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Simultaneous Detection of Cellular Viability and Interleukin-1β Secretion from Single Cells by ELISpot

  • Stephanie A. Conos
  • Lisa M. LindqvistEmail author
  • James E. VinceEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1714)

Abstract

Cell death results in the breakdown of the plasma membrane, which can cause the release of cytosolic proteins. During caspase-1-mediated cell death, termed pyroptosis, pro-inflammatory mediators that lack canonical secretory signal sequences, such as interleukin-1β (IL-1β), are released into the extracellular environment. To define whether cell death is required for the release of IL-1β, or if IL-1β can be actively secreted from viable cells, we have developed a modified IL-1β Enzyme-Linked ImmunoSpot (ELISpot) assay. This assay simultaneously detects cellular viability and IL-1β release at the single-cell level, and is therefore useful to examine how cell death influences IL-1β secretion under different experimental conditions. Cells expressing a surrogate viability marker, such as GFP, are plated onto cellulose filter plates coated with an IL-1β capture antibody. This antibody immobilizes IL-1β as it is released from cells, allowing detection of distinct IL-1β “spots.” Both GFP positive cells and IL-1β spots are detected and quantified using an AID ELISpot Reader, and the captured images are overlaid. Therefore, cell viability and IL-1β release from individual cells can be monitored visually. We have recently used this method to document how individual fibroblasts expressing activated caspase-1 can secrete IL-1β in the absence of cell death. Adaptation of this assay to other experimental conditions may help to define the circumstances where cell death influences IL-1β release and IL-1β-driven inflammatory responses.

Keywords

Interleukin-1β Inflammasome ELISpot Apoptosis Cell death Cytokine Caspase-1 Caspase-8 Pyroptosis Secretion 

Notes

Acknowledgments

We thank Melinda Hardy and Dimitra Zotos for ELISpot protocols, and David Vaux and Kate Lawlor for scintillating discussions and advice. This work was supported by National Health and Medical Research Council (Canberra, Australia) Project grants (1051210), fellowships (JEV [1052598]; LML [1035502]); and operational infrastructure grants through the Australian Government IRISS and the Victorian State Government OIS (361646).

References

  1. 1.
    Menu P, Vince JE (2011) The NLRP3 inflammasome in health and disease: the good, the bad and the ugly. Clin Exp Immunol 166(1):1–15. https://doi.org/10.1111/j.1365-2249.2011.04440.x CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    de Vasconcelos NM, Van Opdenbosch N, Lamkanfi M (2016) Inflammasomes as polyvalent cell death platforms. Cell Mol Life Sci 73(11–12):2335–2347. https://doi.org/10.1007/s00018-016-2204-3 CrossRefPubMedGoogle Scholar
  3. 3.
    Vince JE, Silke J (2016) The intersection of cell death and inflammasome activation. Cell Mol Life Sci 73(11–12):2349–2367. https://doi.org/10.1007/s00018-016-2205-2 CrossRefPubMedGoogle Scholar
  4. 4.
    Monteleone M, Stow JL, Schroder K (2015) Mechanisms of unconventional secretion of IL-1 family cytokines. Cytokine 74(2):213–218. https://doi.org/10.1016/j.cyto.2015.03.022 CrossRefPubMedGoogle Scholar
  5. 5.
    Cullen SP, Kearney CJ, Clancy DM, Martin SJ (2015) Diverse activators of the NLRP3 Inflammasome promote IL-1beta secretion by triggering necrosis. Cell Rep 11(10):1535–1548. https://doi.org/10.1016/j.celrep.2015.05.003 CrossRefPubMedGoogle Scholar
  6. 6.
    Liu T, Yamaguchi Y, Shirasaki Y, Shikada K, Yamagishi M, Hoshino K, Kaisho T, Takemoto K, Suzuki T, Kuranaga E, Ohara O, Miura M (2014) Single-cell imaging of caspase-1 dynamics reveals an all-or-none inflammasome signaling response. Cell Rep 8(4):974–982. https://doi.org/10.1016/j.celrep.2014.1007.1012. Epub 2014 Aug 7CrossRefPubMedGoogle Scholar
  7. 7.
    Shirasaki Y, Yamagishi M, Suzuki N, Izawa K, Nakahara A, Mizuno J, Shoji S, Heike T, Harada Y, Nishikomori R, Ohara O (2014) Real-time single-cell imaging of protein secretion. Sci Rep 4:4736. https://doi.org/10.1038/srep04736 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gaidt MM, Ebert TS, Chauhan D, Schmidt T, Schmid-Burgk JL, Rapino F, Robertson AA, Cooper MA, Graf T, Hornung V (2016) Human monocytes engage an alternative inflammasome pathway. Immunity 44(4):833–846. https://doi.org/10.1016/j.immuni.2016.01.012 CrossRefPubMedGoogle Scholar
  9. 9.
    Chen KW, Gross CJ, Sotomayor FV, Stacey KJ, Tschopp J, Sweet MJ, Schroder K (2014) The neutrophil NLRC4 inflammasome selectively promotes IL-1beta maturation without pyroptosis during acute Salmonella challenge. Cell Rep 8(2):570–582. https://doi.org/10.1016/j.celrep.2014.1006.1028. Epub 2014 Jul 17CrossRefPubMedGoogle Scholar
  10. 10.
    Zanoni I, Tan Y, Di Gioia M, Broggi A, Ruan J, Shi J, Donado CA, Shao F, Wu H, Springstead JR, Kagan JC (2016) An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells. Science 352(6290):1232–1236. https://doi.org/10.1126/science.aaf3036 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhang M, Kenny SJ, Ge L, Xu K, Schekman R (2015) Translocation of interleukin-1beta into a vesicle intermediate in autophagy-mediated secretion. elife 4:e11205. https://doi.org/10.7554/eLife.11205 PubMedPubMedCentralGoogle Scholar
  12. 12.
    Karmakar M, Katsnelson M, Malak HA, Greene NG, Howell SJ, Hise AG, Camilli A, Kadioglu A, Dubyak GR, Pearlman E (2015) Neutrophil IL-1beta processing induced by pneumolysin is mediated by the NLRP3/ASC inflammasome and caspase-1 activation and is dependent on K+ efflux. J Immunol 194(4):1763–1775. https://doi.org/10.4049/jimmunol.1401624. Epub 2015 Jan 21CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Conos SA, Lawlor KE, Vaux DL, Vince JE, Lindqvist LM (2016) Cell death is not essential for caspase-1-mediated interleukin-1beta activation and secretion. Cell Death Differ 23(11):1827–1838. https://doi.org/10.1038/cdd.2016.69 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Czerkinsky CC, Nilsson LA, Nygren H, Ouchterlony O, Tarkowski A (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65(1–2):109–121CrossRefPubMedGoogle Scholar
  15. 15.
    Kalyuzhny AE (ed) (2012) Handbook of elispot: methods and protocols, vol 792, 2nd edn. Humana Press, New York, pp 1–261. https://doi.org/10.1007/978-1-61779-325-7 Google Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical ResearchParkvilleAustralia
  2. 2.Department of Medical BiologyUniversity of MelbourneMelbourneAustralia
  3. 3.Inflammation Division, The Walter and Eliza Hall Institute of Medical ResearchParkvilleAustralia

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