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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., (© 2021 John Wiley & Sons Ltd.)

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 CD62L bright /CD16 dim neutrophils (median: 5.4% and 0.14 × 10 9 /L, respectively), in comparison with healthy (3.54% and 0.12 × 10 9 /L, respectively); p<0.017., 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., Competing Interests: Dr Michael Fricker reports grants from NHMRC, grants from Thoracic Society of Australia and New Zealand, during the conduct of the study. The authors report no other conflicts of interest in this work., (© 2019 Lokwani et al.)

Objectives: The significance of neutrophilic inflammation in obstructive airway disease remains controversial. Recent studies have demonstrated presence of an active neutrophil population in systemic circulation, featuring hypersegmented morphology, with high oxidative burst and functional plasticity in inflammatory conditions. The aim of this study was to characterise neutrophil subsets in bronchial lavage (BL) of obstructive airway disease participants (asthma, chronic obstructive pulmonary disease (COPD) and bronchiectasis) and healthy controls on the basis of nuclear morphology and to assess the association between neutrophil subsets and the clinical parameters of the obstructive airway disease participants., Design: A cross-sectional exploratory study., Setting: John Hunter Hospital and Hunter Medical Research Institute, Australia., Participants: Seventy-eight adults with obstructive airway disease comprised those with stable asthma (n=39), COPD (n=20) and bronchiectasis (n=19) and 20 healthy controls., Materials and Methods: Cytospins were prepared and neutrophil subsets were classified based on nuclear morphology into hypersegmented (>4 lobes), normal (2-4 lobes) and banded (1 lobe) neutrophils and enumerated., Results: Neutrophils from each subset were identified in all participants. Numbers of hypersegmented neutrophils were elevated in participants with airway disease compared with healthy controls (p<0.001). Both the number and the proportion of hypersegmented neutrophils were highest in COPD participants (median (Q1-Q3) of 1073.6 (258.8-2742) × 10 2 /mL and 24.5 (14.0-46.5)%, respectively). An increased proportion of hypersegmented neutrophils in airway disease participants was significantly associated with lower forced expiratory volume in 1 s/forced vital capacity per cent (Spearman's r=-0.322, p=0.004)., Conclusion: Neutrophil heterogeneity is common in BL and is associated with more severe airflow obstruction in adults with airway disease. Further work is required to elucidate the functional consequences of hypersegmented neutrophils in the pathogenesis of disease., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose of Review: Recent advances in both murine models and clinical research of neutrophilic asthma are improving our understanding on the etiology and pathophysiology of this enigmatic endotype of asthma. We here aim at providing an overview of our current and latest insights on the pathophysiology and treatment of neutrophilic asthma., Recent Findings: Activation of the NLRP3 inflammasome pathway with increased IL-1β has been demonstrated in various studies involving patients with asthma. It has been suggested that type 3 innate lymphoid cells are implicated in the inflammatory cascade leading to neutrophilic inflammation. The role of neutrophil extracellular traps is only at the start of being understood and might be an attractive novel therapeutic target. A diverse panel of nonallergic stimuli, such as cigarette smoke, intensive exercise, cold air or saturated fatty acids, have been linked with neutrophilic airway inflammation. Azithromycin treatment could reduce asthma exacerbations and quality of life in patients with persistent asthma., Summary: Research of the last few years has accelerated our insights in mechanisms underlying neutrophilic asthma. This is in stark contrast with the lack of efficacy of different therapies targeting neutrophil chemotaxis and/or signalling cascade, such as IL-17A or CXCR2. Macrolide therapy might be a useful add-on therapy for patients with persistent asthma.

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., (© 2016 Asian Pacific Society of Respirology.)

