In vitro toxicity evaluation in A549 cells of diesel particulate matter from two different particle sampling systems and several resuspension media.

In urban areas, inhalation of fine particles from combustion sources such as diesel engines causes adverse health effects. For toxicity testing, a substantial amount of particulate matter (PM) is needed. Conventional sampling involves collection of PM onto substrates by filtration or inertial impaction. A major drawback to those methodologies is that the extraction process can modify the collected particles and alter their chemical composition. Moreover, prior to toxicity testing, PM samples need to be resuspended, which can alter the PM sample even further. Lastly, the choice of the resuspension medium may also impact the detected toxicological responses. In this study, we compared the toxicity profile of PM obtained from two alternative sampling systems, using in vitro toxicity assays. One system makes use of condensational growth before collection in water in an impinger - BioSampler (CG-BioSampler), and the other, a Dekati® Gravimetric Impactor (DGI), is based on inertial impaction. In addition, various methods for resuspension of DGI collected PM were compared. Tested endpoints included cytotoxicity, formation of cellular reactive oxygen species, and genotoxicity. The alternative collection and suspension methods affected different toxicological endpoints. The water/dimethyl sulfoxide mixture and cell culture medium resuspended particles, along with the CG-BioSampler sample, produced the strongest responses. The water resuspended sample from the DGI appeared least toxic. CG-BioSampler collected PM caused a clear increased response in apoptotic cell death. We conclude that the CG-BioSampler PM sampler is a promising alternative to inertial impaction sampling.

Stratification of asthma by lipidomic profiling of induced sputum supernatant.

BACKGROUND: Asthma is a chronic respiratory disease with significant heterogeneity in its clinical presentation and pathobiology. There is need for improved understanding of respiratory lipid metabolism in asthma patients and its relation to observable clinical features. OBJECTIVE: We performed a comprehensive, prospective, cross-sectional analysis of the lipid composition of induced sputum supernatant obtained from asthma patients with a range of disease severities, as well as from healthy controls. METHODS: Induced sputum supernatant was collected from 211 adults with asthma and 41 healthy individuals enrolled onto the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study. Sputum lipidomes were characterized by semiquantitative shotgun mass spectrometry and clustered using topologic data analysis to identify lipid phenotypes. RESULTS: Shotgun lipidomics of induced sputum supernatant revealed a spectrum of 9 molecular phenotypes, highlighting not just significant differences between the sputum lipidomes of asthma patients and healthy controls, but also within the asthma patient population. Matching clinical, pathobiologic, proteomic, and transcriptomic data helped inform the underlying disease processes. Sputum lipid phenotypes with higher levels of nonendogenous, cell-derived lipids were associated with significantly worse asthma severity, worse lung function, and elevated granulocyte counts. CONCLUSION: We propose a novel mechanism of increased lipid loading in the epithelial lining fluid of asthma patients resulting from the secretion of extracellular vesicles by granulocytic inflammatory cells, which could reduce the ability of pulmonary surfactant to lower surface tension in asthmatic small airways, as well as compromise its role as an immune regulator.

Severe asthma trajectories in adults: findings from the NORDSTAR cohort.

BACKGROUND: There is limited evidence on the pathways leading to severe asthma and we are presently unable to effectively predict the progression of the disease. We aimed to describe the longitudinal trajectories leading to severe asthma and to describe clinical events preceding disease progression in a nationwide population of patients with severe asthma. METHODS: We conducted an observational study based on Swedish data from the NORdic Dataset for aSThmA Research (NORDSTAR) research collaboration platform. We identified adult patients with severe asthma in 2018 according to the European Respiratory Society/American Thoracic Society definition and used latent class analysis to identify trajectories of asthma severity over a 10-year retrospective period from 2018. RESULTS: Among 169 128 asthma patients, we identified 4543 severe asthma patients. We identified four trajectories of severe asthma that were labelled as: trajectory 1 "consistently severe asthma" (n=389 (8.6%)), trajectory 2 "gradual onset severe asthma" (n=942 (20.7%)), trajectory 3 "intermittent severe asthma" (n=1685 (37.1%)) and trajectory 4 "sudden onset severe asthma" (n=1527 (33.6%)). "Consistently severe asthma" had a higher daily inhaled corticosteroid dose and more prevalent osteoporosis compared with the other trajectories. Patients with "gradual onset severe asthma" and "sudden onset severe asthma" developed type 2-related comorbidities concomitantly with development of severe asthma. In the latter group, this primarily occurred within 1-3 years preceding onset of severe asthma. CONCLUSIONS: Four distinct trajectories of severe asthma were identified illustrating different patterns of progression of asthma severity. This may eventually enable the development of better preventive management strategies in severe asthma.

