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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 with 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 colonization 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. [ABSTRACT FROM AUTHOR]

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 H aemophilus influenzae detection associated with higher sputum levels of NE and IL-1β, while S treptococcus 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. [ABSTRACT FROM AUTHOR]

Background: Asthma is a heterogeneous chronic inflammatory disease in which host defense against respiratory viruses such as human rhinovirus (HRV) may be abnormal. This is a matter of some controversy, with some investigators reporting reduced type I interferon (IFN) synthesis and others suggesting that type I IFN synthesis is relatively normal in asthma.Objective: The objective of this study was to examine the responsiveness of circulating mononuclear cells to HRV in a large cohort of participants with poorly controlled asthma and determine whether IFN-α and IFN-β synthesis varies across different inflammatory phenotypes.Methods: Eligible adults with asthma (n = 86) underwent clinical assessment, sputum induction, and blood sampling. Asthma inflammatory subtypes were defined by sputum cell count, and supernatant assessed for IL-1β. Peripheral blood mononuclear cells (PBMCs) were exposed to HRV serotype 1b, and IFN-α and IFN-β release was measured by enzyme-linked immunosorbent assay.Results: Participants (mean age, 59 years; atopy, 76%) had suboptimal asthma control (mean asthma control questionnaire 6, 1.7). In those with neutrophilic asthma (n = 12), HRV1b-stimulated PBMCs produced significantly less IFN-α than PBMCs from participants with eosinophilic (n = 35) and paucigranulocytic asthma (n = 35). Sputum neutrophil proportion and the dose of inhaled corticosteroids were independent predictors of reduced IFN-α production after HRV1b exposure.Conclusions: Antiviral type I IFN production is impaired in those with neutrophilic airway inflammation and in those prescribed high doses of inhaled corticosteroids. Our study is an important step toward identifying those with poorly controlled asthma who might respond best to inhaled IFN therapy during exacerbations. [ABSTRACT FROM AUTHOR]

Background: Chronic Obstructive Pulmonary Disease (COPD) is a progressive airway disease characterised by neutrophilic airway inflammation or bronchitis. Neutrophilic bronchitis is associated with both bacterial colonisation and lung function decline and is common in exacerbations of COPD. Despite current available therapies to control inflammation, neutrophilic bronchitis remains common. This study tested the hypothesis that azithromycin treatment, as an add-on to standard medication, would significantly reduce airway neutrophil and neutrophils chemokine (CXCL8) levels, as well as bacterial load. We conducted a randomised, double-blind, placebo-controlled study in COPD participants with stable neutrophilic bronchitis. Methods: Eligible participants (n = 30) were randomised to azithromycin 250 mg daily or placebo for 12 weeks in addition to their standard respiratory medications. Sputum was induced at screening, randomisation and monthly for a 12 week treatment period and processed for differential cell counts, CXCL8 and neutrophil elastase assessment. Quantitative bacteriology was assessed in sputum samples at randomisation and the end of treatment visit. Severe exacerbations where symptoms increased requiring unscheduled treatment were recorded during the 12 week treatment period and for 14 weeks following treatment. A sub-group of participants underwent chest computed tomography scans (n = 15). Results: Nine participants with neutrophilic bronchitis had a potentially pathogenic bacteria isolated and the median total bacterial load of all participants was 5.22×107 cfu/mL. Azithromycin treatment resulted in a non-significant reduction in sputum neutrophil proportion, CXCL8 levels and bacterial load. The mean severe exacerbation rate was 0.33 per person per 26 weeks in the azithromycin group compared to 0.93 exacerbations per person in the placebo group (incidence rate ratio (95%CI): 0.37 (0.11,1.21), p = 0.062). For participants who underwent chest CT scans, no alterations were observed. Conclusions: In stable COPD with neutrophilic bronchitis, add-on azithromycin therapy showed a trend to reduced severe exacerbations sputum neutrophils, CXCL8 levels and bacterial load. Future studies with a larger sample size are warranted. Trial Registration: Australian New Zealand Clinical Trials Registry ACTRN12609000259246 [ABSTRACT FROM AUTHOR]

Background and objective The non-eosinophilic phenotype of asthma ( NEA) is associated with chronic airway inflammation and airway neutrophilia. An accumulation of apoptotic airway epithelial cells, if not efficiently cleared by efferocytosis, can undergo secondary necrosis, with the potential for inflammation of surrounding tissues. Apoptosis may occur via the T cell granzyme B pathway. The role of granzyme B in NEA is not known. The aim of this study was to investigate production of granzyme B and its inhibitor proteinase inhibitor ( PI)-9 by T cells from induced sputum and compare expression between eosinophilic, NEA and healthy controls. Methods We investigated T cell intracellular granzyme B and its inhibitor, PI-9, in sputum from healthy control subjects ( n = 10), and patients with NEA ( n = 22) or eosinophilic asthma ( EA) ( n = 15) using flow cytometry. Results Granzyme B expression and the ratio of granzyme B to PI-9 positive cells were highest in those with NEA for both CD3+ and CD4+ T cells. The expression of granzyme B was not statistically different between patients with NEA and EA; however, the ratio of granzyme B to PI-9 positive cells for CD3+ T cells was significantly higher in those with NEA compared with EA. Conclusions Induced sputum provides a non-invasive tool for investigating T cell cytotoxic mediators in the various asthma subtypes. Granzyme B expression is increased in NEA and the contribution of granzyme B to chronic inflammation requires further study. [ABSTRACT FROM AUTHOR]

