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Background and objective Low-dose long-term azithromycin is recommended in clinical practice guidelines for obstructive airway diseases (OADs), however, an optimal therapeutic regimen is not yet established. This study aimed to understand the patterns of azithromycin use in OADs, characterise the patients who received it, and evaluate its safety and efficacy using real-world data. METHODS: We audited 91 patients who had received azithromycin for at least 4 weeks for the management of asthma, chronic obstructive pulmonary disease (COPD) or non-cystic fibrosis bronchiectasis. RESULTS: The mean age was 65±18 years, 60% were female, and 48% were ex-smokers. The majority had asthma (75%) either alone (50%) or in combination with COPD (12%) or bronchiectasis (13%). Most (64%) reported cough or sputum at baseline. The most common treatment regimen was azithromycin 250mg daily (73%) for more than 1 year (57%), with only seven adverse events. There was a significant reduction in the proportions of patients requiring emergency department visits (48% versus 32%; p

BACKGROUND Neutrophilic asthma (NA) is a clinically important asthma phenotype, the cellular and molecular basis of which is not completely understood. Airway macrophages are long-lived immune cells that exert important homeostatic and inflammatory functions which are dysregulated in asthma. Unique transcriptomic programmes reflect varied macrophage phenotypes in vitro. We aimed to determine whether airway macrophages are transcriptomically altered in NA. METHODS We performed RNASeq analysis on flow cytometry-isolated sputum macrophages comparing NA (n = 7) and non-neutrophilic asthma (NNA, n = 13). qPCR validation of RNASeq results was performed (NA n = 13, NNA n = 23). Pathway analysis (PANTHER, STRING) of differentially expressed genes (DEGs) was performed. Gene set variation analysis (GSVA) was used to test for enrichment of NA macrophage transcriptomic signatures in whole sputum microarray (cohort 1 - controls n = 16, NA n = 29, NNA n = 37; cohort 2 U-BIOPRED - controls n = 16, NA n = 47, NNA n = 57). RESULTS Flow cytometry-sorting significantly enriched sputum macrophages (99.4% post-sort, 44.9% pre-sort, p

BACKGROUND Airway and systemic eosinophilia are important treatable traits in both severe asthma and COPD. The molecular basis of eosinophilia in COPD is poorly understood but could involve type 2 cytokines (IL5, IL13) and prostaglandin D2 (PGD2 ). METHODS This study included non-obstructive airways disease (OAD) controls (n = 19), a COPD cohort (n = 96) and a severe asthma cohort (n = 84). Demographics, exacerbation history, disease impact (SGRQ) and spirometry were assessed. Participants were categorized as eosinophilic using either sputum eosinophil proportion (≥3%) or blood eosinophil count (≥300/μL). Sputum type 2 inflammatory measures included PGD2 by ELISA and gene expression (qPCR) of IL5, IL13 and the haematopoietic PGD2 synthase (HPGDS). RESULTS Type 2 markers did not differ across groups except HPGDS mRNA which was highest in non-OAD controls and lowest in COPD. IL5 and IL13 mRNA and PGD2 levels were significantly increased in eosinophilic vs non-eosinophilic severe asthma but did not differ between eosinophilic COPD and eosinophilic severe asthma or non-eosinophilic COPD. HPGDS expression was higher in eosinophilic severe asthma compared with eosinophilic COPD. Results were similar using sputum or blood eosinophil cut-offs. Sputum IL5 and IL13 were highly intercorrelated in severe asthma (r = 0.907, p

BACKGROUND Monocytes and macrophages are critical innate immune cells of the airways. Despite their differing functions, few clinical studies discriminate between them and little is known about their regulation in asthma. OBJECTIVE We aimed to distinguish and quantify macrophages, monocytes and monocyte subsets in induced sputum and blood and examine their relationship with inflammatory and clinical features of asthma. METHODS We applied flow cytometry to distinguish macrophages, monocytes and subsets in sputum and blood (n = 53; 45 asthma, 8 non-asthma) and a second asthma sputum cohort (n = 26). Monocyte subsets were identified by surface CD14/CD16 (CD14++ CD16- classical, CD14+ CD16+ intermediate and CD14+ CD16++ non-classical monocytes). Surface CD206, a marker of monocyte tissue differentiation, was measured in sputum. Relationship to airway inflammatory phenotype (neutrophilic n = 9, eosinophilic n = 14, paucigranulocytic n = 22) and asthma severity (severe n = 12, non-severe n = 33) was assessed. RESULTS Flow cytometry- and microscope-quantified sputum differential cell proportions were significantly correlated. Sputum macrophage number was reduced (p = .036), while classical monocyte proportion was increased in asthma vs non-asthma (p = .032). Sputum classical monocyte number was significantly higher in neutrophilic vs paucigranulocytic asthma (p = .013). CD206- monocyte proportion and number were increased in neutrophilic vs eosinophilic asthma (p

Background The 6 gene expression signature (6GS) predicts inflammatory phenotype, exacerbation risk, and corticosteroid responsiveness in asthma. In COPD, patterns of airway inflammation are similar, suggesting the 6GS may be useful. This study determines the diagnostic and prognostic ability of 6GS in predicting inflammatory phenotypes and exacerbation risk in COPD. Methods We performed 2 studies: a cross-sectional phenotype prediction study in stable COPD (total N=132; n=34 eosinophilic (E)-COPD, n=42 neutrophilic (N)-COPD, n=39 paucigranulocytic (PG)-COPD, n=17 mixed-granulocytic (MG)-COPD) that assessed 6GS ability to discriminate phenotypes (eosinophilia≥3%; neutrophilia≥61%); and a prospective cohort study (total n=54, n=8 E-COPD; n=18 N-COPD; n=20 PG-COPD; n=8 MG-COPD, n=21 exacerbation prone (≥2/year)) that investigated phenotype and exacerbation prediction utility. 6GS was measured by qPCR and evaluated using multiple logistic regression and area under the curve (AUC). Short-term reproducibility (intra-class correlation) and phenotyping method agreement (κ statistic) were assessed. Results In the phenotype prediction study, 6GS could accurately identify and discriminate patients with E-COPD from N-COPD (AUC=96.4%; p

Background Mast cells (MCs) are innate immune cells that regulate atopic and non-atopic inflammation in the airways. MCs play a critical role in the pathogenesis of asthma, yet their relationship to airway and systemic inflammation and clinical characteristics of asthma is poorly understood. Objective To quantify MCs in induced sputum samples and understand their relationship to airway and circulatory immune cells, and clinical variables in asthma. Methods We employed flow cytometry of sputum samples to quantify MCs, basophils and other immune cells in 51 participants (45 asthma and 6 non-asthma controls). Relationship of MCs to airway (n = 45) and blood (n = 19) immune cells, participant demographics, asthma history, spirometry and airways hyperresponsiveness (AHR) to hypertonic saline was determined by correlation and comparison of cut-off-based sputum MC high vs low participants. Results Mast cells, basophils and eosinophils were increased in asthma vs non-asthma control sputum. In asthma sputum, MCs, basophils and eosinophils were significantly intercorrelated, and MCs and basophils were elevated in participants with eosinophilic asthma. MCs and basophils, but not eosinophils, correlated with AHR. Sputum MC high asthma was characterized by an increased proportion of participants with uncontrolled asthma and reduced FEV1 and FVC. Trends towards similar clinical associations with elevated MCs were observed in a paucigranulocytic subpopulation (n = 15) lacking airway eosinophilia or neutrophilia. Receiver operator characteristic (ROC) analysis showed peripheral blood eosinophil (PBE) count predicted elevated sputum eosinophils and basophils, but not MCs. Conclusions and clinical relevance Sputum MCs are elevated in asthma, and their measurement may be useful as they relate to key clinical features of asthma (spirometry, asthma control, AHR). PBE count did not predict airway MC status, suggesting direct measurement of airway MCs by sensitive methods such as flow cytometry should be further developed.

