The murine caspase-11 non-canonical inflammasome responds to various bacterial infections. Caspase-11 activation-induced pyroptosis, in response to cytoplasmic lipopolysaccharide (LPS), is critical for endotoxic shock in mice. The mechanism underlying cytosolic LPS sensing and the responsible pattern recognition receptor are unknown. Here we show that human monocytes, epithelial cells and keratinocytes undergo necrosis upon cytoplasmic delivery of LPS. LPS-induced cytotoxicity was mediated by human caspase-4 that could functionally complement murine caspase-11. Human caspase-4 and the mouse homologue caspase-11 (hereafter referred to as caspase-4/11) and also human caspase-5, directly bound to LPS and lipid A with high specificity and affinity. LPS associated with endogenous caspase-11 in pyroptotic cells. Insect-cell purified caspase-4/11 underwent oligomerization upon LPS binding, resulting in activation of the caspases. Underacylated lipid IVa and lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) could bind to caspase-4/11 but failed to induce their oligomerization and activation. LPS binding was mediated by the CARD domain of the caspase. Binding-deficient CARD-domain point mutants did not respond to LPS with oligomerization or activation and failed to induce pyroptosis upon LPS electroporation or bacterial infections. The function of caspase-4/5/11 represents a new mode of pattern recognition in immunity and also an unprecedented means of caspase activation.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Kayagaki, N. et al. Non-canonical inflammasome activation targets caspase-11. Nature 479, 117–121 (2011)
Broz, P. et al. Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1. Nature 490, 288–291 (2012)
Akhter, A. et al. Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization. Immunity 37, 35–47 (2012)
Case, C. L. et al. Caspase-11 stimulates rapid flagellin-independent pyroptosis in response to Legionella pneumophila. Proc. Natl Acad. Sci. USA 110, 1851–1856 (2013)
Aachoui, Y. et al. Caspase-11 protects against bacteria that escape the vacuole. Science 339, 975–978 (2013)
Hagar, J. A., Powell, D. A., Aachoui, Y., Ernst, R. K. & Miao, E. A. Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science 341, 1250–1253 (2013)
Kayagaki, N. et al. Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341, 1246–1249 (2013)
Wang, S. et al. Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 92, 501–509 (1998)
Gurung, P. et al. Toll or interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF)-mediated caspase-11 protease production integrates Toll-like receptor 4 (TLR4) protein- and Nlrp3 inflammasome-mediated host defense against enteropathogens. J. Biol. Chem. 287, 34474–34483 (2012)
Rathinam, V. A. et al. TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by Gram-negative bacteria. Cell 150, 606–619 (2012)
Pilla, D. M. et al. Guanylate binding proteins promote caspase-11-dependent pyroptosis in response to cytoplasmic LPS. Proc. Natl Acad. Sci. USA 111, 6046–6051 (2014)
Meunier, E. et al. Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases. Nature 509, 366–370 (2014)
Raetz, C. R., Reynolds, C. M., Trent, M. S. & Bishop, R. E. Lipid A modification systems in Gram-negative bacteria. Annu. Rev. Biochem. 76, 295–329 (2007)
Whitfield, C. & Trent, M. S. Biosynthesis and export of bacterial lipopolysaccharides. Annu. Rev. Biochem. 83, 99–128 (2014)
Zhao, Y. et al. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477, 596–600 (2011)
Lin, X. Y., Choi, M. S. & Porter, A. G. Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-γ. J. Biol. Chem. 275, 39920–39926 (2000)
Raymond, A. A. et al. Nine procaspases are expressed in normal human epidermis, but only caspase-14 is fully processed. Br. J. Dermatol. 156, 420–427 (2007)
Kobayashi, T. et al. The Shigella OspC3 effector inhibits caspase-4, antagonizes inflammatory cell death, and promotes epithelial infection. Cell Host Microbe 13, 570–583 (2013)
