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

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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.

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Abbreviations

BMDM:

Bone marrow-derived macrophage

Duox:

Dual oxidase

DuoxA:

Duox Activator

MDM:

Monocyte-derived macrophage

Nox5:

NADPH oxidase 5

ROS:

Reactive oxygen species

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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.

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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

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  1. Balázs Rada
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  2. Jonathan J. Park
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  5. Thomas L. Leto
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Correspondence to Thomas L. Leto.

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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

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