Galectins constitute an evolutionary conserved family that bind to β-galactosides. Increasing evidence shows that galectins are involved in many fundamental biological processes such as cellular communication, inflammation, differentiation and apoptosis. Changes in galectin-3 (Gal-3) expression are commonly seen in cancer and pre-cancerous conditions, and Gal-3 may be involved in the regulation of diverse cancer cell activities that contribute to tumourigenesis, cancer progression and metastasis. In addition, Gal-3 is a pro-inflammatory regulator in rheumatoid arthritis. Gal-3 has been shown to be involved in many aspects in allergic inflammation, such as eosinophil recruitment, airway remodeling, development of a Th2 phenotype as well as increased expression of inflammatory mediators. In an in vivo model it was shown that bronchoalveolar lavage (BAL) fluid from ovalbumin-challenged mice contained significantly higher levels of Gal-3 compared to control mice. The molecular mechanisms of Gal-3 in human asthma have not been fully elucidated. This review will focus on what is known about the Gal-3 and its role in the pathophysiological mechanisms of asthma to evaluate the potential of Gal-3 as a biomarker and therapeutic target of asthma. [ABSTRACT FROM AUTHOR]

Objectives Outcome assessment is an important part of the management of airways disease, yet older adults may have difficulty with the burden of testing. This study evaluated the patient perception of tests used for the assessment of airways disease in older people. Data Source Older adults (>55 years) with obstructive airway disease and healthy controls ( N = 56) underwent inhaler technique assessment, skin allergy testing, venepuncture, fractional exhaled nitric oxide ( FENO) and gas diffusion measurement, exercise testing, sputum induction, and questionnaire assessment. They then completed an assessment burden questionnaire across five domains: difficulty, discomfort, pain, symptoms and test duration. Results Test perception was generally favourable. Induced sputum had the greatest test burden perceived as being more difficult (mean 0.83, P = 0.001), associated with more discomfort (mean 1.3, P < 0.001), more painful (0.46, P = 0.019), longer test duration (0.84, P < 0.001) and worsening symptoms (0.55, P = 0.001) than the questionnaires. FENO had a more favourable assessment but was assessed to be difficult to perform. Inhaler technique received the most favourable assessment. Conclusions Older adults hold favourable perceptions to a range of tests that they might encounter in the course of their care for airway disease. The newer tests of sputum induction and FENO have some observed difficulties, in particular sputum induction. The results of this study can inform current practice by including details of the test and its associated adverse effects when conducting the test, as well as providing clear explanations of the utility of tests and how the results might aid in patient care. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is a common and serious respiratory disease, particularly in older individuals, characterised by fixed airway obstruction and persistent airway neutrophilia. The mechanisms that lead to these features are not well established. We investigated the contribution of age, prior smoking, and fixed airflow obstruction on sputum neutrophils, TLR2 expression, and markers of neutrophilic inflammation. Induced sputum from adults with COPD (n = 69) and healthy controls (n = 51) was examined. A sputum portion was dispersed, total, differential cell count and viability recorded, and supernatant assayed for CXCL8, matrix metalloproteinase- (MMP-) 9, neutrophil elastase, and soluble TLR2. Peripheral blood cells (n = 7) were stimulated and TLR2 activation examined. TLR2 levels were increased with ageing, while sputum neutrophils and total sputum MMP-9 levels increased with age, previous smoking, and COPD. In multivariate regression, TLR2 gene expression and MMP-9 levels were significant independent contributors to the proportion of sputum neutrophils after adjustment for age, prior smoking, and the presence of airflow obstruction. TLR2 stimulation led to enhanced release of MMP-9 from peripheral blood granulocytes. TLR2 stimulation activates neutrophils for MMP-9 release. Efforts to understand themechanisms of TLR2 signalling and subsequent MMP-9 production in COPD may assist in understanding neutrophilic inflammation in COPD. [ABSTRACT FROM AUTHOR]

Background 20e30% of patients with asthma have neutrophilic airway inflammation and reduced responsiveness to steroid therapy. They often have chronic airway bacterial colonisation and Haemophilus influenzae is one of the most commonly isolated bacteria. The relationship between chronic airway colonisation and the development of steroid-resistant neutrophilic asthma is unclear. Objectives To investigate the relationship between H influenzae respiratory infection and neutrophilic asthma using mouse models of infection and ovalbumin (OVA)- induced allergic airways disease. Methods BALB/c mice were intratracheally infected with H influenzae (day 10), intraperitoneally sensitised (day 0) and intranasally challenged (day 12e15) with OVA. Treatment groups were administered dexamethasone intranasally during OVA challenge. Infection, allergic airways disease, steroid sensitivity and immune responses were assessed (days 11, 16 and 21). Results The combination of H influenzae infection and allergic airways disease resulted in chronic lung infection that was detected on days 11, 16 and 21 (21, 26 and 31 days after infection). Neutrophilic allergic airways disease and T helper 17 cell development were induced, which did not require active infection. Importantly, all features of neutrophilic allergic airways disease were steroid resistant. Toll-like receptor 4 expression and activation of phagocytes was reduced, but most significantly the influx and/or development of phagocytosing neutrophils and macrophages into the airways was inhibited. Conclusions The combination of infection and allergic airways disease promotes bacterial persistence, leading to the development of a phenotype similar to steroidresistant neutrophilic asthma and which may result from dysfunction in innate immune cells. This indicates that targeting bacterial infection in steroid-resistant asthma may have therapeutic benefit. INSET: Key messages. [ABSTRACT FROM AUTHOR]

Background The well-characterised airway inflammatory phenotypes of asthma include eosinophilic, neutrophilic, mixed eosinophilic/neutrophilic and paucigranulocytic asthma, identified based on the proportion of sputum granulocytes. Systemic inflammation is now recognised as an important part of some airway diseases, but the involvement of systemic inflammation in the pathogenesis of airway inflammatory phenotypes of asthma remains unknown. Methods Induced sputum samples and peripheral blood were collected from participants with asthma (n¼36). Airway inflammatory cell counts were performed from induced sputum and inflammatory phenotype assigned based on the airway eosinophil and neutrophil cut-offs of 3% and 61%, respectively. RNA was extracted from whole blood and gene expression profiles were generated (Illumina Humanref-8 V3) and analysed using GeneSpring GX11. Results There were six genes classified as differentially expressed between the four asthma phenotypes, including the a-defensins (DEFA) 1, 1B, 3 and 4 and neutrophil proteases cathepsin G (CTSG) and elastase (ELA2). Systemic expression of DEFA1,1B,3,4,CTSG and ELA2 was significantly higher in the neutrophilic asthma (NA) phenotype. Microarray results of the a-defensins and neutrophil proteases were successfully validated using real-time PCR. Plasma elastase was significantly increased in people with neutrophilic airway inflammation. Conclusion There is systemic upregulation of a-defensin and neutrophil protease expression in NA, which may represent proinflammatory effects on the bone marrow and the release of immature neutrophils into the circulation. This demonstrates a systemic inflammatory component in NA that further differentiates this from other asthma phenotypes and indicates different mechanisms in NA. [ABSTRACT FROM AUTHOR]

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection- induced neutrophilic inflammation in AAD is mediated by IL-17 responses. [ABSTRACT FROM AUTHOR]

Background: the diagnosis and management of obstructive airway diseases (OADs) such as asthma and chronic obstructive pulmonary disease (COPD) can be challenging in older people.Objective: to assess the clinical, functional, biological and behavioural characteristics relevant to the management of older people with OAD.Methods: a cross-sectional study was conducted in a tertiary teaching hospital. Older people (>55 years) (n = 100) with an OAD underwent a multidimensional assessment (MDA) involving questionnaires, clinical assessments, physiological measurements and biomarkers.Results: the assessment identified a mean (SD) of 11.3 (2.5) clinical management issues and 3.1 (1.8) comorbid conditions per participant. Common problems were: airways hyper-responsiveness (80%); airway inflammation (74%); activity limitation (74%) and systemic inflammation (60.5%). The number and type of issues were similar irrespective of a diagnosis of asthma or COPD (P = 0.2). The degree of health status impairment correlated significantly with the number of clinical management issues detected (r = 0.59; P < 0.0001).Conclusions: older people with OAD experience multiple clinical issues that adversely impact their health status. The number and type are similar irrespective of diagnosis. This MDA identifies significant clinical issues that may not be addressed in a diagnosis centred approach suggesting that a multidisciplinary approach is necessary when assessing and managing older people with OAD. [ABSTRACT FROM AUTHOR]

Potential bacterial pathogens are found in the airways in several diseases that are associated with neutrophilic inflammation. The aim of this study was to characterize subjects with stable asthma, with no symptoms of respiratory infection, to assess whether key potentially pathogenic bacteria were present in significant quantities in the airways and to correlate this with the pattern of airway inflammation and oxidative stress. Subjects with stable asthma ( n = 115) and healthy controls ( n = 8) underwent clinical assessment, including hypertonic saline challenge combined with sputum induction. A significant load of potentially pathogenic bacteria (> 106 cfu/mL) was cultured from the sputum of 17 (15%) subjects with stable asthma and was associated with higher total cell counts, proportion and number of neutrophils, sputum IL-8 and 8-isoprostane concentrations. The role of bacteria in potentiating neutrophilic asthma warrants further investigation. Therapies such as antibiotic and antioxidant treatment may be most effective in this sub-group of patients. [ABSTRACT FROM AUTHOR]

Background: The assessment of eosinophilic airway inflammation using induced sputum identifies a corticosteroid-responsive subtype that can be used to guide anti-inflammatory therapy. The stability of airway inflammation in asthma over time is not known, yet this information is crucial to inflammation-based patient management. Objective: The aim of this study was to determine the reproducibility of non-eosinophilic asthma. Methods: Participants with stable asthma (n = 26) underwent a sputum induction each month for 5 months. Sputum was dispersed and differential cell counts were performed. The reproducibility of inflammatory subtype with different eosinophil cut points (starting at 1% eosinophils) was examined and the minimum number of visits required to determine inflammatory subtype was calculated. Results: One hundred and twenty- two sputum samples were obtained (success 94%). All cut points greater than 2% eosinophils were reproducible and a 3% cut point resulted in the highest agreement with a κ statistic of 0.538. Specificity and sensitivity were high for determining inflammatory subtype after 1 or 3 sputum samples. Conclusions: A cut point of 3% eosinophils should be used to distinguish eosinophilic from non-eosinophilic airway inflammation in asthma. This allows a single sputum sample to be used to reliably determine the presence of eosinophilia. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Oncostatin M, a unique member of the interleukin (IL)-6 cytokine family, is thought to be involved in airway remodeling. The expression of oncostatin M in the lower airways is unknown. The aim of this study was to measure the sputum expression of oncostatin M in patients with asthma with and without irreversible airflow obstruction. Induced sputum was collected from nonsmoking adults with stable asthma (n = 53), 31 with incomplete reversibility of airflow obstruction. Peripheral blood cells were isolated and stimulated with lipopolysaccharide in 10 participants with asthma and irreversible airflow obstruction. Oncostatin M protein levels were determined in supernatant, whereas RNA was extracted to determine Oncostatin M mRNA expression using real-time polymerase chain reaction (PCR). Oncostatin M mRNA expression and protein levels were significantly higher in the sputum of asthmatics with irreversible airflow obstruction. Sputum oncostatin M levels were highest in people with severe airflow obstruction and were localized to airway neutrophils and macrophages. Peripheral blood neutrophils released more oncostatin M when stimulated with lipopolysaccharide compared with unstimulated neutrophils. Sputum oncostatin M is increased in asthma with irreversible airflow obstruction and is present in airway neutrophils and macrophages. Oncostatin M may link airway inflammation to remodeling in asthma. [ABSTRACT FROM AUTHOR]

