Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death

Abstract

Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences. Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis. Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown. Here we identify gasdermin D (Gsdmd) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages. GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands. Interleukin-1β release was also diminished in Gsdmd−/− cells, despite intact processing by caspase-1. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis. The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity. Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis. These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis.

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Figure 1: Genetic screens identify that GSDMD is required for inflammatory-caspase-mediated pyroptosis and its role in caspase-1-induced IL-1β release.
Figure 2: GSDMD is a substrate specifically for inflammatory caspases.
Figure 3: Cleavage of GSDMD by inflammatory caspases is required for pyroptosis.
Figure 4: Interdomain cleavage of GSDMD is sufficient to trigger pyroptosis owing to the intrinsic pyroptosis-inducing activity in its N-terminal domain.
Figure 5: Autoinhibition of the pyroptosis-inducing activity of the gasdermin family.

References

  1. 1

    Lamkanfi, M. & Dixit, V. M. Mechanisms and functions of inflammasomes. Cell 157, 1013–1022 (2014)

    CAS  Article  Google Scholar 

  2. 2

    Henao-Mejia, J., Elinav, E., Thaiss, C. A. & Flavell, R. A. Inflammasomes and metabolic disease. Annu.Rev. Physiol. 76, 57–78 (2014)

    CAS  Article  Google Scholar 

  3. 3

    Kofoed, E. M. & Vance, R. E. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477, 592–595 (2011)

    CAS  Article  ADS  Google Scholar 

  4. 4

    Zhao, Y. et al. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477, 596–600 (2011)

    CAS  Article  ADS  Google Scholar 

  5. 5

    Zhao, Y. & Shao, F. The NAIP-NLRC4 inflammasome in innate immune detection of bacterial flagellin and type III secretion apparatus. Immunol.Rev. 265, 85–102 (2015)

    CAS  Article  Google Scholar 

  6. 6

    Xu, H. et al. Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature 513, 237–241 (2014)

    CAS  Article  ADS  Google Scholar 

  7. 7

    Kayagaki, N. et al. Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341, 1246–1249 (2013)

    CAS  Article  ADS  Google Scholar 

  8. 8

    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)

    CAS  Article  ADS  Google Scholar 

  9. 9

    Shi, J. et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187–192 (2014)

    CAS  Article  ADS  Google Scholar 

  10. 10

    Yang, J., Zhao, Y. & Shao, F. Non-canonical activation of inflammatory caspases by cytosolic LPS in innate immunity. Curr.Opin.Immunol. 32, 78–83 (2015)

    CAS  Article  Google Scholar 

  11. 11

    Kayagaki, N. et al. Non-canonical inflammasome activation targets caspase-11. Nature 479, 117–121 (2011)

    CAS  Article  ADS  Google Scholar 

  12. 12

    Jorgensen, I. & Miao, E. A. Pyroptotic cell death defends against intracellular pathogens. Immunol.Rev. 265, 130–142 (2015)

    CAS  Article  Google Scholar 

  13. 13

    Miao, E. A. et al. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nature Immunol. 11, 1136–1142 (2010)

    CAS  Article  Google Scholar 

  14. 14

    Aachoui, Y. et al. Caspase-11 protects against bacteria that escape the vacuole. Science 339, 975–978 (2013)

    CAS  Article  ADS  Google Scholar 

  15. 15

    Sauer, J. D. et al. Listeria monocytogenes engineered to activate the Nlrc4 inflammasome are severely attenuated and are poor inducers of protective immunity. Proc. Natl Acad. Sci. USA 108, 12419–12424 (2011)

    CAS  Article  ADS  Google Scholar 

  16. 16

    Kovarova, M. et al. NLRP1-dependent pyroptosis leads to acute lung injury and morbidity in mice. J. Immunol. 189, 2006–2016 (2012)

    CAS  Article  Google Scholar 

  17. 17

    Masters, S. L. et al. NLRP1 inflammasome activation induces pyroptosis of hematopoietic progenitor cells. Immunity 37, 1009–1023 (2012)

