Biological function of eosinophil extracellular traps in patients with severe eosinophilic asthma

Abstract

Eosinophil extracellular traps (EETs), a complex of DNA fibers and cytotoxic granule proteins, are implicated in the development of asthma; however, the pathophysiological function of EETs in immune responses has not been fully determined. The present study investigated the characteristics of EETs from patients with non-severe asthma (NSA, n = 20) and severe eosinophilic asthma (SEA, n = 20) and evaluated EET function. The percentage of EET-forming peripheral blood eosinophils stimulated with IL-5 and LPS was significantly higher in patients with SEA than in those with NSA (P = 0.009). This percentage negatively correlated with baseline FEV₁ (r = -0.350, P = 0.027) and positively correlated with serum eosinophil-derived neurotoxin levels in asthmatic subjects (r = 0.437, P = 0.018). In addition, EET formation was markedly associated with reactive oxygen species production (r = 0.750, P < 0.001). These EETs exhibited an autocrine function to induce eosinophil degranulation, which led to granule protein production. Airway epithelial cells stimulated with EETs exhibited increased epithelial detachment and permeability and pro-inflammatory cytokine release. However, EETs were not significantly associated with mast cell activation. The present study suggests that peripheral blood eosinophils from patients with SEA may be more activated to produce EETs than those from patients with NSA, which further induces inflammation in asthmatic airways. Therefore, regulation of EET formation and function may be a novel therapeutic approach for asthma management.

Fig. 1. Characteristics of EETs from peripheral blood eosinophils of patients with NSA and SEA.

a Induction of EET formation (white arrows). Scale bar, 50 µm. b Comparison of the percentage of EET-forming eosinophils with (closed) or without (open) IL-5 and LPS stimulation. Associations between EET formation and c baseline FEV₁/d serum EDN levels. The data are presented as Pearson correlation coefficient r (P value)

Fig. 2. Reactive oxygen species (ROS)-dependent EET production from peripheral blood eosinophils of patients with NSA.

Effects of dexamethasone (Dex), hydroxychloroquine (HCQ) or N-acetyl-l-cysteine (NAC) on a EET formation and b ROS production. Data are presented as the mean ± SD, n = 5. *P < 0.05, **P < 0.01 and ***P < 0.001 obtained using one-way ANOVA with Bonferroni’s post hoc test. n.s., not significant. c A correlation between ROS and EET production. The data are presented as Pearson correlation coefficient r (P value)

Fig. 3. Autocrine function of EETs on eosinophil activation.

a Morphological changes of peripheral blood eosinophils from patients with NSA after 100 nM phorbol myristate acetate (PMA) or 5 µg/mL EET (from NSA) treatment. b Immunofluorescence staining of released EETs (white arrows). Scale bar, 10 µm. c Eosinophil degranulation. d ROS production. e Effect of Dex, HCQ and NAC on eosinophil production of ECP. Data are presented as the mean, n = 5. *P < 0.05, **P < 0.01, and ***P < 0.001 obtained using one-way ANOVA with Bonferroni’s post hoc test. n.s., not significant

Fig. 4. Effect of EETs on airway epithelial cells.

a Changes in A549 cell morphology following EET (from NSA) treatment in a dose-dependent manner. Concentrations of b IL-8 and c IL-6 released from A549, BEAS-2B, and human primary small airway epithelial cells (SAEC) treated with 5 µg/mL EETs. Data are presented as the mean ± SD, n = 5. *P < 0.05, **P < 0.01, and ***P < 0.001 were obtained using one-way ANOVA with Bonferroni’s post hoc test

Fig. 5. Effect of EETs on mast cell activation.

a LAD-2 cell degranulation following EET (from NSA) treatment. b Levels of TNF-α and MCP-1 released from LAD-2 cells. Data are presented as the mean ± SD, n = 3. **P < 0.01 was obtained using one-way ANOVA with Bonferroni’s post hoc test. n.s., not significant