Abstract
Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome ‘specks’ in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- BMM:
-
bone marrow-derived macrophage
- iBMM:
-
immortalised bone marrow-derived macrophage
- CT DNA:
-
calf thymus DNA
- dsDNA:
-
double-stranded DNA
- WT:
-
wild type
- STS:
-
staurosporine
- PBS:
-
phosphate-buffered saline
- Z-VAD-FMK:
-
carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone
- PI:
-
propidium iodide
- MOMP:
-
mitochondrial outer membrane permeability
- LPS:
-
lipopolysaccharide
- HRP:
-
horseradish peroxidase
- siRNA:
-
small interfering RNA
- CARD:
-
caspase recruitment domain
- DED:
-
death effector domain
- DD:
-
death domain
- Spf:
-
specific pathogen-free
- PFA:
-
paraformaldehyde
- Alx:
-
Alexa Fluor dyes
- DAPI:
-
4',6-diamidino-2-phenylindole
- FCS:
-
foetal calf serum
- MTT:
-
thiazolyl blue tetrazolium bromide
References
Schroder K, Tschopp J . The inflammasomes. Cell 2010; 140: 821–832.
Bergsbaken T, Fink SL, Cookson BT . Pyroptosis: host cell death and inflammation. Nat Rev Microbiol 2009; 7: 99–109.
Miao EA, Rajan JV, Aderem A . Caspase-1-induced pyroptotic cell death. Immunol Rev 2011; 243: 206–214.
Fink SL, Cookson BT . Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages. Cell Microbiol 2006; 8: 1812–1825.
Strasser A, Cory S, Adams JM . Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J 2011; 30: 3667–3683.
Pop C, Salvesen GS . Human caspases: activation, specificity, and regulation. J Biol Chem 2009; 284: 21777–21781.
Yazdi AS, Guarda G, D'Ombrain MC, Drexler SK . Inflammatory caspases in innate immunity and inflammation. J Innate Immun 2010; 2: 228–237.
Mace PD, Riedl SJ . Molecular cell death platforms and assemblies. Curr Opin Cell Biol 2010; 22: 828–836.
van Raam BJ, Salvesen GS . Proliferative versus apoptotic functions of caspase-8 Hetero or homo: the caspase-8 dimer controls cell fate. Biochim Biophys Acta 2012; 1824: 113–122.
Burckstummer T, Baumann C, Bluml S, Dixit E, Durnberger G, Jahn H et al. An orthogonal proteomic–genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat Immunol 2009; 10: 266–272.
Fernandes-Alnemri T, Yu JW, Datta P, Wu J, Alnemri ES . AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 2009; 458: 509–513.
Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G, Caffrey DR et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 2009; 458: 514–518.
Roberts TL, Idris A, Dunn JA, Kelly GM, Burnton CM, Hodgson S et al. HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA. Science 2009; 323: 1057–1060.
Cridland JA, Curley EZ, Wykes MN, Schroder K, Sweet MJ, Roberts TL et al. The mammalian PYHIN gene family: phylogeny, evolution and expression. BMC Evol Biol 2012; 12: 140.
Kersse K, Verspurten J, Vanden Berghe T, Vandenabeele P . The death-fold superfamily of homotypic interaction motifs. Trends Biochem Sci 2011; 36: 541–552.
Rathinam VA, Jiang Z, Waggoner SN, Sharma S, Cole LE, Waggoner L et al. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat Immunol 2010; 11: 395–402.
Fernandes-Alnemri T, Yu JW, Juliana C, Solorzano L, Kang S, Wu J et al. The AIM2 inflammasome is critical for innate immunity to Francisella tularensis. Nat Immunol 2010; 11: 385–393.
Jones JW, Kayagaki N, Broz P, Henry T, Newton K, O'Rourke K et al. Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. Proc Natl Acad Sci USA 2010; 107: 9771–9776.
Warren SE, Armstrong A, Hamilton MK, Mao DP, Leaf IA, Miao EA et al. Cutting edge: cytosolic bacterial DNA activates the inflammasome via Aim2. J Immunol 2010; 185: 818–821.
Mariathasan S, Weiss DS, Newton K, McBride J, O'Rourke K, Roose-Girma M et al. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 2006; 440: 228–232.
Pierini R, Juruj C, Perret M, Jones CL, Mangeot P, Weiss DS et al. AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages. Cell Death Differ 2012; 19: 1709–1721.
Kayagaki N, Warming S, Lamkanfi M, Vande Walle L, Louie S, Dong J et al. Non-canonical inflammasome activation targets caspase-11. Nature 2011; 479: 117–121.
Shelton SN, Dillard CD, Robertson JD . Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells. J Biol Chem 2010; 285: 40525–40533.
Zhang XD, Gillespie SK, Hersey P . Staurosporine induces apoptosis of melanoma by both caspase-dependent and -independent apoptotic pathways. Mol Cancer Ther 2004; 3: 187–197.