    CAS  Article  Google Scholar 

  18. 18

    Doitsh, G. et al. Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection. Nature 505, 509–514 (2014)

    CAS  Article  ADS  Google Scholar 

  19. 19

    von Moltke, J. et al. Rapid induction of inflammatory lipid mediators by the inflammasome in vivo. Nature 490, 107–111 (2012)

    CAS  Article  ADS  Google Scholar 

  20. 20

    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)

    CAS  Article  ADS  Google Scholar 

  21. 21

    Akhter, A. et al. Caspase-7 activation by the Nlrc4/Ipaf inflammasome restricts Legionella pneumophila infection. PLoSPathog. 5, e1000361 (2009)

    Google Scholar 

  22. 22

    Sun, L. & Wang, X. A new kind of cell suicide: mechanisms and functions of programmed necrosis. Trends Biochem.Sci. 39, 587–593 (2014)

    CAS  Article  Google Scholar 

  23. 23

    Poreba, M., Strozyk, A., Salvesen, G. S. & Drag, M. Caspase substrates and inhibitors. Cold Spring Harb.Perspect.Biol. 5, a008680 (2013)

    Article  Google Scholar 

  24. 24

    Fujii, T. et al. Gasdermin D (Gsdmd) is dispensable for mouse intestinal epithelium development. Genesis 46, 418–423 (2008)

    CAS  Article  Google Scholar 

  25. 25

    Tanaka, S., Mizushina, Y., Kato, Y., Tamura, M. & Shiroishi, T. Functional conservation of Gsdma cluster genes specifically duplicated in the mouse genome. G3 (Bethesda) 3, 1843–1850 (2013)

    Article  Google Scholar 

  26. 26

    Saeki, N. & Sasaki, H. in Endothelium and epithelium: composition, functions, and pathology (eds J. Carrasco & M. Matheus ) Ch. IX 193–211 (Nova Science Publishers, 2011)

    Google Scholar 

  27. 27

    Kumar, S. et al. Gsdma3I359N is a novel ENU-induced mutant mouse line for studying the function of Gasdermin A3 in the hair follicle and epidermis. J. Dermatol. Sci. 67, 190–192 (2012)

    CAS  Article  Google Scholar 

  28. 28

    Ruge, F. et al. Delineating immune-mediated mechanisms underlying hair follicle destruction in the mouse mutant defolliculated. J. Invest. Dermatol. 131, 572–579 (2011)

    CAS  Article  Google Scholar 

  29. 29

    Zhou, Y. et al. Gsdma3 mutation causes bulge stem cell depletion and alopecia mediated by skin inflammation. Am. J. Pathol. 180, 763–774 (2012)

    CAS  Article  Google Scholar 

  30. 30

    Shi, P. et al. Loss of conserved Gsdma3 self-regulation causes autophagy and cell death. Biochem. J. 468, 325–336 (2015)

    CAS  Article  Google Scholar 

  31. 31

    Agard, N. J., Maltby, D. & Wells, J. A. Inflammatory stimuli regulate caspase substrate profiles. Mol. Cell. Proteomics 9, 880–893 (2010)

    CAS  Article  Google Scholar 

  32. 32

    Crawford, E. D. et al. The DegraBase: a database of proteolysis in healthy and apoptotic human cells. Mol. Cell. Proteomics 12, 813–824 (2013)

    CAS  Article  Google Scholar 

  33. 33

    Koike-Yusa, H., Li, Y., Tan, E.-P., del Castillo Velasco-Herrera, M. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nature Biotechnol. 32, 267–273 (2014)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank J. Ding for recombinant protein purification and X. Wang for reagents. We thank members of the Shao laboratory for helpful discussions and technical assistance. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08020202), the China National Science Foundation Program for Distinguished Young Scholars (31225002) and Program for International Collaborations (31461143006), and the National Basic Research Program of China 973 Program (2012CB518700 and 2014CB849602) to F.S. 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.

