Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling (original) (raw)

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Acknowledgements

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.

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Authors and Affiliations

1. Department of Physiological Chemistry, Genentech Inc., South San Francisco, 94080, California, USA
   Nobuhiko Kayagaki, Irma B. Stowe, Bettina L. Lee, Karen O’Rourke & Vishva M. Dixit
2. Department of Molecular Biology, Genentech Inc., South San Francisco, 94080, California, USA
   Keith Anderson, Søren Warming, Trinna Cuellar, Benjamin Haley & Merone Roose-Girma
3. Department of Protein Chemistry, Genentech Inc., South San Francisco, 94080, California, USA
   Qui T. Phung, Peter S. Liu, Jennie R. Lill, Hong Li & Jiansheng Wu
4. Department of Bioinformatics, Genentech Inc., South San Francisco, 94080, California, USA
   Sarah Kummerfeld
5. Department of Immunology, Genentech Inc., South San Francisco, 94080, California, USA
   Juan Zhang & Wyne P. Lee
6. Program in Cell Death Signaling Networks, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, 92037, California, USA
   Scott J. Snipas & Guy S. Salvesen
7. The Australian Phenomics Facility, The John Curtin School of Medical Research, The Australian National University, Canberra, 2601, Australian Capital Territory, Australia
   Lucy X. Morris, Linda Fitzgerald, Yafei Zhang & Edward M. Bertram
8. Department of Immunology and Infectious Diseases, The John Curtin School of Medical Research, The Australian National University, Canberra, 2601, Australian Capital Territory, Australia
   Edward M. Bertram & Christopher C. Goodnow
9. Garvan Institute of Medical Research, Sydney, 2010, New South Wales, Australia
   Christopher C. Goodnow
10. St. Vincent's Clinical School, UNSW Australia, Darlinghurst, 2010, New South Wales, Australia
    Christopher C. Goodnow

Authors

1. Nobuhiko Kayagaki
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2. Irma B. Stowe
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3. Bettina L. Lee
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4. Karen O’Rourke
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5. Keith Anderson
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6. Søren Warming
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7. Trinna Cuellar
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8. Benjamin Haley
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9. Merone Roose-Girma
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10. Qui T. Phung
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11. Peter S. Liu
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12. Jennie R. Lill
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13. Hong Li
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14. Jiansheng Wu
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15. Sarah Kummerfeld
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16. Juan Zhang
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17. Wyne P. Lee
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18. Scott J. Snipas
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19. Guy S. Salvesen
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20. Lucy X. Morris
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21. Linda Fitzgerald
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22. Yafei Zhang
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23. Edward M. Bertram
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24. Christopher C. Goodnow
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25. Vishva M. Dixit
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Contributions

N.K., I.B.S., B.L.L., K.O., T.C., B.H., P.S.L., Q.T.P., J.R.L., H.L., J.W., S.K., J.Z., W.P.L., S.J.S., L.X.M., L.F., Y.Z. and E.M.B. designed and performed experiments. K.A. and S.W. generated _Gsdmd_−/− mice. S.W. and M.R.-G. generated _Casp1_−/− mice. N.K. and E.M.B. prepared the manuscript. N.K., G.S.S., E.M.B., C.C.G. and V.M.D. contributed to the study design and data analyses.

Corresponding authors

Correspondence toNobuhiko Kayagaki or Vishva M. Dixit.

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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.

Extended Data Figure 2 Gsdmd I105N/I105N BMDMs respond normally to TLR agonists.

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.

Extended Data Figure 3 Non-canonical inflammasome signalling requires Gsdmd.

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.

Extended Data Figure 4 Gsdmd cleavage site.

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.

Extended Data Figure 5 Human GSDMD processing.

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.

Extended Data Figure 8 _Gsdmd_−/− alleles.

_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.

Extended Data Table 1 Bioinformatic analysis of ENU-induced SNVs present in IGL1351 pedigree

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Extended Data Table 2 Adjusted P values of Fig. 6

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Supplementary information

Supplementary Information

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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• Received: 14 July 2015
• Accepted: 04 September 2015
• Published: 16 September 2015
• Issue Date: 29 October 2015
• DOI: https://doi.org/10.1038/nature15541