N-GSDMD trafficking to neutrophil organelles facilitates IL-1β release independently of plasma membrane pores and pyroptosis

TLDR

In inflammasome‑activated macrophages, caspase‑1 cleaves GSDMD to generate N‑GSDMD, which oligomerizes in the plasma membrane to form pores that increase permeability, trigger pyroptosis, and release IL‑1β. In neutrophils, N‑GSDMD traffics to azurophilic granules, where it induces neutrophil elastase leakage and secondary cleavage of GSDMD, and IL‑1β secretion occurs via an autophagy‑dependent pathway. These results show that, unlike macrophages, neutrophil N‑GSDMD does not localize to the plasma membrane or cause pyroptosis but instead associates with granules and autophagosomes, revealing distinct trafficking that underlies neutrophil‑specific inflammasome functions.

Abstract

Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1β release. In contrast, we report that although N-GSDMD is required for IL-1β secretion in NLRP3-activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3+ autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1β via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation.