Pyocyanin-Enhanced Neutrophil Extracellular Trap Formation Requires the NADPH Oxidase

Balázs Rada,Craig P Hayes,Dae-Goon Yoo,Lan Pang,Jonathan J Park,Samuel M Moskowitz,Thomas L Leto,Harry L Malech,Meghan A Jendrysik,Amit Gaggar

doi:10.1371/journal.pone.0054205

Abstract

Beyond intracellular killing, a novel neutrophil-based antimicrobial mechanism has been recently discovered: entrapment and killing by neutrophil extracellular traps (NETs). NETs consist of extruded nuclear DNA webs decorated with granule proteins. Although NET formation is an important innate immune mechanism, uncontrolled NET release damages host tissues and has been linked to several diseases including cystic fibrosis (CF). The major CF airway pathogen Pseudomonas aeruginosa establishes chronic infection. Pseudomonas imbedded within biofilms is protected against the immune system, but maintains chronic inflammation that worsens disease symptoms. Aberrant NET release from recruited neutrophils was found in CF, but the underlying mechanisms remain unclear. One of the most important Pseudomonas virulence factors is pyocyanin, a redox-active pigment that has been associated with diminished lung function in CF. Here we show that pyocyanin promotes NET formation in a time- and dose-dependent manner. Most CF Pseudomonas clinical isolates tested produce pyocyanin in vitro. Pyocyanin-derived reactive oxygen species are required for its NET release. Inhibitor experiments demonstrated involvement of Jun N-terminal Kinase (JNK) and phosphatidylinositol 3-Kinase (PI3K) in pyocyanin-induced NET formation. Pyocyanin-induced NETs also require the NADPH oxidase because NET release in chronic granulomatous disease neutrophils was greatly reduced. Comparison of neutrophils from gp91phox- and p47phox-deficient patients revealed that pyocyanin-triggered NET formation is proportional to their residual superoxide production. Our studies identify pyocyanin as the first secreted bacterial toxin that enhances NET formation. The involvement of NADPH oxidase in pyocyanin-induced NET formation represents a novel mechanism of pyocyanin toxicity.

Highlights

Polymorphonuclear neutrophil granulocytes (PMN) provide the first line of defense against bacteria
[23] pyocyanin has been studied in several laboratories worldwide, and increased levels of both pyocyanin and neutrophil components in the cystic fibrosis (CF) lung have been associated with poor lung function, no reports to date have described any direct effects of pyocyanin on neutrophil extracellular traps (NETs) formation
Contrary to earlier reports showing that pyocyanin induces apoptosis in neutrophils, we found that pyocyanin in shorter time frames and at lower concentrations induces NET formation (Fig. 2 and 3)

Summary

Introduction

Polymorphonuclear neutrophil granulocytes (PMN) provide the first line of defense against bacteria. Neutrophils migrate to the site of infection, engulf and kill the invaders by exposing them to a variety of antimicrobial peptides, proteins and reactive oxygen species. Neutrophils combat pathogens by a recently described novel mechanism, formation of neutrophil extracellular traps (NETs). NETs are composed of a DNA backbone decorated with histones and several antimicrobial neutrophil granule components: myeloperoxidase (MPO), lactoferrin, elastase and bactericidal/permeability-increasing protein. [2] NET formation (NETosis) is a novel form of neutrophil cell death different from apoptosis or necrosis. [5] Reactive oxygen species (ROS) produced by the phagocytic NADPH oxidase are essential for NET formation, since neutrophils of chronic granulomatous disease (CGD) patients are unable to release NETs in response to a variety of stimuli. Bacteria (whole cells, LPS, pilus) or inflammatory mediators (IL-8, IFN I+II, C5a) have been reported to induce NETs. [5] Reactive oxygen species (ROS) produced by the phagocytic NADPH oxidase are essential for NET formation, since neutrophils of chronic granulomatous disease (CGD) patients are unable to release NETs in response to a variety of stimuli. [4] CGD neutrophils produce very little or no superoxide due to genetic deficiencies in any one of several components of the superoxide-producing NADPH oxidase enzyme complex [6]

Methods

Results

Conclusion