Raised sputum extracellular DNA confers lung function impairment and poor symptom control in an exacerbation-susceptible phenotype of neutrophilic asthma

Abstract

Background Extracellular DNA (e-DNA) and neutrophil extracellular traps (NETs) are linked to asthmatics airway inflammation. However, data demonstrating the characterization of airway inflammation associated with excessive e-DNA production and its impact on asthma outcomes are limited. Objective To characterize the airway inflammation associated with excessive e-DNA production and its association with asthma control, severe exacerbations and pulmonary function, particularly, air trapping and small airway dysfunction. Methods We measured e-DNA concentrations in induced sputum from 134 asthma patients and 28 healthy controls. We studied the correlation of e-DNA concentrations with sputum neutrophils, eosinophils and macrophages and the fractional exhaled nitric oxide (FeNO). Lung function was evaluated using spirometry, body plethysmography, impulse oscillometry and inert gas multiple breath washout. We stratified patients with asthma into low-DNA and high-DNA to compare lung function impairments and asthma outcomes. Results Patients with severe asthma had higher e-DNA concentration (54.2 ± 42.4 ng/µl) than patients with mild-moderate asthma (41.0 ± 44.1 ng/µl) or healthy controls (26.1 ± 16.5 ng/µl), (all p values < 0.05). E-DNA concentrations correlated directly with sputum neutrophils (R = 0.49, p < 0.0001) and negatively with sputum macrophages (R = − 0.36, p < 0.0001), but neither with sputum eosinophils (R = 0.10, p = 0.26), nor with FeNO (R = − 0.10, p = 0.22). We found that 29% of asthma patients (n = 39) had high e-DNA concentrations above the upper 95th percentile value in healthy controls (55.6 ng /μl). High-DNA was associated with broad lung function impairments including: airflow obstruction of the large (FEV1) and small airways (FEF50%, FEF25–75), increased air trapping (RV, RV/TLC), increased small airway resistance (R5-20, sReff), decreased lung elasticity (X5Hz) and increased ventilation heterogeneity (LCI), (all P values < 0.05). We also found that high e-DNA was associated with nearly three-fold greater risk of severe exacerbations (OR 2·93 [95% CI 1.2–7.5]; p = 0·012), worse asthma control test (p = 0.03), worse asthma control questionnaire scores (p = 0.01) and higher doses of inhaled corticosteroids (p = 0.026). Conclusion Increased production of extracellular DNA in the airway characterizes a subset of neutrophilic asthma patients who have broad lung function impairments, poor symptom control and increased risk of severe exacerbations.

Sputum alarmin levels delineate distinct T2 cytokine pathways and patient subgroups in asthma

“…This pathway is consistent with transcriptomic studies implicating inflammasome activation in non-T2 asthma, (17,32) and sputum analyses showing elevated IL-1β levels together with caspase activity, neutrophilic inflammation, and neutrophil extracellular traps. (33,34) However, IL-1β mod signaling was not associated with a distinct non-T2 endotype in our study. Instead, IL-1β mod cytokines were co-expressed with T2 cytokines in cluster 3.…”

Longitudinal Impact of Sputum Inflammatory Phenotypes on Small Airway Dysfunction and Disease Outcomes in Asthma

“…We recently reported that increased extracellular DNA production in asthmatic airway, a collateral mechanism of airway neutrophilic inflammation, indicates a broad lung function impairment including SAD. 6 So far, therapies targeting neutrophilic airway inflammation in asthma failed to improve asthma outcomes, 41 leaving the complicated role of airway neutrophilic inflammation in asthma and SAD uncertain.…”

“…4 When considering the count of sputum neutrophils in this classification, eosinophilic asthma can be further subdivided into eosinophilic or mixed granulocytic, whereas noneosinophilic asthma can be subdivided into neutrophilic or paucigranulocytic. 4 Although both eosinophils and neutrophils, separated or combined, have been incriminated in asthmatic airway inflammation, 5,6 they might have different impacts on asthma severity, symptom control, and lung function impairment. 4 For instance, a recent study has shown that predominant mixed granulocytic asthma indicates worse lung function (forced expiratory volume in 1 second [FEV1]) than the other phenotypes.…”

Extracellular Traps: A Novel Therapeutic Target for Severe Asthma

“… 56 In addition, increased levels of extracellular DNA in sputum that negatively correlated with FEV 1 % was noted in NA patients with the exacerbation-susceptible phenotype. 47 Moreover, our group clarified epithelial-derived molecules (S100 calcium-binding protein A9 and serum amyloid A1), which were higher in the sera of NA, could induce NETs, further increasing neutrophilic airway inflammation. 57–59 Taken together, NETs are a key mediator of ongoing neutrophilic activation/degranulation, becoming an additional therapeutic target for NA.…”

“… 46 In response to IL-8, phorbol myristate acetate (PMA), or LPS, activated neutrophils released a web-like structure called NETs, which were made up of DNA and granule proteins. 47 , 48 To date, three major mechanisms have been shown to induce NET formation: NADPH oxidase 2 (Nox2)-dependent pathway with PMA stimulation, Nox2-independent pathway with A23187 or ionomycin stimulation, and mitochondrial ROS-dependent pathway with LPS or complement factor C5a receptor stimulation. 49 Currently, the significance of NETs is emphasized in various diseases including cardiovascular disease, metabolic diseases, certain septic conditions, autoimmune, and inflammatory diseases.…”