Skip to main content
SpringerLink
Log in

ATP induces caspase-3/gasdermin E-mediated pyroptosis in NLRP3 pathway-blocked murine macrophages

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

ATP acts as a canonical activator to induce NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome activation in macrophages, leading to caspase-1/gasdermin D (GSDMD)-mediated pyroptosis. It remains unclear whether ATP can induce pyroptosis in macrophages when the NLRP3 pathway is blocked by pathogenic infection. In this study, we used cellular models to mimic such blockade of NLRP3 activation: bone marrow-derived macrophages (BMDMs) treated with NLRP3-specific inhibitor MCC950 and RAW264.7 cells deficient in ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression. The results showed that ATP treatment induced lytic cell death morphologically resembling canonical pyroptosis in both MCC950-treated BMDMs and RAW264.7 cells, but did not cause the activation of caspase-1 (by detecting caspase-1p10 and mature interleukin-1β) and cleavage of GSDMD. Instead, both apoptotic initiator (caspase-8 and -9) and executioner (caspase-3 and -7) caspases were evidently activated and gasdermin E (GSDME) was cleaved to generate its N-terminal fragment (GSDME-NT) which executes pyroptosis. The GSDME-NT production and lytic cell death induced by ATP were diminished by caspase-3 inhibitor. In BMDMs without MCC950 treatment, ATP induced the formation of ASC specks which were co-localized with caspase-8; with MCC950 treatment, however, ATP did not induced the formation of ASC specks. In RAW264.7 cells, knockdown of GSDME by small interfering RNA attenuated ATP-induced lytic cell death and HMGB1 release into culture supernatants. Collectively, our results indicate that ATP induces pyroptosis in macrophages through the caspase-3/GSDME axis when the canonical NLRP3 pathway is blocked, suggestive of an alternative mechanism for combating against pathogen evasion.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Medzhitov R (2008) Origin and physiological roles of inflammation. Nature 454:428–435. https://doi.org/10.1038/nature07201

    Article  CAS  PubMed  Google Scholar 

  2. Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140:805–820. https://doi.org/10.1016/j.cell.2010.01.022

    Article  CAS  PubMed  Google Scholar 

  3. Lamkanfi M, Dixit VM (2014) Mechanisms and functions of inflammasomes. Cell 157:1013–1022. https://doi.org/10.1016/j.cell.2014.04.007

    Article  CAS  PubMed  Google Scholar 

  4. Kono H, Onda A, Yanagida T (2014) Molecular determinants of sterile inflammation. Curr Opin Immunol 26:147–156. https://doi.org/10.1016/j.coi.2013.12.004

    Article  CAS  PubMed  Google Scholar 

  5. Davies LC, Jenkins SJ, Allen JE, Taylor PR (2013) Tissue-resident macrophages. Nat Immunol 14:986–995. https://doi.org/10.1038/ni.2705

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. de Zoete MR, Palm NW, Zhu S, Flavell RA (2014) Inflammasomes. Cold Spring Harb Perspect Biol 6:a016287. https://doi.org/10.1101/cshperspect.a016287

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. He Y, Hara H, Nunez G (2016) Mechanism and regulation of NLRP3 inflammasome activation. Trends Biochem Sci 41:1012–1021. https://doi.org/10.1016/j.tibs.2016.09.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, Roose-Girma M, Lee WP, Weinrauch Y, Monack DM, Dixit VM (2006) Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440:228–232. https://doi.org/10.1038/nature04515

    Article  CAS  PubMed  Google Scholar 

  9. Di A, Xiong S, Ye Z, Malireddi RKS, Kometani S, Zhong M, Mittal M, Hong Z, Kanneganti TD, Rehman J, Malik AB (2018) The TWIK2 potassium efflux channel in macrophages mediates NLRP3 inflammasome-induced inflammation. Immunity 49(56–65):e54. https://doi.org/10.1016/j.immuni.2018.04.032

    Article  CAS  Google Scholar 

  10. Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, Zhuang Y, Cai T, Wang F, Shao F (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526:660–665. https://doi.org/10.1038/nature15514

    Article  CAS  PubMed  Google Scholar 

  11. Kayagaki N, Stowe IB, Lee BL et al (2015) Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526:666–671. https://doi.org/10.1038/nature15541

    Article  CAS  PubMed  Google Scholar 

  12. Liu X, Zhang Z, Ruan J, Pan Y, Magupalli VG, Wu H, Lieberman J (2016) Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535:153–158. https://doi.org/10.1038/nature18629

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ding J, Wang K, Liu W, She Y, Sun Q, Shi J, Sun H, Wang DC, Shao F (2016) Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535:111–116. https://doi.org/10.1038/nature18590

