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BACKGROUND: In COPD, the balance between matrix metalloproteinases (MMPs) and their natural inhibitors [tissue inhibitors of metalloproteinases (TIMPs)] is shifted towards excessive degradation, reflected in bronchoalveolar lavage (BAL) as increased MMP concentrations. Because of their critical role in lung homeostasis, MMP activity is tightly regulated, but to what extent this regulation occurs through epigenetic mechanisms remains unknown.

METHODS: To explore the interplay between MMPs, TIMPs, and DNA methylation (DNAm) we (1) analysed MMP-9, -12, and TIMP-1 concentrations in BAL fluid, and profiled DNAm in BAL cells from 18 COPD and 30 control subjects, (2) estimated protein-COPD relationships using multivariable regression, (3) identified protein quantitative trait methylation loci (pQTMs) with COPD as a potential modifier in a separate interaction model, and (4) integrated significant interactions with a previous COPD GWAS meta-analysis.

RESULTS: COPD was associated with higher levels of BAL MMP-12 (p = 0.016) but not with MMP-9 or TIMP-1. Further examination of MMP-12 identified association with DNAm at 34 loci (pQTMs), with TGFBR2 (p = 2.25 × 10^-10) and THBS4 (p = 1.11 × 10^-9) among the top ten pQTM genes. The interaction model identified 66 sites where the DNAm-MMP-12 association was significantly different in COPD compared to controls. Of these, one was colocalized with SNPs previously associated with COPD.

CONCLUSIONS: Our findings indicate that airway MMP-12 may partially be regulated by epigenetic mechanisms and that this regulation is disrupted in COPD. Furthermore, integration with COPD GWAS data suggests that this dysregulation is influenced by a combination of environmental factors, disease processes, and genetics, with the latter potentially playing a lesser role.

Background: Exposure to standard petrodiesel exhaust is linked to adverse health effects. Moreover, there is a mounting request to replace fossil-based fuels with renewable and sustainable alternatives and, therefore, rapeseed methyl ester (RME) and other biofuels have been introduced. However, recent toxicological research has indicated that biodiesel exhaust may also induce adverse health-related events.

Aim: To determine whether exposure to 100% RME biodiesel (BD100) exhaust would cause an acute airway neutrophilic recruitment in humans.

Methods: Fourteen healthy subjects underwent exposure to diluted BD100 exhaust and filtered air for 1-h, in a blinded, random fashion. Bronchoscopy with endobronchial mucosal biopsies, bronchial wash (BW) and bronchoalveolar lavage (BAL) was performed six hours after exposure. Differential cell counts and inflammatory markers were determined in the supernatant and biopsies were stained immunohistochemically.

Results: Compared with filtered air, BD100 exhaust exposure increased bronchial mucosal endothelial P-selectin adhesion molecule expression, as well as neutrophil, mast cell and CD68 + macrophage numbers. An increased influx of neutrophils and machrophages was also seen in BW.

Conclusion: Exposure to biodiesel exhaust was associated with an acute airway inflammation that appeared similar to preceding petrodiesel exposure studies. The present findings, together with the recently reported adverse cardiovascular effects after similar biodiesel exposure, indicate that biodiesel is not free of toxicity and may affect human health.

Background: Diesel exhaust (DE) induces neutrophilia and lymphocytosis in experimentally exposed humans. These responses occur in parallel to nuclear migration of NF-κB and c-Jun, activation of mitogen activated protein kinases and increased production of inflammatory mediators. There remains uncertainty regarding the impact of DE on endogenous antioxidant and xenobiotic defences, mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) respectively, and the extent to which cellular antioxidant adaptations protect against the adverse effects of DE.

Methods: Using immunohistochemistry we investigated the nuclear localization of Nrf2 and AhR in the epithelium of endobronchial mucosal biopsies from healthy subjects six-hours post exposure to DE (PM₁₀, 300 µg/m³) versus post-filtered air in a randomized double blind study, as a marker of activation. Cytoplasmic expression of cytochrome P450s, family 1, subfamily A, polypeptide 1 (CYP1A1) and subfamily B, Polypeptide 1 (CYP1B1) were examined to confirm AhR activation; with the expression of aldo–keto reductases (AKR1A1, AKR1C1 and AKR1C3), epoxide hydrolase and NAD(P)H dehydrogenase quinone 1 (NQO1) also quantified. Inflammatory and oxidative stress markers were examined to contextualize the responses observed.

Results: DE exposure caused an influx of neutrophils to the bronchial airway surface (p = 0.013), as well as increased bronchial submucosal neutrophil (p < 0.001), lymphocyte (p = 0.007) and mast cell (p = 0.002) numbers. In addition, DE exposure enhanced the nuclear translocation of the AhR and increased the CYP1A1 expression in the bronchial epithelium (p = 0.001 and p = 0.028, respectively). Nuclear translocation of AhR was also increased in the submucosal leukocytes (p < 0.001). Epithelial nuclear AhR expression was negatively associated with bronchial submucosal CD3 numbers post DE (r = −0.706, p = 0.002). In contrast, DE did not increase nuclear translocation of Nrf2 and was associated with decreased NQO1 in bronchial epithelial cells (p = 0.02), without affecting CYP1B1, aldo–keto reductases, or epoxide hydrolase protein expression.