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 <2.5%) was 54%. In this group, atopy, sputum neutrophil, eosinophil, eosinophil cationic protein (ECP), endotoxin, neutrophil elastase and IL-8 levels were not different from those without asthma. In contrast, eosinophilic asthma (EA) was associated with atopy and sputum ECP and IL-8. The majority of NEA had no evidence of inflammation; only 14% had neutrophilia (≥61% neutrophils), compared with 11% of EA, and 15% of those without asthma. Small differences in FEV1 (NS) were found between EA and NEA, but symptom prevalence and severity was not different (63% of EA and 52% of NEA were classified moderate to severe)., Conclusion: NEA is common in adolescent asthma and has similar clinical characteristics as EA. Neutrophils do not appear to play a role in NEA in adolescents, and underlying mechanisms may not involve airway inflammation., (© 2015 Asian Pacific Society of Respirology.)

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., (Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Background: Galectin-3 (gal-3), a member of the β-galactoside-binding animal lectins, is involved in the recruitment, activation and removal of neutrophils. Neutrophilic asthma is characterized by a persistent elevation of airway neutrophils and impaired efferocytosis. We hypothesized that sputum gal-3 would be reduced in neutrophilic asthma and the expression of gal-3 would be associated with other markers of neutrophilic inflammation., Methods: Adults with asthma (n = 80) underwent a sputum induction following clinical assessment and blood collection. Sputum was dispersed for a differential cell count and ELISA assessment of gal-3, gal-3 binding protein (BP), interleukin (IL)-1β, IL-1 receptor antagonist (RA), IL-8 and IL-6. Gal-3 and gal-3BP immunoreactivity were assessed in mixed sputum cells., Results: Sputum gal-3 (median, (q1,q3)) was significantly reduced in neutrophilic asthma (183 ng/mL (91,287)) compared with eosinophilic (293 ng/mL (188,471), p = 0.021) and paucigranulocytic asthma (399 ng/mL (213,514), p = 0.004). The gal-3/gal-3BP ratio and IL-1RA/IL-1β ratio were significantly reduced, while gal-3BP and IL-1β were significantly elevated in neutrophilic asthma compared with eosinophilic and paucigranulocytic asthma., Conclusion: Patients with neutrophilic asthma have impairment in anti-inflammatory ratio of gal-3/gal-3BP and IL-1RA/IL-1β which provides a further framework for exploration into pathologic mechanisms of asthma phenotypes.

Background: The influence of occupation and ex/passive smoking on inflammatory phenotype is not well understood. The aim of this study was to examine the relationship between occupation, past smoking and current passive smoking and airway inflammation in a population of adults with refractory asthma., Methods: Sixty-six participants with refractory asthma were characterised. Occupational exposure to asthma causing or worsening agents were identified with an asthma-specific job exposure matrix. Exposure to passive cigarette smoke was determined by questionnaire and exhaled carbon monoxide assessment. The carbon content of macrophages was assessed in a sub-group of participants., Results: Nineteen participants had smoked previously with low smoking pack years (median 1.7 years). Ex-smokers more commonly lived with a current smoker (26% vs. 9%, p = 0.11) and were more likely to allow smoking inside their home (26% vs. 4%, p = 0.02) compared to never smokers. Twenty participants had occupations with an identified exposure risk to an asthmagen; thirteen had exposures to irritants such as motor vehicle exhaust and environmental tobacco smoke. Sputum neutrophils were elevated in participants with asthma who had occupational exposures, particularly those who were diagnosed with asthma at a more than 30 years of age., Conclusions: Sputum neutrophils are elevated in refractory asthma with exposure to occupational asthmagens. In addition to older age, exposure to both environmental and occupational particulate matter may contribute to the presence of neutrophilic asthma. This may help explain asthma heterogeneity and geographical variations in airway inflammatory phenotypes in asthma.