    Article  CAS  PubMed  Google Scholar 

  14. He WT, Wan H, Hu L, Chen P, Wang X, Huang Z, Yang ZH, Zhong CQ, Han J (2015) Gasdermin D is an executor of pyroptosis and required for interleukin-1beta secretion. Cell Res 25:1285–1298. https://doi.org/10.1038/cr.2015.139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chen X, He WT, Hu L, Li J, Fang Y, Wang X, Xu X, Wang Z, Huang K, Han J (2016) Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis. Cell Res 26:1007–1020. https://doi.org/10.1038/cr.2016.100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sborgi L, Ruhl S, Mulvihill E, Pipercevic J, Heilig R, Stahlberg H, Farady CJ, Muller DJ, Broz P, Hiller S (2016) GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death. EMBO J 35:1766–1778. https://doi.org/10.15252/embj.201694696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Aglietti RA, Estevez A, Gupta A, Ramirez MG, Liu PS, Kayagaki N, Ciferri C, Dixit VM, Dueber EC (2016) GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes. Proc Natl Acad Sci USA 113:7858–7863. https://doi.org/10.1073/pnas.1607769113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Evavold CL, Ruan J, Tan Y, Xia S, Wu H, Kagan JC (2018) The pore-forming protein gasdermin D regulates interleukin-1 secretion from living macrophages. Immunity 48(35–44):e36. https://doi.org/10.1016/j.immuni.2017.11.013

    Article  CAS  Google Scholar 

  19. Zhang Y, Chen X, Gueydan C, Han J (2018) Plasma membrane changes during programmed cell deaths. Cell Res 28:9–21. https://doi.org/10.1038/cr.2017.133

    Article  CAS  PubMed  Google Scholar 

  20. Wallach D, Kang TB, Dillon CP, Green DR (2016) Programmed necrosis in inflammation: Toward identification of the effector molecules. Science 352:aaf2154. https://doi.org/10.1126/science.aaf2154

    Article  CAS  PubMed  Google Scholar 

  21. Jorgensen I, Miao EA (2015) Pyroptotic cell death defends against intracellular pathogens. Immunol Rev 265:130–142. https://doi.org/10.1111/imr.12287

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shi J, Gao W, Shao F (2017) Pyroptosis: gasdermin-mediated programmed necrotic cell death. Trends Biochem Sci 42:245–254. https://doi.org/10.1016/j.tibs.2016.10.004

    Article  CAS  PubMed  Google Scholar 

  23. Galluzzi L, Vitale I, Aaronson SA et al (2018) Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. Cell Death Differ 25:486–541. https://doi.org/10.1038/s41418-017-0012-4

    Article  PubMed  PubMed Central  Google Scholar 

  24. Wang Y, Gao W, Shi X, Ding J, Liu W, He H, Wang K, Shao F (2017) Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 547:99–103. https://doi.org/10.1038/nature22393

    Article  CAS  PubMed  Google Scholar 

  25. Rogers C, Fernandes-Alnemri T, Mayes L, Alnemri D, Cingolani G, Alnemri ES (2017) Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death. Nat Commun 8:14128. https://doi.org/10.1038/ncomms14128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Stewart MK, Cookson BT (2016) Evasion and interference: intracellular pathogens modulate caspase-dependent inflammatory responses. Nat Rev Microbiol 14:346–359. https://doi.org/10.1038/nrmicro.2016.50

    Article  CAS  PubMed  Google Scholar 

  27. Ulland TK, Ferguson PJ, Sutterwala FS (2015) Evasion of inflammasome activation by microbial pathogens. J Clin Investig 125:469–477. https://doi.org/10.1172/jci75254

    Article  PubMed  PubMed Central  Google Scholar 

  28. LaRock CN, Cookson BT (2012) The Yersinia virulence effector YopM binds caspase-1 to arrest inflammasome assembly and processing. Cell Host Microbe 12:799–805. https://doi.org/10.1016/j.chom.2012.10.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Dorfleutner A, Talbott SJ, Bryan NB, Funya KN, Rellick SL, Reed JC, Shi X, Rojanasakul Y, Flynn DC, Stehlik C (2007) A shope fibroma virus PYRIN-only protein modulates the host immune response. Virus Genes 35:685–694. https://doi.org/10.1007/s11262-007-0141-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Johnston JB, Barrett JW, Nazarian SH, Goodwin M, Ricciuto D, Wang G, McFadden G (2005) A poxvirus-encoded pyrin domain protein interacts with ASC-1 to inhibit host inflammatory and apoptotic responses to infection. Immunity 23:587–598. https://doi.org/10.1016/j.immuni.2005.10.003

    Article  CAS  PubMed  Google Scholar 

  31. Coll RC, Robertson AA, Chae JJ et al (2015) A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med 21:248–255. https://doi.org/10.1038/nm.3806

