Caspase-11 auto-proteolysis is crucial for noncanonical inflammasome activation

Intracellular LPS sensing by caspase-4/5/11 triggers proteolytic activation of pore-forming gasdermin D (GSDMD), leading to pyroptotic cell death in Gram-negative bacteria-infected cells. Involvement of caspase-4/5/11 and GSDMD in inflammatory responses, such as lethal sepsis, makes them highly desirable drug targets. Using knock-in (KI) mouse strains, we herein provide genetic evidence to show that caspase-11 auto-cleavage at the inter-subunit linker is essential for optimal catalytic activity and subsequent proteolytic cleavage of GSDMD. Macrophages from caspase-11–processing dead KI mice (Casp11(Prc D285A/D285A)) exhibit defective caspase-11 auto-processing and phenocopy Casp11(−/−) and caspase-11 enzymatically dead KI (Casp11(Enz C254A/C254A)) macrophages in attenuating responses to cytoplasmic LPS or Gram-negative bacteria infection. Gsdmd(D276A/D276A) KI macrophages also fail to cleave GSDMD and are hypo-responsive to inflammasome stimuli, confirming that the GSDMD Asp(276) residue is a nonredundant and indispensable site for proteolytic activation of GSDMD. Our data highlight the role of caspase-11 self-cleavage as a critical regulatory step for GSDMD processing and response against Gram-negative bacteria.