The role of inflammation in anxiety and depression in the European U-BIOPRED asthma cohorts.

BACKGROUND: Growing evidence indicates high comorbid anxiety and depression in patients with asthma. However, the mechanisms underlying this comorbid condition remain unclear. The aim of this study was to investigate the role of inflammation in comorbid anxiety and depression in three asthma patient cohorts of the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) project. METHODS: U-BIOPRED was conducted by a European Union consortium of 16 academic institutions in 11 European countries. A subset dataset from subjects with valid anxiety and depression measures and a large blood biomarker dataset were analysed, including 198 non-smoking patients with severe asthma (SAn), 65 smoking patients with severe asthma (SAs), 61 non-smoking patients with mild-to-moderate asthma (MMA), and 20 healthy non-smokers (HC). The Hospital Anxiety and Depression Scale was used to measure anxiety and depression and a series of inflammatory markers were analysed by the SomaScan v3 platform (SomaLogic, Boulder, Colo). ANOVA and the Kruskal-Wallis test were used for multiple-group comparisons as appropriate. RESULTS: There were significant group effects on anxiety and depression among the four cohort groups (p < 0.05). Anxiety and depression of SAn and SAs groups were significantly higher than that of MMA and HC groups (p < 0.05. There were significant differences in serum IL6, MCP1, CCL18, CCL17, IL8, and Eotaxin among the four groups (p < 0.05). Depression was significantly associated with IL6, MCP1, CCL18 level, and CCL17; whereas anxiety was associated with CCL17 only (p < 0.05). CONCLUSIONS: The current study suggests that severe asthma patients are associated with higher levels of anxiety and depression, and inflammatory responses may underlie this comorbid condition.

Reduced bronchoalveolar macrophage phagocytosis and cytotoxic effects after controlled short-term exposure to wood smoke in healthy humans.

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.

Plasma proteins elevated in severe asthma despite oral steroid use and unrelated to Type-2 inflammation.

RATIONALE: Asthma phenotyping requires novel biomarker discovery. OBJECTIVES: To identify plasma biomarkers associated with asthma phenotypes by application of a new proteomic panel to samples from two well-characterised cohorts of severe (SA) and mild-to-moderate (MMA) asthmatics, COPD subjects and healthy controls (HCs). METHODS: An antibody-based array targeting 177 proteins predominantly involved in pathways relevant to inflammation, lipid metabolism, signal transduction and extracellular matrix was applied to plasma from 525 asthmatics and HCs in the U-BIOPRED cohort, and 142 subjects with asthma and COPD from the validation cohort BIOAIR. Effects of oral corticosteroids (OCS) were determined by a 2-week, placebo-controlled OCS trial in BIOAIR, and confirmed by relation to objective OCS measures in U-BIOPRED. RESULTS: In U-BIOPRED, 110 proteins were significantly different, mostly elevated, in SA compared to MMA and HCs. 10 proteins were elevated in SA versus MMA in both U-BIOPRED and BIOAIR (alpha-1-antichymotrypsin, apolipoprotein-E, complement component 9, complement factor I, macrophage inflammatory protein-3, interleukin-6, sphingomyelin phosphodiesterase 3, TNF receptor superfamily member 11a, transforming growth factor-ß and glutathione S-transferase). OCS treatment decreased most proteins, yet differences between SA and MMA remained following correction for OCS use. Consensus clustering of U-BIOPRED protein data yielded six clusters associated with asthma control, quality of life, blood neutrophils, high-sensitivity C-reactive protein and body mass index, but not Type-2 inflammatory biomarkers. The mast cell specific enzyme carboxypeptidase A3 was one major contributor to cluster differentiation. CONCLUSIONS: The plasma proteomic panel revealed previously unexplored yet potentially useful Type-2-independent biomarkers and validated several proteins with established involvement in the pathophysiology of SA.