BackgroundChronic airway inflammation is the main driver of pathogenesis in respiratory diseases such as severe asthma, chronic obstructive pulmonary disease, cystic fibrosis (CF) and bronchiectasis. While the role of common pathogens in airway inflammation is widely recognised, the influence of other microbiota members is still poorly understood.MethodsWe hypothesised that the lung microbiota contains bacteria with immunomodulatory activity which modulate net levels of immune activation by key respiratory pathogens. Therefore, we assessed the immunomodulatory effect of several members of the lung microbiota frequently reported as present in CF lower respiratory tract samples.ResultsWe show that Rothia mucilaginosa, a common resident of the oral cavity that is also often detectable in the lower airways in chronic disease, has an inhibitory effect on pathogen- or lipopolysaccharide-induced pro-inflammatory responses, in vitro (three-dimensional cell culture model) and in vivo (mouse model). Furthermore, in a cohort of adults with bronchiectasis, the abundance of Rothia species was negatively correlated with pro-inflammatory markers (interleukin (IL)-8 and IL-1β) and matrix metalloproteinase (MMP)-1, MMP-8 and MMP-9 in sputum. Mechanistic studies revealed that R. mucilaginosa inhibits NF-κB pathway activation by reducing the phosphorylation of IκBα and consequently the expression of NF-κB target genes.ConclusionsThese findings indicate that the presence of R. mucilaginosa in the lower airways potentially mitigates inflammation, which could in turn influence the severity and progression of chronic respiratory disorders.

SummaryVaccinations against SARS-CoV-2 are effective in COVID-19. However, with limited vaccine access, vaccine hesitancy and variant breakthroughs, there is still a need for effective and safe early treatments. Two community-based clinical trials of the inhaled corticosteroid, budesonide, have recently been published showing and improvement in patients with COVID-19 treated early with budesonide1,2. To understand mechanistically how budesonide was beneficial, inflammatory mediators were assessed in the nasal mucosa of patients recruited to the Steroids in COVID (STOIC1) trial and a cohort of SARS-CoV-2 negative individuals. Here we show that in early COVID-19, elevation in viral response proteins and Th1 and Th2 inflammation occurs. Longitudinal sampling in the natural course of COVID-19 showed persistently high interferon levels and elevated concentrations of the eosinophil chemokine, CCL11. In patients who deteriorate, the initial nasal mucosal signal is characterised by a marked suppression of the early inflammatory response, with reduced concentrations of interferon and inflammatory cytokines, but elevated eosinophil chemokines. Systemic inflammation remained altered in COVID-19 patients, implying that even after symptom resolution, changes in immunological mediators do not resolve. Budesonide treatment decreased IL-33 and IFN-γ, implying a reduction in epithelial damage and dampening of the interferon response. Budesonide treatment also increased CCL17 concentrations, suggesting an improved T-cell response; and significantly alters inflammatory pathways giving further insight into how this treatment can accelerate patient recovery.Abstract Figure

Purpose To characterize neutrophils in obstructive airway disease by measuring their surface adhesion molecules and oxidative burst along with characterizing them into different subsets as per their adhesion molecule expression. Patients and methods Peripheral blood from adults with COPD (n=17), asthma (n=20), and healthy participants (n=19) was examined for expression of CD16, CD62L, CD11b, CD11c, and CD54, and analyzed by flow cytometry. For oxidative burst and CD62L shedding analysis, CD16 and CD62L stained leukocytes were loaded with Dihydrorhodamine-123 (DHR-123) and stimulated with N-Formylmethionine-leucyl-phenylalanine (fMLF). Neutrophil subsets were characterized based on CD16 and CD62L expression. Marker surface expression was recorded on CD16+ neutrophils as median fluorescence intensity (MFI). Results Neutrophil surface expression of CD62L was significantly reduced in COPD (median (IQR) MFI: 1156 (904, 1365)) compared with asthma (1865 (1157, 2408)) and healthy controls (2079 (1054, 2960)); p=0.028. COPD neutrophils also demonstrated a significant reduction in CD62L expression with and without fMLF stimulation. Asthma participants had a significantly increased proportion and number of CD62Lbright/CD16dim neutrophils (median: 5.4% and 0.14 × 109/L, respectively), in comparison with healthy (3.54% and 0.12 × 109/L, respectively); p Conclusion Reduced CD62L expression suggests blood neutrophils have undergone priming in COPD but not in asthma, which may be the result of systemic inflammation. The increased shedding of CD62L receptor by COPD blood neutrophils suggests a high sensitivity for activation.

COPD is a seriously disabling respiratory condition that inexorably progresses to disability and mortality. It affects approximately 10% of the population globally with a greater prevalence at advanced ages. Airway bacterial infections complicate the disease course in most COPD patients, leading to increased symptoms, more rapid decline in lung function, acute exacerbations and reduced quality of life. With increasing bacterial resistance to antibiotics and adverse effects of conventional treatments, new effective non-antibiotic antimicrobial therapies are urgently needed to manage COPD. Hypoxia-inducible factor (HIF)-1α is an important transcriptional regulator of cellular responses to hypoxia, oxidants and inflammation, and is overexpressed in the lungs of COPD patients. Recent evidence shows that increased HIF-1α expression can upregulate the platelet-activating factor receptor (PAFR) on the airway epithelial surface that is increased in smokers and particularly COPD patients. The receptor is utilized by PAFR-dependent bacteria (Streptococcus pneumoniae, Haemophilus influenzae and Pseudomonas aeruginosa) to induce infection in both the respiratory and gastrointestinal (GI) tracts. However, the importance and mechanism of HIF-1α in augmenting PAFR-dependent bacterial infections in COPD are poorly understood. Here, we review the evidence for the roles of local tissue hypoxia-induced inflammation, HIF-1α and PAFR in facilitating bacterial infections in COPD. Blocking PAFR may provide a novel antimicrobial approach to manage bacterial infections in COPD.

Background Both obesity and high dietary fat intake activate the nucleotide oligomerization domain–like receptor protein 3 (NLRP3) inflammasome. Objective We aimed to examine NLRP3 inflammasome activity in the airways of obese asthmatic patients after macronutrient overload and in immune cells challenged by inflammasome triggers. Methods Study 1 was a cross-sectional observational study of nonobese (n = 51) and obese (n = 76) asthmatic adults. Study 2 was a randomized, crossover, acute feeding study in 23 asthmatic adults (n = 12 nonobese and n = 11 obese subjects). Subjects consumed 3 isocaloric meals on 3 separate occasions (ie, saturated fatty acid, n-6 polyunsaturated fatty acid, and carbohydrate) and were assessed at 0 and 4 hours. For Studies 1 and 2, airway inflammation was measured based on sputum differential cell counts, IL-1β protein levels (ELISA), and sputum cell gene expression (Nanostring nCounter). In Study 3 peripheral blood neutrophils and monocytes were isolated by using Ficoll density gradient and magnetic bead separation and incubated with or without palmitic acid, LPS, or TNF-α for 24 hours, and IL-1β release was measured (ELISA). Results In Study 1 NLRP3 and nucleotide oligomerization domain 1 (NOD1) gene expression was upregulated, and sputum IL-1β protein levels were greater in obese versus nonobese asthmatic patients. In Study 2 the saturated fatty acid meal led to increases in sputum neutrophil percentages and sputum cell gene expression of Toll-like receptor 4 (TLR4) and NLRP3 at 4 hours in nonobese asthmatic patients. In Study 3 neutrophils and monocytes released IL-1β when challenged with a combination of palmitic acid and LPS or TNF-α. Conclusion The NLRP3 inflammasome is a potential therapeutic target in asthmatic patients. Behavioral interventions that reduce fatty acid exposure, such as weight loss and dietary saturated fat restriction, warrant further exploration.

