrease in sputum neutrophil numbers after 3 hours. In mice, neutrophil counts were significantly increased 16 hours after repeated CS exposure but reduced 2 hours after an additional exposure. In vitro, CS induced necrotic neutrophil cell death, as indicated by mitochondrial dysfunction, inhibition of apoptosis, and DAMP release. Supernatants from CS-treated neutrophils significantly increased the release of CXCL8 in normal human bronchial epithelial cells. Together, these observations show, for the first time, that CS exposure induces neutrophil necrosis, leading to DAMP release, which may amplify CS-induced airway inflammation by promoting airway epithelial proinflammatory responses.

Genetic variation associates with susceptibility for cigarette smoke-induced neutrophilia in mice.

Neutrophilic airway inflammation is one of the major hallmarks of chronic obstructive pulmonary disease and is also seen in steroid resistant asthma. Neutrophilic airway inflammation can be induced by different stimuli including cigarette smoke (CS). Short-term exposure to CS induces neutrophilic airway inflammation in both mice and humans. Since not all individuals develop extensive neutrophilic airway inflammation upon smoking, we hypothesized that this CS-induced innate inflammation has a genetic component. This hypothesis was addressed by exposing 30 different inbred mouse strains to CS or control air for 5 consecutive days, followed by analysis of neutrophilic lung inflammation. By genomewide haplotype association mapping, we identified four susceptibility genes with a significant association to lung tissue levels of the neutrophil marker myeloperoxidase under basal conditions and an additional five genes specifically associated with CS-induced tissue MPO levels. Analysis of the expression levels of the susceptibility genes by quantitative RT-PCR revealed that three of the four genes associated with CS-induced tissue MPO levels had CS-induced changes in gene expression levels that correlate with CS-induced airway inflammation. Most notably, CS exposure induces an increased expression of the coiled-coil domain containing gene, Ccdc93, in mouse strains susceptible for CS-induced airway inflammation whereas Ccdc93 expression was decreased upon CS exposure in nonsusceptible mouse strains. In conclusion, this study shows that CS-induced neutrophilic airway inflammation has a genetic component and that several genes contribute to the susceptibility for this response.

Glycogen synthase kinase-3ß modulation of glucocorticoid responsiveness in COPD.

In chronic obstructive pulmonary disease (COPD), oxidative stress regulates the inflammatory response of bronchial epithelium and monocytes/macrophages through kinase modulation and has been linked to glucocorticoid unresponsiveness. Glycogen synthase-3ß (GSK3ß) inactivation plays a key role in mediating signaling processes upon reactive oxygen species (ROS) exposure. We hypothesized that GSK3ß is involved in oxidative stress-induced glucocorticoid insensitivity in COPD. We studied levels of phospho-GSK3ß-Ser9, a marker of GSK3ß inactivation, in lung sections and cultured monocytes and bronchial epithelial cells of COPD patients, control smokers, and nonsmokers. We observed increased levels of phospho-GSK3ß-Ser9 in monocytes, alveolar macrophages, and bronchial epithelial cells from COPD patients and control smokers compared with nonsmokers. Pharmacological inactivation of GSK3ß did not affect CXCL8 or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression but resulted in glucocorticoid insensitivity in vitro in both inflammatory and structural cells. Further mechanistic studies in monocyte and bronchial epithelial cell lines showed that GSK3ß inactivation is a common effector of oxidative stress-induced activation of the MEK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways leading to glucocorticoid unresponsiveness. In primary monocytes, the mechanism involved modulation of histone deacetylase 2 (HDAC2) activity in response to GSK3ß inactivation. In conclusion, we demonstrate for the first time that ROS-induced glucocorticoid unresponsiveness in COPD is mediated through GSK3ß, acting as a ROS-sensitive hub.

Profiling over 1500 lipids in induced lung sputum and the implications in studying lung diseases.

Induced lung sputum is a valuable matrix in the study of respiratory diseases. Although the methodology of sputum collection has evolved to a point where it is repeatable and responsive to inflammation, its use in molecular profiling studies is still limited. Here, an in-depth lipid profiling of induced lung sputum using high-resolution liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) is described. An enormous complexity in lipid composition could be revealed. Over 1500 intact lipids, originating from 6 major lipid classes, have been accurately identified in 120 µL of induced sputum. By number and measured intensity, glycerophospholipids represent the largest lipid class, followed by sphingolipids, glycerolipids, fatty acyls, sterol lipids, and prenol lipids. Several prenol lipids, originating from tobacco, could be detected in the lung sputum of smokers. To illustrate the utility of the methodology in studying respiratory diseases, a comparative lipid screening was performed on lung sputum extracts in order to study the effect of Chronic Obstructive Pulmonary Disease (COPD) on the lung barrier lipidome. Results show that sphingolipid expression in induced sputum significantly differs between smokers with and without COPD.