Cold air exposure at - 15 °C induces more airway symptoms and epithelial stress during heavy exercise than rest without aggravated airway constriction.

PURPOSE: Exposure to cold air may harm the airways. It is unclear to what extent heavy exercise adds to the cold-induced effects on peripheral airways, airway epithelium, and systemic immunity among healthy individuals. We investigated acute effects of heavy exercise in sub-zero temperatures on the healthy airways. METHODS: Twenty-nine healthy individuals underwent whole body exposures to cold air in an environmental chamber at - 15 °C for 50 min on two occasions; a 35-min exercise protocol consisting of a 5-min warm-up followed by 2 × 15 min of running at 85% of VO2max vs. 50 min at rest. Lung function was measured by impulse oscillometry (IOS) and spirometry before and immediately after exposures. CC16 in plasma and urine, and cytokines in plasma were measured before and 60 min after exposures. Symptoms were surveyed pre-, during and post-trials. RESULTS: FEV1 decreased after rest (- 0.10 ± 0.03 L, p < 0.001) and after exercise (- 0.06 ± 0.02 L, p = 0.012), with no difference between trials. Exercise in - 15 °C induced greater increases in lung reactance (X5; p = 0.023), plasma CC16 (p < 0.001) as well as plasma IL-8 (p < 0.001), compared to rest. Exercise induced more intense symptoms from the lower airways, whereas rest gave rise to more general symptoms. CONCLUSION: Heavy exercise during cold air exposure at - 15 °C induced signs of an airway constriction to a similar extent as rest in the same environment. However, biochemical signs of airway epithelial stress, cytokine responses, and symptoms from the lower airways were more pronounced after the exercise trial.

Acute cardiovascular effects of controlled exposure to dilute Petrodiesel and biodiesel exhaust in healthy volunteers: a crossover study.

BACKGROUND: Air pollution derived from combustion is associated with considerable cardiorespiratory morbidity and mortality in addition to environmental effects. Replacing petrodiesel with biodiesel may have ecological benefits, but impacts on human health remain unquantified. The objective was to compare acute cardiovascular effects of blended and pure biodiesel exhaust exposure against known adverse effects of petrodiesel exhaust (PDE) exposure in human subjects. In two randomized controlled double-blind crossover studies, healthy volunteers were exposed to PDE or biodiesel exhaust for one hour. In study one, 16 subjects were exposed, on separate occasions, to PDE and 30% rapeseed methyl ester biodiesel blend (RME30) exhaust, aiming at PM10 300 µg/m3. In study two, 19 male subjects were separately exposed to PDE and exhaust from a 100% RME fuel (RME100) using similar engine load and exhaust dilution. Generated exhaust was analyzed for physicochemical composition and oxidative potential. Following exposure, vascular endothelial function was assessed using forearm venous occlusion plethysmography and ex vivo thrombus formation was assessed using a Badimon chamber model of acute arterial injury. Biomarkers of inflammation, platelet activation and fibrinolysis were measured in the blood. RESULTS: In study 1, PDE and RME30 exposures were at comparable PM levels (314 ± 27 µg/m3; (PM10 ± SD) and 309 ± 30 µg/m3 respectively), whereas in study 2, the PDE exposure concentrations remained similar (310 ± 34 µg/m3), but RME100 levels were lower in PM (165 ± 16 µg/m3) and PAHs, but higher in particle number concentration. Compared to PDE, PM from RME had less oxidative potential. Forearm infusion of the vasodilators acetylcholine, bradykinin, sodium nitroprusside and verapamil resulted in dose-dependent increases in blood flow after all exposures. Vasodilatation and ex vivo thrombus formation were similar following exposure to exhaust from petrodiesel and the two biodiesel formulations (RME30 and RME100). There were no significant differences in blood biomarkers or exhaled nitric oxide levels between exposures. CONCLUSIONS: Despite differences in PM composition and particle reactivity, controlled exposure to biodiesel exhaust was associated with similar cardiovascular effects to PDE. We suggest that the potential adverse health effects of biodiesel fuel emissions should be taken into account when evaluating future fuel policies. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01337882 /NCT01883466. Date of first enrollment March 11, 2011, registered April 19, 2011, i.e. retrospectively registered.

Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation.

BACKGROUND: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear. OBJECTIVE: We sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients. METHODS: An IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens. RESULTS: Activation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS-high asthma with increased epithelial expression of IL-6TS-inducible genes in the absence of systemic inflammation. The IL-6TS-high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1ß, IL-8, and IL-1ß. CONCLUSIONS: Local lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.

IL-17-high asthma with features of a psoriasis immunophenotype.

BACKGROUND: The role of IL-17 immunity is well established in patients with inflammatory diseases, such as psoriasis and inflammatory bowel disease, but not in asthmatic patients, in whom further study is required. OBJECTIVE: We sought to undertake a deep phenotyping study of asthmatic patients with upregulated IL-17 immunity. METHODS: Whole-genome transcriptomic analysis was performed by using epithelial brushings, bronchial biopsy specimens (91 asthmatic patients and 46 healthy control subjects), and whole blood samples (n = 498) from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. Gene signatures induced in vitro by IL-17 and IL-13 in bronchial epithelial cells were used to identify patients with IL-17-high and IL-13-high asthma phenotypes. RESULTS: Twenty-two of 91 patients were identified with IL-17, and 9 patients were identified with IL-13 gene signatures. The patients with IL-17-high asthma were characterized by risk of frequent exacerbations, airway (sputum and mucosal) neutrophilia, decreased lung microbiota diversity, and urinary biomarker evidence of activation of the thromboxane B2 pathway. In pathway analysis the differentially expressed genes in patients with IL-17-high asthma were shared with those reported as altered in psoriasis lesions and included genes regulating epithelial barrier function and defense mechanisms, such as IL1B, IL6, IL8, and ß-defensin. CONCLUSION: The IL-17-high asthma phenotype, characterized by bronchial epithelial dysfunction and upregulated antimicrobial and inflammatory response, resembles the immunophenotype of psoriasis, including activation of the thromboxane B2 pathway, which should be considered a biomarker for this phenotype in further studies, including clinical trials targeting IL-17.

Stratification of asthma phenotypes by airway proteomic signatures.

BACKGROUND: Stratification by eosinophil and neutrophil counts increases our understanding of asthma and helps target therapy, but there is room for improvement in our accuracy in prediction of treatment responses and a need for better understanding of the underlying mechanisms. OBJECTIVE: We sought to identify molecular subphenotypes of asthma defined by proteomic signatures for improved stratification. METHODS: Unbiased label-free quantitative mass spectrometry and topological data analysis were used to analyze the proteomes of sputum supernatants from 246 participants (206 asthmatic patients) as a novel means of asthma stratification. Microarray analysis of sputum cells provided transcriptomics data additionally to inform on underlying mechanisms. RESULTS: Analysis of the sputum proteome resulted in 10 clusters (ie, proteotypes) based on similarity in proteomic features, representing discrete molecular subphenotypes of asthma. Overlaying granulocyte counts onto the 10 clusters as metadata further defined 3 of these as highly eosinophilic, 3 as highly neutrophilic, and 2 as highly atopic with relatively low granulocytic inflammation. For each of these 3 phenotypes, logistic regression analysis identified candidate protein biomarkers, and matched transcriptomic data pointed to differentially activated underlying mechanisms. CONCLUSION: This study provides further stratification of asthma currently classified based on quantification of granulocytic inflammation and provided additional insight into their underlying mechanisms, which could become targets for novel therapies.

