Maaike de Vries

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.

Long-term Air Pollution Exposure, Genome-wide DNA Methylation and Lung Function in the LifeLines Cohort Study

Background: Long-term air pollution exposure is negatively associated with lung function, yet the mechanisms underlying this association are not fully clear. Differential DNA methylation may explain this association. Objectives: Our main aim was to study the association between long-term air pollution exposure and DNA methylation. Methods: We performed a genome-wide methylation study using robust linear regression models in 1,017 subjects from the LifeLines cohort study to analyze the association between exposure to nitrogen dioxide (NO2) and particulate matter (PM2.5, fine particulate matter with aerodynamic diameter ≤2.5μm; PM10, particulate matter with aerodynamic diameter ≤10μm) and PM2.5absorbance, indicator of elemental carbon content (estimated with land-use-regression models) with DNA methylation in whole blood (Illumina® HumanMethylation450K BeadChip). Replication of the top hits was attempted in two independent samples from the population-based Cooperative Health Research in the Region of Augsburg studies (KORA). Results: Depending on the p-value threshold used, we found significant associations between NO2 exposure and DNA methylation for seven CpG sites (Bonferroni corrected threshold p<1.19×10−7) or for 4,980 CpG sites (False Discovery Rate<0.05). The top associated CpG site was annotated to the PSMB9 gene (i.e., cg04908668). None of the seven Bonferroni significant CpG-sites were significantly replicated in the two KORA-cohorts. No associations were found for PM exposure. Conclusions: Long-term NO2 exposure was genome-wide significantly associated with DNA methylation in the identification cohort but not in the replication cohort. Future studies are needed to further elucidate the potential mechanisms underlying NO2-exposure–related respiratory disease. https://doi.org/10.1289/EHP2045

Occupational exposure to pesticides is associated with differential DNA methylation

Objectives Occupational pesticide exposure is associated with a wide range of diseases, including lung diseases, but it is largely unknown how pesticides influence airway disease pathogenesis. A potential mechanism might be through epigenetic mechanisms, like DNA methylation. Therefore, we assessed associations between occupational exposure to pesticides and genome-wide DNA methylation sites. Methods 1561 subjects of LifeLines were included with either no (n=1392), low (n=108) or high (n=61) exposure to any type of pesticides (estimated based on current or last held job). Blood DNA methylation levels were measured using Illumina 450K arrays. Associations between pesticide exposure and 420 938 methylation sites (CpGs) were assessed using robust linear regression adjusted for appropriate confounders. In addition, we performed genome-wide stratified and interaction analyses by gender, smoking and airway obstruction status, and assessed associations between gene expression and methylation for genome-wide significant CpGs (n=2802). Results In total for all analyses, high pesticide exposure was genome-wide significantly (false discovery rate P<0.05) associated with differential DNA methylation of 31 CpGs annotated to 29 genes. Twenty of these CpGs were found in subjects with airway obstruction. Several of the identified genes, for example, RYR1, ALLC, PTPRN2, LRRC3B, PAX2 and VTRNA2-1, are genes previously linked to either pesticide exposure or lung-related diseases. Seven out of 31 CpGs were associated with gene expression levels. Conclusions We show for the first time that occupational exposure to pesticides is genome-wide associated with differential DNA methylation. Further research should reveal whether this differential methylation plays a role in the airway disease pathogenesis induced by pesticides.

Inhibition of Pim1 kinase reduces viral replication in primary bronchial epithelial cells

Respiratory viral infections are responsible for 85% of asthma exacerbations, which in turn lead to increased morbidity and mortality, causing a high societal and economic burden [1, 2]. Asthma patients have been found to be more susceptible to viral infections compared with non-asthmatic individuals [3]. It has been postulated that a reduced induction of apoptosis in virally infected airway epithelial cells from asthmatic patients might play a role in this increased susceptibility to viral infections [4]. Building on the role of apoptosis in respiratory viral infections, we tested whether the inhibition of a well-known survival pathway active in airway epithelial cells could reduce viral replication. To this end, we used an in vitro approach and infected primary bronchial epithelial cells (PBECs) from healthy volunteers with human rhinovirus (HRV)-16 in the absence or presence of a highly specific pharmacological inhibitor for Pim1 kinase [5]. Pim1 kinase is a constitutively active serine/threonine kinase known to be involved in cell survival by increasing the threshold for apoptosis [6, 7]. We have shown previously that Pim1 kinase is highly expressed in the bronchial airway epithelium and that pharmacological inhibition of Pim1 kinase increases the sensitivity of bronchial epithelial cells to cell death upon challenge with cigarette smoke extract [8]. Therefore, we hypothesised that inhibition of Pim1 kinase activity in virally infected PBECs would enhance the onset of cell death, resulting in reduced viral replication. Inhibition of Pim1 kinase activity reduces viral replication by inducing the onset of bronchial epithelial cell death http://ow.ly/IJWGD

Traumatic Perforation of the Lamina Cribrosa During Nasal Intubation of a Preterm Infant

Traumatic perforation of the lamina cribrosa and penetration of the brain occurred during nasotracheal intubation of a preterm infant requiring resuscitation. This rare complication is specifically associated with the nasal route of intubation. The complication resulted in significant morbidity. The infant developed an extensive intracranial hemorrhage and posthemorrhagic hydrocephalus that required ventricular drainage. We recommend that nasotracheal intubation be performed with utmost care. We confirm Cameron and Lupton’s recommendation of using a small feeding tube over which to slide the endotracheal tube. Despite extensive iatrogenic damage, the patient’s neurodevelopmental follow-up at 2 years 9 months appeared relatively mild.

