Search

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: 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]

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]

Background: Asthma pathophysiology and treatment responsiveness are predicted by inflammatory phenotype. However, the relationship between airway microbiology and asthma phenotype is poorly understood. Objective: We aimed to characterize the airway microbiota in patients with symptomatic stable asthma and relate composition to airway inflammatory phenotype and other phenotypic characteristics. Methods: Themicrobial composition of induced sputum specimens collected from adult patients screened for amulticenter randomized controlled trialwas determined by using 16SrRNAgene sequencing. Inflammatory phenotypes were defined by sputum neutrophil and eosinophil cell proportions. Microbiota were defined by usinga- and β-diversity measures, and interphenotype differences were identified by using similarity of percentages, network analysis, and taxon fold change. Phenotypic predictors of airway microbiology were identified by using multivariate linear regression. Results: Microbiota composition was determined in 167 participants and classified as eosinophilic (n 5 84), neutrophilic (n 5 14), paucigranulocytic (n 5 60), or mixed neutrophiliceosinophilic (n 5 9) asthma phenotypes. Airway microbiology was significantly less diverse (P 5 .022) and more dissimilar (P 5 .005) in neutrophilic compared with eosinophilic participants. Sputum neutrophil proportions, but not eosinophil proportions, correlated significantly with these diversity measures (a-diversity: Spearman r 5 20.374, P < .001; β-diversity: r 5 0.238, P 5 .002). Interphenotype differences were characterized by a greater frequency of pathogenic taxa at high relative abundance and reduced Streptococcus, Gemella, and Porphyromonas taxa relative abundance in patients with neutrophilic asthma. Multivariate regression confirmed that sputum neutrophil proportion was the strongest predictor of microbiota composition. Conclusions: Neutrophilic asthma is associated with airway microbiology that is significantly different fromthat seen in patients with other inflammatory phenotypes, particularly eosinophilic asthma. Differences in microbiota composition might influence the response to antimicrobial and steroid therapies and the risk of lung infection. [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: 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: 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 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. [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]

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]