Bettina Levänen

Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients

BACKGROUNDnAsthma is characterized by increased airway narrowing in response to nonspecific stimuli. The disorder is influenced by both environmental and genetic factors. Exosomes are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in asthma. In this study we characterized the microRNA (miRNA) content of exosomes in healthy control subjects and patients with mild intermittent asthma both at unprovoked baseline and in response to environmental challenge.nnnOBJECTIVEnTo investigate alterations in bronchoalveolar lavage fluid (BALF) exosomal miRNA profiles due to asthma, and following subway air exposure.nnnMETHODSnExosomes were isolated from BALF from healthy control subjects (nxa0=xa010) and patients with mild intermittent asthma (nxa0=xa010) after subway and control exposures. Exosomal RNA was analyzed by using microarrays containing probes for 894 human miRNAs, and selected findings were validated with quantitative RT-PCR. Results were analyzed by using multivariate modeling.nnnRESULTSnThe presence of miRNAs was confirmed in exosomes from BALF of both asthmatic patients and healthy control subjects. Significant differences in BALF exosomal miRNA was detected for 24 miRNAs with a subset of 16 miRNAs, including members of the let-7 and miRNA-200 families, providing robust classification of patients with mild nonsymptomatic asthma from healthy subjects with 72% cross-validated predictive power (Q(2)xa0=xa00.72). In contrast, subway exposure did not cause any significant alterations in miRNA profiles.nnnCONCLUSIONnThese studies demonstrate substantial differences in exosomal miRNA profiles between healthy subjects and patients with unprovoked, mild, stable asthma. These changes might be important in the inflammatory response leading to bronchial hyperresponsiveness and asthma.

Asthmatics Exhibit Altered Oxylipin Profiles Compared to Healthy Individuals after Subway Air Exposure

Background Asthma is a chronic inflammatory lung disease that causes significant morbidity and mortality worldwide. Air pollutants such as particulate matter (PM) and oxidants are important factors in causing exacerbations in asthmatics, and the source and composition of pollutants greatly affects pathological implications. Objectives This randomized crossover study investigated responses of the respiratory system to Stockholm subway air in asthmatics and healthy individuals. Eicosanoids and other oxylipins were quantified in the distal lung to provide a measure of shifts in lipid mediators in association with exposure to subway air relative to ambient air. Methods Sixty-four oxylipins representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of bronchoalveolar lavage (BAL)-fluid. Validations through immunocytochemistry staining of BAL-cells were performed for 15-LOX-1, COX-1, COX-2 and peroxisome proliferator-activated receptor gamma (PPARγ). Multivariate statistics were employed to interrogate acquired oxylipin and immunocytochemistry data in combination with patient clinical information. Results Asthmatics and healthy individuals exhibited divergent oxylipin profiles following exposure to ambient and subway air. Significant changes were observed in 8 metabolites of linoleic- and α-linolenic acid synthesized via the 15-LOX pathway, and of the COX product prostaglandin E2 (PGE2). Oxylipin levels were increased in healthy individuals following exposure to subway air, whereas asthmatics evidenced decreases or no change. Conclusions Several of the altered oxylipins have known or suspected bronchoprotective or anti-inflammatory effects, suggesting a possible reduced anti-inflammatory response in asthmatics following exposure to subway air. These observations may have ramifications for sensitive subpopulations in urban areas.

Interleukin-26 in Antibacterial Host Defense of Human Lungs. Effects on Neutrophil Mobilization