Background: Protracted bacterial bronchitis (PBB) is a common and treatable cause of chronic wet cough in children in which the mechanisms are not understood. This study investigates the IL-1 pathway and a neutrophil gene expression signature in PBB., Methods: BAL was collected from children in an experimental cohort (n = 21, PBB; n = 33, control subjects), and a second validation cohort (n = 36, PBB; n = 11, control subjects). IL-1β, IL-1 receptor antagonist (IL-1RA), and α-defensins 1-3 were assayed by enzyme-linked immunosorbent assay, western blot, and quantitative real-time polymerase chain reaction, together with selected IL-1 pathway members and neutrophil-related molecules., Results: In the experimental cohort, children with symptomatic PBB had significantly higher levels of IL-1β and α-defensin gene and protein expression. Expression of the neutrophil chemokine receptor C-X-C motif receptor 2 was also higher in PBB. IL-1RA protein was higher, however, the IL-1RA:IL-1β ratio was lower in children with PBB than control subjects. In the validation cohort, protein and gene expression of IL-1β and α-defensins 1-3 were confirmed higher, as was gene expression of IL-1 pathway members and C-X-C motif receptor 2. IL-1β and α-defensin 1-3 levels lowered when PBB was treated and resolved. In children with recurrent PBB, gene expression of the IL-1β signaling molecules pellino-1 and IL-1 receptor-associated kinase 2 was significantly higher. IL-1β protein levels correlated with BAL neutrophilia and the duration and severity of cough symptoms. IL-1β and α-defensin 1-3 levels were highly correlated., Conclusions: PBB is characterized by increased IL-1β pathway activation. IL-1β and related mediators were associated with BAL neutrophils, cough symptoms, and disease recurrence, providing insight into PBB pathogenesis.

Asthma is a heterogeneous inflammatory airways disorder where interleukin (IL)-1β is thought to be a key mediator, especially in the neutrophilic subtype of asthma. The generation of active IL-1β requires proteolytic cleavage typically mediated through the formation of a caspase-1-containing inflammasome. This study hypothesised that an IL-1β endotype associated with the nucleotide-binding domain, leucine-rich repeat-containing family protein (NLRP)3/apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)/caspase-1 inflammasome is characteristic of patients with the neutrophilic subtype of asthma. Participants with asthma (n=85) and healthy controls (n=27) underwent clinical assessment, spirometry and sputum induction. Sputum was processed for differential cell count, gene expression and protein mediators. NLRP3 and caspase-1 expression was also determined by immunocytochemistry. Sputum macrophages were isolated (n=8) and gene expression of NLRP3 and IL-1β determined. There was significantly elevated gene expression of NLRP3, caspase-1, caspase-4, caspase-5 and IL-1β in participants with neutrophilic asthma. Protein levels of IL-1β were significantly higher in those with neutrophilic asthma and correlated with sputum IL-8 levels. Sputum macrophages, as well as sputum neutrophils in neutrophilic asthma, expressed NLRP3 and caspase-1 protein. NLRP3 inflammasome is upregulated in neutrophilic asthma and may regulate the inflammation process observed in this asthma phenotype through production of IL-1β.

Background: Asthma is a heterogeneous inflammatory disease and eosinophilic, non-eosinophilic and neutrophilic forms are recognised. While clinically similar to eosinophilic asthma, patients with non-eosinophilic asthma have different responses to treatment and little is known about the triggers of symptoms and inflammation., Objective: This study sought to characterise asthma control, exacerbation frequency and potential triggers of non-eosinophilic and specifically neutrophilic asthma such as infection, gastroesophageal reflux disease, and rhinosinusitis., Methods: Adults with asthma (n=65; doctor's diagnosis plus demonstrated response to bronchodilator and/or airways hyperresponsiveness to hypertonic saline) were recruited from the Respiratory and Sleep Medicine Ambulatory Care Service at John Hunter Hospital, NSW, Australia. Questionnaires were administered to assess gastroesophageal reflux disease, rhinosinusitis and asthma control. A sputum induction was performed and sputum was processed for assessment of inflammatory cells, infection, and lipid laden macrophages (Oil Red O)., Results: Participants with neutrophilic asthma (n=11, 23%) had a higher frequency of primary care doctor visits for asthma exacerbations and a high prevalence (> 70%) of chest infections in the previous 12 months. There was also an increased prevalence of rhinosinusitis (64%) and increased symptoms of gastroesophageal reflux disease compared to those with eosinophilic asthma., Conclusions: The clinical pattern of neutrophilic asthma is different from paucigranulocytic and eosinophilic asthma with evidence of abnormal upper airways responses. Specific and targeted treatment of these airway problems may assist in the control and management of neutrophilic asthma.

