Skip to main content

Advertisement

Springer Nature Link
Log in

NLRP3 inflammasome activation and interleukin-1β release in macrophages require calcium but are independent of calcium-activated NADPH oxidases

  • Original Research Paper
  • Published:
Inflammation Research Aims and scope Submit manuscript

Abstract

Objective and design

We studied the involvement of calcium and calcium-activated NADPH oxidases in NLRP3 inflammasome activation and IL-1β release to better understand inflammasome signaling in macrophages.

Material or subjects

Human volunteer blood donors were recruited to isolate monocytes to differentiate them into macrophages. Wild-type or DUOX1-deficient C57/B6 mice were used to prepare bone marrow-derived macrophages.

Treatment

Murine or human macrophages were treated in vitro with NLRP3 inflammasome agonists (ATP, silica crystals) or calcium agonists (thapsigargin, ionomycin) in calcium-containing or calcium-free medium.

Methods

Intracellular calcium changes were followed by measuring FURA2-based fluorescence. Gene expression changes were measured by quantitative real-time PCR. Protein expression was assessed by western blotting. Enzymatic activity was measured by fluorescence caspase-1 activity assay. IL-1β release was determined by ELISA. ELISA data were analyzed by ANOVA and Tukey’s post hoc test.

Results

Our data show that calcium is essential for IL-1β release in human macrophages. Increases in cytosolic calcium alone lead to IL-1β secretion. Calcium removal blocks caspase-1 activation. Human macrophages express Duox1, a calcium-regulated NADPH oxidase that produces reactive oxygen species. However, Duox1-deficient murine macrophages show normal IL-1β release.

Conclusions

Human macrophage inflammasome activation and IL-1β secretion requires calcium but does not involve NADPH oxidases.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

BMDM:

Bone marrow-derived macrophage

Duox:

Dual oxidase

DuoxA:

Duox Activator

MDM:

Monocyte-derived macrophage

Nox5:

NADPH oxidase 5

ROS:

Reactive oxygen species

References

  1. Murakami T, Ockinger J, Yu J, Byles V, McColl A, Hofer AM, et al. Critical role for calcium mobilization in activation of the NLRP3 inflammasome. Proc Natl Acad Sci USA. 2012;109(28):11282–7. doi:10.1073/pnas.1117765109.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  2. Carta S, Tassi S, Pettinati I, Delfino L, Dinarello CA, Rubartelli A. The rate of interleukin-1beta secretion in different myeloid cells varies with the extent of redox response to Toll-like receptor triggering. J Biol Chem. 2011;286(31):27069–80. doi:10.1074/jbc.M110.203398.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Thastrup O, Dawson AP, Scharff O, Foder B, Cullen PJ, Drobak BK, et al. Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents Actions. 1989;27(1–2):17–23.

    Article  PubMed  CAS  Google Scholar 

  4. Brayden DJ, Hanley MR, Thastrup O, Cuthbert AW. Thapsigargin, a new calcium-dependent epithelial anion secretagogue. Br J Pharmacol. 1989;98(3):809–16.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  5. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell. 2002;10(2):417–26.

    Article  PubMed  CAS  Google Scholar 

  6. Liu C, Hermann TE. Characterization of ionomycin as a calcium ionophore. J Biol Chem. 1978;253(17):5892–4.

    PubMed  CAS  Google Scholar 

  7. Joosten LA, Ea HK, Netea MG, Busso N. Interleukin-1beta activation during acute joint inflammation: a limited role for the NLRP3 inflammasome in vivo. Joint, Bone, Spine : Revue du Rhumatisme. 2011;78(2):107–10. doi:10.1016/j.jbspin.2010.11.004.

    Article  CAS  Google Scholar 

  8. Oosting M, van de Veerdonk FL, Kanneganti TD, Sturm P, Verschueren I, Berende A, et al. Borrelia species induce inflammasome activation and IL-17 production through a caspase-1-dependent mechanism. Eur J Immunol. 2011;41(1):172–81. doi:10.1002/eji.201040385.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. Stienstra R, Joosten LA, Koenen T, van Tits B, van Diepen JA, van den Berg SA, et al. The inflammasome-mediated caspase-1 activation controls adipocyte differentiation and insulin sensitivity. Cell Metab. 2010;12(6):593–605. doi:10.1016/j.cmet.2010.11.011.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  10. van de Veerdonk FL, Smeekens SP, Joosten LA, Kullberg BJ, Dinarello CA, van der Meer JW, et al. Reactive oxygen species-independent activation of the IL-1beta inflammasome in cells from patients with chronic granulomatous disease. Proc Natl Acad Sci USA. 2010;107(7):3030–3. doi:10.1073/pnas.0914795107.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Lee GS, Subramanian N, Kim AI, Aksentijevich I, Goldbach-Mansky R, Sacks DB, et al. The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP. Nature. 2012;492(7427):123–7. doi:10.1038/nature11588.

