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Activation of the Non-canonical Inflammasome in Mouse and Human Cells

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The Inflammasome

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2459))

Abstract

The non-canonical inflammasome is a signaling platform that allows for the detection of cytoplasmic lipopolysaccharides (LPS) in immune and non-immune cells. Upon detection of LPS, this inflammasome activates the signaling proteases caspase-4 and -5 (in humans) and caspase-11 (in mice). Inflammatory caspases activation leads to caspase self-processing and the cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD N-terminal fragments oligomerize and form pores at the plasma membranes, leading to an inflammatory form of cell death called pyroptosis. Here, we describe a simple method to activate the non-canonical inflammasome in myeloid and epithelial cells and to measure its activity using cell death assay and immunoblotting.

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References

  1. Schroder K, Tschopp J (2010) The inflammasomes. Cell 140(6):821–832

    Article  CAS  Google Scholar 

  2. Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol 16(7):407–420

    Article  CAS  Google Scholar 

  3. Man SM, Karki R, Kanneganti T-D (2017) Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol Rev 277(1):61–75

    Article  CAS  Google Scholar 

  4. Liston A, Masters SL (2017) Homeostasis-altering molecular processes as mechanisms of inflammasome activation. Nat Rev Immunol 17(3):208–214

    Article  CAS  Google Scholar 

  5. Boucher D, Monteleone M, Coll RC, Chen KW, Ross CM, Teo JL et al (2018) Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity. J Exp Med 215(3):827–840

    Article  CAS  Google Scholar 

  6. Chan AH, Schroder K (2020) Inflammasome signaling and regulation of interleukin-1 family cytokines. J Exp Med 217(1):e20190314

    Article  Google Scholar 

  7. Broz P, Pelegrín P, Shao F (2020) The gasdermins, a protein family executing cell death and inflammation. Nat Rev Immunol 20(3):143–157

    Article  CAS  Google Scholar 

  8. Kayagaki N, Warming S, Lamkanfi M, Vande Walle L, Louie S, Dong J et al (2011) Non-canonical inflammasome activation targets caspase-11. Nature 479(7371):117–121

    Article  CAS  Google Scholar 

  9. Shi J, Zhao Y, Wang Y, Gao W, Ding J, Li P et al (2014) Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514(7521):187–192

    Article  CAS  Google Scholar 

  10. Kayagaki N, Wong MT, Stowe IB, Ramani SR, Gonzalez LC, Akashi-Takamura S et al (2013) Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341(6151):1246–1249

    Article  CAS  Google Scholar 

  11. Hagar JA, Powell DA, Aachoui Y, Ernst RK, Miao EA (2013) Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science 341(6151):1250–1253

    Article  CAS  Google Scholar 

  12. Santos JC, Boucher D, Schneider LK, Demarco B, Dilucca M, Shkarina K et al (2020) Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria. Nat Commun 11(1):3276

    Article  CAS  Google Scholar 

  13. Wandel MP, Kim B-H, Park E-S, Boyle KB, Nayak K, Lagrange B et al (2020) Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms. Nat Immunol 21(8):880–891

    Article  CAS  Google Scholar 

  14. Kutsch M, Sistemich L, Lesser CF, Goldberg MB, Herrmann C, Coers J (2020) Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions. EMBO J 39(13):e104926

    Article  CAS  Google Scholar 

  15. Fisch D, Clough B, Domart M-C, Encheva V, Bando H, Snijders AP et al (2020) Human GBP1 differentially targets salmonella and toxoplasma to license recognition of microbial ligands and caspase-mediated death. Cell Rep 32(6):108008

    Article  CAS  Google Scholar 

  16. Ross C, Chan AH, Von Pein J, Boucher D, Schroder K (2018) Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome. Life Sci Alliance 1(6):e201800237

    Article  Google Scholar 

  17. Casson CN, Yu J, Reyes VM, Taschuk FO, Yadav A, Copenhaver AM et al (2015) Human caspase-4 mediates noncanonical inflammasome activation against gram-negative bacterial pathogens. Proc Natl Acad Sci U S A 112(21):6688–6693

    Article  CAS  Google Scholar 

  18. Lee BL, Stowe IB, Gupta A, Kornfeld OS, Roose-Girma M, Anderson K et al (2018) Caspase-11 auto-proteolysis is crucial for noncanonical inflammasome activation. J Exp Med 215(9):2279–2288

    Article  CAS  Google Scholar 

  19. Baker PJ, Boucher D, Bierschenk D, Tebartz C, Whitney PG, D’Silva DB et al (2015) NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5. Eur J Immunol 45(10):2918–2926

    Article  CAS  Google Scholar 

  20. Rühl S, Broz P (2015) Caspase-11 activates a canonical NLRP3 inflammasome by promoting K(+) efflux. Eur J Immunol 45(10):2927–2936

    Article  Google Scholar 

  21. Wannamaker W, Davies R, Namchuk M, Pollard J, Ford P, Ku G et al (2007) (S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18. J Pharmacol Exp Ther 321(2):509–516

    Article  CAS  Google Scholar 

  22. Coll RC, Hill JR, Day CJ, Zamoshnikova A, Boucher D, Massey NL et al (2019) MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition. Nat Chem Biol 15(6):556–559

    Article  CAS  Google Scholar 

  23. Chen KW, Monteleone M, Boucher D, Sollberger G, Ramnath D, Condon ND et al (2018) Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps. Sci Immunol 3(26):eaar6676

    Article  Google Scholar 

  24. Gross O (2012) Measuring the inflammasome. Methods Mol Biol 844:199–222

    Article  Google Scholar 

Download references

Acknowledgments

Figures were created with BioRender (www.biorender.com). J.S.B. is supported by Kennedy Trust, KTRR start-up fellowship (KENN 15 16 06), and M.R.C. New Investigator Grant (MR/S000623/1). R.C. is supported by an Academy of Medical Sciences Springboard Award (SBF005\1104), a Royal Society Research Grant (RGS\R1\201127), and institutional funding from the Wellcome-Wolfson Institute for Experimental Medicine and Queen’s University Belfast. D.B. is supported by a Springboard award (SBF006\1025) from the Academy for Medical Science and institutional funding from the University of York.

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Authors and Affiliations

  1. Medical Sciences Division, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Kennedy Institute of Rheumatology Research, University of Oxford, Oxford, UK

    Jelena S. Bezbradica

  2. The Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast, UK

    Rebecca C. Coll

  3. Department of Biology, York Biomedical Research Institute, University of York, Heslington, York, UK

    Dave Boucher

Authors
  1. Jelena S. Bezbradica
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  2. Rebecca C. Coll
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  3. Dave Boucher
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Corresponding author

Correspondence to Dave Boucher .

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Editors and Affiliations

  1. School of Kinesiology and Health Science, York University, Toronto, ON, Canada

    Ali A. Abdul-Sater

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Bezbradica, J.S., Coll, R.C., Boucher, D. (2022). Activation of the Non-canonical Inflammasome in Mouse and Human Cells. In: Abdul-Sater, A.A. (eds) The Inflammasome. Methods in Molecular Biology, vol 2459. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2144-8_5

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  • DOI: https://doi.org/10.1007/978-1-0716-2144-8_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2143-1

  • Online ISBN: 978-1-0716-2144-8

  • eBook Packages: Springer Protocols

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