Abstract: Obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are major global health issues. Although considered as distinct diseases, airway inflammation is a key underlying pathophysiological process in asthma, COPD and bronchiectasis. Persistent neutrophilic airway inflammation (neutrophilic bronchitis) occurs with innate immune activation and is a feature of each of these airway diseases. Little is known about the mechanisms leading to neutrophilic bronchitis and few treatments are effective in reducing neutrophil accumulation in the airways. There is a similar pattern of inflammatory mediator release and toll like receptor 2 expression in asthma, COPD and bronchiectasis. We propose the existence of an active amplification mechanism, an effector arm of the innate immune system, involving toll like receptor 2, operating in persistent neutrophilic bronchitis. Neutrophil persistence in the airways can occur through a number of mechanisms such as impaired apoptosis, efferocytosis and mucus hypersecretion, all of which are impaired in airways disease. Impairment of neutrophil clearance results in a reduced ability to respond to bacterial infection. Persistent activation of airway neutrophils may result in the persistent activation of the innate immune system resulting in further airway insult. Current therapies are limited for the treatment of neutrophilic bronchitis; possible treatments being investigated include theophylline, statins, antagonists of pro-inflammatory cytokines and macrolide antibiotics. Macrolides have shown great promise in their ability to reduce airway inflammation, and can reduce airway neutrophils, levels of CXCL8 and neutrophil proteases in the airways. Studies also show improvements in quality of life and exacerbation rates in airways diseases. [Copyright &y& Elsevier]

Neutrophilic inflammation is a key effector arm of the innate immune response. Neutrophils may contribute significantly to airway inflammation in certain asthma subtypes. The objective of this study is to investigate the innate immune responses of isolated airway and circulating neutrophils in asthma. Participants with asthma (n = 17) and healthy volunteers (n = 11) underwent induced sputum and blood collection. Neutrophils were isolated from dispersed selected sputum and blood granulocytes using magnetic cell separation. Neutrophils were cultured with or without lipopolysaccharide (100 ng/mL) for 24 hours. Innate immune mediators were measured by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Airway neutrophils from participants with asthma spontaneously released lower levels of interleukin (IL)-8, IL-1β and tumor necrosis factor-α proteins, and had lower levels of cytokine gene expression compared to healthy controls. Toll-like receptor 4 (TLR4) gene expression was significantly decreased in airway neutrophils from participants with asthma compared to healthy volunteers. Resting and lipopolysaccharide (LPS)-stimulated circulating neutrophils had lower levels of TLR2 and IL-1β gene expression in asthma, but were otherwise similar to healthy controls. No differences were seen for matrix metalloproteinase (MMP)-9 release in asthma. Innate immune responses of airway neutrophil cells are impaired in asthmatic subjects on prophylactic therapy and may impact on susceptibility to, and severity of, airway infections. [ABSTRACT FROM AUTHOR]

Background: The role of the innate immune system in the pathogenesis of asthma is unclear. Activation of innate immune receptors in response to bacterial lipopolysaccharide, viral infection and particulate matter triggers a pre-programmed inflammatory response, which involves interleukin (IL)8 and neutrophil influx. The inflammatory response in asthma is heterogeneous. Aim: To test the hypothesis that innate immune activation may be a relevant inflammatory mechanism in neutrophilic asthma where 1L8 levels are increased. Methods: Induced sputum was obtained from non-smoking adults with asthma (n = 49), healthy controls (n = 1 3) and a positive reference group with bronchiectasis (n = 9). Subjects with asthma were classified into inflammatory subtypes using induced sputum cell counts. Sputum was examined for mRNA expression of the innate immune receptors toll-like receptor (TLR)2, TLR4 and CD1 4, and inflammatory cytokines. A separate sputum portion was dispersed and the supernatant assayed for surfactant protein A, 1L8, soluble CD14 and endotoxin. Results: Expression of innate immune receptors was increased in subjects with bronchiectasis and neutrophilic asthma compared with other asthma subtypes and controls. Increased expression of the receptors TLR2, TLR4 and CD14, as well as the pro-inflammatory cytokines 1L8 and IL1β, was observed. Subjects with neutrophilic asthma had higher airway levels of endotoxin than the other groups studied. Conclusion: There is evidence of activation of the innate immune system in asthma which results in the production of pro-inflammatory cytokines and may contribute to the pathogenesis of neutrophilic asthma. [ABSTRACT FROM AUTHOR]

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Objective: The authors sought to investigate the detection of non-eosinophilic asthma using induced sputum. Although this is an important subtype of clinical asthma, its recognition is not standardized. Methods: Adult non-smokers with asthma and healthy controls underwent sputum induction and hypertonic saline challenge. Non-eosinophilic asthma was defined as symptomatic asthma with normal sputum eosinophil counts. The normal range for sputum eosinophil count was determined using the 95th percentile from the healthy control group as a cut-off point. Results: The recognition of non-eosinophilic asthma using eosinophil proportion was in agreement with a definition based on absolute eosinophil count (kappa 0.67). Non-eosinophilic asthma was a stable subtype over both the short term (4 weeks) and longer term (5 years, kappa 0.77). Airway inflammation in asthma could be categorized into four inflammatory subtypes based on sputum eosinophil and neutrophil proportions. These subtypes were neutrophilic asthma, eosinophilic asthma, mixed granulocytic asthma and paucigranulocytic asthma. Subjects with increased neutrophils (neutrophilic asthma and mixed granulocytic asthma) were older and had an increased total cell count and cell viability compared with other subtypes. Conclusion: Induced sputum eosinophil proportion is a good discriminator for eosinophilic asthma, providing a reproducible definition of a homogenous group. The remaining non-eosinophilic subjects are heterogeneous and can be further classified based on the presence of neutrophils. These inflammatory subtypes have important implications for the investigation and characterization of airway inflammation in asthma. [ABSTRACT FROM AUTHOR]