Identification and prospective stability of electronic nose (eNose)-derived inflammatory phenotypes in patients with severe asthma.

BACKGROUND: Severe asthma is a heterogeneous condition, as shown by independent cluster analyses based on demographic, clinical, and inflammatory characteristics. A next step is to identify molecularly driven phenotypes using "omics" technologies. Molecular fingerprints of exhaled breath are associated with inflammation and can qualify as noninvasive assessment of severe asthma phenotypes. OBJECTIVES: We aimed (1) to identify severe asthma phenotypes using exhaled metabolomic fingerprints obtained from a composite of electronic noses (eNoses) and (2) to assess the stability of eNose-derived phenotypes in relation to within-patient clinical and inflammatory changes. METHODS: In this longitudinal multicenter study exhaled breath samples were taken from an unselected subset of adults with severe asthma from the U-BIOPRED cohort. Exhaled metabolites were analyzed centrally by using an assembly of eNoses. Unsupervised Ward clustering enhanced by similarity profile analysis together with K-means clustering was performed. For internal validation, partitioning around medoids and topological data analysis were applied. Samples at 12 to 18 months of prospective follow-up were used to assess longitudinal within-patient stability. RESULTS: Data were available for 78 subjects (age, 55 years [interquartile range, 45-64 years]; 41% male). Three eNose-driven clusters (n = 26/33/19) were revealed, showing differences in circulating eosinophil (P = .045) and neutrophil (P = .017) percentages and ratios of patients using oral corticosteroids (P = .035). Longitudinal within-patient cluster stability was associated with changes in sputum eosinophil percentages (P = .045). CONCLUSIONS: We have identified and followed up exhaled molecular phenotypes of severe asthma, which were associated with changing inflammatory profile and oral steroid use. This suggests that breath analysis can contribute to the management of severe asthma.

Severe asthma-A population study perspective.

BACKGROUND: Severe asthma is a considerable challenge for patients, health-care professionals and society. Few studies have estimated the prevalence of severe asthma according to modern definitions of which none based on a population study. OBJECTIVE: To describe characteristics and estimate the prevalence of severe asthma in a large adult population-based asthma cohort followed for 10-28 years. METHODS: N = 1006 subjects with asthma participated in a follow-up during 2012-14, when 830 (mean age 59 years, 56% women) still had current asthma. Severe asthma was defined according to three internationally well-known criteria: the ATS workshop definition from 2000 used in the US Severe Asthma Research Programme (SARP), the 2014 ATS/ERS Task force definition and the GINA 2017. All subjects with severe asthma according to any of these criteria were undergoing respiratory specialist care and were also contacted by telephone to verify treatment adherence. RESULTS: The prevalence of severe asthma according to the three definitions was 3.6% (US SARP), 4.8% (ERS/ATS Taskforce), and 6.1% (GINA) among subjects with current asthma. Although all were using high ICS doses and other maintenance treatment, >90% did not have controlled asthma according to the asthma control test. Severe asthma was related to age >50 years, nasal polyposis, impaired lung function, sensitization to aspergillus, and tended to be more common in women. Further, neutrophils in blood significantly discriminated severe asthma from other asthma. CONCLUSIONS AND CLINICAL RELEVANCE: Severe asthma differed significantly from other asthma in terms of demographic, clinical and inflammatory characteristics, results suggesting possibilities for improved treatment regimens of severe asthma. The prevalence of severe asthma in this asthma cohort was 4%-6%, corresponding to approximately 0.5% of the general population.

Contribution of airway eosinophils in airway wall remodeling in asthma: Role of MMP-10 and MET.