Untargeted lipidomic analysis in chronic obstructive pulmonary disease. Uncovering sphingolipids.

RATIONALE: Cigarette smoke is the major risk factor in the development of chronic obstructive pulmonary disease (COPD). Lipidomics is a novel and emerging research field that may provide new insights in the origins of chronic inflammatory diseases, such as COPD. OBJECTIVES: To investigate whether expression of the sputum lipidome is affected by COPD or cigarette smoking. METHODS: Lipid expression was investigated with liquid chromatography and high-resolution quadrupole time-of-flight mass spectrometry in induced sputum comparing smokers with and without COPD, and never-smokers. Changes in lipid expression after 2-month smoking cessation were investigated in smokers with and without COPD. MEASUREMENTS AND MAIN RESULTS: More than 1,500 lipid compounds were identified in sputum. The class of sphingolipids was significantly higher expressed in smokers with COPD than in smokers without COPD. At single compound level, 168 sphingolipids, 36 phosphatidylethanolamine lipids, and 5 tobacco-related compounds were significantly higher expressed in smokers with COPD compared with smokers without COPD. The 13 lipids with a high fold change between smokers with and without COPD showed high correlations with lower lung function and inflammation in sputum. Twenty (glyco)sphingolipids and six tobacco-related compounds were higher expressed in smokers without COPD compared with never-smokers. Two-month smoking cessation reduced expression of 26 sphingolipids in smokers with and without COPD. CONCLUSIONS: Expression of lipids from the sphingolipid pathway is higher in smokers with COPD compared with smokers without COPD. Considering their potential biologic properties, they may play a role in the pathogenesis of COPD.

Glucocorticoids induce the production of the chemoattractant CCL20 in airway epithelium.

Th17-mediated neutrophilic airway inflammation has been implicated in decreased response to glucocorticoids in asthma. We aimed to investigate the effect of glucocorticoids on the airway epithelial release of the neutrophilic and Th17-cell chemoattractant CCL20. We studied CCL20 and CXCL8 sputum levels in asthmatic subjects using inhaled glucocorticoids or not, and the effect of budesonide on CCL20 and CXCL8 production in primary bronchial epithelial cells. The mechanism behind the effect of budesonide-induced CCL20 production was studied in 16HBE14o- cells using inhibitors for the glucocorticoid receptor, intracellular pathways and metalloproteases. We observed higher levels of CCL20, but not CXCL8, in the sputum of asthmatics who used inhaled glucocorticoids. CCL20 levels correlated with inhaled glucocorticoid dose and sputum neutrophils. Budesonide increased tumour necrosis factor (TNF)-α-induced CCL20 by primary bronchial epithelium, while CXCL8 was suppressed. In 16HBE14o- cells, similar effects were observed at the CCL20 protein and mRNA levels, indicating transcriptional regulation. Although TNF-α-induced CCL20 release was dependent on the ERK, p38 and STAT3 pathways, the increase by budesonide was not. Inhibition of glucocorticoid receptor or ADAM17 abrogated the budesonide-induced increase in CCL20 levels. We show that glucocorticoids enhance CCL20 production by bronchial epithelium, which may constitute a novel mechanism in Th17-mediated glucocorticoid-insensitive inflammation in asthma.

Increased activation of blood neutrophils after cigarette smoking in young individuals susceptible to COPD.

BACKGROUND: Cigarette smoking is the most important risk factor for Chronic Obstructive Pulmonary Disease (COPD). Only a subgroup of smokers develops COPD and it is unclear why these individuals are more susceptible to the detrimental effects of cigarette smoking. The risk to develop COPD is known to be higher in individuals with familial aggregation of COPD. This study aimed to investigate if acute systemic and local immune responses to cigarette smoke differentiate between individuals susceptible or non-susceptible to develop COPD, both at young (18-40 years) and old (40-75 years) age. METHODS: All participants smoked three cigarettes in one hour. Changes in inflammatory markers in peripheral blood (at 0 and 3 hours) and in bronchial biopsies (at 0 and 24 hours) were investigated. Acute effects of smoking were analyzed within and between susceptible and non-susceptible individuals, and by multiple regression analysis. RESULTS: Young susceptible individuals showed significantly higher increases in the expression of FcγRII (CD32) in its active forms (A17 and A27) on neutrophils after smoking (p = 0.016 and 0.028 respectively), independently of age, smoking status and expression of the respective markers at baseline. Smoking had no significant effect on mediators in blood or inflammatory cell counts in bronchial biopsies. In the old group, acute effects of smoking were comparable between healthy controls and COPD patients. CONCLUSIONS: We show for the first time that COPD susceptibility at young age associates with an increased systemic innate immune response to cigarette smoking. This suggests a role of systemic inflammation in the early induction phase of COPD. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00807469.