Background Severe asthma is a complex disease. Transcriptomic profiling has contributed to understanding the pathogenesis of asthma, especially type-2 inflammation. However, there is still poor understanding of non-type-2 asthma, and consequently there are limited treatment options. Objective The aim of this study was to identify differentially expressed genes (DEGs) and pathways in endobronchial biopsies associated with inflammatory phenotypes of severe asthma. Methods This cross-sectional study examined endobronchial biopsies from 47 adults with severe asthma (neutrophilic asthma (NA) n=9, eosinophilic asthma (EA) n=22 and paucigranulocytic asthma (PGA) n=16), and 13 healthy controls (HC). RNA was extracted and transcriptomic profiles generated (Illumina Humanref-12 V4) and analysed using GeneSpring GX14.9.1. Pathway identification using Ingenuity Pathway Analysis. Results NA had the most distinct profile, with signature of 60 top-ranked DEGs (FC >±2) including genes associated to innate immunity response, neutrophil degranulation, and IL-10 signalling. NA presented enrichment to pathways previously linked to neutrophilic inflammation; dendritic cell maturation, Th1, TREM1, inflammasome, Th17, and p38 MAPK, as well as novel links to neuroinflammation, NFAT, and PKCθ signalling. EA presented similar transcriptomic profiles to PGA and HC. Despite the higher proportion of bacterial colonisation in NA, no changes were observed in the transcriptomic profiles of severe asthma culture positive compared with severe asthma culture negative. Conclusions & clinical relevance NA features a distinct transcriptomic profile with seven pathways enriched in NA compared to EA, PGA and HC. All those with severe asthma had significant enrichment for SUMOylation, basal cell carcinoma signalling, and Wnt/β-catenin pathways compared to HC, despite high dose inhaled corticosteroids. These findings contribute to the understanding of mechanistic pathways in endobronchial biopsies associated with NA and identify potential novel treatment targets for severe asthma.

Monoclonal antibodies directed against interleukin (IL)‐5, such as mepolizumab and benralizumab, are an effective and established treatment for severe eosinophilic asthma. Here, we present a patient with eosinophilic asthma with a partial clinical response to mepolizumab initially, as measured by these biomarkers, who when investigated was found to have refractory airway eosinophilia. Escalation of the mepolizumab dose led to further but still only partial response. A treatment trial with benralizumab was more successful and led to suppression of airway eosinophilia. We review the literature, focusing on eosinophil biology at the tissues and the different mechanisms of action of the two agents., Monoclonal antibodies directed against interleukin (IL)‐5, such as mepolizumab and benralizumab, are an effective and established treatment for severe eosinophilic asthma. Here, we present a patient with eosinophilic asthma with a partial clinical response to mepolizumab, although with refractory airway eosinophilia, who did not fully respond to dose escalation. A treatment trial with benralizumab was more successful and led to suppression of airway eosinophilia.

Background Dysregulation of tumour necrosis factor-α (TNF-α) signalling is implicated in neutrophilic asthma. TNF-α signalling involves membrane-bound and soluble ligand (TNF-α) and receptors (TNFRs); however, little is known about how these proteins are altered in asthma. We hypothesised that intercompartment-, immune cell- and/or asthma inflammatory phenotype-dependent regulation could relate to TNF dysregulation in neutrophilic asthma. Methods Measurements were made in 45 adults with asthma (36 non-neutrophilic, 9 neutrophilic) and 8 non-asthma controls. Soluble TNF-α, TNF receptor 1 (TNFR1) and TNFR2 were quantified in plasma and sputum supernatant by ELISA, and membrane-bound TNF-α/TNFR1/TNFR2 measured on eosinophils, neutrophils, monocytes, and macrophages in blood and sputum by flow cytometry. Marker expression was compared between inflammatory phenotypes and compartments, and relationship of membrane-bound and soluble TNF markers and immune cell numbers tested by correlation. Results Soluble sputum TNFR1 and TNFR2 were increased in neutrophilic versus non-neutrophilic asthma (p=0.010 and p=0.029). Membrane-bound TNF-α expression was upregulated on sputum versus blood monocytes, while TNFR1 and TNFR2 levels were reduced on airway versus blood monocytes and neutrophils. Soluble TNFR1 and TNFR2 in sputum significantly correlated with the number of airway monocytes (p=0.016, r=0.358 and p=0.029, r=0.327). Conclusion Our results imply that increased sputum soluble TNF receptor levels observed in neutrophilic asthma relate to the increased recruitment of monocytes and neutrophils into the airways and their subsequent receptor shedding. Monocytes also increase TNF-α ligand expression in the airways. These results suggest an important contribution of airway monocytes to the altered inflammatory milieu in neutrophilic asthma., Monocytes modify the inflammatory milieu in neutrophilic asthma. Blood and sputum analysis reveal TNF pathway dysregulation relating to altered TNF-α ligand and receptor expression by monocytes and their increased airway recruitment in neutrophilic asthma. https://bit.ly/3onJ1s7

Introduction Asthma is a common, chronic lung disease marked by reversible airflow obstruction driven by inflammation. There is increasing evidence for a neutrophil-dominant inflammatory endotype1 with clear mechanistic potential for neutrophil elastase (NE) to cause clinical manifestations of disease. However, the relationship between neutrophil proteinases and asthma pathophysiology remains uncertain. We hypothesised that patients with a neutrophilic-endotype would have increased NE activity compared to other asthma endotypes and healthy smokers. Methods Patients with respiratory physician-confirmed asthma diagnosis were recruited. Controls without asthma were age-matched with normal lung function and no symptoms suggestive of asthma/alternative respiratory disease Asthma patients were divided into endotypes by analysis of sputum cellular composition as previously described.1 NE activity was measured in plasma using an ELISA based on specific fibrinogen-cleavage product Aα–Val360.2 Results Forty-five asthma patients (Mean (SD): age 60.6 years (10.3); 40% male) and 45 controls (age 60.2 years (10.1); 51% male) were recruited. Asthma patients had evidence of airflow obstruction (mean (SD): FEV1/FVC% predicted asthma 64.3 (11.7) versus controls 77.5 (6.3), p When considered together, patients with asthma had a significantly higher NE activity footprint than controls (mean (SD): asthma 12.7 nM (5.7), control 9.4 nM (3.1), p=0.0011) When divided into phenotypes, patients with neutrophil-dominant asthma (n=15) had increased activity footprint compared to a specifically age-matched sub-group of controls (n=15, Mean (SD): asthma 15.0 nM (5.9), control 8.9 nM (2.9), p=0.0034). There were no differences between the NE activity footprint comparing patients with eosinophilic (n=11, 11.4 nM (5.5) p>0.9999) or paucigranulocytic (n=14, 10.3 nM (5.4) p>0.9999) endotypes to controls. In neutrophilic asthma, there was no correlation between NE activity and neutrophils in sputum (%) (R2=0.02677, p=0.5601), age (R2=0.08626, p=0.2880), FEV1/FVC% (R2=0.04410, p=0.4911), or smoking exposure (R2=0.5515, p=0.4766). Conclusions These findings suggest that there is a differing systemic proteolytic enzyme activity in neutrophil–dominant asthma patients compared to controls and between neutrophilic asthma and paucigranulocytic asthma, which may influence disease pathophysiology. References Simpson JL, et al. ERJ 2014. Carter RI, et al. Thorax 2015.

Background The AMAZES randomized controlled trial demonstrated that long-term low-dose azithromycin treatment reduces exacerbations of poorly controlled asthma, but the therapeutic mechanisms remain unclear. Dysregulation of the inflammatory tumour necrosis factor (TNF) pathway is implicated in asthma and could be suppressed by azithromycin. We aimed to determine the inflammatory and clinical associations of soluble TNF signalling proteins (TNF receptors [TNFR] 1 and 2, TNF) in sputum and serum, and to test the effect of 48 weeks of azithromycin vs placebo on TNF markers. Methods Sputum supernatant and serum TNFR1, TNFR2 (n = 142; 75 azithromycin-treated, 67 placebo-treated) and TNF (n = 48; 22 azithromycin-treated, 26 placebo-treated) were measured by ELISA in an AMAZES trial sub-population at baseline and end of treatment. Baseline levels were compared between sputum inflammatory phenotypes, severe/non-severe asthma and frequent/non-frequent exacerbators. Effect of azithromycin on markers was tested using linear mixed models. Results Baseline sputum TNFR1 and TNFR2 were significantly increased in neutrophilic vs non-neutrophilic asthma phenotypes, while serum markers did not differ. Sputum TNFR1 and TNFR2 were increased in severe asthma and correlated with poorer lung function, worse asthma control and increasing age. Serum TNFR1 was also increased in severe asthma. Sputum and serum TNFR2 were increased in frequent exacerbators. Azithromycin treatment significantly reduced sputum TNFR2 and TNF relative to placebo, specifically in non-eosinophilic participants. Conclusions We demonstrate dysregulation of TNF markers, particularly in the airways, that relates to clinically important phenotypes of asthma including neutrophilic and severe asthma. Suppression of dysregulated TNF signalling by azithromycin could contribute to its therapeutic mechanism.