Varfolomeev EE, Schuchmann M, Luria V, Chiannilkulchai N, Beckmann JS, Mett IL et al. Targeted disruption of the mouse caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 1998; 9: 267–276.
Kang TB, Ben-Moshe T, Varfolomeev EE, Pewzner-Jung Y, Yogev N, Jurewicz A et al. Caspase-8 serves both apoptotic and nonapoptotic roles. J Immunol 2004; 173: 2976–2984.
Denault JB, Eckelman BP, Shin H, Pop C, Salvesen GS . Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)-mediated inhibition of caspase 9. Biochem J 2007; 405: 11–19.
McDonnell MA, Wang D, Khan SM, Vander Heiden MG, Kelekar A . Caspase-9 is activated in a cytochrome c-independent manner early during TNFalpha-induced apoptosis in murine cells. Cell Death Differ 2003; 10: 1005–1015.
Vince JE, Wong WW, Gentle I, Lawlor KE, Allam R, O'Reilly L et al. Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation. Immunity 2012; 36: 215–227.
Gringhuis SI, Kaptein TM, Wevers BA, Theelen B, van der Vlist M, Boekhout T et al. Dectin-1 is an extracellular pathogen sensor for the induction and processing of IL-1beta via a noncanonical caspase-8 inflammasome. Nat Immunol 2012; 13: 246–254.
Maelfait J, Vercammen E, Janssens S, Schotte P, Haegman M, Magez S et al. Stimulation of Toll-like receptor 3 and 4 induces interleukin-1beta maturation by caspase-8. J Exp Med 2008; 205: 1967–1973.
Bossaller L, Chiang PI, Schmidt-Lauber C, Ganesan S, Kaiser WJ, Rathinam VA et al. Cutting edge: FAS (CD95) mediates noncanonical IL-1beta and IL-18 maturation via caspase-8 in an RIP3-independent manner. J Immunol 2012; 189: 5508–5512.
Wewers MD, Winnard AV, Dare HA . Endotoxin-stimulated monocytes release multiple forms of IL-1 beta, including a proIL-1 beta form whose detection is affected by export. J Immunol 1999; 162: 4858–4863.
Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D et al. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol 2009; 183: 787–791.
Schroder K, Sagulenko V, Zamoshnikova A, Richards AA, Cridland JA, Irvine KM et al. Acute lipopolysaccharide priming boosts inflammasome activation independently of inflammasome sensor induction. Immunobiology 2012; 217: 1325–1329.
Masumoto J, Dowds TA, Schaner P, Chen FF, Ogura Y, Li M et al. ASC is an activating adaptor for NF-kappa B and caspase-8-dependent apoptosis. Biochem Biophys Res Commun 2003; 303: 69–73.
Abdelaziz DH, Gavrilin MA, Akhter A, Caution K, Kotrange S, Khweek AA et al. Asc-dependent and independent mechanisms contribute to restriction of Legionella pneumophila infection in murine macrophages. Front Microbiol 2011; 2: 18.
Puri AW, Broz P, Shen A, Monack DM, Bogyo M . Caspase-1 activity is required to bypass macrophage apoptosis upon Salmonella infection. Nat Chem Biol 2012; 8: 745–747.
Nam YJ, Mani K, Ashton AW, Peng CF, Krishnamurthy B, Hayakawa Y et al. Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death-fold interactions. Mol Cell 2004; 15: 901–912.
Inohara N, Koseki T, del Peso L, Hu Y, Yee C, Chen S et al. Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J Biol Chem 1999; 274: 14560–14567.
Masumoto J, Taniguchi S, Ayukawa K, Sarvotham H, Kishino T, Niikawa N et al. ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J Biol Chem 1999; 274: 33835–33838.
Conway KE, McConnell BB, Bowring CE, Donald CD, Warren ST, Vertino PM . TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers. Cancer Res 2000; 60: 6236–6242.
McConnell BB, Vertino PM . TMS1/ASC: the cancer connection. Apoptosis 2004; 9: 5–18.
Ohtsuka T, Ryu H, Minamishima YA, Macip S, Sagara J, Nakayama KI et al. ASC is a Bax adaptor and regulates the p53–Bax mitochondrial apoptosis pathway. Nat Cell Biol 2004; 6: 121–128.
Motani K, Kushiyama H, Imamura R, Kinoshita T, Nishiuchi T, Suda T . Caspase-1 protein induces apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-mediated necrosis independently of its catalytic activity. J Biol Chem 2011; 286: 33963–33972.
Kumar Y, Radha V, Swarup G . Interaction with Sug1 enables Ipaf ubiquitination leading to caspase 8 activation and cell death. Biochem J 2010; 427: 91–104.
Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM, Salvesen GS . Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J Biol Chem 1997; 272: 7797–7800.