Conclusion: These in vivo human data confirm earlier cell and animal-based observations of the induction of the AhR and CYP1A1 by diesel exhaust. The induction of phase I xenobiotic response occurred in the absence of the induction of antioxidant or phase II xenobiotic defences at the investigated time point 6 h post-exposures. This suggests DE-associated compounds, such as polycyclic aromatic hydrocarbons (PAHs), may induce acute inflammation and alter detoxification enzymes without concomitant protective cellular adaptations in human airways.

Background: Exposure to wood smoke has been shown to contribute to adverse respiratory health effects including airway infections, but the underlying mechanisms are unclear. A preceding study failed to confirm any acute inflammation or cell influx in bronchial wash (BW) or bronchoalveolar lavage (BAL) 24 h after wood smoke exposure but showed unexpected reductions in leukocyte numbers. The present study was performed to investigate responses at an earlier phase, regarding potential development of acute inflammation, as well as indications of cytotoxicity.

Methods: In a double-blind, randomised crossover study, 14 healthy participants were exposed for 2 h to filtered air and diluted wood smoke from incomplete wood log combustion in a common wood stove with a mean particulate matter concentration of 409 µg/m3. Bronchoscopy with BW and BAL was performed 6 h after exposure. Differential cell counts, assessment of DNA-damage and ex vivo analysis of phagocytic function of phagocytosing BAL cells were performed. Wood smoke particles were also collected for in vitro toxicological analyses using bronchial epithelial cells (BEAS-2B) and alveolar type II-like cells (A549).

Results: Exposure to wood smoke increased BAL lactate dehydrogenase (LDH) (p = 0.04) and reduced the ex vivo alveolar macrophage phagocytic capacity (p = 0.03) and viability (p = 0.02) vs. filtered air. BAL eosinophil numbers were increased after wood smoke (p = 0.02), while other cell types were unaffected in BW and BAL. In vitro exposure to wood smoke particles confirmed increased DNA-damage, decreased metabolic activity and cell cycle disturbances.

Conclusions: Exposure to wood smoke from incomplete combustion did not induce any acute airway inflammatory cell influx at 6 h, apart from eosinophils. However, there were indications of a cytotoxic reaction with increased LDH, reduced cell viability and impaired alveolar macrophage phagocytic capacity. These findings are in accordance with earlier bronchoscopy findings at 24 h and may provide evidence for the increased susceptibility to infections by biomass smoke exposure, reported in population-based studies.

DNA methylation patterns in chronic pulmonary obstructive disease (COPD) might offer new insights into disease pathogenesis. To assess methylation profiles in the main COPD target organ, we performed an epigenome-wide association study on BAL cells. Bronchoscopies were performed in 18 subjects with COPD and 15 control subjects (ex- and current smokers). DNA methylation was measured using the Illumina MethylationEPIC BeadChip Kit, covering more than 850,000 CpGs. Differentially methylated positions (DMPs) were examined for 1) enrichment in pathways and functional gene relationships using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology, 2) accelerated aging using Horvath's epigenetic clock, 3) correlation with gene expression, and 4) colocalization with genetic variation. We found 1,155 Bonferroni-significant (P < 6.74 × 10-8) DMPs associated with COPD, many with large effect sizes. Functional analysis identified biologically plausible pathways and gene relationships, including enrichment for transcription factor activity. Strong correlation was found between DNA methylation and chronological age but not between COPD and accelerated aging. For 79 unique DMPs, DNA methylation correlated significantly with gene expression in BAL cells. Thirty-nine percent of DMPs were colocalized with COPD-associated SNPs. To the best of our knowledge, this is the first epigenome-wide association study of COPD on BAL cells, and our analyses revealed many differential methylation sites. Integration with mRNA data showed a strong functional readout for relevant genes, identifying sites where DNA methylation might directly affect expression. Almost half of DMPs were colocated with SNPs identified in previous genome-wide association studies of COPD, suggesting joint genetic and epigenetic pathways related to disease.

Background: Snus usage is commonly touted as a safer alternative to cigarette smoking. However, recent studies have demonstrated possible adverse cardiovascular effects in chronic snus users. The present study evaluates the effects of chronic snus use on vascular function by assessing central arterial stiffness and endothelial vasodilatory function in healthy chronic snus users as compared to matched non-users.

Methods and results: Fifty healthy males (24 snus users, 26 age-matched controls) with a mean age of 44 years were included in the study. Arterial stiffness was assessed employing both pulse wave velocity and pulse wave analysis. Endothelial vasodilatory function was measured by venous occlusion plethysmography, utilizing intra-arterial administration of acetylcholine, glyceryl trinitrate and bradykinin to further gauge endothelium-dependent and -independent vasodilatory function. Arterial stiffness was significantly higher in chronic snus users as compared to controls: pulse wave velocity [m/s]: 6.6±0.8 vs 7.1±0.9 resp. (p = 0.026), augmentation index corrected for heart rate [%]: 0.1±13.2 vs 7.3±7.8 resp. (p = 0.023). Endothelial independent vasodilation, i.e. the reaction to glyceryl trinitrate, was significantly lower in snus users as measured by venous occlusion plethysmography.