Introduction: Biomarkers are used to select patients for therapy in severe asthma, but not to regularly adjust therapy, especially oral corticosteroids (OCS). Our goal was to test the efficacy of an algorithm to guide the titration of OCS in adults with severe asthma using blood eosinophil count and fraction of exhaled nitric oxide levels (FeNO). Methods: This proof-of-concept randomized controlled trial assigned severe asthma participants (n=32) to an inflammation arm; where OCS dose was adjusted based on blood eosinophil count and FeNO, or a best-practice clinical care arm. The primary outcome was number of severe exacerbations and time to first severe exacerbation assessed over 12 months. Results: The relative risk of a severe exacerbation in the inflammation versus control arms was 0.88 (Adj.; 95%CI:0.47, 1.62; p=0.675) with a mean exacerbation rate per year of 2.5 and 3.5, respectively. There was a trend toward a longer median time to first severe exacerbation in the inflammation arm (73 vs. 32 days, Adj. HR:0.714; 95%CI:0.25, 2.06; p=0.533). The odds ratio of an emergency department (ED) or hospital admission in the inflammation management arm was 0.177 (Adj; 95%CI: 0.023, 1.361; p=0.0961). There was no significant difference in mean OCS dose used over the course of the study between the two groups, with both groups decreasing their doses by 0.1mg/day/visit. Conclusion: A treatment algorithm to adjust oral corticosteroids using blood eosinophil count and FeNO is feasible in a clinical setting and resulted in a longer time to exacerbation and reduced odds of a hospital admission or ED visit. This warrants further study to optimize the use of oral corticosteroids in the future.

Sputum mast cells associate with clinical and inflammatory features of asthma: Despite their known pathogenic role, the relationship of mast cells (MCs) to inflammatory and clinical features of asthma is poorly understood. We aimed to quantify sputum MCs and examine their relationship to airway and circulatory immune cells and clinical variables in asthma. We conducted flow cytometric analysis of induced sputum to quantify MCs, basophils and other immune cells (n=45 asthma, n=6 non-asthma). Relationship of MCs to sputum and blood (n=19) immune cells in asthma, participant demographics, asthma history, spirometry and dose response slope (DRS) to hypertonic saline was determined. Eosinophilic asthma (EA) was defined by ≥3% sputum eosinophils. Sputum MCs, basophils and eosinophils were significantly intercorrelated and increased in asthma vs non-asthma. MCs and basophils were elevated in EA. MCs and basophils, but not eosinophils, correlated with DRS. Cutoff-based comparison of MC high vs low asthma showed more uncontrolled asthma and reduced FEV1 and FVC in MC high asthma. Similar clinical associations with MC high status were observed in a subpopulation (n = 15) lacking airway eosinophilia or neutrophilia. ROC analysis showed peripheral blood eosinophil (PBE) count predicted elevated sputum eosinophils (AUC=86.7%, P=0.02) or basophils (AUC=82.8%, P=0.016), but not MCs (AUC=52.4%, P=0.87). Sputum MCs are elevated in asthma and they relate to key clinical features of asthma (asthma control, DRS, spirometry). Discord between MCs and PBE suggests MCs are regulated by discrete mechanisms to eosinophils. The clinical significance of elevated MCs in a subset of non-eosinophilic asthma merits further investigation.

Background Severe asthma is a complex heterogeneous disease typically requiring advanced therapies. Underlying the treatment of all asthma, however, is the consistent recommendation across international guidelines to ensure that adherence to therapy is adequate. Currently, there is no consensus on an objective marker of adherence. Methods We performed a prospective observational study of 17 participants taking oral prednisolone using serum prednisolone levels as a marker of adherence, and sputum eosinophilia as a marker of control of type 2 airway inflammation. Based on these biomarkers, we classified participants into a non-adherent and an adherent cohort, and further stratified by the presence of ongoing sputum eosinophilia. Results We identified 3 non-adherent participants and 14 who were adherent, based on their serum prednisolone levels. Stratification using sputum eosinophil counts identified one participant as having ongoing sputum eosinophilia in the setting of non-adherence, while six were identified as steroid resistant with ongoing sputum eosinophilia despite adherence to oral prednisolone therapy. Conclusion Serum prednisolone can be used an objective marker of adherence in those patients with severe asthma taking daily oral prednisolone. In combination with sputum eosinophil counts, a steroid resistant cohort can be distinguished from one with ongoing inflammation in the setting of non-adherence. This information can then be used by clinicians to differentiate the optimal next steps for treatment in these specific populations. Trial registration Participants were recruited as part of the Markers of Inflammation in the Management of Severe Asthma (MIMOSA) study, trial registration ACTRN12616001015437, 02 August 2016.

Summary Background Bronchiectasis is predominantly a neutrophilic inflammatory disease. There are no established therapies that directly target neutrophilic inflammation because little is understood of the underlying mechanisms leading to severe disease. Neutrophil extracellular trap (NET) formation is a method of host defence that has been implicated in multiple inflammatory diseases. We aimed to investigate the role of NETs in disease severity and treatment response in bronchiectasis. Methods In this observational study, we did a series of UK and international studies to investigate the role of NETs in disease severity and treatment response in bronchiectasis. First, we used liquid chromatography-tandem mass spectrometry to identify proteomic biomarkers associated with disease severity, defined using the bronchiectasis severity index, in patients with bronchiectasis (n=40) in Dundee, UK. Second, we validated these biomarkers in two cohorts of patients with bronchiectasis, the first comprising 175 patients from the TAYBRIDGE study in the UK and the second comprising 275 patients from the BRIDGE cohort study from centres in Italy, Spain, and UK, using an immunoassay to measure NETs. Third, we investigated whether pathogenic bacteria had a role in NET concentrations in patients with severe bronchiectasis. In a separate study, we enrolled patients with acute exacerbations of bronchiectasis (n=20) in Dundee, treated with intravenous antibiotics for 14 days and proteomics were used to identify proteins associated with treatment response. Findings from this cohort were validated in an independent cohort of patients who were admitted to the same hospital (n=20). Fourth, to assess the potential use of macrolides to reduce NETs in patients with bronchiectasis, we examined two studies of long-term macrolide treatment, one in patients with bronchiectasis (n=52 from the UK) in which patients were given 250 mg of azithromycin three times a week for a year, and a post-hoc analysis of the Australian AMAZES trial in patients with asthma (n=47) who were given 500 mg of azithromycin 3 times per week for a year. Findings Sputum proteomics identified that NET-associated proteins were the most abundant and were the proteins most strongly associated with disease severity. This finding was validated in two observational cohorts, in which sputum NETs were associated with bronchiectasis severity index, quality of life, future risk of hospital admission, and mortality. In a subgroup of 20 patients with acute exacerbations, clinical response to intravenous antibiotic treatment was associated with successfully reducing NETs in sputum. Patients with Pseudomonas aeruginosa infection had a lessened proteomic and clinical response to intravenous antibiotic treatment compared with those without Pseudomonas infections, but responded to macrolide therapy. Treatment with low dose azithromycin was associated with a significant reduction in NETs in sputum over 12 months in both bronchiectasis and asthma. Interpretation We identified NETs as a key marker of disease severity and treatment response in bronchiectasis. These data support the concept of targeting neutrophilic inflammation with existing and novel therapies. Funding Scottish Government, British Lung Foundation, and European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC).