Background: Influenza (Flu) and respiratory syncytial virus (RSV) are important viral pathogens that cause lower respiratory tract infections and severe exacerbations of asthma. Molecular biological techniques are permitting a rapid and accurate diagnosis of infections caused by respiratory pathogens, and have typically been applied to upper respiratory samples. Sputum induction provides an opportunity to directly sample secretions from the lower respiratory tract. Objectives/study design: To determine the role of induced sputum reverse-transcription polymerase chain reaction (RT-PCR) in the detection of respiratory pathogens and compare this with detection using serology and immunofluorescent antigen (IFA) testing, we recruited 49 adults from emergency room with exacerbations of asthma. After a medical assessment and spirometry, sputum was induced using ultrasonically nebulised normal saline. Sputum was assayed using IFA and RT-PCR for flu and RSV. Flu serology was performed acutely and at convalescence, 4–5 weeks later. Results: Influenza A or B was detected in 24% of the samples by PCR, significantly more than the nine cases detected using serology and the one case using IFA (P<0.05). RSV was detected in 37% of samples using PCR and 20% by IFA (P<0.05). Conclusion: The combination of induced sputum and RT-PCR provides a useful means of detecting respiratory infection. The technique is safe in both adults and children, and RT-PCR is more sensitive than conventional serology and IFA. The improved sensitivity of induced sputum RT-PCR also permits a more rapid diagnosis and the opportunity of early administration of effective treatments. [Copyright &y& Elsevier]

Background: Interleukin (IL)-5 measurement in sputum samples has produced variable results that appear to be due to methodological problems. The aim of the present study was to investigate the effects of dithiothreitol (DTT), sputum protease inhibition and sample storage on IL-5 recovery in order to develop a method to accurately measure IL-5 in dispersed sputum supernatant. Methodology: Measurement of IL-5 in sputum was performed in 22 subjects with airway disease. Interleukin-5 recovery was measured in samples spiked with recombinant human IL-5 using a commercial ELISA. A mix of four protease inhibitors (PI) was added to sputum processed using the selection method with dispersion using DTT and stored with and without inhibitors. Results: The addition of PI to sputum resulted in a 24% increase in IL-5 recovery. Recovery was not further increased with the addition of a blocking protein. Storage of IL-5-spiked sputum gave significantly less recovery. The addition of PI to sputum processed with DTT had no effect on total cell count, viability or cell differential. Conclusion: Interleukin-5 recovery is increased by the addition of PI to samples processed using the selected portion method with DTT dispersion. A protease inhibitor cocktail should be added to sputum for IL-5 assay. [ABSTRACT FROM AUTHOR]

Study objectives: To identify the characteristics of airway inflammation in persistent asthma and to examine the role of neutrophilic inflammation in noneosinophilic persistent asthma. Methods: Nonsmoking adults (n = 56) with persistent asthma and healthy control subjects (n = 8) underwent hypertonic saline solution challenge and sputum induction. Selected sputum portions were dispersed with dithiothreitol and assayed for total cell count, cellular differential, supernatant eosinophil cationic protein (ECP), myeloperoxidase, and interleukin (IL)-8. Results: We identified two distinct inflammatory patterns. Typical eosinophilic inflammation occurred in 41% of subjects, whereas the remainder exhibited noneosinophilic asthma (59%). Both neutrophil percentage and absolute neutrophil counts were increased in subjects with noneosinophilic asthma (64%, 283 × 10[sup 6]/mL) compared to eosinophillc asthma (14%, 41 × 10[sup 6]/ mL) and control subjects (34%, 49 × 10[sup 6]/mL; p = 0.0001). Myeloperoxidase was elevated in both noneosinophilic (280 ng/mL) and eosinophillc groups (254 ng/mL) compared with control subjects (82 ng/mL; p = 0.002). Sputum IL-8 levels were highest in subjects with noneosinophilic asthma (45 ng/mL) compared to eosinophilic asthma (9.6 ng/mL) and control subjects (3.5 ng/mL; p = 0.0001). Neutrophils correlated with IL-8 levels (r = 0.72). ECP was highest in subjects with eosinophilic asthma (2,685 ng/mL) compared with noneosinophillc asthma (1,081 ng/mL) and control subjects (110 ng/mL; p = 0.0001). Conclusion: Induced-sputum analysis in persistent asthma identifies two different inflammatory patterns. The most common pattern is noneosinophilic, associated with a neutrophil influx and activation, which may be mediated by IL-8 secretion. There is heterogeneity of airway inflammation in persistent asthma, which indicates differing mechanisms and may impact on treatment responses. [ABSTRACT FROM AUTHOR]

Background: Low‐dose long‐term azithromycin is recommended in clinical practice guidelines for obstructive airway diseases (OAD); however, an optimal therapeutic regimen is not yet established. Aim: To understand the patterns of azithromycin use in OAD, 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 250 mg 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% vs 32%; P < 0.001) and hospital admissions (35% vs 31%; P < 0.001) after starting azithromycin. In 88% of cases, physicians favoured the use of azithromycin. Conclusion: Physicians are currently using low‐dose azithromycin for a long duration of more than 1 year for the management of OAD. The typical case definition is an older non‐smoking adult with persistent asthma, often in combination with another OAD and presenting with bothersome cough or sputum. Azithromycin was well tolerated and led to reduced healthcare utilisation. Further research is required to establish an optimal dosage regimen of azithromycin in OAD. [ABSTRACT FROM AUTHOR]