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li CG, Yan L, Jing YY, Xu LH, Liang YD, Wei HX, Hu B, Pan H, Zha QB, Ouyang DY, He XH (2017) Berberine augments ATP-induced inflammasome activation in macrophages by enhancing AMPK signaling. Oncotarget 8:95–109. https://doi.org/10.18632/oncotarget.13921

    Article  PubMed  Google Scholar 

  33. Py BF, Jin M, Desai BN, Penumaka A, Zhu H, Kober M, Dietrich A, Lipinski MM, Henry T, Clapham DE, Yuan J (2014) Caspase-11 controls interleukin-1beta release through degradation of TRPC1. Cell Rep 6:1122–1128. https://doi.org/10.1016/j.celrep.2014.02.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Li CG, Yan L, Mai FY, Shi ZJ, Xu LH, Jing YY, Zha QB, Ouyang DY, He XH (2017) Baicalin inhibits NOD-like receptor family, pyrin containing domain 3 inflammasome activation in murine macrophages by augmenting protein kinase a signaling. Front Immunol 8:1409. https://doi.org/10.3389/fimmu.2017.01409

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kayagaki N, Warming S, Lamkanfi M, Vande Walle L, Louie S, Dong J, Newton K, Qu Y, Liu J, Heldens S, Zhang J, Lee WP, Roose-Girma M, Dixit VM (2011) Non-canonical inflammasome activation targets caspase-11. Nature 479:117–121. https://doi.org/10.1038/nature10558

    Article  CAS  PubMed  Google Scholar 

  36. Liu Y, Jing YY, Zeng CY, Li CG, Xu LH, Yan L, Bai WJ, Zha QB, Ouyang DY, He XH (2017) Scutellarin suppresses NLRP3 inflammasome activation in macrophages and protects mice against bacterial sepsis. Front Pharmacol 8:975. https://doi.org/10.3389/fphar.2017.00975

    Article  CAS  PubMed  Google Scholar 

  37. Mascarenhas DPA, Cerqueira DM, Pereira MSF, Castanheira FVS, Fernandes TD, Manin GZ, Cunha LD, Zamboni DS (2017) Inhibition of caspase-1 or gasdermin-D enable caspase-8 activation in the Naip5/NLRC4/ASC inflammasome. PLoS Pathog 13:e1006502. https://doi.org/10.1371/journal.ppat.1006502

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Pelegrin P, Barroso-Gutierrez C, Surprenant A (2008) P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage. J Immunol 180:7147–7157. https://doi.org/10.4049/jimmunol.180.11.7147

    Article  CAS  PubMed  Google Scholar 

  39. Schneider KS, Gross CJ, Dreier RF et al (2017) The inflammasome drives GSDMD-independent secondary pyroptosis and IL-1 release in the absence of caspase-1 protease activity. Cell Rep 21:3846–3859. https://doi.org/10.1016/j.celrep.2017.12.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Van Opdenbosch N, Van Gorp H, Verdonckt M et al (2017) Caspase-1 engagement and TLR-induced c-FLIP expression suppress ASC/caspase-8-dependent apoptosis by inflammasome sensors NLRP1b and NLRC4. Cell Rep 21:3427–3444. https://doi.org/10.1016/j.celrep.2017.11.088

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lee BL, Mirrashidi KM, Stowe IB, Kummerfeld SK, Watanabe C, Haley B, Cuellar TL, Reichelt M, Kayagaki N (2018) ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages. Sci Rep 8:3788. https://doi.org/10.1038/s41598-018-21998-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Feltham R, Vince JE, Lawlor KE (2017) Caspase-8: not so silently deadly. Clin Transl Immunology 6:e124. https://doi.org/10.1038/cti.2016.83

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Munoz-Planillo R, Kuffa P, Martinez-Colon G, Smith BL, Rajendiran TM, Nunez G (2013) K+ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38:1142–1153. https://doi.org/10.1016/j.immuni.2013.05.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tschopp J (2011) Mitochondria: sovereign of inflammation? Eur J Immunol 41:1196–1202. https://doi.org/10.1002/eji.201141436

    Article  CAS  PubMed  Google Scholar 

  45. Zhong Z, Liang S, Sanchez-Lopez E, He F, Shalapour S, Lin XJ, Wong J, Ding S, Seki E, Schnabl B, Hevener AL, Greenberg HB, Kisseleva T, Karin M (2018) New mitochondrial DNA synthesis enables NLRP3 inflammasome activation. Nature. https://doi.org/10.1038/s41586-018-0372-z

    Article  PubMed  PubMed Central  Google Scholar 

  46. Shimada K, Crother TR, Karlin J et al (2012) Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 36:401–414. https://doi.org/10.1016/j.immuni.2012.01.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ly JD, Grubb DR, Lawen A (2003) The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis 8:115–128. https://doi.org/10.1023/a:1022945107762

