NETs persisting in vasculature undergo self-renewal with consequences for subsequent infection: a mouse model study

• NET protein components adhere to vascular endothelium for 5–8 months, whereas DNA is detached within days but circulates for up to a year

• In vivo physiological removal of NETs induces second-wave traps, altering the severity of subsequent infection, depending on its timing

While key for pathogen immobilization, neutrophil extracellular traps (NETs) often cause severe bystander cell/tissue damage. This was hypothesized to depend on their prolonged presence in the vasculature, leading to cytotoxicity. Imaging of NETs (histones, neutrophil elastase, extracellular DNA) with intravital microscopy in blood vessels of mouse livers in a pathogen-replicative-free environment (endotoxemia) led to detection of NET proteins attached to the endothelium for months despite the early disappearance of extracellular DNA. Intravascular liver macrophages (Kupffer cells) and neutrophils, but not monocytes, were involved in NET removal. They utilized scavenger receptors (SR-A) and Toll-like receptors (TLR2/4) to recognize NET components. Despite the absence of further stimuli, 14 days later a second wave of NET formation occurred, initiated by remnants of NETs from the first wave. The second burst of NET production was triggered by histones, which induced an inflammatory milieu (IL-1β) and activated platelets and coagulation-related events, including factor VII-activating protease (FSAP) activity. This in turn recruited and activated neutrophils to release the second wave of NETs. In peptidyl arginine deiminase (PAD4)-deficient mice, not forming NETs, inflammation and liver damage were reduced in comparison to their wild-type counterparts. When mice were challenged with methicillin-resistant Staphylococcus aureus (MRSA) 14 or 165 days post the second NETs, the course of infection/injury was diminished and exacerbated, respectively. Our study demonstrates that the complete removal of NETs in vivo takes much longer than hypothesized, and a vicious cycle of NET formation/disassembly impacts subsequent infection, depending on the time elapsed since its occurrence.