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

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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Correspondence to Thomas L. Leto.

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Responsible Editor: Bernhard Gibbs.

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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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Keywords

  • Calcium
  • Duox
  • Macrophage
  • Inflammasome
  • NADPH oxidase