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.
Highlights
Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments
No studies have directly examined if N-GSDMD forms pores in the neutrophil plasma membrane, following activation of NLRP3 inflammasomes by nigericin or ATP
Using two different antibody clones that target murine GSDMD, we found that p52 pro-GSDMD levels were similar in LPS-primed neutrophils and macrophages prior to NLRP3 inflammasome activation (Fig. 2a and Supplementary Fig. 6a); there was less p31 N-GSDMD in NLRP3-activated neutrophils relative to macrophages, which correlated with lower production of cleaved caspase-1 (Fig. 2a)
Summary
Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1β release. 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. N-GSDM effector moiety maintains pro-GSDM in an autoinhibited conformation Disruption of this interface by proteolytic cleavage of linker loops or mutation of key residues induces conformational rearrangement of N-GSDM subunits Caspase-1 cleaves proIL-1β to the 17 kDa bioactive cytokine, and cleaves the 52 kDa pro-GSDMD to 31 kDa N-GSDMD products, which oligomerize at the macrophage plasma membrane to generate pores that function as direct conduits for IL-1β efflux and mediators of pyroptosis[10]. Accumulation of functional N-GSDMD pores in the neutrophil plasma membrane or roles for membrane repair in limiting pore numbers in neutrophils have not been explicitly evaluated
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