    Article  PubMed  CAS  Google Scholar 

  12. Tschopp J, Schroder K. NLRP3 inflammasome activation: the convergence of multiple signalling pathways on ROS production? Nat Rev Immunol. 2010;10(3):210–5. doi:10.1038/nri2725.

    Article  PubMed  CAS  Google Scholar 

  13. Jones JW, Broz P, Monack DM. Innate immune recognition of francisella tularensis: activation of type-I interferons and the inflammasome. Frontiers Microbiol. 2011;2:16. doi:10.3389/fmicb.2011.00016.

    CAS  Google Scholar 

  14. Conforti-Andreoni C, Beretta O, Licandro G, Qian HL, Urbano M, Vitulli F, et al. Synergism of NOD2 and NLRP3 activators promotes a unique transcriptional profile in murine dendritic cells. J Leukoc Biol. 2010;88(6):1207–16. doi:10.1189/jlb.1009652.

    Article  PubMed  CAS  Google Scholar 

  15. Mortellaro A, Wong SC, Fric J, Ricciardi-Castagnoli P. The need to identify myeloid dendritic cell progenitors in human blood. Trends Immunol. 2010;31(1):18–23. doi:10.1016/j.it.2009.09.010.

    Article  PubMed  CAS  Google Scholar 

  16. Zhou R, Yazdi AS, Menu P, Tschopp J. A role for mitochondria in NLRP3 inflammasome activation. Nature. 2011;469(7329):221–5. doi:10.1038/nature09663.

    Article  PubMed  CAS  Google Scholar 

  17. Rada B, Leto TL. Oxidative innate immune defenses by Nox/Duox family NADPH oxidases. Contribut Microbiol. 2008;15:164–87. doi:10.1159/000136357.

    Article  CAS  Google Scholar 

  18. Orient A, Donko A, Szabo A, Leto TL, Geiszt M. Novel sources of reactive oxygen species in the human body. Nephrol, Dialysis, Transpl : Off Pub Eur Dialysis Transpl Assoc Eur Renal Assoc. 2007;22(5):1281–8. doi:10.1093/ndt/gfm077.

    Article  CAS  Google Scholar 

  19. Meissner F, Seger RA, Moshous D, Fischer A, Reichenbach J, Zychlinsky A. Inflammasome activation in NADPH oxidase defective mononuclear phagocytes from patients with chronic granulomatous disease. Blood. 2010;116(9):1570–3. doi:10.1182/blood-2010-01-264218.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  20. van Bruggen R, Koker MY, Jansen M, van Houdt M, Roos D, Kuijpers TW, et al. Human NLRP3 inflammasome activation is No1–4 independent. Blood. 2010;115(26):5398–400. doi:10.1182/blood-2009-10-250803.

    Article  PubMed  Google Scholar 

  21. Banfi B, Molnar G, Maturana A, Steger K, Hegedus B, Demaurex N, et al. A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes. J Biol Chem. 2001;276(40):37594–601. doi:10.1074/jbc.M103034200.

    Article  PubMed  CAS  Google Scholar 

  22. Ameziane-El-Hassani R, Morand S, Boucher JL, Frapart YM, Apostolou D, Agnandji D, et al. Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity. J Biol Chem. 2005;280(34):30046–54. doi:10.1074/jbc.M500516200.

    Article  PubMed  CAS  Google Scholar 

  23. Morand S, Ueyama T, Tsujibe S, Saito N, Korzeniowska A, Leto TL. Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation. FASEB J Off Pub Fed Am Soc Exp Biol. 2009;23(4):1205–18. doi:10.1096/fj.08-120006.

    CAS  Google Scholar 

  24. Yoo DG, Winn M, Pang L, Moskowitz SM, Malech HL, Leto TL, et al. Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation. J Immunol. 2014;192(10):4728–38. doi:10.4049/jimmunol.1301589.