E-cadherin: gatekeeper of airway mucosa and allergic sensitization.

The airway epithelium plays a role in immune regulation during environmental challenge, which is intertwined with its barrier function and capacity to limit submucosal access of environmental factors. In asthma, mucosal barrier function is often compromised, with disrupted expression of the adhesion molecule E-cadherin. Recent progress suggests that E-cadherin contributes to the structural and immunological function of airway epithelium, through the regulation of epithelial junctions, proliferation, differentiation, and production of growth factors and proinflammatory mediators that can modulate the immune response. Here, we discuss this novel role for E-cadherin in mediating the crucial immunological decision between maintenance of tolerance versus induction of innate and adaptive immunity.

Caveolin-1 controls airway epithelial barrier function. Implications for asthma.

The molecular basis for airway epithelial fragility in asthma has remained unclear. We investigated whether the loss of caveolin-1, the major component of caveolae and a known stabilizer of adherens junctions, contributes to epithelial barrier dysfunction in asthma. We studied the expression of caveolin-1 and adhesion molecules E-cadherin and ß-catenin in airway sections, and we cultured bronchial epithelial cells from patients with asthma and from healthy control subjects. To determine the functional role of caveolin-1, we investigated the effects of caveolin-1 up-regulation and down-regulation on E-cadherin expression, barrier function, and proallergic activity in the human bronchial epithelial cell lines 16HBE and BEAS-2B. The membrane expression of caveolin-1 was significantly lower in airway epithelia from patients with asthma than from subjects without asthma, and this lower expression was maintained in vitro upon air-liquid interface and submerged culturing. Importantly, reduced caveolin-1 expression was accompanied by a loss of junctional E-cadherin and ß-catenin expression, disrupted epithelial barrier function, and increased levels of the proallergic cytokine thymic stromal lymphopoietin (TSLP). Furthermore, E-cadherin redistribution upon exposure to epidermal growth factor or house dust mite was paralleled by the internalization of caveolin-1 in 16HBE cells. These effects appear to be causally related, because the short, interfering RNA down-regulation of caveolin-1 resulted in the delocalization of E-cadherin and barrier dysfunction in 16HBE cells. Moreover, caveolin-1 overexpression improved barrier function and reduced TSLP expression in BEAS-2B cells. Together, our data demonstrate a crucial role for caveolin-1 in epithelial cell-cell adhesion, with important consequences for epithelial barrier function and the promotion of Th2 responses in asthma.

Role of aberrant WNT signalling in the airway epithelial response to cigarette smoke in chronic obstructive pulmonary disease.

BACKGROUND: WNT signalling is activated during lung tissue damage and inflammation. We investigated whether lung epithelial expression of WNT ligands, receptors (frizzled; FZD) or target genes is dysregulated on cigarette smoking and/or in chronic obstructive pulmonary disease (COPD). METHODS: We studied this in human lung epithelial cell lines and primary bronchial epithelial cells (PBEC) from COPD patients and control (non-)smokers, at baseline and on cigarette smoke extract (CSE) exposure. RESULTS: CSE significantly decreased WNT-4, WNT-10B and FZD2 and increased WNT-5B mRNA expression in 16HBE, but did not affect WNT-4 protein. The mRNA expression of WNT-4, but not other WNT ligands, was lower in PBEC from smokers than non-smokers and downregulated by CSE in PBEC from all groups, yet higher in PBEC from COPD patients than control smokers. Moreover, PBEC from COPD patients displayed higher WNT-4 protein expression than both smokers and non-smokers. Exogenously added WNT-4 significantly increased CXCL8/IL-8, IL-6, CCL5/RANTES, CCL2/MCP-1 and vascular endothelial growth factor (VEGF) secretion in 16HBE, but did not affect the canonical WNT target genes MMP-2, MMP-9, fibronectin, ß-catenin, Dickkopf and axin-2, and induced activation of the non-canonical signalling molecule p38. Moreover, WNT-4 potentiated the CSE-induced upregulation of IL-8 and VEGF. CONCLUSIONS: WNT-4 mRNA and protein levels are higher in PBEC from COPD patients than control (non-)smokers, while cigarette smoke downregulates airway epithelial WNT-4 mRNA, but not protein expression. As WNT-4 further increases CSE-induced pro-inflammatory cytokine release in bronchial epithelium, we propose that higher epithelial WNT-4 levels in combination with cigarette smoking may have important implications for the development of airway inflammation in COPD.