Differential cell counts is a challenging task when applying computer vision algorithms to pathology. Existing approaches to train cell recognition require high availability of multi-class segmentation and/or bounding box annotations and suffer in performance when objects are tightly clustered. We present differential count network ("DCNet"), an annotation efficient modality that utilises keypoint detection to locate in brightfield images the centre points of cells (not nuclei) and their cell class. The single centre point annotation for DCNet lowered burden for experts to generate ground truth data by 77.1% compared to bounding box labeling. Yet centre point annotation still enabled high accuracy when training DCNet on a multi-class algorithm on whole cell features, matching human experts in all 5 object classes in average precision and outperforming humans in consistency. The efficacy and efficiency of the DCNet end-to-end system represents a significant progress toward an open source, fully computationally approach to differential cell count based diagnosis that can be adapted to any pathology need. [ABSTRACT FROM AUTHOR]

See article, page 671 See article, page 671 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 <.001, p =.013). Increased sputum classical and CD206− monocyte numbers in neutrophilic asthma were confirmed in the second cohort. Blood monocytes did not vary with airway inflammatory phenotype, but blood classical monocyte proportion and number were increased in severe vs non‐severe asthma (p =.022, p =.011). Conclusion and Clinical relevance: Flow cytometry allowed distinction of sputum macrophages, monocytes and subsets, revealing compartment‐specific dysregulation of monocytes in asthma. We observed an increase in classical and CD206− monocytes in sputum in neutrophilic asthma, suggesting co‐recruitment of monocytes and neutrophils to the airways in asthma. Our data suggest further investigation of how airway monocyte dysregulation impacts on asthma‐related disease activity is merited. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Galectins constitute an evolutionary conserved family that bind to β-galactosides. Increasing evidence shows that galectins are involved in many fundamental biological processes such as cellular communication, inflammation, differentiation and apoptosis. Changes in galectin-3 (Gal-3) expression are commonly seen in cancer and pre-cancerous conditions, and Gal-3 may be involved in the regulation of diverse cancer cell activities that contribute to tumourigenesis, cancer progression and metastasis. In addition, Gal-3 is a pro-inflammatory regulator in rheumatoid arthritis. Gal-3 has been shown to be involved in many aspects in allergic inflammation, such as eosinophil recruitment, airway remodeling, development of a Th2 phenotype as well as increased expression of inflammatory mediators. In an in vivo model it was shown that bronchoalveolar lavage (BAL) fluid from ovalbumin-challenged mice contained significantly higher levels of Gal-3 compared to control mice. The molecular mechanisms of Gal-3 in human asthma have not been fully elucidated. This review will focus on what is known about the Gal-3 and its role in the pathophysiological mechanisms of asthma to evaluate the potential of Gal-3 as a biomarker and therapeutic target of asthma. [ABSTRACT FROM AUTHOR]

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 OVAsensitized/ 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. [ABSTRACT FROM AUTHOR]

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 (e DNA) 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 e DNA and NET neutrophil antimicrobial proteins were significantly elevated in asthma and COPD compared with healthy controls. Levels of e DNA and NET components were significantly higher in neutrophilic versus non-neutrophilic asthma and COPD. NETs were clearly visualized in sputum smears. PAD4 m RNA was upregulated in neutrophilic COPD. The level of e DNA was higher in severe asthma. High e DNA 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 e DNA 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. [ABSTRACT FROM AUTHOR]

Background. Innate immune antimicrobial peptides, including β-defensin-1, promote the chemotaxis and activation of several immune cells. The role of β-defensin-1 in asthma and chronic obstructive pulmonary disease (COPD) remains unclear. Methods. Induced sputum was collected from healthy controls and individuals with asthma or COPD. β-defensin-1 protein in sputum supernatantwas quantified by ELISA. Biomarker potentialwas examined using receiver operating characteristic curves. β-defensin-1 release fromprimary bronchial epithelial cells (pBECs) was investigated in culture with andwithout cigarette smoke extract (CSE). Results. Airway β-defensin-1 protein was elevated in COPD participants compared to asthma participants and healthy controls. Inflammatory phenotype had no effect on β-defensin-1 levels in asthma or COPD. β-defensin-1 protein was significantly higher in severe asthma compared to controlled and uncontrolled asthma. β-defensin-1 protein could predict the presence of COPD from both healthy controls and asthma patients. Exposure of pBECs to CSE decreased β-defensin-1 production in healthy controls; however in pBECs from COPD participants the level of β-defensin-1 remanied unchanged. Conclusions. Elevated β-defensin-1 protein is a feature of COPD and severe asthma regardless of inflammatory phenotype. β-defensin-1 production is dysregulated in the epithelium of patients with COPD and may be an effective biomarker and potential therapeutic target. [ABSTRACT FROM AUTHOR]

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 <.05). RNASeq analysis confirmed macrophage purity and identified DEGs in NA macrophages. Selected DEGs (SLAMF7, DYSF, GPR183, CSF3, PI3, CCR7, all p <.05 NA vs. NNA) were confirmed by qPCR. Pathway analysis of NA macrophage DEGs was consistent with responses to bacteria, contribution to neutrophil recruitment and increased expression of phagocytosis and efferocytosis factors. GSVA demonstrated neutrophilic macrophage gene signatures were significantly enriched in whole sputum microarray in NA vs. NNA and controls in both cohorts. Conclusions: We demonstrate a pathophysiologically relevant sputum macrophage transcriptomic programme in NA. The finding that there is transcriptional activation of inflammatory programmes in cell types other than neutrophils supports the concept of NA as a specific endotype. [ABSTRACT FROM AUTHOR]