Background: Bronchiectasis is predominantly a neutrophilic inflammatory disease. There are no established therapies that directly target neutrophilic inflammation because of a lack of understanding of the underlying mechanisms leading to severe disease. Neutrophil extracellular trap (NET) formation is a mode of host defence that has been implicated in multiple inflammatory diseases. Methods: We conducted a series of UK and international studies to investigate the role of NETs in disease severity and treatment response in bronchiectasis. Nano LC/MS-MS was used to identify proteomic markers associated with disease severity, defined using the bronchiectasis severity index (BSI) (n=40). These were validated in two cohorts (n=175 and n=275) using a validated immunoassay to measure NETs. 20 patients with acute exacerbations of bronchiectasis were treated with intravenous antibiotics for 14 days and proteomics used to identify proteins associated with treatment response. Two studies of long-term macrolide treatment, one in bronchiectasis and a post-hoc analysis of the AMAZES trial in asthma, investigated the effect of macrolide treatment on NETs. Findings: 96 proteins were differentially expressed in sputum between severe and mild bronchiectasis. The most abundant and most discriminating were proteins known to be NET proteins. This was validated in two observational cohorts where sputum NETs were associated with BSI, quality of life, lung function, future risk of hospital admissions and mortality. Antibiotic treatment in 20 patients was associated with 23 significantly down-regulated and 16 up-regulated proteins, with the “neutrophil degranulation” pathway being most strongly implicated in response. Patients with Pseudomonas aeruginosa infection had a blunted proteomic and clinical response to treatment. Treatment with low dose azithromycin was associated with a significant reduction in NETs in sputum over 12 months in both bronchiectasis and asthma. Interpretation: NETs are identified as a key marker of disease severity and treatment response in bronchiectasis. Funding: Supported by the Scottish Government Chief Scientist Office (Senior Clinical Fellowship to JDC) and British Lung Foundation through the BLF Chair of Respiratory Research. Supported by the European Bronchiectasis Network (EMBARC), a European Respiratory Society Clinical Research Collaboration. Declaration of Interests: None to declare Ethics Approval Statement: The studies were approved by local research ethics committees and patients provided informed consent.

Long-term, low-dose azithromycin reduces exacerbation frequency in chronic obstructive pulmonary disease (COPD), yet the mechanism remains unclear. This study characterised genome-wide gene expression changes in patients with neutrophilic COPD following long-term, low-dose azithromycin treatment. Patients with neutrophilic COPD (>61% or >162×104 cells per mL sputum neutrophils) were randomised to receive either azithromycin or placebo for 12 weeks. Sputum and blood were obtained before and after 12 weeks of treatment. Gene expression was defined using microarrays. Networks were analysed using the Search Tool for the Retrieval of Interacting Gene database. In sputum, 403 genes were differentially expressed following azithromycin treatment (171 downregulated and 232 upregulated), and three following placebo treatment (one downregulated and two upregulated) compared to baseline (adjusted p1.5). In blood, 138 genes were differentially expressed with azithromycin (121 downregulated and 17 upregulated), and zero with placebo compared to baseline (adjusted p1.3). Network analysis revealed one key network in both sputum (14 genes) and blood (46 genes), involving interferon-stimulated genes, human leukocyte antigens and genes regulating T-cell responses. Long-term, low-dose azithromycin is associated with downregulation of genes regulating antigen presentation, interferon and T-cell responses, and numerous inflammatory pathways in the airways and blood of neutrophilic COPD patients., Azithromycin modifies gene expression in COPD http://ow.ly/Mqpv30lLXUM

Background Both the six gene signature (6GS: CPA3, DNASE1L3, CLC, IL1B, ALPL, and CXCR2) and T‐helper 2 signature (TH2S: CLCA1, SERPINB2, and POSTN) are proposed as biomarkers in the identification of inflammatory phenotypes of asthma in induced sputum and epithelial brushings, respectively. The aim of this study was to explore patterns of gene expression of known signatures, 6GS and TH2S in endobronchial biopsies. Methods This was an exploratory cross‐sectional study of gene expression in endobronchial biopsies of 55 adults with asthma and 9 healthy controls (HC). The expression of the 6GS and TH2S was determined by quantitative polymerase chain reaction. Correlations with clinical and cellular characteristics were performed, and receiver operating characteristic was utilized to assess signatures' ability to predict asthma from HC and inflammatory phenotypes. Results Gene expression of DNASE1L3 (P = .045) was upregulated in asthma compared with HC, and IL1B (P = .017) was upregulated in neutrophilic asthma compared with non‐neutrophilic asthma. In asthma, the expression of CPA3 was negatively associated with ICS daily dose (r = −.339; P = .011), IL1B expression was positively associated with bronchial lavage fluid (BLF) total cell count (r = .340; P = .013) and both CLC and POSTN expression were associated with lymphocytes percentage in BLF (r = −.355, P = .009; r = −.300, P = .025, respectively). Both 6GS (area under curve [AUC] = 86.3%; P = .017) and TH2S (AUC = 72.7%; P = .037) could significantly predict asthma from HC. In addition, 6GS can identify neutrophilic (AUC = 93.2%; P = .005) and TH2S identifies eosinophilic (AUC = 62.7%; P = .033) asthma. Conclusions and Clinical Relevance There was increased expression of DNASE1L3 in asthma and IL1B in neutrophilic asthma. These results show similar upregulated patterns of expression in two genes of the 6GS in endobronchial biopsies, previously identified in sputum. The upregulation of DNASE1L3 and IL1B suggests that common mechanisms may be at play throughout the airway., Similar patterns of expression of known gene signatures were observed in endobronchial biopsies characterized by the upregulation of DNASE1L3 in asthma and IL1B in neutrophilic asthma suggesting these pathways are relevant in both biopsy and sputum in asthma.

Macrolide antibiotics have a range of immunomodulatory properties, which may influence clinical outcomes such as reduced exacerbations or attacks in patients with airways disease. While add-on azithromycin (AZM) reduces asthma attacks, the mechanisms remain unclear. We tested the hypothesis that add-on AZM therapy would reduce innate immune cytokines in sputum following 48 weeks of treatment. Induced sputum was collected at baseline and end of treatment from adults with symptomatic asthma in a randomized double-blind placebo controlled trial of oral AZM (500mg x 3 times weekly). Supernatant was assessed for IL-1β, IL-6, TNF-α and galectin-3 using ELISA and extracellular DNA (eDNA) using SYBR Green absorbance assay. Serum IL-6 was also assessed by ELISA. Despite no change in sputum cell counts, there were significantly lower levels of sputum IL-1β, TNF-α, IL-6 and eDNA following AZM therapy. This reduction was only observed in the non-eosinophilic participants with the exception of sputum IL-6, which was also reduced in those with eosinophilic asthma. Sputum galectin-3 and serum IL-6 were not altered with AZM therapy. AZM reduces innate immune inflammatory markers and eDNA but not systemic inflammation in poorly controlled asthma. Whether this is independent of changes in bacterial composition requires further study.

Introduction: Dysfunction of the bronchial epithelium plays an important role in asthma, however its measurement is challenging. Columnar epithelial cells are often quantified, yet rarely analyzed, in induced sputum studies. Hypothesis: We hypothesized that sputum columnar epithelial cell count would be associated with clinical and inflammatory variables in asthma, and that sputum analysis could provide a relatively non-invasive measure of airway epithelial activation status. Methods: We examined the relationship of sputum columnar epithelial cells with clinical and inflammatory variables of asthma in a retrospective cross-sectional cohort (901 participants with asthma and 138 healthy controls). In smaller cohorts we used flow cytometry, microarray, qPCR and ELISA to characterize sputum columnar epithelial cells and products. Results: Multivariate analysis and generation of 90th centile cut-offs (≥11% or ≥18.1x104/mL) for columnar epithelial cell “high” asthma revealed a significant relationship between elevated sputum columnar cells and male gender, severe asthma and non-neutrophilic airway inflammation. Flow cytometry showed viable columnar epithelial cells were present in all sputum samples tested. A gene signature (SCGB3A1, LDLRAD1, FOXJ1, DNALI1, CFAP157, CFAP53) was detected in columnar epithelial cell-high sputum. CLCA1 mRNA and periostin protein, previously identified biomarkers of IL-13-mediated epithelial activation, were elevated in columnar epithelial cell-high sputum samples, but only when accompanied by eosinophilia. Conclusions: Measurement of epithelial biomarkers in sputum samples has potential value in non-invasive assessment of altered bronchial epithelium status in asthma.

