Phagocyte extracellular traps in children with neutrophilic airway inflammation

Abstract

Childhood lung infection is often associated with prominent neutrophilic airway inflammation and excess production of proteases such as neutrophil elastase (NE). The mechanisms responsible for this inflammation are not well understood. One potentially relevant pathway is the production of extracellular traps by neutrophils (NETs) and macrophages (METs). The aim of this study was to measure NET and MET expression in children and the effect of deoxyribonculease (DNase) 1 and α₁-antitrypsin (AAT) on this process. We studied 76 children (median age of 4.0 years) with cystic fibrosis or chronic cough who underwent investigational bronchoscopy. NETs, METs and neutrophil elastase activity in bronchoalveolar lavage (BAL) samples were measured using confocal microscopy and functional assays. The effects of DNase 1 and AAT on NET/MET expression and neutrophil elastase activity were examined in vitro. Both subject groups had airway neutrophilia with prominent BAL production of NETs with neutrophil elastase co-expression; the mean %±standard error of the mean of neutrophils expressing NETs in the cystic fibrosis group was 23.3±2.8% and in the non-cystic fibrosis group was 28.4±3.9%. NET expression was higher in subjects who had detectable neutrophil elastase activity (p≤0.0074). The percentage of macrophages expressing METs in the cystic fibrosis group was 10.7±1.2% and in the non-cystic fibrosis group was 13.2±1.9%. DNase 1 decreased NET/MET expression (p<0.0001), but increased neutrophil elastase activity (p≤0.0137). The combination of AAT and DNase 1 reduced neutrophil elastase activity (p≤0.0049). We observed prominent extracellular trap formation in symptomatic children with and without cystic fibrosis. This innate inflammatory response was down-regulated by a combination of currently available therapeutics.

FIGURE 1

Phagocyte extracellular trap expression. Neutrophil (NET) and macrophage (MET) extracellular trap expression was assessed in both the cystic fibrosis (CF) and non-CF groups using confocal microscopy; at baseline and with the addition of the bacterium nontypeable Haemophilus influenzae (NTHi). a) NET expression and b) MET expression. Images shown are merged with chromatin (blue), citrullinated histone (H3Cit) (red), matrix metalloproteinase (MMP) 9 (green) and neutrophil elastase (NE) (white). c) NET expression in CF group and d) NET expression in non-CF group. e) MET expression in CF group and f) MET expression in non-CF group. Bronchoalveolar lavage (BAL) neutrophil elastase (NE) activity was assessed in both g) CF and h) non-CF groups. The number of neutrophils expressing NETs was compared between subjects without detectable NE and with detectable NE. Results shown as mean±sem or median.

FIGURE 2

Measurement of interleukin (IL) 6 and alpha-1 antitrypsin (AAT) levels. Potential biomarkers/inflammatory mediators were assessed in the BAL fluid. Both childhood groups had detectable BAL IL-6 that was higher than the adult control group (a). BAL macrophage production of IL-6 with nontypeable Haemophilus influenzae (NTHi) infection in cystic fibrosis (CF) (b) and non-CF (c) groups. BAL levels of AAT (measured with ELISA) were compared between CF, non-CF and adult control groups (d). Results expressed as medians.

FIGURE 3

Effect of deoxyribonuclease (DNase) 1 and alpha-1 antitrypsin (AAT). The effect of DNase 1 on neutrophil extracellular trap (NET) expression was assessed in both the cystic fibrosis (CF) and non-CF groups. a) Extracellular trap expression in NTHi stimulated cells and b) the effect of the addition of DNase 1. c) NET expression in CF group and d) NET expression in non-CF group. The addition of AAT decreased NE activity compared to control in e) CF subjects and similar results were noted in f) non-CF subjects. DNase 1 increases NE activity which is reduced by the addition of AAT in g) CF subjects and h) non-CF subjects. The addition of AAT to BAL reduces NE activity in i) CF and j) non-CF populations. Results shown as mean±sem or median.

FIGURE 4

Potential relation between key results. Summary of the possible relation between the airway and systemic immune responses. Infection/inflammation in the airway causes the expression of neutrophil extracellular traps (NETs) with neutrophil elastase (NE); as well as activating macrophages to produce inflammatory mediators such as interleukin (IL)-6. One of the potential effects of inflammatory mediators is to act on the liver to induce acute phase reactants such as C-reactive protein (CRP) and α₁-antitrypsin (AAT). Circulating AAT may then come back to the airway. DNase 1 breaks down NETs and AAT inactivates NE. Both DNase 1 and AAT may be given exogeneously.