Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd−/− mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd−/− mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.
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We thank the staff of the Australian Phenomics Facility, Genentech Transgenic Technology and FACS cores, and K. Bowman, J. Payandeh, E. Dueber, R. Aglietti, A. Gupta and A. Peterson for technical expertise and discussion, K. Newton for manuscript editing, A. Muszyński, L. S. Forsberg, and R. W. Carlson for S. typhimurium LPS. Most authors were employees of Genentech, Inc.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Homozygosity for a Gsdmd point mutation correlates with unresponsiveness to cytoplasmic LPS.
Gsdmd genotypes and screen phenotypes of mice derived from IGL1351 pedigree (top). Only the identification numbers of screened animals are shown. Thirty-nine SNV genotypes mutated in the pedigree and their phenotypes (bottom). Hits represent mice whose peritoneal macrophages showed attenuated LPS-induced IL-1β secretion to a similar extent as Casp11 mutant 129 strain. Mutant, homozygous.
a, RANTES production from BMDMs cultured for 16 h with medium alone (cont) or the TLR stimulants indicated. b, Western blots of BMDM extracts and supernatants at 6 h after LPS electroporation. Graph shows the mean ± s.d. of triplicate wells and represents three independent experiments. For source gel, see Supplementary Fig. 2.
a, Western blots of BMDMs cultured with or without Pam3CSK4 for 6 h. Gsdmd−/−1 and Gsdmd−/−2 are independent knockout strains. b–d, IL-1β and LDH released from BMDMs after 16 h. P. aeruginosa infection was analysed at 4 h. e, RANTES production from BMDMs after 16 h. f, IL-1β and LDH released from BMDMs at 16 h after LPS electroporation, LPS plus cholera toxin B (Ctb) complex, or S. typhimurium LPS transfection. g, i, Western blots of EA.hy926 (g) or THP-1 cells (i). h, LDH released from control (parental or luciferase gRNA), GSDMD knockout (KO), or CASP4 knockout THP-1 cells at 16 h after LPS electroporation or dsDNA transfection. Graphs show mean ± s.d. of triplicate wells and represent three independent experiments. For source gels of a, g and i, see Supplementary Information Fig. 2.
a, Alignment of Gsdmd amino acid sequences. The conserved Gsdmd cleavage site (D276 in mouse) is boxed in red. b, Immunoblots of BMDM extracts and supernatants at 16 h after LPS/Ctb treatment. A non-specific band is indicated with an asterisk. Cont, Ctb alone. For source gel, see Supplementary Fig. 3.
a, Western blots of THP-1 extracts and supernatants at 3 h after LPS electroporation. b, Western blots of human GSDMD/HEK293T stable transfectants at 24 h after transient transfection of the indicated plasmids. A non-specific band is indicated with an asterisk. c, Cytotoxicity of the human proteins indicated at 24 h after transient transfection of HEK293T cells. Numbers indicate nanograms of plasmid transfected. Western blots (right) indicate protein expression. Expression of the N-terminal fragment (1–275) was below detection levels, presumably due to its potent toxicity. Graph shows mean ± s.d. of triplicate wells and represents three independent experiments. For source gels of a, b and c, see Supplementary Fig. 3.
Extended Data Figure 6 BMDMs stimulated with cytoplasmic LPS do not release NLRP3-stimulating damage-associated molecular pattern activity.
a, Paracrine signalling hypothesis. DAMP, damage-associated molecular pattern. b, LDH released from BMDMs at 16 h after LPS transfection or electroporation. c, IL-1β released from Casp11−/− BMDMs at 16 h after stimulation with ATP or incubation with Il1b−/− BMDM culture supernatants derived in b. d, LDH released from 1:1 mixed cultures of the indicated BMDMs at 16 h after stimulation. Graphs show mean ± s.d. of triplicate wells and represent three independent experiments.
Extended Data Figure 7 Canonical inflammasome stimuli induce caspase-1-dependent processing of Gsdmd.
a, Western blots of BMDM extracts and supernatants at 8 h after stimulation. Cont, medium alone. LPS, LPS + Ctb. Asterisk indicates a non-specific band. b, Western blots of Gsdmd−/− BMDMs. Gsdmd−/−3 and Gsdmd−/−4 are independent knockout strains (Extended Data Fig. 8). c, IL-1β released from BMDMs. LPS, LPS electroporation. Graphs show mean ± s.d. of triplicate wells and represent three independent experiments. d, Immunoblots of Casp1−/− BMDMs stimulated with Pam3CSK4 for 5 h. For source gels of a, b and d, see Supplementary Fig. 3.
Gsdmd−/− animals used in this study were compound or homozygous F1 and F2 knockouts generated from mosaic F0 founder and F1 crosses, respectively. gRNA target sequences are highlighted in bold. Deleted bases are indicated by red hyphens. Inserted nucleic acids are highlighted in red.
This file contains the Source Gels as follows: Supplementary Figure 1 - Main Figures 1c, 1d, 2b, 3a, 3c, 3e, 4a; Supplementary Figure 2 - Main Figures 5a, 5b, and Extended Data Figures 2b, 3a, 3g 3i; Supplementary Figure 3 – Extended Data Figures 4b, 5a, 5b, 5c, 7a, 7b, 7d. (PDF 2562 kb)
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Kayagaki, N., Stowe, I., Lee, B. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666–671 (2015). https://doi.org/10.1038/nature15541
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