Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death (original) (raw)

References

  1. Lamkanfi, M. & Dixit, V. M. Mechanisms and functions of inflammasomes. Cell 157, 1013–1022 (2014)
    Article CAS Google Scholar
  2. Henao-Mejia, J., Elinav, E., Thaiss, C. A. & Flavell, R. A. Inflammasomes and metabolic disease. Annu.Rev. Physiol. 76, 57–78 (2014)
    Article CAS Google Scholar
  3. Kofoed, E. M. & Vance, R. E. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477, 592–595 (2011)
    Article CAS ADS Google Scholar
  4. Zhao, Y. et al. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477, 596–600 (2011)
    Article CAS ADS Google Scholar
  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)
    Article CAS Google Scholar
  6. Xu, H. et al. Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature 513, 237–241 (2014)
    Article CAS ADS Google Scholar
  7. Kayagaki, N. et al. Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341, 1246–1249 (2013)
    Article CAS ADS Google Scholar
  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)
    Article CAS ADS Google Scholar
  9. Shi, J. et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187–192 (2014)
    Article CAS ADS Google Scholar
  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)
    Article CAS Google Scholar
  11. Kayagaki, N. et al. Non-canonical inflammasome activation targets caspase-11. Nature 479, 117–121 (2011)
    Article CAS ADS Google Scholar
  12. Jorgensen, I. & Miao, E. A. Pyroptotic cell death defends against intracellular pathogens. Immunol.Rev. 265, 130–142 (2015)
    Article CAS Google Scholar
  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)
    Article CAS Google Scholar
  14. Aachoui, Y. et al. Caspase-11 protects against bacteria that escape the vacuole. Science 339, 975–978 (2013)
    Article CAS ADS Google Scholar
  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)
    Article CAS ADS Google Scholar
  16. Kovarova, M. et al. NLRP1-dependent pyroptosis leads to acute lung injury and morbidity in mice. J. Immunol. 189, 2006–2016 (2012)
    Article CAS Google Scholar
  17. Masters, S. L. et al. NLRP1 inflammasome activation induces pyroptosis of hematopoietic progenitor cells. Immunity 37, 1009–1023 (2012)
    Article CAS Google Scholar
  18. Doitsh, G. et al. Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection. Nature 505, 509–514 (2014)
    Article CAS ADS Google Scholar
  19. von Moltke, J. et al. Rapid induction of inflammatory lipid mediators by the inflammasome in vivo. Nature 490, 107–111 (2012)
    Article CAS ADS Google Scholar
  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)
    Article CAS ADS Google Scholar
  21. Akhter, A. et al. Caspase-7 activation by the Nlrc4/Ipaf inflammasome restricts Legionella pneumophila infection. PLoSPathog. 5, e1000361 (2009)
    Google Scholar
  22. Sun, L. & Wang, X. A new kind of cell suicide: mechanisms and functions of programmed necrosis. Trends Biochem.Sci. 39, 587–593 (2014)
    Article CAS Google Scholar
  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. Fujii, T. et al. Gasdermin D (Gsdmd) is dispensable for mouse intestinal epithelium development. Genesis 46, 418–423 (2008)
    Article CAS Google Scholar
  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. 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. 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)
    Article CAS Google Scholar
  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)
    Article CAS Google Scholar
  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)
    Article CAS Google Scholar
  30. Shi, P. et al. Loss of conserved Gsdma3 self-regulation causes autophagy and cell death. Biochem. J. 468, 325–336 (2015)
    Article CAS Google Scholar
  31. Agard, N. J., Maltby, D. & Wells, J. A. Inflammatory stimuli regulate caspase substrate profiles. Mol. Cell. Proteomics 9, 880–893 (2010)
    Article CAS Google Scholar
  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)
    Article CAS Google Scholar
  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)
    Article CAS Google Scholar

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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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Author notes

  1. Jianjin Shi and Yue Zhao: These authors contributed equally to this work.

Authors and Affiliations

  1. Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, Tsinghua University, 100084, China
    Jianjin Shi
  2. National Institute of Biological Sciences, Beijing, 102206, China
    Jianjin Shi, Yue Zhao, Kun Wang, Xuyan Shi, Yue Wang, Huanwei Huang, Yinghua Zhuang, Tao Cai, Fengchao Wang & Feng Shao
  3. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
    Feng Shao
  4. National Institute of Biological Sciences, Beijing, Collaborative Innovation Center for Cancer Medicine, Beijing, 102206, China
    Feng Shao

Authors

  1. Jianjin Shi
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  2. Yue Zhao
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  3. Kun Wang
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  4. Xuyan Shi
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  5. Yue Wang
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  6. Huanwei Huang
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  7. Yinghua Zhuang
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  8. Tao Cai
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  9. Fengchao Wang
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  10. Feng Shao
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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 toFeng Shao.

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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, d–f) 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 (b–f). 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.

a–c, 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 (b–f). 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 Δ_sipD 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 (a–c) 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 (Δ_sipD_, Δ_bipB_ and Δ_escN_, 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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