RATIONALEnThe role of the presumed Th17 cytokine IL-26 in antibacterial host defense of the lungs is not known.nnnOBJECTIVESnTo characterize the role of IL-26 in antibacterial host defense of human lungs.nnnMETHODSnIntrabronchial exposure of healthy volunteers to endotoxin and vehicle was performed during bronchoscopy and bronchoalveolar lavage (BAL) samples were harvested. Intracellular IL-26 was detected using immunocytochemistry and immunocytofluorescence. This IL-26 was also detected using flow cytometry, as was its receptor complex. Cytokines and phosphorylated signal transducer and activator of transcription (STAT) 1 plus STAT3 were quantified using ELISA. Gene expression was analyzed by real-time polymerase chain reaction and neutrophil migration was assessed in vitro.nnnMEASUREMENTS AND MAIN RESULTSnExtracellular IL-26 was detected in BAL samples without prior exposure in vivo and was markedly increased after endotoxin exposure. Alveolar macrophages displayed gene expression for, contained, and released IL-26. Th and cytotoxic T cells also contained IL-26. In the BAL samples, IL-26 concentrations and innate effector cells displayed a correlation. Recombinant IL-26 potentiated neutrophil chemotaxis induced by IL-8 and fMLP but decreased chemokinesis for neutrophils. Myeloperoxidase in conditioned media from neutrophils was decreased. The IL-26 receptor complex was detected in neutrophils and IL-26 decreased phosphorylated STAT3 in these cells. In BAL and bronchial epithelial cells, IL-26 increased gene expression of the IL-26 receptor complex and STAT1 plus STAT3. Finally, IL-26 increased the release of neutrophil-mobilizing cytokines in BAL but not in epithelial cells.nnnCONCLUSIONSnThis study implies that alveolar macrophages produce IL-26, which stimulates receptors on neutrophils and focuses their mobilization toward bacteria and accumulated immune cells in human lungs.

Linoleic acid-derived lipid mediators increase in a female-dominated subphenotype of COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality; however, the role of inflammatory mediators in its pathobiology remains unclear. The aim of this study was to investigate the influence of gender in COPD on lipid mediator levels. Bronchoalveolar lavage fluid (BALF) and serum were obtained from healthy never-smokers, smokers and COPD patients (Global Initiative for Chronic Obstructive Lung Disease stage I–II/A–B) (n=114). 94 lipid mediators derived from the cytochrome-P450, lipoxygenase, and cyclooxygenase pathways were analysed by liquid chromatography-mass spectrometry. Multivariate modelling identified a 9-lipid panel in BALF that classified female smokers with COPD from healthy female smokers (p=6×10−6). No differences were observed for the corresponding male population (p=1.0). These findings were replicated in an independent cohort with 92% accuracy (p=0.005). The strongest drivers were the cytochrome P450-derived epoxide products of linoleic acid (leukotoxins) and their corresponding soluble epoxide hydrolase (sEH)-derived products (leukotoxin-diols). These species correlated with lung function (r=0.87; p=0.0009) and mRNA levels of enzymes putatively involved in their biosynthesis (r=0.96; p=0.003). Leukotoxin levels correlated with goblet cell abundance (r=0.72; p=0.028). These findings suggest a mechanism by which goblet cell-associated cytochrome-P450 and sEH activity produce elevated leukotoxin-diol levels, which play a putative role in the clinical manifestations of COPD in a female-dominated disease sub-phenotype. Linoleic acid-derived lipids in BALF may indicate the transition from healthy smoker to COPD in a female subphenotype http://ow.ly/XowqN

The cytokine interleukin-26 as a biomarker in pediatric asthma

In this pilot study, we examined associations between local interleukin (IL)-26, disease severity and biomarkers of Th2-mediated inflammation in a well-defined cohort of pediatric patients (14xa0years median age, 41xa0% females) with controlled (nu2009=u200928) or uncontrolled (nu2009=u200948) asthma. Sputum IL-26 protein concentrations (ELISA) reflected disease control in patients without local (low exhaled nitric oxide) or systemic (low blood eosinophils) signs of eosinophilic inflammation. Moreover, sputum-IL-26 concentrations correlated with those of blood neutrophils. Our study indicates that IL-26 is a potential biomarker of disease severity in pediatric asthma without signs of Th2-mediated inflammation.

Systemic cytokine signaling via IL-17 in smokers with obstructive pulmonary disease: a link to bacterial colonization?