BACKGROUND: Eosinophils play an important role in the pathophysiology of asthma being implicated in airway epithelial damage and airway wall remodeling. We determined the genes associated with airway remodeling and eosinophilic inflammation in patients with asthma. METHODS: We analyzed the transcriptomic data from bronchial biopsies of 81 patients with moderate-to-severe asthma of the U-BIOPRED cohort. Expression profiling was performed using Affymetrix arrays on total RNA. Transcription binding site analysis used the PRIMA algorithm. Localization of proteins was by immunohistochemistry. RESULTS: Using stringent false discovery rate analysis, MMP-10 and MET were significantly overexpressed in biopsies with high mucosal eosinophils (HE) compared to low mucosal eosinophil (LE) numbers. Immunohistochemical analysis confirmed increased expression of MMP-10 and MET in bronchial epithelial cells and in subepithelial inflammatory and resident cells in asthmatic biopsies. Using less-stringent conditions (raw P-value < 0.05, log2 fold change > 0.5), we defined a 73-gene set characteristic of the HE compared to the LE group. Thirty-three of 73 genes drove the pathway annotation that included extracellular matrix (ECM) organization, mast cell activation, CC-chemokine receptor binding, circulating immunoglobulin complex, serine protease inhibitors, and microtubule bundle formation pathways. Genes including MET and MMP10 involved in ECM organization correlated positively with submucosal thickness. Transcription factor binding site analysis identified two transcription factors, ETS-1 and SOX family proteins, that showed positive correlation with MMP10 and MET expression. CONCLUSION: Pathways of airway remodeling and cellular inflammation are associated with submucosal eosinophilia. MET and MMP-10 likely play an important role in these processes.

Large-Scale Label-Free Quantitative Mapping of the Sputum Proteome.

Analysis of induced sputum supernatant is a minimally invasive approach to study the epithelial lining fluid and, thereby, provide insight into normal lung biology and the pathobiology of lung diseases. We present here a novel proteomics approach to sputum analysis developed within the U-BIOPRED (unbiased biomarkers predictive of respiratory disease outcomes) international project. We present practical and analytical techniques to optimize the detection of robust biomarkers in proteomic studies. The normal sputum proteome was derived using data-independent HDMSE applied to 40 healthy nonsmoking participants, which provides an essential baseline from which to compare modulation of protein expression in respiratory diseases. The "core" sputum proteome (proteins detected in ≥40% of participants) was composed of 284 proteins, and the extended proteome (proteins detected in ≥3 participants) contained 1666 proteins. Quality control procedures were developed to optimize the accuracy and consistency of measurement of sputum proteins and analyze the distribution of sputum proteins in the healthy population. The analysis showed that quantitation of proteins by HDMSE is influenced by several factors, with some proteins being measured in all participants' samples and with low measurement variance between samples from the same patient. The measurement of some proteins is highly variable between repeat analyses, susceptible to sample processing effects, or difficult to accurately quantify by mass spectrometry. Other proteins show high interindividual variance. We also highlight that the sputum proteome of healthy individuals is related to sputum neutrophil levels, but not gender or allergic sensitization. We illustrate the importance of design and interpretation of disease biomarker studies considering such protein population and technical measurement variance.

Urban particulate matter stimulation of human dendritic cells enhances priming of naive CD8 T lymphocytes.

Epidemiological studies have consistently shown associations between elevated concentrations of urban particulate matter (UPM) air pollution and exacerbations of asthma and chronic obstructive pulmonary disease, which are both associated with viral respiratory infections. The effects of UPM on dendritic cell (DC) -stimulated CD4 T lymphocytes have been investigated previously, but little work has focused on CD8 T-lymphocyte responses despite their importance in anti-viral immunity. To address this, we examined the effects of UPM on DC-stimulated naive CD8 T-cell responses. Expression of the maturation/activation markers CD83, CCR7, CD40 and MHC class I on human myeloid DCs (mDCs) was characterized by flow cytometry after stimulation with UPMin vitro in the presence/absence of granulocyte-macrophage colony-stimulating factor (GM-CSF). The capacity of these mDCs to stimulate naive CD8 T-lymphocyte responses in allogeneic co-culture was then assessed by measuring T-cell cytokine secretion using cytometric bead array, and proliferation and frequency of interferon-γ (IFN-γ)-producing T lymphocytes by flow cytometry. Treatment of mDCs with UPM increased expression of CD83 and CCR7, but not MHC class I. In allogeneic co-cultures, UPM treatment of mDCs enhanced CD8 T-cell proliferation and the frequency of IFN-γ+ cells. The secretion of tumour necrosis factor-α, interleukin-13, Granzyme A and Granzyme B were also increased. GM-CSF alone, and in concert with UPM, enhanced many of these T-cell functions. The PM-induced increase in Granzyme A was confirmed in a human experimental diesel exposure study. These data demonstrate that UPM treatment of mDCs enhances priming of naive CD8 T lymphocytes and increases production of pro-inflammatory cytokines. Such UPM-induced stimulation of CD8 cells may potentiate T-lymphocyte cytotoxic responses upon concurrent airway infection, increasing bystander damage to the airways.

Sputum proteomics and airway cell transcripts of current and ex-smokers with severe asthma in U-BIOPRED: an exploratory analysis.

Severe asthma patients with a significant smoking history have airflow obstruction with reported neutrophilia. We hypothesise that multi-omic analysis will enable the definition of smoking and ex-smoking severe asthma molecular phenotypes.The U-BIOPRED cohort of severe asthma patients, containing current-smokers (CSA), ex-smokers (ESA), nonsmokers and healthy nonsmokers was examined. Blood and sputum cell counts, fractional exhaled nitric oxide and spirometry were obtained. Exploratory proteomic analysis of sputum supernatants and transcriptomic analysis of bronchial brushings, biopsies and sputum cells was performed.Colony-stimulating factor (CSF)2 protein levels were increased in CSA sputum supernatants, with azurocidin 1, neutrophil elastase and CXCL8 upregulated in ESA. Phagocytosis and innate immune pathways were associated with neutrophilic inflammation in ESA. Gene set variation analysis of bronchial epithelial cell transcriptome from CSA showed enrichment of xenobiotic metabolism, oxidative stress and endoplasmic reticulum stress compared to other groups. CXCL5 and matrix metallopeptidase 12 genes were upregulated in ESA and the epithelial protective genes, mucin 2 and cystatin SN, were downregulated.Despite little difference in clinical characteristics, CSA were distinguishable from ESA subjects at the sputum proteomic level, with CSA patients having increased CSF2 expression and ESA patients showing sustained loss of epithelial barrier processes.

Lipid phenotyping of lung epithelial lining fluid in healthy human volunteers.

BACKGROUND: Lung epithelial lining fluid (ELF)-sampled through sputum induction-is a medium rich in cells, proteins and lipids. However, despite its key role in maintaining lung function, homeostasis and defences, the composition and biology of ELF, especially in respect of lipids, remain incompletely understood. OBJECTIVES: To characterise the induced sputum lipidome of healthy adult individuals, and to examine associations between different ELF lipid phenotypes and the demographic characteristics within the study cohort. METHODS: Induced sputum samples were obtained from 41 healthy non-smoking adults, and their lipid compositions analysed using a combination of untargeted shotgun and liquid chromatography mass spectrometry methods. Topological data analysis (TDA) was used to group subjects with comparable sputum lipidomes in order to identify distinct ELF phenotypes. RESULTS: The induced sputum lipidome was diverse, comprising a range of different molecular classes, including at least 75 glycerophospholipids, 13 sphingolipids, 5 sterol lipids and 12 neutral glycerolipids. TDA identified two distinct phenotypes differentiated by a higher total lipid content and specific enrichments of diacyl-glycerophosphocholines, -inositols and -glycerols in one group, with enrichments of sterols, glycolipids and sphingolipids in the other. Subjects presenting the lipid-rich ELF phenotype also had significantly higher BMI, but did not differ in respect of other demographic characteristics such as age or gender. CONCLUSIONS: We provide the first evidence that the ELF lipidome varies significantly between healthy individuals and propose that such differences are related to weight status, highlighting the potential impact of (over)nutrition on lung lipid metabolism.

Multi-cellular human bronchial models exposed to diesel exhaust particles: assessment of inflammation, oxidative stress and macrophage polarization.