Background: The progression of obstructive airway diseases (OADs) including asthma, chronic obstructive pulmonary disease (COPD) and asthma-COPD overlap syndrome in older adults is not well understood. Objective: To examine the prognosis of OADs and to identify potential determinants for longitudinal changes in clinical outcomes. Methods: We consecutively recruited 99 older adults (>55 years) with OADs who underwent a multidimensional assessment at baseline and 4 years which involved spirometry, 6-min walk distance (6MWD), assessments of health status (Saint George's Respiratory Questionnaire, SGRQ), comorbidity, and serum and sputum biomarkers. All-cause mortality and respiratory hospitalisation during the follow-up period were recorded. Clinical outcomes were compared between basal and final visits, and changes in clinical outcomes were compared among asthma, COPD and asthma-COPD overlap groups. Associations between clinical parameters, biomarkers and prognosis were examined. Results: After a median follow-up of 4.2 years, outcome data were available for 75 (75.8%) patients. There were 16 (16.2%) deaths. The BODE index predicted all-cause mortality in older people with OADs. While spirometry, 6MWD and SGRQ deteriorated significantly over the 4 years, there was significant heterogeneity in the longitudinal changes in these clinical outcomes. Participants with COPD had a significant decline in FEV1 (p = 0.003), SGRQ (p = 0.030) and 6MWD [decline of 75.5 (93.4) m, p = 0.024]. The change in 6MWD was lower in the asthma-COPD overlap group. Airflow reversibility was associated with a reduced decline in 6MWD. Conclusion: COPD patients had a poor prognosis compared with asthma and asthma-COPD overlap patients. The BODE index is a useful prognostic indicator in older adults with OADs. Both airway disease diagnosis and BODE index warrant specific attention in clinical practice. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The role of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in immune responses mediated by T-helper 2 (TH2) lymphocytes is unknown. Here we characterize the development of allergic airway disease in TRAIL-deficient (Tnfsf10−/−) mice and in mice exposed to short interfering RNA targeting TRAIL. We show that TRAIL is abundantly expressed in the airway epithelium of allergic mice and that inhibition of signaling impairs production of the chemokine CCL20 and homing of myeloid dendritic cells and T cells expressing CCR6 and CD4 to the airways. Attenuated homing limits TH2 cytokine release, inflammation, airway hyperreactivity and expression of the transcriptional activator STAT6. Activation of STAT6 by interleukin-13 restores airway hyperreactivity in Tnfsf10−/− mice. Recombinant TRAIL induces pathognomic features of asthma and stimulates the production of CCL20 in primary human bronchial epithelium cells. TRAIL is also increased in sputum of asthmatics. The function of TRAIL in the airway epithelium identifies this molecule as a target for the treatment of asthma. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 < 0.001) and COPD (r = 0.824, p < 0.001), were moderately correlated with sputum eosinophils in severe asthma (IL5r = 0.440, p < 0.001; IL13r = 0.428, p < 0.001) and were weakly correlated in COPD (IL5r = 0.245, p < 0.05; IL13r = 0.317, p < 0.05). Conclusions: Molecular markers of type 2 airway inflammation do not differ between eosinophilic asthma and eosinophilic COPD; however, the relationship between eosinophilia and type 2 airway markers appears weaker in COPD than in severe asthma. [ABSTRACT FROM AUTHOR]

Background and objective: Long‐term data on children with PBB has been identified as a research priority. We describe the 5‐year outcomes for children with PBB to ascertain the presence of chronic respiratory disease (bronchiectasis, recurrent PBB and asthma) and identify the risk factors for these. Methods: Prospective cohort study was undertaken at the Queensland Children's Hospital, Brisbane, Australia, of 166 children with PBB and 28 controls (undergoing bronchoscopy for symptoms other than chronic wet cough). Monitoring was by monthly contact via research staff. Clinical review, spirometry and CT chest were performed as clinically indicated. Results: A total of 194 children were included in the analysis. Median duration of follow‐up was 59 months (IQR: 50–71 months) post‐index PBB episode, 67.5% had ongoing symptoms and 9.6% had bronchiectasis. Significant predictors of bronchiectasis were recurrent PBB in year 1 of follow‐up (ORadj = 9.6, 95% CI: 1.8–50.1) and the presence of Haemophilus influenzae in the BAL (ORadj = 5.1, 95% CI: 1.4–19.1). Clinician‐diagnosed asthma at final follow‐up was present in 27.1% of children with PBB. A significant BDR (FEV1 improvement >12%) was obtained in 63.5% of the children who underwent reversibility testing. Positive allergen‐specific IgE (ORadj = 14.8, 95% CI: 2.2–100.8) at baseline and bronchomalacia (ORadj = 5.9, 95% CI: 1.2–29.7) were significant predictors of asthma diagnosis. Spirometry parameters were in the normal range. Conclusion: As a significant proportion of children with PBB have ongoing symptoms at 5 years, and outcomes include bronchiectasis and asthma, they should be carefully followed up clinically. Defining biomarkers, endotypes and mechanistic studies elucidating the different outcomes are now required. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Diverging susceptibility and severity in respiratory diseases is prevalent between males and females. Sex hormones have inconclusively been attributed as the cause of these differences, however, strong evidence exists promoting genetic factors leading to sexual dimorphism. As such, we investigate differential proinflammatory cytokine (interleukin (IL)‐6 and CXCL8) release from TNF‐α stimulated primary human lung fibroblasts in vitro. We present, for the first time, in vitro evidence supporting clinical findings of differential production of IL‐6 between males and females across various respiratory diseases. IL‐6 was found to be produced approximately two times more from fibroblasts derived from females compared to males. As such we demonstrate sexual dimorphism in cytokine production of IL‐6 outside the context of biological factors in the human body. As such, our data highlight that differences exist between males and females in the absence of sex hormones. We, for the first time, demonstrate inherent in vitro differences exist between males and females in pulmonary fibroblasts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The composition of the airway microbiome in patients with chronic airway diseases, such as severe asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis and cystic fibrosis (CF), has the potential to inform a precision model of clinical care. Patients with these conditions share overlapping disease characteristics, including airway inflammation and airflow limitation. The clinical management of chronic respiratory conditions is increasingly moving away from a one‐size‐fits‐all model based on primary diagnosis, towards care targeting individual disease traits, and is particularly useful for subgroups of patients who respond poorly to conventional therapies. Respiratory microbiome analysis is an important potential contributor to such a 'treatable traits' approach, providing insight into both microbial drivers of airways disease, and the selective characteristics of the changing lower airway environment. We explore the potential to integrate respiratory microbiome analysis into a treatable traits model of clinical care and provide a practical guide to the application and clinical interpretation of respiratory microbiome analysis. [ABSTRACT FROM AUTHOR]

