A Distinct c-Raf–MEK1/2–p38 MAPK–Caspase Signaling Pathway Mediates TNF-α-Induced Rapid and Non-Lytic Neutrophil Extracellular Trap Formation in Human Neutrophil-Like Cells

Neutrophil extracellular traps (NETs) contribute to host defense by trapping pathogens. However, excessive or dysregulated NET formation is implicated in the pathogenesis of various conditions, including thrombosis, autoimmune diseases, and cancer. Despite their clinical significance, the specific intracellular signaling pathways underlying NET formation in response to proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), remain incompletely defined. Thus, in the present study, we aimed to elucidate the mechanisms underlying TNF-α-induced NET formation in human neutrophil-like differentiated HL-60 (dHL-60) cells. TNF-α induced rapid, non-lytic NET formation in dHL-60 cells without compromising cell viability. Mechanistically, TNF-α activated a signaling cascade involving c-Raf, MEK1/2, and p38 mitogen-activated protein kinase (MAPK). This signaling cascade regulated the sequential caspase activation, with caspase-2 acting upstream of caspase-1 cleavage to promote NET formation. Collectively, these findings suggest that TNF-α induces non-lytic NET formation via a defined c-Raf–MEK1/2–p38 MAPK–caspase pathway and a distinct caspase hierarchy. This mechanism offers a valuable experimental model for studying NET-mediated pathologies in proinflammatory environments.