    Article  PubMed  CAS  Google Scholar 

  25. Rada B, Jendrysik MA, Pang L, Hayes CP, Yoo DG, Park JJ, et al. Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase. PLoS One. 2013;8(1):e54205. doi:10.1371/journal.pone.0054205.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  26. Donko A, Ruisanchez E, Orient A, Enyedi B, Kapui R, Peterfi Z, et al. Urothelial cells produce hydrogen peroxide through the activation of Duox1. Free Radical Biol Med. 2010;49(12):2040–8. doi:10.1016/j.freeradbiomed.2010.09.027.

    Article  CAS  Google Scholar 

  27. Rubartelli A, Gattorno M, Netea MG, Dinarello CA. Interplay between redox status and inflammasome activation. Trends Immunol. 2011;32(12):559–66. doi:10.1016/j.it.2011.08.005.

    Article  PubMed  CAS  Google Scholar 

  28. Bauernfeind F, Ablasser A, Bartok E, Kim S, Schmid-Burgk J, Cavlar T, et al. Inflammasomes: current understanding and open questions. Cell Mol Life Sci CMLS. 2011;68(5):765–83. doi:10.1007/s00018-010-0567-4.

    Article  CAS  Google Scholar 

  29. Leto TL, Geiszt M. Role of Nox family NADPH oxidases in host defense. Antioxid Redox Signal. 2006;8(9–10):1549–61. doi:10.1089/ars.2006.8.1549.

    Article  PubMed  CAS  Google Scholar 

  30. Geiszt M, Witta J, Baffi J, Lekstrom K, Leto TL. Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense. FASEB J Off Pub Fed Am Soc Exp Biol. 2003;17(11):1502–4. doi:10.1096/fj.02-1104fje.

    CAS  Google Scholar 

  31. Broz P, Monack DM. Molecular mechanisms of inflammasome activation during microbial infections. Immunol Rev. 2011;243(1):174–90. doi:10.1111/j.1600-065X.2011.01041.x.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  32. Conforti-Andreoni C, Ricciardi-Castagnoli P, Mortellaro A. The inflammasomes in health and disease: from genetics to molecular mechanisms of autoinflammation and beyond. Cell Mol Immunol. 2011;8(2):135–45. doi:10.1038/cmi.2010.81.

    Article  PubMed  CAS  Google Scholar 

  33. Jin C, Flavell RA. Molecular mechanism of NLRP3 inflammasome activation. J Clin Immunol. 2010;30(5):628–31. doi:10.1007/s10875-010-9440-3.

    Article  PubMed  CAS  Google Scholar 

  34. Schroder K, Tschopp J. The inflammasomes. Cell. 2010;140(6):821–32. doi:10.1016/j.cell.2010.01.040.

    Article  PubMed  CAS  Google Scholar 

  35. Arlehamn CS, Petrilli V, Gross O, Tschopp J, Evans TJ. The role of potassium in inflammasome activation by bacteria. J Biol Chem. 2010;285(14):10508–18. doi:10.1074/jbc.M109.067298.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  36. Petrilli V, Papin S, Dostert C, Mayor A, Martinon F, Tschopp J. Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ. 2007;14(9):1583–9. doi:10.1038/sj.cdd.4402195.

    Article  PubMed  CAS  Google Scholar 

  37. Gao YD, Hanley PJ, Rinne S, Zuzarte M, Daut J. Calcium-activated K(+) channel (K(Ca)3.1) activity during Ca(2+) store depletion and store-operated Ca(2+) entry in human macrophages. Cell Calcium. 2010;48(1):19–27. doi:10.1016/j.ceca.2010.06.002.

    Article  PubMed  CAS  Google Scholar 

  38. Brough D, Le Feuvre RA, Wheeler RD, Solovyova N, Hilfiker S, Rothwell NJ, et al. Ca2+ stores and Ca2+ entry differentially contribute to the release of IL-1 beta and IL-1 alpha from murine macrophages. J Immunol. 2003;170(6):3029–36.

    Article  PubMed  CAS  Google Scholar 

  39. Choi EM. Regulation of intracellular Ca(2+) by reactive oxygen species in osteoblasts treated with antimycin A. J Appl Toxicol JAT. 2012;32(2):118–25. doi:10.1002/jat.1642.

    Article  CAS  Google Scholar 

  40. Rada BK, Geiszt M, Van Bruggen R, Nemet K, Roos D, Ligeti E. Calcium signalling is altered in myeloid cells with a deficiency in NADPH oxidase activity. Clin Exp Immunol. 2003;132(1):53–60.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  41. Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol. 2010;11(2):136–40. doi:10.1038/ni.1831.