Chen IF, Ou-Yang F, Hung JY, Liu JC, Wang H, Wang SC et al. AIM2 suppresses human breast cancer cell proliferation in vitro and mammary tumor growth in a mouse model. Mol Cancer Ther 2006; 5: 1–7.
Woerner SM, Kloor M, Schwitalle Y, Youmans H, Doeberitz M, Gebert J et al. The putative tumor suppressor AIM2 is frequently affected by different genetic alterations in microsatellite unstable colon cancers. Genes Chromosomes Cancer 2007; 46: 1080–1089.
Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS et al. Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science 1995; 267: 2000–2003.
Mariathasan S, Newton K, Monack DM, Vucic D, French DM, Lee WP et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 2004; 430: 213–218.
Wang S, Miura M, Jung YK, Zhu H, Li E, Yuan J . Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 1998; 92: 501–509.
Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J . Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 2006; 440: 237–241.
Ogilvy S, Metcalf D, Print CG, Bath ML, Harris AW, Adams JM . Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival. Proc Natl Acad Sci USA 1999; 96: 14943–14948.
O'Reilly LA, Ekert P, Harvey N, Marsden V, Cullen L, Vaux DL et al. Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is dependent on both Apaf-1 and caspase-9. Cell Death Differ 2002; 9: 832–841.
Sester DP, Brion K, Trieu A, Goodridge HS, Roberts TL, Dunn J et al. CpG DNA activates survival in murine macrophages through TLR9 and the phosphatidylinositol 3-kinase–Akt pathway. J Immunol 2006; 177: 4473–4480.
Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 2008; 9: 847–856.
Stacey KJ, Young GR, Clark F, Sester DP, Roberts TL, Naik S et al. The molecular basis for the lack of immunostimulatory activity of vertebrate DNA. J Immunol 2003; 170: 3614–3620.
Stacey KJ, Ross IL, Hume DA . Electroporation and DNA-dependent cell death in murine macrophages. Immunol Cell Biol 1993; 71 (Pt 2): 75–85.
O'Reilly LA, Divisekera U, Newton K, Scalzo K, Kataoka T, Puthalakath H et al. Modifications and intracellular trafficking of FADD/MORT1 and caspase-8 after stimulation of T lymphocytes. Cell Death Differ 2004; 11: 724–736.
Masumoto J, Taniguchi S, Sagara J . Pyrin N-terminal homology domain- and caspase recruitment domain-dependent oligomerization of ASC. Biochem Biophys Res Commun 2001; 280: 652–655.
Acknowledgements
We thank Ben Croker for supplying Casp11−/− and Asc−/− mice; Odilia Wijburg for Casp1−/− mice; Jerry Adams and Mark Smyth for vav-Bcl-2 mice; and Richard Flavell, Vishva Dixit (Genentech) and Junying Yuan for permission to use Casp1−/−, ASC−/− and Casp11−/− mice, respectively. Immortalised BMMs were provided by Kate Fitzgerald and Eicke Latz. We acknowledge the support received from the Australian Infectious Diseases Research Centre to undertake this work. KJS is supported by Australian Research Council Future Fellowship FT0991576. The research was supported by National Health and Medical Research Council grants 631472 and 1010887.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Edited by G Salvesen
Author Contributions
All authors read and approved the manuscript. VS: Project direction, cell death assays, western blots, gene knockdown, immunofluorescence and confocal microscopy, writing of the manuscript; ST: Western blots, gene knockdown, cell death assays; DPS: Project direction, cell death assays; AI: caspase-2, Bcl-2, temperature dependence data; JAC: Protein interaction studies, western blots, cell death assays, immunofluorescence, confocal microscopy; JEV, PRV and JMH: Protein interaction studies; TLR, KS and JS: Supervision, intellectual input; KJS: Project conception and design, cell death assays, gene knockdown, manuscript preparation.
Supplementary Information accompanies this paper on Cell Death and Differentiation website
Supplementary information
Rights and permissions
About this article
Cite this article
Sagulenko, V., Thygesen, S., Sester, D. et al. AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC. Cell Death Differ 20, 1149–1160 (2013). https://doi.org/10.1038/cdd.2013.37
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cdd.2013.37
Keywords
This article is cited by
-
Gasdermins and pyroptosis in the kidney
Nature Reviews Nephrology (2023)
-
Pyroptosis in renal inflammation and fibrosis: current knowledge and clinical significance
Cell Death & Disease (2023)
-
Apoptotic caspase-7 activation inhibits non-canonical pyroptosis by GSDMB cleavage
Cell Death & Differentiation (2023)
-
IL-1α-releasing TH17 cells live long and prosper
Nature Immunology (2023)
-
The Pro-tumor and Anti-tumor Effects of NLRP3 Inflammasome as a New Therapeutic Option for Colon Cancer: a Meta-analysis of Pre-clinical Studies
Journal of Gastrointestinal Cancer (2023)
