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

Lee et al. generate knock-in mice with critical residues mutated in caspase-11 and GSDMD. This is the first compelling genetic evidence to demonstrate the critical roles of caspase-11 catalytic and auto-proteolytic activities, as well as GSDMD cleavage in a physiological response to LPS.
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 (Casp11Prc D285A/D285A) exhibit defective caspase-11 auto-processing and phenocopy Casp11−/− and caspase-11 enzymatically dead KI (Casp11Enz C254A/C254A) macrophages in attenuating responses to cytoplasmic LPS or Gram-negative bacteria infection. GsdmdD276A/D276A KI macrophages also fail to cleave GSDMD and are hypo-responsive to inflammasome stimuli, confirming that the GSDMD Asp276 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.