Introduction: Severe asthma (SA) is a complex disease with diverse clinical and inflammatory presentations. Current efforts focus on understanding mechanisms of pathogenesis associated with SA phenotypes in order to improve treatment responsiveness. Objectives: We aimed to identify mechanisms of pathogenesis by identifying differentially expressed genes (DEGs) from endobronchial biopsies in different granulocytic inflammatory phenotypes of SA. Methods: SA was defined according to ERS/ATS guidelines and inflammatory phenotypes based on cell count in in bronchial lavage fluid (BLF). Severe eosinophilic asthma (SEA):>3% eosinophils; severe neutrophilic asthma (SNA):>61% neutrophils; and paucigranulocytic (SPGA) Results: We identified 353 DEGs between SEA and HC. Top upregulated genes include NOS2A implicated in production of NO during inflammation, and AKRIC2 reported upregulated in smokers. We identified 550 DEGs between SNA and HC. Top genes include IDO1, SAA1 part of the acute phase response signalling, CD163 expressed by monocytes following stimuli as well as S100A9 previously reported for driving mucin5AC production in SA. We identified 165 DEGs between SPGA and HC. Top genes include DEFB1 and FKBP5 studied for its response following corticosteroids exposure in asthma. Conclusions: This study identified a number of distinct candidate genes playing potential roles in the pathogenesis and contribute to the understanding of mechanisms involved in these phenotypes.

Introduction: The regulation of adhesion molecules present on the surface of neutrophils plays an important role in their influx to the airways during inflammation. The pattern of this regulation and its association with neutrophil function is not well known in obstructive airway disease (OAD) and understanding these patterns may provide insight into neutrophil activation and recruitment to the airways. Aim: To measure blood neutrophil adhesion molecule (CD62L, CD11b, CD11c, CD54) surface expression in OAD and healthy participants. Methods: Peripheral, whole blood from 20 asthma, 16 COPD and 20 healthy participants was incubated with CD16-PE-Cy7, CD62L-BV421, CD11b-PE, CD11c-APC, and CD54-BV650 and cell suspension analysed on a Fortessa X20 cytometer. Marker surface expression was recorded on CD16+ neutrophils as median fluorescence intensity (MFI). Results: Both age and gender were similar between asthma (median age 65, with 55% males) and COPD (median age 72, with 50% males), while the healthy were predominantly females (75%) and were younger (median age 59) compared with COPD, p=0.003. The cell surface expression of CD62L was significantly reduced in COPD with a MFI (q1, q3) of 1138 (902, 1353) compared with asthma (1865 [1157, 2408]) and healthy controls (1946 [1041, 2688]), p=0.022. No significant differences were found for the other adhesion molecules tested. Conclusion: Blood neutrophils in COPD display reduced CD62L expression in comparison with asthma and healthy. The reduction in CD62L is suggestive of activation-associated shedding of CD62L, a process that takes place during the movement of neutrophils to the site of inflammation.

Introduction: The identification of asthma phenotypes is an evolving area. Analysis of sputum transcriptomics in U-BIOPRED defined three transcriptome-associated clusters (TACs) (Kuo C-HS, et al. Eur Respir J 2017) while Australia (NC-AU) proposed a six-gene signature (6GS) to identify inflammatory phenotypes (Baines K, et al. J Allergy Clin Immunol. 2014). Both studies identify different granulocytic inflammatory phenotypes in subjects with asthma in their corresponding populations. Objective: We aimed to assess the reproducibility of the TACs signatures in NC-AU cohort and the 6GS in U-BIOPRED. Methods: Microarray data from subjects with different granulocytic inflammatory phenotypes from two cohorts of asthma was analysed (U-BIOPRED: healthy control (HC) n=16; eosinophilic asthma (EA) n=42; neutrophilic asthma (NA) n=20 and paucigranulocytic asthma (PGA) n=26 | NC-AU: HC n=16; EA n=19; NA n=16; PGA n=18. We used gene set variation analysis to perform the comparison. Results: Adults with different granulocytic inflammatory phenotypes presented significantly different enrichment for the TACs and 6GS. In NC-AU, we confirmed that TAC1 signature was significantly enriched in EA compared to other phentoypes; TAC2 was enriched in NA compared to other phenotypes and HC; and TAC3 signature was significantly enriched in PGA compared to other phenotypes and HC. The inflammatory phenotypes from U-BIOPRED presented significant enrichment for the 6GS; EAvsNA(p Conclusion: Differences in gene expression in distinct inflammatory phenotypes can be reproduced in two distinct well-characterised asthma cohorts, providing validation across independent cohorts.

Background and objective Severe asthma is responsible for a disproportionate burden of illness and healthcare costs spent on asthma. This study analyses sputum transcriptomics to investigate the mechanisms and novel treatment targets of severe asthma. Methods Induced sputum samples were collected in a cross-sectional study from participants with severe asthma (n = 12, defined as per GINA criteria), non-severe uncontrolled (n = 21) and controlled asthma (n = 21) and healthy controls (n = 15). Sputum RNA was extracted and transcriptomic profiles were generated (Illumina HumanRef-8 V2) and analysed (GeneSpring). Sputum protein lysates were analysed for p38 activation in a validation study (n = 24 asthma, n = 8 healthy) by western blotting. Results There were 2166 genes differentially expressed between the four groups. In severe asthma, the expression of 1875, 1308 and 563 genes was altered compared to healthy controls, controlled and uncontrolled asthma, respectively. Of the 1875 genes significantly different to healthy controls, 123 were >2-fold change from which four networks were identified. Thirty genes (>2-fold change) were significantly different in severe asthma compared to both controlled asthma and healthy controls. There was enrichment of genes in the p38 signalling pathway that were associated with severe asthma. Phosphorylation of p38 was increased in a subset of severe asthma samples, correlating with neutrophilic airway inflammation. Conclusion Severe asthma is associated with substantial differences in sputum gene expression that underlie unique cellular mechanisms. The p38 signalling pathway may be important in the pathogenesis of severe asthma, and future investigations into p38 inhibition are warranted as a 'non-Th2' therapeutic option.

Background Dysfunction of the bronchial epithelium plays an important role in asthma; however, its measurement is challenging. Columnar epithelial cells are often quantified, yet rarely analysed, in induced sputum studies. Objective We aimed to test whether sputum columnar epithelial cell proportion and count are altered in asthma, and whether they are associated with clinical and inflammatory variables. We aimed to test whether sputum-based measures could provide a relatively non-invasive means through which to monitor airway epithelial activation status. Methods We examined the relationship of sputum columnar epithelial cells with clinical and inflammatory variables of asthma in a large retrospective cross-sectional cohort (901 participants with asthma and 138 healthy controls). In further studies, we used flow cytometry, microarray, qPCR and ELISA to characterize sputum columnar epithelial cells and their products. Results Multivariate analysis and generation of 90th centile cut-offs (≥11% or ≥18.1 × 104 /mL) to identify columnar epithelial cell "high" asthma revealed a significant relationship between elevated sputum columnar cells and male gender, severe asthma and non-neutrophilic airway inflammation. Flow cytometry showed viable columnar epithelial cells were present in all sputum samples tested. An epithelial gene signature (SCGB3A1, LDLRAD1, FOXJ1, DNALI1, CFAP157, CFAP53) was detected in columnar epithelial cell-high sputum. CLCA1 mRNA and periostin protein, previously identified biomarkers of IL-13-mediated epithelial activation, were elevated in columnar epithelial cell-high sputum samples, but only when accompanied by eosinophilia. Conclusions & clinical relevance Sputum columnar epithelial cells are related to important clinical and inflammatory variables in asthma. Measurement of epithelial biomarkers in sputum samples could allow non-invasive assessment of altered bronchial epithelium status in asthma.