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Contributions

F.S. and J.S. conceived the study; J.S. performed the CRISPR-Cas9 screens; J.S. and Y.Zha. designed and performed the majority of experiments, assisted by K.W. and X. S.; H.H. and T.C. performed the deep sequencing; J.S., Y.W., Y.Zhu. and F.W. generated the knockout mice. J.S., Y.Zha. and F.S. analysed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Feng Shao.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 siRNA knockdown validation of the requirement of GSDMD for LPS- and LFn–BsaK-induced pyroptosis.

a, LPS electroporation-induced pyroptosis in the absence of priming. Primary BMDMs (priBMDM) (wild-type (WT) or the Casp1 and Casp11 double knockout) or iBMDM cells (wild-type or Tlr4−/−) were assayed. Tlr4−/−iBMDMs were used for the CRISPR-Cas9 screen in this study. b, Reverse-transcription PCR analyses of caspase-5 expression in HeLa and 293T cells. Plasmid harbouring caspase-5 cDNA serves as the positive control. c, siRNA knockdown validation of the CRISPR-Cas9 screen of LPS-induced pyroptosis. HeLa cells were used to validate the selected top hits from the screen. Mixtures of two independent siRNA pairs targeting each gene were transfected into the cells. siRNAs targeting CASP4 and luciferase were used as the positive and negative control, respectively. d, Effects of GSDMD siRNA knockdown on LPS-induced pyroptosis in HeLa cells. e, f, Effects of Gsdmd siRNA knockdown on LPS and LFn–BsaK-induced pyroptosis in iBMDM cells. The knockdown efficiency (d, e) was measured by qRT–PCR analyses. ATP-based cell viability (a, c, d, f) and siRNA knockdown efficiency (d, e) were expressed as mean values ± s.d. from three technical replicates. Data shown are representative of two (c) or three (a, b, df) independent experiments.

Extended Data Figure 2 The gasdermin family of proteins in human and mouse.

a, Multiple sequence alignment of human GSDMA, GSDMB, GSDMC, GSDMD and mouse GSDMD. The alignment was performed by using the ClustalW2 algorithm and displayed with ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Identical residues are highlighted by the dark red background and conserved residues are indicated by red font. The black box marks the caspase-1/4/11 cleavage motifs in human and mouse GSDMD. The residue number is indicated on the left of the sequence. b, Phylogenetic tree of all the gasdermin family of proteins in human and mouse. ClustalW alignment was carried out to generate the phylogenetic tree by using the ‘Neighbor Joining’ method. DFNA5 and DFNB59 are distantly related to the gasdermins, and the latter only contains gasdermin-N domain.

Extended Data Figure 3 Generation of GSDMD-deficient cell lines and assays for cytosolic LPS- and LFn–BsaK-triggered pyroptosis.

a, Generation of GSDMD−/− HeLa cells and Gsdmd−/− iBMDM cells by CRISPR-Cas9-mediated targeting. Shown are the sequence mutations of the three HeLa cell clones and two iBMDM clones used in the study. b, c, f, Effects of GSDMD−/− on LPS electroporation-induced pyroptosis in HeLa (b) and iBMDM cells (c) and LFn–BsaK-induced pyroptosis in iBMDM cells (f). d, e, Complementation of GSDMD−/− HeLa cells and Gsdmd−/− iBMDM cells by stably expressed mouse GSDMD–3×Flag. The accompanying blots show the expression of exogenous GSDMD by anti-Flag immunoblotting with anti-tubulin blots serving as the loading control. ATP-based cell viability is expressed as mean values ± s.d. from three technical replicates (bf). Data shown are representative of at least three independent experiments.

