Clusters of GSDMD and GSDME mediate neutrophil pyroptosis in cryopyrin-associated periodic syndrome

The Nod-like receptor family pyrin domain–containing 3 (NLRP3) inflammasome is a cytosolic multiprotein immune sensor that is restrained from assembling at steady state. Mutations in NLRP3 associated with cryopyrin-associated periodic syndrome (CAPS) enhance inflammasome formation and activation, resulting in neutrophil-predominant systemic and tissue inflammation. Here, we investigated how gasdermin D (GSDMD) and GSDME drive interleukin-1β (IL-1β) release, pyroptosis, and autoinflammation during constitutive and canonical NLRP3 activation in neutrophils. Mass cytometry analysis of neutrophils from patients with CAPS revealed heterogeneous subtypes with differing amounts of intracellular IL-1β. In mice, neutrophil-specific expression of activating Nlrp3 mutations orthologous to those in patients with CAPS uncovered nonredundant roles for GSDMD and GSDME downstream of inflammasome activation. Combined deletion of Gsdmd and Gsdme in these CAPS models reduced IL-1β secretion, prevented pyroptosis, and mitigated autoinflammation. Loss of both GSDMD and GSDME reduced caspase-1 cleavage and pro–IL-1β processing downstream of NLRP3 activation, whereas GSDMD deficiency alone increased cleavage of GSDME and caspase-8. Clusters of GSDMD and GSDME were closely associated with one another at the plasma membrane of activated neutrophils from CAPS mice. These findings indicate that the coordinated actions of membrane-associated GSDMD and GSDME reinforce cytosolic NLRP3 inflammasome activation to promote IL-1β secretion, lytic cell death, and autoinflammation.