Conclusions: The results of this study show an increased arterial stiffness and an underlying endothelial dysfunction in daily snus users as compared to matched non-tobacco controls. These findings indicate that long-term use of snus may alter the function of the endothelium and therefore reinforces the assertion that chronic snus use is correlated to an increased risk of development of cardiovascular disease.

Inflammatory signaling pathways involving eicosanoids and other regulatory lipid mediators are a subject of intensive study, and a role for these in acute lung injury is not yet well understood. We hypothesized that oxylipin release from lung injury could be detected in bronchoalveolar lavage fluid and in plasma. In a porcine model of surfactant depletion, ventilation with hyperinflation was assessed. Bronchoalveolar lavage and plasma samples were analyzed for 37 different fatty acid metabolites (oxylipins). Over time, hyperinflation altered concentrations of 4 oxylipins in plasma (TXB₂, PGE₂, 15-HETE and 11-HETE), and 9 oxylipins in bronchoalveolar lavage fluid (PGF_2α, PGE₂, PGD₂, 12,13-DiHOME, 11,12-DiHETrE, 13-HODE, 9-HODE, 15-HETE, 11-HETE). Acute lung injury caused by high tidal volume ventilation in this porcine model was associated with rapid changes in some elements of the oxylipin profile, detectable in lavage fluid, and plasma. These oxylipins may be relevant in the pathogenesis of acute lung injury by hyperinflation.

Background: Pulmonary sarcoidosis is an inflammatory disease characterised by granuloma formation and heterogeneous clinical outcome. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine contributing to granuloma formation and high levels of TNF have been shown to associate with progressive disease. Mononuclear phagocytes (MNPs) are potent producers of TNF and highly responsive to inflammation. In sarcoidosis, alveolar macrophages have been well studied. However, MNPs also include monocytes/monocyte-derived cells and dendritic cells, which are poorly studied in sarcoidosis, despite their central role in inflammation.

Objective: To determine the role of pulmonary monocyte-derived cells and dendritic cells during sarcoidosis.

Methods: We performed in-depth phenotypic, functional and transcriptomic analysis of MNP subsets from blood and bronchoalveolar lavage (BAL) fluid from 108 sarcoidosis patients and 30 healthy controls. We followed the clinical development of patients and assessed how the repertoire and function of MNP subsets at diagnosis correlated with 2-year disease outcome.

Results: Monocytes/monocyte-derived cells were increased in blood and BAL of sarcoidosis patients compared to healthy controls. Interestingly, high frequencies of blood intermediate monocytes at time of diagnosis associated with chronic disease development. RNA sequencing analysis showed highly inflammatory MNPs in BAL of sarcoidosis patients. Furthermore, frequencies of BAL monocytes/monocyte-derived cells producing TNF without exogenous stimulation at time of diagnosis increased in patients that were followed longitudinally. In contrast to alveolar macrophages, the frequency of TNF-producing BAL monocytes/monocyte-derived cells at time of diagnosis was highest in sarcoidosis patients that developed progressive disease.

Conclusion: Our data show that pulmonary monocytes/monocyte-derived cells are highly inflammatory and can be used as a predictor of disease outcome in sarcoidosis patients.

Background: Differences in the expression of regulatory T cells (Tregs) have been suggested to explain why some smokers develop COPD and some do not. Upregulation of Tregs in response to smoking would restrain airway inflammation and thus the development of COPD; while the absense of such upregulation would over time lead to chronic inflammation and COPD. We hypothesized that—among COPD patients—the same mechanism would affect rate of decline in lung function; specifically, that a decreased expression of Tregs would be associated with a more rapid decline in FEV1.

Methods: Bronchoscopy with BAL was performed in 52 subjects recruited from the longitudinal OLIN COPD study; 12 with COPD and a rapid decline in lung function (loss of FEV1 ≥ 60 ml/year), 10 with COPD and a non-rapid decline in lung function (loss of FEV1 ≤ 30 ml/year), 15 current and ex-smokers and 15 non-smokers with normal lung function. BAL lymphocyte subsets were determined using flow cytometry.

Results: The proportions of Tregs with regulatory function (FoxP3+/CD4+CD25bright) were significantly lower in COPD subjects with a rapid decline in lung function compared to those with a non-rapid decline (p = 0.019). This result was confirmed in a mixed model regression analysis in which adjustments for inhaled corticosteroid usage, smoking, sex and age were evaluated. No significant difference was found between COPD subjects and smokers or non-smokers with normal lung function.

Conclusions: COPD subjects with a rapid decline in lung function had lower proportions of T cells with regulatory function in BAL fluid, suggesting that an inability to suppress the inflammatory response following smoking might lead to a more rapid decline in FEV1.