Fungal sensitization and its relationship to mepolizumab response in patients with severe eosinophilic asthma

Letter to the Editor: In asthma, sensitization to fungal, perennial or seasonal allergens increases the risk of uncontrolled symptoms, exacerbations and poor disease outcomes.1 In severe asthma, typically 20%-29% of patients show sensitization to ≥1 fungal allergen, with Aspergillus being one of the most common.2-4 These patients have worse lung function, increased risk of oral corticosteroid use, hospitalization and a greater degree of airflow obstruction than patients non-sensitized to fungal allergens. [opening paragraph

Impaired P2X1 Receptor–Mediated Adhesion in Eosinophils from Asthmatic Patients

Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at −60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP–evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin–dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration

Impaired P2X1 Receptor–Mediated Adhesion in Eosinophils from Asthmatic Patients

Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at −60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP–evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin–dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration

NADPH oxidase 4 over-expression is associated with epithelial ciliary dysfunction in neutrophilic asthma

BACKGROUND: Bronchial epithelial ciliary dysfunction is an important feature of asthma. We sought to determine the role in asthma of neutrophilic inflammation and NADPH oxidases in ciliary dysfunction. METHODS: Bronchial epithelial ciliary function was assessed by video-microscopy in fresh ex vivo epithelial strips from asthmatics stratified by their sputum cell differentials and in cultures from healthy controls and asthmatics. Bronchial epithelial oxidative damage was determined by 8-oxo-dG expression. NOX/DUOX expression was assessed in bronchial epithelial cells by microarrays, with NOX4, DUOX1/2 expression assessed in bronchial biopsies. Ciliary dysfunction following NADPH oxidase inhibition, using GKT137831, was evaluated in fresh epithelial strips from asthmatics and a murine model of ovalbumin sensitisation and challenge. RESULTS: Ciliary beat frequency was impaired in asthmatics with sputum neutrophilia (n=11) versus those without (n=10) (5.8 [0.6] versus 8.8 [0.5]Hz; P=0.003) and was correlated with sputum neutrophil count (r=-0.70; P<0.001). Primary bronchial epithelial cells expressed DUOX1/2 and NOX4. 8-oxo-dG and NOX4, were elevated in neutrophilic versus non-neutrophilic asthmatics, DUOX1 was elevated in both, and DUOX2 was elevated in non-neutrophilic asthma in vivo. In primary epithelial cultures ciliary dysfunction did not persist, although NOX4 expression and reactive oxygen species generation was increased from subjects with neutrophilic asthma. GKT137831 both improved ciliary function in ex vivo epithelial strips (n=13), relative to the intensity of neutrophilic inflammation, and abolished ciliary dysfunction in the murine asthma model without a reduction in inflammation. CONCLUSIONS: Ciliary dysfunction is increased in neutrophilic asthma associated with increased NOX4 expression and is attenuated by NADPH oxidase inhibition

Airway Pathological Heterogeneity in Asthma: Visualisation of Disease Micro-Clusters using Topological Data Analysis

Background: Asthma is a complex chronic disease underpinned by pathological changes within the airway wall. How variations in structural airway pathology and cellular inflammation contribute to expression and severity of asthma are poorly understood. Objectives: We therefore evaluated pathological heterogeneity using topological data analysis (TDA) with the aim of visualizing disease clusters and microclusters. Methods: A discovery population of 202 adult patients [142 asthma, 60 healthy] and an external replication population [59 severe asthma] were evaluated. Pathology and gene expression were examined in bronchial biopsy samples. TDA was applied using pathological variables alone to create pathology-driven visual networks. Results: In the discovery cohort, TDA identified four groups/networks with multiple micro clusters/regions of interest that were masked by group level statistics. Specifically, TDA group 1 consisted of a high proportion of healthy subjects with a microcluster representing a topological continuum connecting healthy subjects to patients with mild-moderate asthma. Three additional moderate to severe asthma TDA groups (airway smooth muscleHIGH , reticular basement membraneHIGH and RemodellingLOW) were identified and contained numerous microclusters with varying pathological and clinical features. Mutually exclusive Th2 and Th17 tissue gene expression signatures were identified in all pathological groups. Discovery and external replication applied to the severe asthma subgroup only, identified highly similar 'pathological data shapes' via analyses of persistent homology. Conclusions: We have identified and replicated novel pathological phenotypes of asthma using topological data analysis. Our methodology is applicable to other complex chronic disease