    Article  PubMed  CAS  Google Scholar 

  42. Cruz CM, Rinna A, Forman HJ, Ventura AL, Persechini PM, Ojcius DM. ATP activates a reactive oxygen species-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages. J Biol Chem. 2007;282(5):2871–9. doi:10.1074/jbc.M608083200.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  43. Cassel SL, Eisenbarth SC, Iyer SS, Sadler JJ, Colegio OR, Tephly LA, et al. The Nalp3 inflammasome is essential for the development of silicosis. Proc Natl Acad Sci USA. 2008;105(26):9035–40. doi:10.1073/pnas.0803933105.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  44. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science. 2008;320(5876):674–7. doi:10.1126/science.1156995.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  45. Guerra AN, Gavala ML, Chung HS, Bertics PJ. Nucleotide receptor signalling and the generation of reactive oxygen species. Purinergic Signal. 2007;3(1–2):39–51. doi:10.1007/s11302-006-9035-x.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  46. Pfeiffer ZA, Guerra AN, Hill LM, Gavala ML, Prabhu U, Aga M, et al. Nucleotide receptor signaling in murine macrophages is linked to reactive oxygen species generation. Free Radical Biol Med. 2007;42(10):1506–16. doi:10.1016/j.freeradbiomed.2007.02.010.

    Article  CAS  Google Scholar 

  47. Geiszt M. NADPH oxidases: new kids on the block. Cardiovasc Res. 2006;71(2):289–99. doi:10.1016/j.cardiores.2006.05.004.

    Article  PubMed  CAS  Google Scholar 

  48. Bauernfeind F, Bartok E, Rieger A, Franchi L, Nunez G, Hornung V. Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome. J Immunol. 2011;187(2):613–7. doi:10.4049/jimmunol.1100613.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  49. Tassi S, Carta S, Delfino L, Caorsi R, Martini A, Gattorno M, et al. Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1beta secretion. Proc Natl Acad Sci USA. 2010;107(21):9789–94. doi:10.1073/pnas.1000779107.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  50. Boots AW, Hristova M, Kasahara DI, Haenen GR, Bast A, van der Vliet A. ATP-mediated activation of the NADPH oxidase DUOX1 mediates airway epithelial responses to bacterial stimuli. J Biol Chem. 2009;284(26):17858–67. doi:10.1074/jbc.M809761200.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  51. Rada B, Lekstrom K, Damian S, Dupuy C, Leto TL. The Pseudomonas toxin pyocyanin inhibits the dual oxidase-based antimicrobial system as it imposes oxidative stress on airway epithelial cells. J Immunol. 2008;181(7):4883–93.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  52. Razzell W, Evans IR, Martin P, Wood W. Calcium flashes orchestrate the wound inflammatory response through DUOX activation and hydrogen peroxide release. Curr Biol CB. 2013;23(5):424–9. doi:10.1016/j.cub.2013.01.058.

    Article  CAS  Google Scholar 

  53. Sham D, Wesley UV, Hristova M, van der Vliet A. ATP-mediated transactivation of the epidermal growth factor receptor in airway epithelial cells involves DUOX1-dependent oxidation of Src and ADAM17. PLoS One. 2013;8(1):e54391. doi:10.1371/journal.pone.0054391.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  54. Kwon J, Shatynski KE, Chen H, Morand S, de Deken X, Miot F et al. The nonphagocytic NADPH oxidase Duox1 mediates a positive feedback loop during T cell receptor signaling. Science signaling. 2010;3(133):ra59. doi:10.1126/scisignal.2000976.

Download references

Acknowledgments

This study was supported by funds from the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.

Author information

Authors and Affiliations

  1. Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD, 20852, USA

    Balázs Rada, Jonathan J. Park & Thomas L. Leto

  2. Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, 501 DW Brooks Drive, Athens, GA, 30602, USA

    Balázs Rada & Payel Sil

  3. Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary

    Miklós Geiszt

  4. “Lendület” Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, Budapest, Hungary

    Miklós Geiszt

Authors
  1. Balázs Rada
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jonathan J. Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Payel Sil
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miklós Geiszt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thomas L. Leto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas L. Leto.

Additional information

Responsible Editor: Bernhard Gibbs.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rada, B., Park, J.J., Sil, P. et al. NLRP3 inflammasome activation and interleukin-1β release in macrophages require calcium but are independent of calcium-activated NADPH oxidases. Inflamm. Res. 63, 821–830 (2014). https://doi.org/10.1007/s00011-014-0756-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00011-014-0756-y

Keywords

Search

Navigation