We examined whether systemic cytokine signaling via interleukin (IL)-17 and growth-related oncogene-α (GRO-α) is impaired in smokers with obstructive pulmonary disease including chronic bronchitis (OPD-CB). We also examined how this systemic cytokine signaling relates to bacterial colonization in the airways of the smokers with OPD-CB. Currently smoking OPD-CB patients (n=60, corresponding to Global initiative for chronic Obstructive Lung Disease [GOLD] stage I–IV) underwent recurrent blood and sputum sampling over 60 weeks, during stable conditions and at exacerbations. We characterized cytokine protein concentrations in blood and bacterial growth in sputum. Asymptomatic smokers (n=10) and never-smokers (n=10) were included as control groups. During stable clinical conditions, the protein concentrations of IL-17 and GRO-α were markedly lower among OPD-CB patients compared with never-smoker controls, whereas the asymptomatic smoker controls displayed intermediate concentrations. Notably, among OPD-CB patients, colonization by opportunistic pathogens was associated with markedly lower IL-17 and GRO-α, compared with colonization by common respiratory pathogens or oropharyngeal flora. During exacerbations in the OPD-CB patients, GRO-α and neutrophil concentrations were increased, whereas protein concentrations and messenger RNA for IL-17 were not detectable in a reproducible manner. In smokers with OPD-CB, systemic cytokine signaling via IL-17 and GRO-α is impaired and this alteration may be linked to colonization by opportunistic pathogens in the airways. Given the potential pathogenic and therapeutic implications, these findings deserve to be validated in new and larger patient cohorts.

Metabolomics analysis identifies sex-associated metabotypes of oxidative stress and the autotaxin–lysoPA axis in COPD

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease and a leading cause of mortality and morbidity worldwide. The aim of this study was to investigate the sex dependency of circulating metabolic profiles in COPD. Serum from healthy never-smokers (healthy), smokers with normal lung function (smokers), and smokers with COPD (COPD; Global Initiative for Chronic Obstructive Lung Disease stages I–II/A–B) from the Karolinska COSMIC cohort (n=116) was analysed using our nontargeted liquid chromatography–high resolution mass spectrometry metabolomics platform. Pathway analyses revealed that several altered metabolites are involved in oxidative stress. Supervised multivariate modelling showed significant classification of smokers from COPD (p=2.8×10−7). Sex stratification indicated that the separation was driven by females (p=2.4×10−7) relative to males (p=4.0×10−4). Significantly altered metabolites were confirmed quantitatively using targeted metabolomics. Multivariate modelling of targeted metabolomics data confirmed enhanced metabolic dysregulation in females with COPD (p=3.0×10−3) relative to males (p=0.10). The autotaxin products lysoPA (16:0) and lysoPA (18:2) correlated with lung function (forced expiratory volume in 1u2005s) in males with COPD (r=0.86; p<0.0001), but not females (r=0.44; p=0.15), potentially related to observed dysregulation of the miR-29 family in the lung. These findings highlight the role of oxidative stress in COPD, and suggest that sex-enhanced dysregulation in oxidative stress, and potentially the autotaxin–lysoPA axis, are associated with disease mechanisms and/or prevalence. Oxidative stress and the autotaxin–lysoPA axis evidence sex-associated metabotypes in the serum of COPD patients http://ow.ly/kAeE309MpdI

Troubleshooting Image Analysis in 2DE

The image analysis part of gel-based proteome research plays an important role in the overall success of the experiment. The main purpose of software-assisted 2DE gel analysis is to detect the protein spots, match them between gels within an experiment, and identify any differences in protein expression between sets of samples. Efficient analysis of protein expression relies on automated image processing techniques. There are several factors to consider in the choice of software product, as well as in the implementation of the analysis itself. Successful quantification of protein expression levels is largely dependent on the algorithms for spot matching, normalization, and background subtraction provided by the 2DE analysis software. In addition to generic protocols for image acquisition and subsequent 2DE image analysis (using Progenesis PG200), this chapter describes methods for quantitative and qualitative evaluation of the quality of the image analysis.

