Hypochlorous acid regulates neutrophil extracellular trap release in humans

Abstract

Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine.

Fig. 1

Metabolism of oxygen radicals produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.

Fig. 2

Fluorometric quantification of neutrophil extracellular trap (NET)-DNA from phorbol myristate acetate (PMA) (25 nM)-stimulated neutrophils after 3 h incubation in the absence or presence of (a) diphenylene iodonium (DPI) 25 µM and (b) superoxide dismutase (SOD) 95 units/ml. Data represent six independent experiments ± standard deviation (s.d.). Fluorometric quantification of NET-DNA (c) and reactive oxygen species (ROS) generation (d) from PMA (25 nM) or opsonized Staphylococcus aureus (300 per neutrophil)-stimulated neutrophils after 3 h incubation in the absence or presence of cytochalasin B (cytoB; 10 µg/ml). Data represent at least five independent experiments ± s.d. Luminol measures total ROS, isoluminol measures extracellular ROS and lucigenin measures extracellular superoxide. **P ≤ 0·01 by two-tailed paired t-test.

Fig. 3

(a) Fluorometric quantification of neutrophil extracellular trap (NET)-DNA from phorbol myristate acetate (PMA) 25 nM-stimulated neutrophils after 3 h incubation in the absence or presence of catalase inhibitor, 3-aminotriazole (3-AT) 1 mM. Data represent six independent experiments ± standard deviation (s.d.) (b) Peak PMA 25 nM stimulated total reactive oxygen species (ROS) production measured by luminol. Data represent three independent experiments ± standard deviation (s.d.). *P ≤ 0·05 by two-tailed paired t-test. (c) Effect of 3-AT 1 mM on the activity of purified myeloperoxidase (MPO) 100 ng/ml compared to the specific MPO inhibitor, aminobenzoic acid hydrazide (4-ABAH) 1 mM (positive control). Experiment performed in triplicate ± s.d. Fluorometric quantification of NET-DNA from PMA 25 nM-stimulated neutrophils after 3 h incubation in the absence or presence of (d) glutathione precursor, NAC 10 mM or (e) 4-ABAH 0·1 mM. Data represent six independent experiments ± s.d. **P ≤ 0·01 by two-tailed paired t-test.

Fig. 4

(a) Fluorometric quantification of neutrophil extracellular trap (NET)-DNA from hypochlorous acid (HOCl)-stimulated neutrophils after 3 h incubation. Data represent six independent experiments ± standard deviation (s.d.). (b) Images of SYTOX-stained NET-DNA released after 3 h in the absence or presence of HOCl 0·75 mM. Bar represents 50 µm. (c) Fluorometric quantification of NET-DNA from hydrochloric acid (HCl) stimulated neutrophils after 3 h incubation. Experiment performed in quadruplicate ± s.d.; phorbol myristate acetate (PMA) 25 nM stimulation included as positive control. (d) Fluorometric quantification of NET-DNA released from chronic granulomatous disease (CGD) neutrophils after 3 h incubation with HOCl 0·75 mM. Data representative of three independent experiments ± s.d. *P ≤ 0·05, **P ≤ 0·01, ***P ≤ 0·001 by two-tailed paired t-test in comparison to unstimulated cells. (e) Fluorometric quantification of NET-DNA from PMA 25 nM or HOCl 0·75 mM-stimulated neutrophils after 3 h incubation in the absence or presence of taurine. Data represent four independent experiments ± s.d. **P ≤ 0·01, ***P ≤ 0·01 by two-tailed paired t-test in comparison to 0 mM taurine.

Fig. 5

Time–course fluorescence photomicrographs (SYTOX staining) of neutrophils stimulated with (a) 0·75 mM hypochlorous acid (HOCl); (b) 1% Triton X-100 to induce cell lysis; (c) phosphate-buffered saline (PBS) negative control. PBS does not affect neutrophil viability to release nuclear DNA. Triton lyses cells immediately (0 min) to release DNA without neutrophil extracellular trap (NET) structure formation. HOCl stimulates NET-DNA release between 30 and 70 min post-stimulation. Bar represents 100 µm.