BACKGROUND: Diesel exhaust particles (DEP) are a major component of outdoor air pollution. DEP mediated pulmonary effects are plausibly linked to inflammatory and oxidative stress response in which macrophages (MQ), epithelial cells and their cell-cell interaction plays a crucial role. Therefore, in this study we aimed at studying the cellular crosstalk between airway epithelial cells with MQ and MQ polarization following exposure to aerosolized DEP by assessing inflammation, oxidative stress, and MQ polarization response markers. METHOD: Lung mucosa models including primary bronchial epithelial cells (PBEC) cultured at air-liquid interface (ALI) were co-cultured without (PBEC-ALI) and with MQ (PBEC-ALI/MQ). Cells were exposed to 12.7 µg/cm2 aerosolized DEP using XposeALI®. Control (sham) models were exposed to clean air. Cell viability was assessed. CXCL8 and IL-6 were measured in the basal medium by ELISA. The mRNA expression of inflammatory markers (CXCL8, IL6, TNFα), oxidative stress (NFKB, HMOX1, GPx) and MQ polarization markers (IL10, IL4, IL13, MRC1, MRC2 RETNLA, IL12 andIL23) were measured by qRT-PCR. The surface/mRNA expression of TLR2/TLR4 was detected by FACS and qRT-PCR. RESULTS: In PBEC-ALI exposure to DEP significantly increased the secretion of CXCL8, mRNA expression of inflammatory markers (CXCL8, TNFα) and oxidative stress markers (NFKB, HMOX1, GPx). However, mRNA expressions of these markers (CXCL8, IL6, NFKB, and HMOX1) were reduced in PBEC-ALI/MQ models after DEP exposure. TLR2 and TLR4 mRNA expression increased after DEP exposure in PBEC-ALI. The surface expression of TLR2 and TLR4 on PBEC was significantly reduced in sham-exposed PBEC-ALI/MQ compared to PBEC-ALI. After DEP exposure surface expression of TLR2 was increased on PBEC of PBEC-ALI/MQ, while TLR4 was decreased in both models. DEP exposure resulted in similar expression pattern of TLR2/TLR4 on MQ as in PBEC. In PBEC-ALI/MQ, DEP exposure increased the mRNA expression of anti-inflammatory M2 macrophage markers (IL10, IL4, IL13, MRC1, MRC2). CONCLUSION: The cellular interaction of PBEC with MQ in response to DEP plays a pivotal role for MQ phenotypic alteration towards M2-subtypes, thereby promoting an efficient resolution of the inflammation. Furthermore, this study highlighted the fact that cell-cell interaction using multicellular ALI-models combined with an in vivo-like inhalation exposure system is critical in better mimicking the airway physiology compared with traditional cell culture systems.

U-BIOPRED clinical adult asthma clusters linked to a subset of sputum omics.

BACKGROUND: Asthma is a heterogeneous disease in which there is a differential response to asthma treatments. This heterogeneity needs to be evaluated so that a personalized management approach can be provided. OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinicophysiologic parameters and performed an omics analysis of sputum. METHODS: Partition-around-medoids clustering was applied to a training set of 266 asthmatic participants from the European Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes (U-BIOPRED) adult cohort using 8 prespecified clinic-physiologic variables. This was repeated in a separate validation set of 152 asthmatic patients. The clusters were compared based on sputum proteomics and transcriptomics data. RESULTS: Four reproducible and stable clusters of asthmatic patients were identified. The training set cluster T1 consists of patients with well-controlled moderate-to-severe asthma, whereas cluster T2 is a group of patients with late-onset severe asthma with a history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of nonsmokers. Cluster T4 is predominantly composed of obese female patients with uncontrolled severe asthma with increased exacerbations but with normal lung function. The validation set exhibited similar clusters, demonstrating reproducibility of the classification. There were significant differences in sputum proteomics and transcriptomics between the clusters. The severe asthma clusters (T2, T3, and T4) had higher sputum eosinophilia than cluster T1, with no differences in sputum neutrophil counts and exhaled nitric oxide and serum IgE levels. CONCLUSION: Clustering based on clinicophysiologic parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.