Extracellular cadmium in the bronchoalveolar space of long-term tobacco smokers with and without COPD and its association with inflammation

Tobacco contains cadmium, and this metal has been attributed a causative role in pulmonary emphysema among smokers, although extracellular cadmium has not to date been quantified in the bronchoalveolar space of tobacco smokers with or without COPD. We determined whether cadmium is enhanced in the bronchoalveolar space of long-term tobacco smokers with or without COPD in vivo, its association with inflammation, and its effect on chemokine release in macrophage-like cells in vitro. Bronchoalveolar lavage (BAL), sputum, and blood samples were collected from current, long-term smokers with and without COPD and from healthy nonsmokers. Cadmium concentrations were determined in cell-free BAL fluid using inductively coupled plasma mass spectrometry. Blood monocyte-derived macrophages were exposed to cadmium chloride in vitro. Depending upon the type of sample, molecular markers of inflammation were quantified either as protein (enzyme-linked immunosorbent assay) or as mRNA (real-time polymerase chain reaction). Cadmium concentrations were markedly increased in cell-free BAL fluid of smokers compared to that of nonsmokers (n=19–29; P<0.001), irrespective of COPD. In these smokers, the measured cadmium displayed positive correlations with macrophage TNF-α mRNA in BAL, neutrophil and CD8+ cell concentrations in blood, and finally with IL-6, IL-8, and MMP-9 protein in sputum (n=10–20; P<0.05). The cadmium chloride exposure caused a concentration-dependent increase in extracellular IL-8 protein in monocyte-derived macrophages in vitro. In conclusion, extracellular cadmium is enhanced in the bronchoalveolar space of long-term smokers and displays pro-inflammatory features. Its pathogenic role in tobacco-induced disease deserves further evaluation.

Impact of tobacco smoking on cytokine signaling via interleukin-17A in the peripheral airways

There is excessive accumulation of neutrophils in the airways in chronic obstructive pulmonary disease (COPD) but the underlying mechanisms remain poorly understood. It is known that extracellular cytokine signaling via interleukin (IL)-17A contributes to neutrophil accumulation in the airways but nothing is known about the impact of tobacco smoking on extracellular signaling via IL-17A. Here, we characterized the impact of tobacco smoking on extracellular cytokine signaling via IL-17A in the peripheral airways in long-term smokers with and without COPD and in occasional smokers before and after short-term exposure to tobacco smoke. We quantified concentrations of IL-17A protein in cell-free bronchoalveolar lavage (BAL) fluid samples (Immuno-quantitative PCR) and cytotoxic T-cells (immunoreactivity for CD8+ and CD3+) in bronchial biopsies. Matrix metalloproteinase-8 and human beta defensin 2 proteins were also quantified (enzyme-linked immunosorbent assay) in the BAL samples. The concentrations of IL-17A in BAL fluid were higher in long-term smokers without COPD compared with nonsmoking healthy controls, whereas those with COPD did not differ significantly from either of the other groups. Short-term exposure to tobacco smoke did not induce sustained alterations in these concentrations in occasional smokers. Long-term smokers displayed higher concentrations of IL-17A than did occasional smokers. Moreover, these concentrations correlated with CD8+ and CD3+ cells in biopsies among long-term smokers with COPD. In healthy nonsmokers, BAL concentrations of matrix metalloproteinase-8 and IL-17A correlated, whereas this was not the case in the pooled group of long-term smokers with and without COPD. In contrast, BAL concentrations of human beta defensin 2 and IL-17A correlated in all study groups. This study implies that long-term but not short-term exposure to tobacco smoke increases extracellular cytokine signaling via IL-17A in the peripheral airways. In the smokers with COPD, this signaling may involve cytotoxic T-cells. Long-term exposure to tobacco smoke leads to a disturbed association of extracellular IL-17A signaling and matrix metalloproteinase-8, of potential importance for the coordination of antibacterial activity.