Background Galectin-3 is a 32 kDa protein secreted by macrophages involved in processes such as cell activation, chemotaxis and phagocytosis. Galectin-3 has previously been shown to improve the ability of airway macrophages to ingest apoptotic cells (efferocytosis) in chronic obstructive pulmonary disease (COPD) and may be of interest in non-eosinophilic asthma (NEA) which is also characterised by impaired efferocytosis. It was hypothesised that the addition of exogenous galectin-3 to monocyte-derived macrophages (MDMs) derived from donors with NEA would enhance their ability to engulf apoptotic granulocytes. Methods Eligible non-smoking adults with asthma (n = 19), including 7 with NEA and healthy controls (n = 10) underwent a clinical assessment, venepuncture and sputum induction. MDMs were co-cultured with apoptotic granulocytes isolated from healthy donors with or without exogenous recombinant galectin-3 (50 μg/mL) and efferocytosis was assessed by flow cytometry. Galectin-3 expression and localisation in MDMs was visualised by immunofluorescence staining and fluorescence microscopy. Galectin-3, interleukin (IL)-6 and CXCL8 secretion were measured in cell culture supernatants by ELISA and cytometric bead array. Results Baseline efferocytosis (mean (±standard deviation)) was lower in participants with asthma (33.2 (±17.7)%) compared with healthy controls (45.3 (±15.9)%; p = 0.081). Efferocytosis did not differ between the participants with eosinophilic asthma (EA) (31.4 (±19.2)%) and NEA (28.7 (±21.5)%; p = 0.748). Addition of galectin-3 significantly improved efferocytosis in asthma, particularly in NEA (37.8 (±18.1)%) compared with baseline (30.4 (±19.7)%; p = 0.012). Efferocytosis was not associated with any of the clinical outcomes but was negatively correlated with sputum macrophage numbers (Spearman r = − 0.671; p = 0.017). Galectin-3 was diffusely distributed in most MDMs but formed punctate structures in 5% of MDMs. MDM galectin-3 secretion was lower in asthma (9.99 (2.67, 15.48) ng/mL) compared with the healthy controls (20.72 (11.28, 27.89) ng/mL; p = 0.044) while IL-6 and CXCL8 levels were similar. Conclusions Galectin-3 modulates macrophage function in asthma, indicating a potential role for galectin-3 to reverse impaired efferocytosis in NEA. Electronic supplementary material The online version of this article (10.1186/s12931-018-0967-9) contains supplementary material, which is available to authorized users.

Viral respiratory infections trigger severe exacerbations of asthma, worsen disease symptoms, and impair lung function. To investigate the mechanisms underlying viral exacerbation, we established a mouse model of respiratory syncytial virus (RSV)–induced exacerbation after allergen sensitization and challenge. RSV infection of OVA-sensitized/challenged BALB/c mice resulted in significantly increased airway hyperresponsiveness (AHR) and macrophage and neutrophil lung infiltration. Exacerbation was accompanied by increased levels of inflammatory cytokines (including TNF-α, MCP-1, and keratinocyte-derived protein chemokine [KC]) compared with uninfected OVA-treated mice or OVA-treated mice exposed to UV-inactivated RSV. Dexamethasone treatment completely inhibited all features of allergic disease, including AHR and eosinophil infiltration, in uninfected OVA-sensitized/challenged mice. Conversely, dexamethasone treatment following RSV-induced exacerbation only partially suppressed AHR and failed to dampen macrophage and neutrophil infiltration or inflammatory cytokine production (TNF-α, MCP-1, and KC). This mimics clinical observations in patients with exacerbations, which is associated with increased neutrophils and often poorly responds to corticosteroid therapy. Interestingly, we also observed increased TNF-α levels in sputum samples from patients with neutrophilic asthma. Although RSV-induced exacerbation was resistant to steroid treatment, inhibition of TNF-α and MCP-1 function or depletion of macrophages suppressed features of disease, including AHR and macrophage and neutrophil infiltration. Our findings highlight critical roles for macrophages and inflammatory cytokines (including TNF-α and MCP-1) in viral-induced exacerbation of asthma and suggest examination of these pathways as novel therapeutic approaches for disease management.

Background and objective Neutrophil extracellular traps (NETs) are web-like structures comprising DNA and antimicrobial proteins, expelled from neutrophils during NETosis. Persistence of NETs can be pro-inflammatory, yet their role in respiratory disease remains unclear. This study aimed to investigate the presence of NETs in sputum from patients with asthma and COPD, and the relationship of NETs with inflammatory phenotype and disease severity. Methods Induced sputum was collected from healthy controls, asthma and COPD patients. Extracellular DNA (eDNA) was quantified by PicoGreen. LL-37, α-defensins1–3, NE, IL-1β and CXCL8 were quantified by ELISA. PAD4 and NLRP3 gene expression was performed using qPCR. NETs were imaged in sputum smears using immunofluorescence microscopy. Results Sputum eDNA and NET neutrophil antimicrobial proteins were significantly elevated in asthma and COPD compared with healthy controls. Levels of eDNA and NET components were significantly higher in neutrophilic versus non-neutrophilic asthma and COPD. NETs were clearly visualized in sputum smears. PAD4 mRNA was upregulated in neutrophilic COPD. The level of eDNA was higher in severe asthma. High eDNA levels were associated with heightened innate immune responses, including elevated CXCL8 and IL-1β, and NLRP3 gene expression in both COPD and asthma. Antimicrobial proteins and eDNA were positively correlated with airway neutrophils, and negatively correlated with lung function and symptoms. Conclusion NETs are present in the airways of subjects with asthma and COPD. Accumulation of excessive NETs was associated with activation of innate immune responses contributing to disease pathogenesis in chronic airway disease.

Background and objective Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and inflammation. Airway bacterial colonization is increased in COPD; however, the role of potentially pathogenic and non-pathogenic bacteria in the pathogenesis of disease is unclear. This study characterized the presence of bacteria in a well-characterized cohort of adults with COPD and healthy controls. Methods Adults with COPD (n = 70) and healthy controls (n = 51) underwent clinical assessment and sputum induction. Sputum was dispersed, and total and differential cell counts were performed. Bacteria were cultured, identified and enumerated. Supernatants were assessed for neutrophil elastase (NE) and IL-1β. Common respiratory pathogens were also determined using real-time PCR. Results Participants with COPD had a typical neutrophilic inflammatory profile. The total load of bacteria was increased in COPD and was associated with poorer respiratory health status, as measured by the St George's Respiratory Questionnaire (Spearman's r = 0.336, P = 0.013). Significantly lower levels of culturable Bacillus species were identified compared with healthy controls. PCR analyses revealed increased rates of detection of potentially pathogenic bacteria with Haemophilus influenzae detection associated with higher sputum levels of NE and IL-1β, while Streptococcus pneumoniae was more common in male ex-smokers with emphysema and a deficit in diffusion capacity. Conclusion Non-pathogenic and pathogenic bacteria were altered in the sputum of patients with COPD. These observations highlight the potential to identify treatment and management strategies that both target specific bacterial pathogens and restore the microbial balance, which may lead to reductions in inflammation and subsequent improvements in lung health.