Lung tissue gene-expression signature for the ageing lung in COPD

Introduction COPD is a chronic, progressive, inflammatory disease of the lungs and the third leading cause of death worldwide. The current knowledge of the pathophysiology of COPD is limited and novel insights in underlying disease mechanisms are urgently needed. Since there are clear parallels between ageing and COPD, we investigated genes underlying lung ageing in general and abnormal lung ageing in COPD. Methods Whole genome mRNA profiling was performed on lung tissue samples (n=1197) and differential gene expression with increasing age was analysed using an adjusted linear regression model. Subsequent pathway analysis was performed using GeneNetwork and the gene-expression signature was compared with lung ageing in the Genotype-Tissue Expression (GTEx) project. In a subset of patients with COPD (n=311) and non-COPD controls (n=270), we performed an interaction analysis between age and COPD to identify genes differentially expressed with age in COPD compared with controls, followed by gene set enrichment pathway analysis. Results We identified a strong gene-expression signature for lung ageing with 3509 differentially expressed genes, of which 33.5% were found nominal significant in the GTEx project. Interestingly, we found EDA2R as a strong candidate gene for lung ageing. The age*COPD interaction analysis revealed 69 genes significantly differentially expressed with age between COPD and controls. Conclusions Our study indicates that processes related to lung development, cell-cell contacts, calcium signalling and immune responses are involved in lung ageing in general. Pathways related to extracellular matrix, mammalian target of rapamycin signalling, splicing of introns and exons and the ribosome complex are proposed to be involved in abnormal lung ageing in COPD.

Lung ageing and COPD: is there a role for ageing in abnormal tissue repair?

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide, with increasing prevalence, in particular in the elderly. COPD is characterised by abnormal tissue repair resulting in (small) airways disease and emphysema. There is accumulating evidence that ageing hallmarks are prominent features of COPD. These ageing hallmarks have been described in different subsets of COPD patients, in different lung compartments and also in a variety of cell types, and thus might contribute to different COPD phenotypes. A better understanding of the main differences and similarities between normal lung ageing and the pathology of COPD may improve our understanding of the mechanisms driving COPD pathology, in particular in those patients that develop the most severe form of COPD at a relatively young age, i.e. severe early-onset COPD patients. In this review, after introducing the main concepts of lung ageing and COPD pathology, we focus on the role of (abnormal) ageing in lung remodelling and repair in COPD. We discuss the current evidence for the involvement of ageing hallmarks in these pathological features of COPD. We also highlight potential novel treatment strategies and opportunities for future research based on our current knowledge of abnormal lung ageing in COPD. Several ageing hallmarks are present in COPD and indicate a role for (abnormal) ageing in tissue repair in COPD http://ow.ly/OLtW30gE3ct

Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair.

No association between DNA methylation and COPD in never and current smokers

Introduction Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory lung disease with cigarette smoke as the main risk factor for its development. Since not every smoker develops COPD, other factors likely underlie differences in susceptibility to develop COPD. Here, we tested if DNA methylation may be such a factor by assessing the association between DNA methylation levels and COPD in never and current smokers from the general population. Methods For the current study, 1561 subjects were non-randomly selected from the LifeLines cohort study. We included 903 never smokers and 658 current smokers with and without COPD, defined as pre-bronchodilator forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC) <70%. Subsequently, we performed robust regression analysis on whole blood DNA methylation levels of 420 938 CpG sites with COPD as outcome. Results None of the CpG sites in both the never and the current smokers were genome-wide significantly associated with COPD. CpG site cg14972228 annotated to SIPAL3 was most significant (p=5.66×10−6) in the never smokers, while CpG site cg08282037 annotated to EPS8L1 was most significant (p=1.45×10−5) in the current smokers. Conclusion In contrast to a previous, smaller study, we did not observe any significant association between DNA methylation levels and the presence of COPD, independent of smoking status. Apparently, DNA methylation studies are highly variable.

DNA methylation mediates the association between occupational exposures and lung function

Introduction: Occupational exposures, such as biological dust, mineral dust and gases/fumes, are associated with lower lung function levels and attribute to 15-20% of all Chronic Obstructive Pulmonary Disease (COPD) cases. Epigenetic mechanisms such as DNA methylation have been suggested to play a role in these associations. Aim: To assess if the association between occupational exposures and lung function (FEV1/FVC) is mediated by DNA methylation. Methods: We included 1,561 subjects of the LifeLines cohort with either no, low, or high occupational exposure to biological dust, mineral dust and gases/fumes based on the current or last held job. Associations between the three exposures and 420,938 blood DNA methylation sites (CpGs, Illumina 450K array) were assessed using robust linear regression adjusted for appropriate confounders. Differentially methylated regions (DMRs) were identified using comb-p in python. Mediation of the top site per region was assessed using bootstrapping in R. Results: Using p<10−5 4, 5 and 6 single CpGs were associated with biological dust, mineral dust and gases/fumes, respectively, but none were genome-wide significant. In total 7, 8, and 30 genome-wide significant DMRs were identified, respectively. The CpG cg06462684 in the promoter of YWHAH, who binds to the glucocorticoid receptor, significantly mediated the association between FEV1/FVC and mineral dust. In addition, cg14870271 in the promoter of LGALS3BP, known to play a role in immune responses, mediated the association between FEV1/FVC and gases/fumes. Conclusion: We show that the association between lung function levels and occupational exposure to mineral dust or gases/fumes is mediated by DNA methylation.