Critical role of aldehydes in cigarette smoke-induced acute airway inflammation.

BACKGROUND: Cigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation. METHODS: BALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs. RESULTS: Compared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 µM, WF-CS; 58.5 ± 8.2 µM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p<0.01) and lung tissue (p<0.01) were significantly increased in the CS- and WF-CS-exposed mice compared to air control mice. Interestingly, the numbers of neutrophils (p<0.001) in BALF and neutrophils and eosinophils (p<0.05) in lung tissue were significantly increased in the CS-exposed but not in WF-CS-exposed mice as compared to air control mice. Levels of the neutrophil and eosinophil chemoattractants KC, MCP-1, MIP-1α and IL-5 were all significantly increased in lung tissue from CS-exposed mice compared to both WF-CS-exposed and air control mice. Interestingly, depletion of aldehydes in WF-CS extract significantly reduced IL-8 production in Beas-2b as compared to CSE, which could be restored by the aldehyde acrolein. CONCLUSION: Aldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation.

Characterization of protocadherin-1 expression in primary bronchial epithelial cells: association with epithelial cell differentiation.

Protocadherin-1 (PCDH1) is a novel susceptibility gene for asthma that is expressed in airway epithelium. We aimed to characterize PCDH1 mRNA transcripts and protein expression in primary bronchial epithelial cells and to determine regulation of PCDH1 during mucociliary differentiation. Total RNA and protein were isolated from human primary bronchial epithelial cells. PCDH1 transcripts were characterized by rapid amplification of cDNA ends in bronchial epithelial cells of 4 subjects. PCDH1 expression was quantified by quantitative RT-PCR and Western blotting in bronchial epithelial cells directly ex vivo and after air liquid interface (ALI) or submerged culture. We identified 5 novel exons on the 5' end and 1 exon on the 3' end of PCDH1. Novel transcripts showed major variation in expression of intracellular conserved motifs. Expression levels of PCDH1 transcripts encoding exon 1-2 were 4-fold higher, and transcripts encoding exon 3-4 were 15-fold higher in freshly isolated bronchial epithelial cells than in submerged cultures. PCDH1 mRNA (3- to 8-fold) and protein levels (2- to 3-fold) were strongly up-regulated during mucociliary differentiation of primary bronchial epithelial cells in ALI cultures. In summary, PCDH1 transcripts display remarkable variability in expression of conserved intracellular signaling domains. Enhanced PCDH1 expression levels strongly correlate with differentiation of bronchial epithelial cells.

Cigarette smoke induces endoplasmic reticulum stress response and proteasomal dysfunction in human alveolar epithelial cells.

Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease. Cigarette smoke (CS) causes oxidative stress and severe damage to proteins in the lungs. One of the main systems to protect cells from the accumulation of damaged proteins is the ubiquitin-proteasome pathway. In the present study, we aimed to find out whether exposure of alveolar epithelial cells to CS induces an endoplasmic reticulum (ER) stress response by accumulation of damaged proteins that are inefficiently degraded by the proteasomes. The hypothesis was tested in a human alveolar epithelial cell line (A549) exposed to gas-phase CS. Exposure to gas-phase CS for 5 min caused an increase in the amount of ubiquitin-protein conjugates within 4 h. Cigarette smoke exposure also induced the ER stress response marker eIF2α, followed by a significant reduction of nascent protein synthesis and increase in the level of free intracellular amino acids. Moreover, CS exposure significantly reduced all three proteasomal activities (caspase-, trypsin- and chymotrypsin-like activity) within 4 h, which was still present after 24 h. It can be concluded that gas-phase CS induces ER stress in A549 alveolar epithelial cells, leading to inadequate protein turnover caused by an accumulation of damaged proteins, reduction in nascent protein synthesis and inhibition of the proteasome. We suggest that prolonged ER stress may lead to excessive cell death with disruption of the epithelial barrier, contributing to development of chronic obstructive pulmonary disease.