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 (<i>Casp11^{Prc D285A/D285A}</i> ) exhibit defective caspase-11 auto-processing and phenocopy <i>Casp11^-/-</i> and caspase-11 enzymatically dead KI (<i>Casp11^{Enz C254A/C254A}</i> ) macrophages in attenuating responses to cytoplasmic LPS or Gram-negative bacteria infection. <i>Gsdmd^D276A/D276A</i> 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.