Dynamic NETosis is Carried Out by Live Neutrophils in Human and Mouse Bacterial Abscesses and During Severe Gram-Positive Infection

Gram-positive bacteria such as Staphylococcus aureus1–4 and Streptococcus pyogenes5 are highly invasive pathogens causing severe skin infections and sepsis in humans. Neutrophils are critical for host survival and NETs may represent a novel defense mechanism6–9. In vitro NETs are a cell-death event occurring hours after initial stimulation10,11; however questions remain concerning NETosis in vivo. Conventional PMN killing involves cell recruitment, emigration, chemotax and phagocytosis of microbes12,13. However chasing individual microbes is ineffective to deal with rapidly disseminating Gram-positive pathogens. Utilizing both phagocytosis and NETosis might be counter-productive if neutrophils engulf bacteria and then lyse releasing all their bacterial contents. Thus non-lytic NETosis may prevent bacteria from escaping following phagocytosis. To date NETosis has only been observed in immobile and incapacitated cells. In vitro, we described rapid NET release (< 10 min) without cell lysis, however the fate and function of these PMN could not be evaluated14,15. In this study we hypothesize that rapid NET release may be carried out in vivo by live viable PMN. To understand the relationship between conventional PMN-mediated and NET-mediated host defense, we directly visualize PMN behavior during Gram-positive skin infections in mice and humans. Using spinning-disk confocal intravital microscopy (SDCIM) surprising insights, not in line with the current NETosis paradigm, were revealed. Cell death and lytic release of NETs were not observed in vivo, nor was nuclear condensation and budding off en bloc, observed in red blood cell enucleation16. Instead, live functional PMN crawled, phagocytosed and rapidly NETosed simultaneously, resulting in widespread tissue NETs that limited bacterial dissemination. NETosis occurred in emigrated PMN during localized infection and this was tightly regulated through Tlr2 and complement mediated opsonization. NETosing PMN crawled with erratic pseudopod formation and extreme cell morphologies due to the loss of the nuclear structure acting as a fulcrum for the cytoskeleton17,18. Importantly, human neutrophils released NETs in an in vivo model and patients with acute Gram-positive abscesses demonstrated intact NETosing PMN identical to the animal model. Here we describe an alternate fate of in vivo NETosing PMN that is distinct from cell-death NETosis.