Mechanisms of NET formation. (A) PMA and other stimuli induce lytic-NET formation. Stimulation of neutrophils with PMA resulted in the activation of NADPH oxidase, via PKC and Raf-MEK-ERK signaling pathway and consequent ROS generation. Afterward, PAD4 is activated and citrullinates arginine on histones causing chromatin decondensation. MPO and NE are released from cytoplasmic azurophilic granules and then translocated to the nucleus contributing to unfolding of chromatin. Subsequently, the nuclear envelope broke down, releasing the chromatin in the cytosol, which mixed with cytosolic proteins. NE also cleaves GSDMD in the cytosol to its active form (GSDMD-NT), which, besides forming pores in the plasma membrane, also mediates pore formation in nuclear and granule membranes, enhancing NE and other granular content release. Finally, NETs are released, and the neutrophil dies. (B) Nonlytic NET formation is induced by the recognition of stimuli through Toll-like receptor 2 (TLR2), TLR4, or complement receptors independent of NAPDH oxidase activation. S aureus and C albicans activate TLR2 and complement receptors, respectively, and E coli or LPS-activated platelets activate TLR4. Along with PAD4 activation and NE translocation to the nucleus, chromatin decondensation proceeds and protein-decorated chromatin is expelled via vesicles without plasma membrane disruption. After the release of NETs, neutrophils are still alive for further functions.