Wang, S. et al. Identification and characterization of Ich-3, a member of the interleukin-1β converting enzyme (ICE)/Ced-3 family and an upstream regulator of ICE. J. Biol. Chem. 271, 20580–20587 (1996)
Shin, H. J. et al. Kinetics of binding of LPS to recombinant CD14, TLR4, and MD-2 proteins. Mol. Cells 24, 119–124 (2007)
Qureshi, N., Jarvis, B. W. & Takayama, K. in Endotoxin in Health and Disease (eds Brade H., Opal, S. M., Vogel, S. N. & Morrison, D. C. ) 687 (Marcel Dekker, 1999)
Ferguson, A. D. et al. A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins. Structure 8, 585–592 (2000)
Koshiba, T., Hashii, T. & Kawabata, S. A structural perspective on the interaction between lipopolysaccharide and factor C, a receptor involved in recognition of Gram-negative bacteria. J. Biol. Chem. 282, 3962–3967 (2007)
Ohto, U., Fukase, K., Miyake, K. & Satow, Y. Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa. Science 316, 1632–1634 (2007)
Park, B. S. et al. The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex. Nature 458 1191–1195 (2009)
Yoon, S. I., Hong, M., Han, G. W. & Wilson, I. A. Crystal structure of soluble MD-1 and its interaction with lipid IVa. Proc. Natl Acad. Sci. USA 107, 10990–10995 (2010)
Boatright, K. M. & Salvesen, G. S. Mechanisms of caspase activation. Curr. Opin. Cell Biol. 15, 725–731 (2003)
McIlwain, D. R., Berger, T. & Mak, T. W. Caspase functions in cell death and disease. Cold Spring Harb. Perspect. Biol. 5, a008656 (2013)
Riedl, S. J. & Shi, Y. Molecular mechanisms of caspase regulation during apoptosis. Nature Rev. Mol. Cell Biol. 5, 897–907 (2004)
Iwanaga, S. & Lee, B. L. Recent advances in the innate immunity of invertebrate animals. J. Biochem. Mol. Biol. 38, 128–150 (2005)
Gong, Y. N. et al. Chemical probing reveals insights into the signaling mechanism of inflammasome activation. Cell Res. 20, 1289–1305 (2010)
Zhu, Y. et al. Structural mechanism of host Rab1 activation by the bifunctional Legionella type IV effector SidM/DrrA. Proc. Natl Acad. Sci. USA 107, 4699–4704 (2010)
Fujimoto, Y. et al. Synthesis of lipid A and its analogues for investigation of the structural basis for their bioactivity. J. Endotoxin Res. 11, 341–347 (2005)
Yang, J., Zhao, Y., Shi, J. & Shao, F. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation. Proc. Natl Acad. Sci. USA 110, 14408–14413 (2013)
He, S. et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α. Cell 137, 1100–1111 (2009)
Boatright, K. M. et al. A unified model for apical caspase activation. Mol. Cell 11, 529–541 (2003)
Li, H., Willingham, S. B., Ting, J. P. & Re, F. Cutting edge: inflammasome activation by alum and alum’s adjuvant effect are mediated by NLRP3. J. Immunol. 181, 17–21 (2008)
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013)
Ge, J., Gong, Y. N., Xu, Y. & Shao, F. Preventing bacterial DNA release and absent in melanoma 2 inflammasome activation by a Legionella effector functioning in membrane trafficking. Proc. Natl Acad. Sci. USA 109, 6193–6198 (2012)
Brumell, J. H., Tang, P., Zaharik, M. L. & Finlay, B. B. Disruption of the Salmonella-containing vacuole leads to increased replication of Salmonella enterica serovar typhimurium in the cytosol of epithelial cells. Infect. Immun. 70, 3264–3270 (2002)
We thank Y. Fujimoto and K. Fukase for providing biotin-conjugated lipid A and lipid IVa and B. Finlay for S. typhimurium ΔsifA strain. We also thank W. Li at Tsinghua University for assistance in SLS and AU analyses and members of the Shao laboratory for discussions and technical assistance. The research was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute and the Beijing Scholar Program to F.S. This work was also supported by the National Basic Research Program of China 973 Program (2012CB518700), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08020202) and China National Science Foundation Program for Distinguished Young Scholars (31225002) to F.S.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Cytoplasmic LPS induces ASC/caspase-1-independent but caspase-4-dependent necrosis in human cells.