Purpose of Review: To review recent developments in the mechanisms of chronic cough., Recent Findings: Although cough prevalence is similar across the world, there are significant regional differences in the diseases associated with cough. This may represent regional differences in diet, environmental exposure, and diagnostic assessment and labelling. Cough can be reliably measured using several validated questionnaires and objective tools that monitor cough frequency and cough reflex sensitivity. Cough with eosinophilic bronchitis responds to anti-inflammatory therapy with corticosteroids and montelukast. The absence of airway hyperresponsiveness may be because of the release of a bronchoprotective mediator, prostaglandin E2. Cough with neutrophilic bronchitis is associated with rhinitis and gastroesophageal reflux disease. Laryngeal hypersensitivity (LHS) is an important new concept that explains cough mechanisms, identifies potentially new pharmacotherapies that target neural pathways and also transient receptor channels, and demonstrates how behavioural therapies can be effective in refractory cough. The concept also explains symptoms in a variety of related syndromes such as vocal cord dysfunction., Summary: New insights into cough mechanisms that focus on LHS help explain cough symptoms and the place of effective therapies including speech pathology intervention and neuromodulators.

Background and Objective: Increased sputum neutrophilia has been observed in asthma, but also during normal ageing in asthmatics and non-asthmatics. It remains unclear what constitutes 'normal' neutrophil levels in different age groups., Methods: We assessed the relationship between age and airway neutrophils of 194 asthmatics and 243 non-asthmatics (age range: 6-80 years). Regression analyses were used to assess this relationship adjusted for confounders including asthma status, atopy, gender, smoking and current use of inhaled corticosteroids (ICS). Age-corrected reference values for different age groups were determined using the 95th percentile of non-asthmatic participants., Results: Age was positively associated with sputum neutrophils in both asthmatic and non-asthmatic adults (0.46% neutrophil increase/year (95% confidence interval (CI) 0.18, 0.73) and 0.44%/year (0.25, 0.64, respectively), but no association was found in the <20-year age category. Individuals with high sputum neutrophil counts (>95th percentile of non-asthmatic counts for any given age group) were significantly more likely to be asthmatic (odds ratio = 2.5; 95% CI: 1.3, 5.0), with the greatest effect observed in the older age group. Other factors that independently associated with increased sputum neutrophil levels included atopy in non-asthmatic adults, male gender and current use of ICS in asthmatic adults. Age-specific reference values for neutrophil percentage were under 20 years-76%, 20-40 years-62%, 40-60 years-63% and over 60 years-67%., Conclusions: Airway neutrophilia is related to age in adults, with a neutrophilic asthma phenotype present in older adults. The use of appropriate age-specific reference values is recommended for future studies aimed at elucidating the role of neutrophils in asthma., (© 2013 The Authors. Respirology © 2013 Asian Pacific Society of Respirology.)

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 the mechanisms of TLR2 signalling and subsequent MMP-9 production in COPD may assist in understanding neutrophilic inflammation in COPD.

Background: 20-30% 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 12-15) 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 steroid-resistant 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.

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 (n=36). 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 α-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 α-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 α-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.

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.

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 (> 10(6) 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.

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.

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-1beta and tumor necrosis factor-alpha 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-1beta 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.