    Article  CAS  PubMed  Google Scholar 

  48. Antonopoulos C, El Sanadi C, Kaiser WJ, Mocarski ES, Dubyak GR (2013) Proapoptotic chemotherapeutic drugs induce noncanonical processing and release of IL-1beta via caspase-8 in dendritic cells. J Immunol 191:4789–4803. https://doi.org/10.4049/jimmunol.1300645

    Article  CAS  PubMed  Google Scholar 

  49. Pierini R, Juruj C, Perret M, Jones CL, Mangeot P, Weiss DS, Henry T (2012) AIM2/ASC triggers caspase-8-dependent apoptosis in francisella-infected caspase-1-deficient macrophages. Cell Death Differ 19:1709–1721. https://doi.org/10.1038/cdd.2012.51

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sagulenko V, Thygesen SJ, Sester DP, Idris A, Cridland JA, Vajjhala PR, Roberts TL, Schroder K, Vince JE, Hill JM, Silke J, Stacey KJ (2013) AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC. Cell Death Differ 20:1149–1160. https://doi.org/10.1038/cdd.2013.37

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Bossaller L, Chiang PI, Schmidt-Lauber C, Ganesan S, Kaiser WJ, Rathinam VA, Mocarski ES, Subramanian D, Green DR, Silverman N, Fitzgerald KA, Marshak-Rothstein A, Latz E (2012) Cutting edge: FAS (CD95) mediates noncanonical IL-1beta and IL-18 maturation via caspase-8 in an RIP3-independent manner. J Immunol 189:5508–5512. https://doi.org/10.4049/jimmunol.1202121

    Article  CAS  PubMed  Google Scholar 

  52. Vince JE, De Nardo D, Gao W et al (2018) The mitochondrial apoptotic effectors BAX/BAK activate caspase-3 and -7 to trigger NLRP3 inflammasome and caspase-8 driven IL-1beta activation. Cell Rep 25(2339–2353):e2334. https://doi.org/10.1016/j.celrep.2018.10.103

    Article  CAS  Google Scholar 

  53. Rauch I, Deets KA, Ji DX, von Moltke J, Tenthorey JL, Lee AY, Philip NH, Ayres JS, Brodsky IE, Gronert K, Vance RE (2017) NAIP-NLRC4 inflammasomes coordinate intestinal epithelial cell expulsion with eicosanoid and IL-18 release via activation of caspase-1 and -8. Immunity 46:649–659. https://doi.org/10.1016/j.immuni.2017.03.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Orning P, Weng D, Starheim K et al (2018) Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death. Science 362:1064–1069. https://doi.org/10.1126/science.aau2818

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Sarhan J, Liu BC, Muendlein HI, Li P, Nilson R, Tang AY, Rongvaux A, Bunnell SC, Shao F, Green DR, Poltorak A (2018) Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection. Proc Natl Acad Sci USA. https://doi.org/10.1073/pnas.1809548115

    Article  PubMed  PubMed Central  Google Scholar 

  56. Di Virgilio F, Dal Ben D, Sarti AC, Giuliani AL, Falzoni S (2017) The P2X7 receptor in infection and inflammation. Immunity 47:15–31. https://doi.org/10.1016/j.immuni.2017.06.020

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the grants from the National Natural Science Foundation of China (Grant Nos. 81773965, 81673664 and 81873064).

Author information

Author notes

  1. Chen-Ying Zeng, Chen-Guang Li and Jun-Xiang Shu are contributed equally to this work.

Authors and Affiliations

  1. Department of Immunobiology, College of Life Science and Technology, Jinan University, Guangzhou, China

    Chen-Ying Zeng, Chen-Guang Li, Jun-Xiang Shu, Dong-Yun Ouyang, Feng-Yi Mai, Qiong-Zhen Zeng, Cheng-Cheng Zhang & Xian-Hui He

  2. Department of Cell Biology, College of Life Science and Technology, Jinan University, Guangzhou, China

    Li-Hui Xu

  3. Department of Gynecology and Obstetrics, The First Affiliated Hospital of Jinan University, Guangzhou, China

    Rui-Man Li

Authors
  1. Chen-Ying Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chen-Guang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jun-Xiang Shu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Li-Hui Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dong-Yun Ouyang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Feng-Yi Mai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Qiong-Zhen Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Cheng-Cheng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Rui-Man Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xian-Hui He
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Rui-Man Li or Xian-Hui He.

Ethics declarations

Conflicts of interest

The authors declare no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 821 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zeng, CY., Li, CG., Shu, JX. et al. ATP induces caspase-3/gasdermin E-mediated pyroptosis in NLRP3 pathway-blocked murine macrophages. Apoptosis 24, 703–717 (2019). https://doi.org/10.1007/s10495-019-01551-x

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-019-01551-x

Keywords

Search

Navigation