A Novel Mechanism of Rapid Nuclear Neutrophil Extracellular Trap Formation in Response to Staphylococcus aureus

Abstract

Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3–4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5–60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton–Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton–Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.

Hypoxic regulation of neutrophil function and consequences for Staphylococcus aureus infection

“…Whether NETs kill [8,95] or merely trap [11] S. aureus, it seems likely that a reduction in NETosis under hypoxia (as discussed in Section 4.6.) would allow Staphylococcus to thrive.…”

Angiotensin converting enzyme inhibitors and angiotensin II receptor antagonist attenuate tumor growth via polarization of neutrophils toward an antitumor phenotype

“…[51][52][53] After stimulation with PMA (1 mg/mL) for 4 h, neutrophils were stained with Sytox Green (3 mM) for 30 min. Sytox Green fluorescence was measured using fluorescence microplate reader (Spectramax M2/e, Molecular Devices) at an excitation wavelength of 492 nm, and an emission wavelength of 530 nm.…”

A Thermonuclease ofNeisseria gonorrhoeaeEnhances Bacterial Escape From Killing by Neutrophil Extracellular Traps

“…These features share similarity to certain mechanisms of NET release in response to S. aureus [11], but N. gonorrhoeae does not possess leukotoxins, like those in S. aureus, that are associated with alternative modes of NET formation, leaving the open question of what N. gonorrhoeae factor(s) stimulate NET release. Formation of NETs also did not require phagocytosis of N. gonorrhoeae, suggesting that early events in the interaction between N. gonorrhoeae and neutrophils drive this process.…”

“…NETs have been observed in vivo in models of acute inflammation and infection, and NETs capture and can kill bacteria and fungi [8,9]. NETs may be released by dying cells by NETosis or from live, infected cells; mitochondrial DNA can also contribute to NETs [10][11][12].…”