With the development of culture-independent techniques, numerous studies have demonstrated that the lower airway is not sterile in health and harbors diverse microbial communities. Furthermore, new evidence suggests that there is a distinct lower airway microbiome in those with chronic respiratory disease. To understand the role of lower airway dysbiosis in the pathogenesis of asthma, in this article, we review the published reports about the lung microbiome of healthy controls, provide an outlook on the contribution of lower airway dysbiosis to asthma, especially steroid-resistant asthma, and discuss the potential therapies targeted for lower airway dysbiosis. [ABSTRACT FROM AUTHOR]

Background: Exacerbations of asthma cause a substantial global illness burden. Adults with uncontrolled persistent asthma despite maintenance treatment require additional therapy. Since macrolide antibiotics can be used to treat persistent asthma, we aimed to assess the efficacy and safety of oral azithromycin as add-on therapy in patients with uncontrolled persistent asthma on medium-to-high dose inhaled corticosteroids plus a long-acting bronchodilator.Methods: We did a randomised, double-blind, placebo controlled parallel group trial to determine whether oral azithromycin decreases the frequency of asthma exacerbations in adults (≥18 years) with symptomatic asthma despite current use of inhaled corticosteroid and long-acting bronchodilator, and who had no hearing impairment or abnormal prolongation of the corrected QT interval. Patients were randomly assigned (1:1) to receive azithromycin 500 mg or placebo three times per week for 48 weeks. Patients were centrally allocated using concealed random allocation from a computer-generated random numbers table with permuted blocks of 4 or 6 and stratification for centre and past smoking. Primary efficacy endpoints were the rate of total (severe and moderate) asthma exacerbations over 48 weeks and asthma quality of life. Data were analysed on an intention-to-treat basis. The trial is registered at the Australian and New Zealand Clinical Trials Registry (ANZCTR), number 12609000197235.Findings: Between June 12, 2009, and Jan 31, 2015, 420 patients were randomly assigned (213 in the azithromycin group and 207 in the placebo group). Azithromycin reduced asthma exacerbations (1·07 per patient-year [95% CI 0·85-1·29]) compared with placebo (1·86 per patient-year [1·54-2·18]; incidence rate ratio [IRR] 0·59 [95% CI 0·47-0·74]; p<0·0001). The proportion of patients experiencing at least one asthma exacerbation was reduced by azithromycin treatment (127 [61%] patients in the placebo group vs 94 [44%] patients in the azithromycin group, p<0·0001). Azithromycin significantly improved asthma-related quality of life (adjusted mean difference, 0·36 [95% CI 0·21-0·52]; p=0·001). Diarrhoea was more common in azithromycin-treated patients (72 [34%] vs 39 [19%]; p=0·001).Interpretation: Adults with persistent symptomatic asthma experience fewer asthma exacerbations and improved quality of life when treated with oral azithromycin for 48 weeks. Azithromycin might be a useful add-on therapy in persistent asthma.Funding: National Health and Medical Research Council of Australia, John Hunter Hospital Charitable Trust. [ABSTRACT FROM AUTHOR]

Severe, steroid-resistant asthma is clinically and economically important since affected individuals do not respond to mainstay corticosteroid treatments for asthma. Patients with this disease experience more frequent exacerbations of asthma, are more likely to be hospitalized, and have a poorer quality of life. Effective therapies are urgently required, however, their development has been hampered by a lack of understanding of the pathological processes that underpin disease. A major obstacle to understanding the processes that drive severe, steroid-resistant asthma is that the several endotypes of the disease have been described that are characterized by different inflammatory and immunological phenotypes. This heterogeneity makes pinpointing processes that drive disease difficult in humans. Clinical studies strongly associate specific respiratory infections with severe, steroid-resistant asthma. In this review, we discuss key findings from our studies where we describe the development of representative experimental models to improve our understanding of the links between infection and severe, steroid-resistant forms of this disease. We also discuss their use in elucidating the mechanisms, and their potential for developing effective therapeutic strategies, for severe, steroid-resistant asthma. Finally, we highlight how the immune mechanisms and therapeutic targets we have identified may be applicable to obesity-or pollution-associated asthma. [ABSTRACT FROM AUTHOR]

Viral infection is a common trigger for acute exacerbations of chronic obstructive pulmonary disease (AECOPD). The aim of this study is to investigate the expression of cytokines in AECOPD. Patients with AECOPD requiring hospitalization were recruited. Meanwhile healthy volunteers of similar age that accepted routine check-ups and showed no clinical symptoms of inflammatory diseases were also recruited. Induced sputum and serum were collected. Induced sputum of participants was processed and tested for thirteen viruses and bacteria. Forty cytokines were assayed in serum using the Quantibody Human Inflammation Array 3 (Ray Biotech, Inc.). The most common virus detected in virus positive AECOPD (VP) was influenza A (16%). No virus was found in controls. Circulating levels of IL-6, TNF-α, and MCP-1 were elevated in VP and coinfection subjects (p < 0.05), while the levels of 37 other cytokines showed no difference, compared with virus negative groups and controls (p > 0.05). Additionally, VP patients were less likely to have received influenza vaccination. VP patients had a systemic inflammation response involving IL-6, TNF-α, and MCP-1 which may be due to virus-induced activation of macrophages. There are important opportunities for further investigating AECOPD mechanisms and for the development of better strategies in the management and prevention of virus-related AECOPD. [ABSTRACT FROM AUTHOR]