Protracted bacterial bronchitis (PBB) in young children is characterised by prolonged wet cough, prominent airway interleukin (IL)-1β expression and infection, often with nontypeable Haemophilus influenzae (NTHi). The mechanisms responsible for IL-1-driven inflammation in PBB are poorly understood. We hypothesised that the inflammation in PBB involves the NLRP3 and/or AIM2 inflammasome/IL-1β axis. Lung macrophages obtained from bronchoalveolar lavage (BAL), peripheral blood mononuclear cells (PBMCs), blood monocytes and monocyte-derived macrophages from patients with PBB and age-matched healthy controls were cultured in control medium or exposed to live NTHi. In healthy adult PBMCs, CD14+ monocytes contributed to 95% of total IL-1β-producing cells upon NTHi stimulation. Stimulation of PBB PBMCs with NTHi significantly increased IL-1β expression (p, Airway IL-1β activation in protracted bacterial bronchitis http://ow.ly/ut9r30iqim2

Mast cells are a resident inflammatory cell of the airways, involved in both the innate and adaptive immune response. The relationship between mast cells and inflammatory phenotypes and treatment response of asthma is not clear.Clinical characteristics of subjects with stable asthma (n=55), inflammatory cell counts and gene expression microarrays in induced sputum were analysed. Sputum mast cell subtypes were determined by molecular phenotyping based on expression of mast cell biomarkers (tryptase (TPSAB1), chymase (CMA1) and carboxypeptidase A3 (CPA3)). Effects of mast cell subtypes on steroid response were observed in a prospective cohort study (n=50).MCT (n=18) and MCT/CPA3 (mRNA expression of TPSAB1 and CPA3; n=29) subtypes were identified, as well as a group without mast cell gene expression (n=8). The MCT/CPA3 subtype had elevated exhaled nitric oxide fraction, sputum eosinophils, bronchial sensitivity and reactivity, and poorer asthma control. This was accompanied by upregulation of 13 genes. Multivariable logistic regression identified CPA3 (OR 1.21, p=0.004) rather than TPSAB1 (OR 0.92, p=0.502) as a determinant of eosinophilic asthma. The MCT/CPA3 subtype had a better clinical response and reduced signature gene expression with corticosteroid treatment.Sputum mast cell subtypes of asthma can be defined by a molecular phenotyping approach. The MCT/CPA3 subtype demonstrated increased bronchial sensitivity and reactivity, and signature gene expression, which was associated with airway eosinophilia and greater corticosteroid responsiveness.

Background and objective Neutrophilic inflammation has been implicated in non-eosinophilic asthma (NEA) in adults, but little is known about NEA in children/adolescents. We assessed clinical and inflammatory characteristics of NEA in adolescent asthma. Methods Airway inflammation, sputum endotoxin, airway hyper-reactivity, atopy and lung function were assessed in 77 adolescents with asthma and 68 without asthma (12–17 years). Asthma was identified on the basis of wheeze and asthma history. Results The proportion of NEA (sputum eosinophils

Introduction Sputum colour is associated with neutrophilic inflammation in chronic bronchitis and chronic obstructive pulmonary disease (COPD). Neutrophilia and sputum expectoration is notable in asthma, but whether sputum colour is associated with and predicts the presence of neutrophilic inflammation in asthma is unknown. The objective of the study is to assess the ability of sputum colour in distinguishing asthma inflammatory phenotypes. Methods Induced sputum samples collected from 271 adults with stable asthma were retrospectively assessed. Sputum colour was determined using the BronkoTest sputum colour chart and correlated to differential cell counts and CXCL-8 concentration. Neutrophilic inflammation was defined as an age-corrected sputum neutrophil proportion (≥61.6% for age 20–40 years; ≥63.2% for age 40–60 and ≥67.2% for age >60 years), whereas neutrophilic bronchitis (NB) was defined as high total cell count (≥5.1×106 cells/mL) plus an increased age-corrected neutrophil proportion. The optimal cut-off for sputum colour to predict neutrophilic inflammation and NB was determined using receiver operator characteristic curve analysis. Results A sputum colour score of ≥3 represented and predicted neutrophilic inflammation with modest accuracy (area under the curve (AUC)=0.64; p

Macrolide antibiotics have a range of immunomodulatory properties, which may influence clinical outcomes such as reduced exacerbations in patients with airways disease. Azithromycin (AZM) has been shown to reduce IL-1β release from blood monocytes, however little is known about the impact of AZM therapy on airway IL-1β. We tested the hypothesis that AZM would reduce sputum IL-1β and that this reduction would be associated with a reduction in sputum neutrophils. Induced Sputum collected from a randomized double-blind placebo controlled trial of oral AZM (500mg, 3 times weekly, 48 weeks) in adults with symptomatic asthma was dispersed using dithiothreitol and supernatant collected and assessed for IL-1β using ELISA in 195 paired samples. There was a significant reduction in sputum IL-1β following AZM therapy (median 398ng/mL at baseline to 168ng/mL following treatment, p=0.003). A significant reduction was observed in both eosinophilic (EA) and non-eosinophilic (NEA) participants, however, those with NEA had a three-fold larger reduction in IL-1β following AZM compared with EA, median change 234 ng/mL vs. 72 ng/mL, p=0.028. Baseline IL-1β was significantly positively associated with baseline neutrophil number in all participants (r = 0.499, p Sputum IL-1β was significantly reduced following AZM add-on therapy in poorly controlled asthma irrespective of baseline asthma phenotype.

Analysis of the proportions of eosinophils and neutrophils in induced sputum of asthmatic patients allows inflammatory phenotyping which can inform prognosis and treatment. Other immune cell types (e.g. monocytes, mast cells) are present in the airway but are not easily or commonly quantified by conventional analyses. Therefore more extensive enumeration of sputum immune cell populations could identify novel (and refine existing) inflammatory phenotypes in asthma. We evaluated the suitability of flow cytometry (FC) as a tool for improved sputum cell quantification, and tested the suitability of FC-based cell sorting to purify subpopulations of cells for downstream functional analyses. Sputum cell suspensions were prepared using conventional dispersion methods and stained (panel: CD45-APC, CD16-PE, CD14-FITC, CD206-BV421, HLA-DR-BV786, viability dye 7-AAD). Analysis was performed using a BD FACSAria III and cell sorting was performed on suitable samples. Sorted cell populations were prepared by cytospin for quantification by microscope. FC-based quantification of macrophages, neutrophils, eosinophils and lymphocytes showed agreement with that obtained by conventional microscopy (Bland-Altman analysis). Cell sorting of macrophages, neutrophils and monocytes yielded highly pure populations. We conclude FC provides a suitable and flexible platform for extensive quantification of sputum immune cell populations and downstream functional analyses. Cell sorting was successful when performed on relatively abundant cell types macrophages, neutrophils and monocytes. Further studies will quantify immune cells overlooked by microscope analysis and correlate these with clinical and inflammatory aspects of asthma.

Background: Interleukin-27 (IL-27) may contribute to steroid-resistant asthma, however, the mechanisms mediating these effects and association of IL-27 with asthma endotypes are unclear. AIM: The study aims to examine sputum IL-27 mRNA and protein levels in asthma endotypes and inflammation. Methods: Induced sputum was collected from 80 adults with stable asthma; 30 neutrophilic asthma (NA), 30 eosinophilic asthma (EA) and 20 paucigranulocytic asthma (PGA). Total and differential cell counts, mRNA and protein expression of IL-27 and TNF-α were assessed. Results: Patients had a mean age of 55 years and 59% were female. Gene expression for IL-27EBI3 and TNF-α were increased in NA compared with EA and PGA (p 1 % predicted (r=-0.28, p=0.04). No significant correlation existed between mRNA and protein levels for IL-27 or TNF-α (p>0.05). Conclusion: Lack of any correlation between IL-27R, IL-27p28 and IL-27EBI3 mRNA with IL-27 protein suggests differential translation of IL-27 in asthma endotypes. Association between eosinophil% and IL-27 protein suggests a functional role of IL-27 in EA.

Sputum basophil numbers are increased in allergic asthmatics, but it is unclear what role airway basophils play in "TH2-low" asthma phenotypes. Using flow cytometry, we found that basophils were significantly increased in all asthmatics (n=26) compared with healthy controls (n=8) (P=0.007) with highest levels observed in eosinophilic asthma (EA); median 0.22%, IQR 0.11%-0.47%; n=14) compared with non-EA (NEA) (0.06%, 0.00%-0.20%; n=12; P