Auto-amplification and spatial propagation of neutrophil extracellular traps

The release of cellular DNA as neutrophil extracellular traps (NETs) plays a pivotal role in the immune response to pathogens by physically entrapping and killing microbes. NET release occurs at a greater frequency within neutrophil clusters and swarms, indicating a potential for collective behavior. However, little is known about how dense clustering of cells influences the frequency of NET release. Using an image-based assay for NETosis in nanowells, we show that the frequency of NETosis increases with cell density. We then co-incubate NETotic neutrophils with naive neutrophils and find that NETotic neutrophils can induce secondary NETosis in naive neutrophils in a cell density-dependent manner. Further mechanistic studies show that secondary NETosis is caused by a combination of DNA and protein factors. Finally, we immobilize NETotic neutrophils in a plaque, and then place the plaque near naive neutrophils to characterize the spatial propagation of secondary NETosis. We find that secondary NETosis from naive neutrophils increases over time, but remains spatially restricted to the periphery of the plaque. Together, we show that NETosis is an auto-amplified process, but that the spatial propagation of NET release is strictly regulated. This study shows that NETosis is an auto-amplified process whereby NETotic neutrophils can induce secondary NETosis in proximal naive neutrophils, resulting in spatial propagation of NETs.