Extended Data Figure 4 CRISPR-Cas9 screen of LFn–BsaK-triggered pyroptosis and effects of Gsdmd knockout on LFn–BsaK-induced and caspase-1-mediated caspase-3/7 cleavage.

a, gRNA hits from a genome-wide CRISPR-Cas9 screen of LFn–BsaK-induced pyroptosis in mouse Tlr4−/− iBMDM cells. Shown are those genes with multiple gRNA hits. The ranking, the average fold increase and the sequences for each gRNA are listed. Genes highlighted in red encode known components in the pathway (Antxr2 encodes the endocytosis receptor for the LFn tag) and were hit by multiple gRNAs. b, siRNA knockdown validation of screen hits. Mixtures of two independent siRNA pairs targeting each gene were transfected into the iBMDM cells before stimulation with LFn–BsaK. siRNAs targeting Nlrc4 and luciferase were used as the positive and negative control, respectively. ATP-based cell viability is expressed as mean values ± s.d. from three technical replicates. c, Caspase-3/7 activation upon prolonged LFn–BsaK treatment in wild-type and Gsdmd−/− (the KO-1 clone; KO, knockout) iBMDM cells. Cell lysates were analysed by anti-caspase-3/7 and tubulin immunoblotting. Data shown are representative of two (b) and three (c) independent experiments.

Extended Data Figure 5 GSDMD is not required for and not cleaved in TNFα-induced necroptosis and apoptosis.

ac, Effects of GSDMD knockdown on LPS electroporation-induced pyroptosis and TSZ-induced necroptosis in HT-29 cells. Two independent GSDMD-targeting siRNAs (#1 and #2) were assayed and the immunoblots in a show the knockdown efficiency. d, e, Primary BMDM cells from Gsdmd−/− or other indicated mouse strains were stimulated with TSZ (d) or LPS + zVAD (e) to trigger necroptosis. f, The absence of GSDMD cleavage in TNFα-induced apoptosis and necroptosis. 2×Flag–HA–GSDMD was stably expressed in HeLa and HT-29 cells. Apoptosis was induced by TNFα + CHX treatment in HeLa cells and necroptosis was induced by TSZ stimulation of HT-29 cells. Lysates of stimulated cells were analysed by anti-Flag and anti-tubulin immunoblotting to examine possible GSDMD cleavage. GSDMD-FL, full-length GSDMD. ATP-based cell viability is expressed as mean values ± s.d. from three technical replicates (bf). Data shown are representative of at least two independent experiments.

Extended Data Figure 6 Generation of Gsdmd−/− mice and assays for inflammasome-mediated caspase-1 autoprocessing and secretion.

a, Gsdmd−/− mice were generated by CRISPR-Cas9-mdiated targeting. Shown are the sequence mutations in the two homozygous F1 lines (F1-1 and F1-2) used in the study. b, Anti-caspase-1/caspase-11 immunoblots of lysates of unstimulated primary BMDM cells derived from wild-type and Gsdmd−/− mice. c, Primary BMDMs derived from wild-type or Gsdmd−/− mice were stimulated with indicated canonical inflammasome stimuli or infected with S. typhimurium (wild type or the T3SS-deficient ΔsipD mutant). Total cell lysates or the culture supernatants were subjected to anti-caspase-1 or anti-tubulin immunoblotting. Data shown (b, c) are representative of two independent experiments.

Extended Data Figure 7 Specific cleavage of GSDMD by inflammatory caspases.

a, Effects of Gsdmd knockout on caspase-1 activation by the AIM2 inflammasome. Indicated iBMDM cells were stimulated by poly(dA:dT) transfection. b, Effects of the pan-caspase inhibitor zVAD on LPS electroporation- and LFn–BsaK-induced GSDMD cleavage in HeLa and iBMDM cells, respectively. ATP-based cell viability is expressed as mean values ± s.d. from three technical replicates. c, Assays of GSDMD cleavage by inflammatory and apoptotic caspases overexpressed in cells. 3×Flag–GSDMD was co-transfected with indicated Myc–caspase into 293T cells. Total cell lysates were analysed by anti-caspase-1 (a, b), anti-Flag (b, c), anti-Myc (c) and anti-tubulin (ac) immunoblotting. Data shown are representative of three independent experiments.