Rationale: Patients with refractory asthma have persistent symptoms despite maximal treatment with inhaled corticosteroids and long-acting bronchodilators. The availability of add-on therapies is limited, and effective add-on therapies that target noneosinophilic airway inflammation are needed. Macrolide antibiotics, such as clarithromycin, have in vitro efficacy against IL-8 and neutrophils, key inflammatory mediators in noneosinophilic asthma., Objectives: To determine the efficacy of clarithromycin in patients with severe refractory asthma and specifically in a subgroup of patients with noneosinophilic asthma., Methods: Subjects with severe refractory asthma (n = 45) were randomized to receive clarithromycin (500 mg twice daily) or placebo for 8 weeks., Measurements and Main Results: The primary outcome for this study was sputum IL-8 concentration. Other inflammatory outcomes assessed included sputum neutrophil numbers and concentrations of neutrophil elastase and matrix metalloproteinase (MMP)-9. Clinical outcomes were also assessed, including lung function, airway hyperresponsiveness to hypertonic saline, asthma control, quality of life, and symptoms. Clarithromycin therapy significantly reduced airway concentrations of IL-8 and neutrophil numbers and improved quality-of-life scores compared with placebo. Reductions in neutrophil elastase and MMP-9 concentrations were also observed. These reductions in inflammation were most marked in those with refractory noneosinophilic asthma., Conclusions: Clarithromycin therapy can modulate IL-8 levels and neutrophil accumulation and activation in the airways of patients with refractory asthma. Macrolide therapy may be an important additional therapy that could be used to reduce noneosinophilic airway inflammation, particularly neutrophilic inflammation, in asthma. Clinical trial registered with the Australian Clinical Trials Registry www.actr.org.au (No. 12605000318684).

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.

Introduction: The significance of endoplasmic reticulum (ER) stress in asthma is unclear. Here, we demonstrate that ER stress and the unfolded protein response (UPR) are related to disease severity and inflammatory phenotype.Methods: Induced sputum (n=47), bronchial lavage (n=23) and endobronchial biopsies (n=40) were collected from participants with asthma with varying disease severity, inflammatory phenotypes and from healthy controls. Markers for ER stress and UPR were assessed. These markers were also assessed in established eosinophilic and neutrophilic murine models of asthma.Results: Our results demonstrate increased ER stress and UPR pathways in asthma and these are related to clinical severity and inflammatory phenotypes. Genes associated with ER protein chaperone (BiP, CANX, CALR), ER-associated protein degradation (EDEM1, DERL1) and ER stress-induced apoptosis (DDIT3, PPP1R15A) were dysregulated in participants with asthma and are associated with impaired lung function (forced expiratory volume in 1 s) and active eosinophilic and neutrophilic inflammation. ER stress genes also displayed a significant correlation with classic Th2 (interleukin-4, IL-4/13) genes, Th17 (IL-17F/CXCL1) genes, proinflammatory (IL-1b, tumour necrosis factor α, IL-8) genes and inflammasome activation (NLRP3) in sputum from asthmatic participants. Mice with allergic airway disease (AAD) and severe steroid insensitive AAD also showed increased ER stress signalling in their lungs.Conclusion: Heightened ER stress is associated with severe eosinophilic and neutrophilic inflammation in asthma and may play a crucial role in the pathogenesis of asthma. [ABSTRACT FROM AUTHOR]

