Pyocyanin-Enhanced Neutrophil Extracellular Trap Formation Requires the NADPH Oxidase

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.

Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity

“…Consistent with investigations using other pharmacological agents to stimulate NET production (19,24), we found anacardic acid-induced NET production to be both ROS-and PI3K-dependent; this is in keeping with reports describing coupling of the S1P4 receptor to G␣ 12/13 (25), which has been shown to trigger cell death pathways via activation of PI3K (26). Given the diverse array of GPCRs acting through G␣ 12/13 , our findings could help guide future efforts to identify novel and potentially more selective/potent, immune-boosting compounds.…”

NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

“…The requirement for NADPH oxidase in NETosis is dependent on the stimulus (38,52,(56)(57)(58)(59). H 2 O 2 and hypochlorous acid, ROI metabolites generated by NADPH oxidase activation in neutrophils, trigger NET release (17,(60)(61)(62).…”

Pseudogout-Associated Inflammatory Calcium Pyrophosphate Dihydrate Microcrystals Induce Formation of Neutrophil Extracellular Traps

“…Pelleted neutrophils were stored in a 1:1 mix of autologous serum and RPMI 1640 medium (Life Technologies, Grand Island, NY) until use. Serum was isolated from 10 ml coagulated blood by centrifugation and sterile filtration (28). Before use, required number of neutrophils was washed twice in calcium-, magnesium-containing HBSS (Mediatech, Manassas, VA), and experiments were performed on neutrophils resuspended in HBSS without serum or albumin as assay buffer.…”

“…NET formation stimulated by bacteria or PMA require reactive oxygen species (ROS) produced by the Nox2-based NADPH oxidase (28,35,39,40). Treatment of neutrophils with the NADPH oxidase inhibitor DPI abolishes NET formation induced by bacteria or PMA (35,39).…”

“…Chronic granulomatous disease (CGD) patients lack a fully functional respiratory burst because of mutations in one of the subunits of the NADPH oxidase enzyme complex and suffer from recurrent fungal and bacterial infections (41). CGD neutrophils do not produce NETs in response to bacteria or PMA (28,35). Therefore, we asked whether neutrophils respond to CPPD crystal exposure with superoxide production.…”