Non-apoptotic Cell Death Control of Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are networks of chromatin and microbicidal proteins released by neutrophils in response to infection and tissue damage. Although classically viewed as a discrete biochemical and cellular process in neutrophils, the effector pathways integrating diverse upstream activating signals to control the formation of NETs (NETosis) are poorly defined. Cell death is one such common unifying endpoint of neutrophils, with several bona fide non-apoptotic cell death agonists now described to initiate NETosis. Integrating these new genetic findings into our existing knowledge of NETosis will likely reveal varied cellular and biochemical processes controlling NET release and specific anti-microbial and inflammatory effector functions of NETs triggered by specific non-apoptotic cell death. To facilitate investigation of regulated cell death pathways in NETosis, we offer a detailed protocol for neutrophil purification from mouse bone marrow and human blood, analysis of NETs by flow cytometry, and validation by immunogold electron microscopy. Future studies may better define cell death-specific forms of NETosis and their influence on inflammation and autoimmunity.