a, Viability of U937 cells upon electroporation with lipid A, MDP, LPS-RS or wild-type LPS derived from the indicated bacterial strains. b, Effects of ASC knockdown and CASP1 knockout on LPS electroporation-induced pyroptosis in U937 cells. ASC knockdown U937 cells were generated by lentivirus-mediated shRNA transduction. CASP1 knockout U937 cells were generated by CRISPR/Cas9-mediated targeting. c, Effects of caspase-1 (YVAD) or pan-caspase inhibitor (zVAD) on LPS electroporation-induced cytotoxicity in U937 and THP1 cells. d, e, Effects of CASP4 knockdown on LPS electroporation-induced cytotoxicity in U937 and HeLa cells. Control or a CASP4-specific shRNA (CASP4-1/2/3) was stably expressed in U937 cells in d; a CASP4-specific (CASP4-1/2) or CASP3-targeting or control siRNA was transfected into HeLa cells in e. f, Quantification of cytosolic LPS upon electroporation or bacterial infection. 5 × 105 CASP4−/− HeLa cells were either electroporated with 0.5 μg of LPS or infected with S. typhimurium ΔsifA. Cells were washed with cold PBS 5 times and lysed in a buffer containing 1% Triton X-100. ATP-based cell viability was measured in a–e. The immunoblots show the knockdown of ASC and CASP4 in b, d, e and knockout of CASP1 in b. Graphs show the mean values ± s.d. from three technical replicates. All data shown are representative of at least three independent experiments.
Extended Data Figure 2 Caspase-11-mediated cell death in response to cytoplasmic LPS and complementation by caspase-4.
a, Effects of Ripk3 and Mlkl deficiency on LPS-induced necrosis in mouse BMDMs. b, Wild-type (WT) or the catalytically inactive caspase-11(C254A) or caspase-4(C258A) mutant was stably expressed in Casp11−/− iBMDM cells generated by CRISPR/Cas9-mediated targeting. c, IFN-γ stimulates caspase-11 expression and increase the sensitivity of primary BMDMs to LPS electroporation. d, Wild-type (WT) or the catalytically inactive caspase-11(C254A) mutant was stably expressed in 293T cells. LPS was delivered by electroporation. ATP-based cell viability in a–d was expressed as mean values ± s.d. from three technical replicates. The accompanying immunoblots show the expression of caspase-4 or caspase-11. Data shown in a, c are representative of two independent experiments and those in other panels are of at least three independent experiments.
Extended Data Figure 3 Assays of the ability of known CARD-domain proteins in inducing caspase-11 activation.
Each of the 18 indicated CARD-domain proteins was overexpressed in 293T cells stably expressing caspase-11. As a control (#19), 293T-caspase-11 cells were subjected to LPS electroporation. The top shows the ATP-based cell viability expressed as mean values ± s.d. from three technical replicates. Anti-Flag, Myc and HA immunoblots confirm the expression of indicated CARD-domain proteins. LE, long exposure. Data shown are representative of three independent experiments.
Extended Data Figure 4 LPS stimulates the oligomerization of caspase-4/11 in the CARD domain-dependent manner.
a, b, Recombinant caspase-4/11 purified from E. coli and insect cells exhibit different oligomerization states. The left and right show the gel filtration chromatography and Coomassie blue stained pore-limit native gels of indicated recombinant caspase-11(C254A) (a) and caspase-4(C258A) protein (b), respectively. c, Molecular weight determination of insect-cell purified caspase-4 and caspase-11 by static light scattering (SLS) and analytic ultracentrifugation (AU). Shown are mean values ± s.d. d, Responses of insect-cell purified caspase-11 (before and after LPS incubation) to crosslinking agents (BS3 and DSS). Shown are Coomassie blue-stained SDS–PAGE gels. e, Immunoblotting of Flag tagged caspase-11–Myc expressed in 293T cells. f, Gel filtration chromatography of LPS-incubated full-length (FL) and the N-terminal 59-residue deletion (ΔN59) mutant caspase-11. Insect-cell purified caspase-11 (FL and ΔN59, both in the C254A background) was incubated with LPS-Rc overnight at 4°C. The proteins were further purified by affinity chromatography using Ni-NTA beads and subjected to Superdex 200 gel filtration chromatography. The LPS contents in each elution fraction (0.5 ml) were plotted against the corresponding elution volume. g, Pore-limit native gel analysis of lipid A induced oligomerization of insect-cell purified caspase-11 FL and the ΔN59 mutant (both in the catalytic-cysteine intact background). Data shown in c, f, are representative of two independent experiments and those in other panels are of at least three independent experiments.