Extended Data Figure 8 Resistance of the GSDMD D/A mutant to inflammatory-caspase cleavage.

a, b, Assays of proteolytic cleavage of the GSDMD D/A mutant by overexpression-activated inflammatory caspases. 3×Flag-tagged mouse (a) or human (b) GSDMD (wild-type or the D/A mutant) was co-transfected with Myc-tagged caspase-1/11 (a) or caspase1/4/5/11 (b) into 293T cells. Cell lysates were analysed by anti-Flag, anti-Myc and anti-tubulin immunoblotting. c, d, Assays of proteolytic cleavage of GSDMD D/A mutant by bacterial-infection-activated caspase-1. Wild-type, Gsdmd knockout (the KO-1 clone), or Gsdmd KO-1 complemented with 2×Flag–HA–GSDMD (wild-type or the D/A mutant) iBMDM cells were infected with wild-type S. typhimurium (c), B. thailandensis or EPEC (d) to induce caspase-1 activation (by the NAIP–NLRC4 inflammasome), or their T3SS-deficient mutant strains (ΔsipD, ΔbipB and ΔescN, respectively) as controls. Cell lysates were analysed by anti-caspase-1, anti-tubulin and anti-Flag immunoblotting. p10, mature caspase-1. GSDMD-FL, full-length GSDMD; GSDMD-N, the N-terminal cleavage product of GSDMD. The D/A mutants refer to D275A for human GSDMD and D276A for mouse GSDMD. Data shown are representative of three independent experiments.

Extended Data Figure 9 Characterization of GSDMA3 and other gasdermin family members.

a, Flag-tagged GSDMA, GSDMB, GSDMC and GSDMD were co-transfected with caspase-1 (upper panel) or caspase-11 (lower panel) into 293T cells. Cell lysates were analysed by anti-Flag, anti-caspase-1, anti-Myc or anti-tubulin immunoblotting. b, Wild-type GSDMA3 or a GSDMA3-mutant harbouring a PPase cleavage site between its gasdermin-N and -C domain was expressed in 293T cells. Recombinant PPase was transfected into the cells by electroporation. The upper panel shows the immunoblots of cell lysates to examine GSDMA3 cleavage and the lower panel shows ATP-based cell viability expressed as mean values ± s.d. from three technical replicates. c, The absence of GSDMA3 cleavage in TNFα-induced apoptosis and necroptosis. Flag–GSDMA3 was expressed in HeLa and L929 cells. Apoptosis was induced by TNFα + CHX treatment in HeLa cells and necroptosis was induced by TSZ stimulation of L929 cells. Lysates of stimulated cells were analysed by anti-Flag and anti-tubulin immunoblotting. Data shown are representative of three independent experiments.

Supplementary information

Supplementary Information

This file contains figures of the uncropped immunoblots for key data presented in the main text and Extended Data sections of the manuscript. It also contains Supplementary Table 1 listing the sequences of siRNAs and primers used in the study. (PDF 1192 kb)

TNFα-induced apoptosis in GSDMD-/- HeLa cells expressing wild-type GSDMD

Flag-GSDMD was stably expressed in HeLa GSDMD-/- HeLa cells. Cells were treated with TNFα+CHX. Cells were recorded 20 min after stimulation for the duration of time indicated on the upper right corner (h : min : s: ms). Scale bar, 10 μm. Also see Fig. 4c. (MOV 8058 kb)

TNFα-induced pyroptosis in GSDMD-/- HeLa cells expressing caspase-3-sensitive GSDMD mutant

The GSDMD mutant was generated by replacing the FLTD site with the caspase-3 cleavage site DEVD. The mutant Flag-GSDMD was stably expressed in GSDMD-/- HeLa cells. Cells were treated with TNFα+CHX. Cells were recorded 20 min after stimulation for the duration of time indicated on the upper right corner (h : min : s: ms). Scale bar, 10 μm. Also see Fig. 4c. (MOV 8414 kb)

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Shi, J., Zhao, Y., Wang, K. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015). https://doi.org/10.1038/nature15514

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