Airway inflammation plays a key role in asthma pathogenesis but is heterogeneous in nature. There has been significant scientific discovery with regard to type 2-driven, eosinophil-dominated asthma, with effective therapies ranging from inhaled corticosteroids to novel biologics. However, studies suggest that approximately 1 in 5 adults with asthma have an increased proportion of neutrophils in their airways. These patients tend to be older, have potentially pathogenic airway bacteria and do not respond well to classical therapies. Currently, there are no specific therapeutic options for these patients, such as neutrophil-targeting biologics.Neutrophils comprise 70% of the total circulatory white cells and play a critical defence role during inflammatory and infective challenges. This makes them a problematic target for therapeutics. Furthermore, neutrophil functions change with age, with reduced microbial killing, increased reactive oxygen species release and reduced production of extracellular traps with advancing age. Therefore, different therapeutic strategies may be required for different age groups of patients.The pathogenesis of neutrophil-dominated airway inflammation in adults with asthma may reflect a counterproductive response to the defective neutrophil microbial killing seen with age, resulting in bystander damage to host airway cells and subsequent mucus hypersecretion and airway remodelling. However, in children with asthma, neutrophils are less associated with adverse features of disease, and it is possible that in children, neutrophils are less pathogenic.In this review, we explore the mechanisms of neutrophil recruitment, changes in cellular function across the life course and the implications this may have for asthma management now and in the future. We also describe the prevalence of neutrophilic asthma globally, with a focus on First Nations people of Australia, New Zealand and North America. [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: Galectin-3 (gal-3), a member of the β-galactoside-binding animal lectins, is involved in the recruitment, activation and removal of neutrophils. Neutrophilic asthma is characterized by a persistent elevation of airway neutrophils and impaired efferocytosis. We hypothesized that sputum gal-3 would be reduced in neutrophilic asthma and the expression of gal-3 would be associated with other markers of neutrophilic inflammation. Methods: Adults with asthma (n = 80) underwent a sputum induction following clinical assessment and blood collection. Sputum was dispersed for a differential cell count and ELISA assessment of gal-3, gal-3 binding protein (BP), interleukin (IL)-1β, IL-1 receptor antagonist (RA), IL-8 and IL-6. Gal-3 and gal-3BP immunoreactivity were assessed in mixed sputum cells. Results: Sputum gal-3 (median, (q1,q3)) was significantly reduced in neutrophilic asthma (183 ng/mL (91,287)) compared with eosinophilic (293 ng/mL (188,471), p = 0.021) and paucigranulocytic asthma (399 ng/mL (213,514), p = 0.004). The gal-3/gal-3BP ratio and IL-1RA/IL-1β ratio were significantly reduced, while gal-3BP and IL-1β were significantly elevated in neutrophilic asthma compared with eosinophilic and paucigranulocytic asthma. Conclusion: Patients with neutrophilic asthma have impairment in anti-inflammatory ratio of gal-3/gal-3BP and IL-1RA/IL-1β which provides a further framework for exploration into pathologic mechanisms of asthma phenotypes. [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: Airway inflammation is associated with asthma exacerbation risk, treatment response, and disease mechanisms. Objective: This study aimed to identify and validate a sputum gene expression signature that discriminates asthma inflammatory phenotypes. Methods: An asthma phenotype biomarker discovery study generated gene expression profiles from induced sputum of 47 asthmatic patients. A clinical validation study (n = 59 asthmatic patients) confirmed differential expression of key genes. A 6-gene signature was identified and evaluated for reproducibility (n = 30 asthmatic patients and n = 20 control subjects) and prediction of inhaled corticosteroid (ICS) response (n = 71 asthmatic patients). Receiver operating characteristic curves were calculated, and area under the curve (AUC) values were reported. Results: From 277 differentially expressed genes between asthma inflammatory phenotypes, we identified 23 genes that showed highly significant differential expression in both the discovery and validation populations. A signature of 6 genes, including Charcot-Leydon crystal protein (CLC); carboxypeptidase A3 (CPA3); deoxyribonuclease I-like 3 (DNASE1L3); IL-1β (IL1B); alkaline phosphatase, tissue-nonspecific isozyme (ALPL); and chemokine (C-X-C motif) receptor 2 (CXCR2), was reproducible and could significantly (P < .0001) discriminate eosinophilic asthma from other phenotypes, including patients with noneosinophilic asthma (AUC, 89.6%), paucigranulocytic asthma (AUC, 92.6%), or neutrophilic asthma (AUC, 91.4%) and healthy control subjects (AUC, 97.6%), as well as discriminating patients with neutrophilic asthma from those with paucigranulocytic asthma (AUC, 85.7%) and healthy control subjects (AUC, 90.8). The 6-gene signature predicted ICS response (>12% change in FEV1; AUC, 91.5%). ICS treatment reduced the expression of CLC, CPA3, and DNASE1L3 in patients with eosinophilic asthma. Conclusions: A sputum gene expression signature of 6 biomarkers reproducibly and significantly discriminates inflammatory phenotypes of asthma and predicts ICS treatment response. This signature has the potential to become a useful diagnostic tool to assist in the clinical diagnosis and management of asthma. [Copyright &y& Elsevier]

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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]