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. 2018 Sep 3;215(9):2279-2288.
doi: 10.1084/jem.20180589. Epub 2018 Aug 22.

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

Bettina L Lee  1 , Irma B Stowe  1 , Aaron Gupta  1 , Opher S Kornfeld  1 , Merone Roose-Girma  2 , Keith Anderson  2 , Søren Warming  2 , Juan Zhang  3 , Wyne P Lee  3 , Nobuhiko Kayagaki  4
Affiliations

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

Bettina L Lee et al. J Exp Med. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
Induction of cleaved caspase-11 fragment in macrophages in response to cytoplasmic LPS. (A) Schematic of caspase-11 representing the CARD domain and large and small catalytic subunits. Predicted cleavage sites D59, D80, and D285 are indicated. Asterisk (*) marks critical cysteine residue in caspase-11 catalytic site (C254). M61 is a predicted alternative start site. (B and C) BMDMs were treated with 1 µg/ml Pam3CSK4 for 5 h before stimulation. (B) Immunoblot of caspase-11, GSDMD, and actin in combined cell extract (ext) and supernatant (sup) from WT BMDMs 30, 60, and 120 min after LPS electroporation or control. (C) LDH release time course in WT BMDMs 30, 60, and 120 min after LPS stimulation from cells in B. Data are presented as mean ± SD (n = 3) in C and representative of at least two independent experiments (B and C).
Figure 2.
Figure 2.
Caspase-11 auto-processing at Asp285 is essential for GSDMD processing, pyroptosis, and IL-1β release. (A–D) BMDMs were treated with 1 µg/ml Pam3CSK4 for 5 h. (A) Immunoblot of caspase-11, GSDMD, and actin in extract (ext) + supernatant (sup) from WT, Casp11Enz C254A/C254A, and Casp11Prc D285A/D285A BMDMs at 60 min after LPS electroporation or control. (B) Cell death measured by percent YOYO-1+ cells from live cell images taken every 30 min over a 16-h time course following LPS electroporation of BMDMs from two mice per genotype. (C) LDH, IL-1β, and IL-18 release from BMDMs stimulated with transfected LPS (5 µg/ml plus FuGENE HD), ATP (5 mM), or nigericin (10 µg/ml) after 16 h, 3 h, or 30 min, respectively. (D) LDH, IL-1β, and IL-18 release measured from supernatants of BMDMs infected with indicated strains of bacteria. Data are representative of at least two independent experiments (A and B). Data are presented as mean ± SD (n = 3) and representative of at least three independent experiments (C and D).
Figure 3.
Figure 3.
GSDMD Asp276 residue is the critical nonredundant cleavage site following caspase-1/11 activation. (A–D) BMDMs were treated with 1 µg/ml Pam3CSK4 for 5 h before stimulation. (A) Immunoblot of GSDMD and actin in combined cell extract (ext) + supernatant (sup) from WT and GsdmdD276A/D276A BMDMs after 60 min following control treatment, LPS electroporation, or ATP stimulation for detection of GSDMD full-length (pro), GSDMD NT (p30), and p43. (B) Cell death measured by percent YOYO-1+ cells from live cell images taken every 30 min over a 16 h time course following LPS electroporation of BMDMs from two mice per genotype. (C) LDH, IL-1β, and IL-18 release measured from supernatants of BMDMs stimulated with transfected LPS (5 µg/ml plus FuGENE HD), ATP (5 mM), or nigericin (10 µg/ml) after 16 h, 3 h, or 30 min, respectively. (D) LDH, IL-1β, and IL-18 release measured from supernatants of BMDMs infected with indicated strains of bacteria. Data are representative of at least two independent experiments (A and B). Data are presented as mean ± SD (n = 3) and representative of at least three independent experiments (C and D).
Figure 4.
Figure 4.
Caspase-11 auto-processing and GSDMD cleavage are required to induce acute septic shock. (A and C) Kaplan-Meier survival plots for mice (n = 10 per genotype) challenged with 54 mg/kg LPS. Adjusted P values are supplied in Figs. S3 (A and B). (B) Serum IL-1β and IL-18 levels 12 h after intraperitoneal injection with 20 mg/kg LPS. Each dot represents a single animal (n = 5); error bars represent mean ± SD. Data are representative of at least two independent experiments.
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