Extended Data Figure 5 Caspase-5 can bind to LPS and complement Casp11−/− iBMDMs in cytoplasmic LPS-induced cell death.
a, Streptavidin pulldown assays of biotin-conjugated lipid A, lipid IVa, LPS, Pam3CSK4, and MDP binding to Flag tagged CASP5a (C315A) and CASP5f (C328A) in transfected 293T cell lysates. b, Caspase-5 complementation of Casp11−/− iBMDMs in cytoplasmic LPS-induced pyroptosis. Wild-type caspase-5f (C5f) was stably expressed in Casp11−/− iBMDMs generated by CRISPR/Cas9-mediated targeting. ATP-based cell viability was expressed as mean values ± s.d. from three technical replicates. All data shown are representative of at least three independent experiments.
a, Assays of the caspase-1/11 CARD-domain chimaeric protein in sensing cytoplasmic LPS. The chimaeric protein (Flag tagged C1/C11) was generated by replacing the CARD domain in Flag tagged caspase-1 with that of caspase-11. CASP4 knockout HeLa cells, Casp11 knockout iBMDM cells and 293T cells stably expressing Flag-C1/C11 (WT or the catalytic cysteine mutant (C/A)) were electroporated with LPS. ATP-based cell viability was expressed as mean values ± s.d. from three technical replicates. Expression of the chimaeric proteins were shown by anti-Flag immunoblotting. b, Streptavidin pulldown assays of the binding of biotinylated LPS to the C1/C11 chimaera or indicated caspase-11 variants in lysates of transfected 293T cells. c, e, LPS-induced oligomerization of the Flag tagged C1/C11 chimaera and the caspase-11 CARD domain itself. Lysates of 293T cells stably expressing the chimaeric protein (c) or caspase-11 CARD domain (e) were subjected to incubation with LPS and then analysed on pore-limit native gels. Cells were lysed in the HBS buffer (50 mM HEPES, pH 7.5, 150 mM NaCl and 3 mM EDTA) supplemented with 0.005% Tween-20. d, Oligomerization assays of caspase-11 CARD domain overexpressed in 293T cells. TRADD and TRADD death domain (DD) were included as positive controls and Blue Native gel was employed to examine the oligomerization. Shown in b–e are anti-Flag immunoblots. All data shown are representative of at least three independent experiments.
Insect-cell purified full-length caspases were subjected to incubation with MDP, lipid A, lipid IVa, LPS or the indicated LPS variants. The caspase activity was determined by measuring the fluorescence intensity of free AMC hydrolyzed from the zVAD-AMC substrate. Shown in a, b are the Michaelis–Menten kinetic parameters (kcat and Km) (values ± standard errors; ND, not determined). Graphs in c, d, show fluorescence intensity values determined with indicated enzyme and substrate concentration as mean values ± s.d. from three replicates. All data shown are representative of at least three independent experiments.
a, b, Effects of Tween-20 on LPS binding-induced oligomerization and catalytic activation of caspase-4. LPS-incubated recombinant caspase-4 was subjected to further incubation with increasing concentrations of Tween-20 as indicted. The fluorescence intensities in b are mean values ± s.d. from three replicates. c, Assays of lipid IVa and LPS-RS in stimulating oligomerization of caspase-4/11. Insect-cell purified caspase-11(C254A) and caspase-4(C258A) were subjected to incubation with indicated agonists, and the samples were analysed by the pore-limit native PAGE gel electrophoresis. All data shown are representative of at least three independent experiments.
Extended Data Figure 9 Multiple sequence alignment of caspase-11 and caspase-4 derived from various mammals.
Indicated caspase-sequences were aligned by using the ClustalW2 algorithm and the alignment was generated in ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Residues important for LPS binding to caspase-11 are highlighted in orange.
Extended Data Figure 10 Point mutations in caspase-11 CARD domain disrupt LPS binding to both caspase-11 CARD domain and full-length caspase-11.
a, Streptavidin pulldown assays of the binding of biotinylated LPS to wild-type or indicated point mutants of Flag tagged caspase-11 CARD domain in lysates of transfected 293T cells. b, Effects of the CARD-domain point mutants on transient overexpression-induced caspase-11 autoprocessing in 293T cells. c, Surface plasmon resonance measurements of the binding between LPS and the CARD-domain point mutants of insect-cell purified caspase-11. Colour indicated caspase-11 proteins were immobilized on the chips and shown are the corresponding sensorgrams expressed in RU (response unit) versus time after subtracting the control signal. All data shown are representative of two independent experiments.
About this article
Cite this article
Shi, J., Zhao, Y., Wang, Y. et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187–192 (2014). https://doi.org/10.1038/nature13683
The EMBO Journal (2020)
Nature Communications (2020)
Inactivation of the Cytoprotective Major Vault Protein by Caspase-1 and -9 in Epithelial Cells during Apoptosis
Journal of Investigative Dermatology (2020)
LPS Induces Active HMGB1 Release From Hepatocytes Into Exosomes Through the Coordinated Activities of TLR4 and Caspase-11/GSDMD Signaling
Frontiers in Immunology (2020)