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

• DOI: 10.1007/s00011-014-0756-y
• Publication Date: 2014-07-21T10:57:31Z
• Authors: Balázs Rada; Jonathan J. Park; Payel Sil; Miklós Geiszt; Thomas L. Leto
• Keywords: 0303 health sciences; Inflammasomes; Ionomycin; Macrophages; Interleukin-1beta; 610; NADPH Oxidases; Mice, Transgenic; Silicon Dioxide; Dual Oxidases; Mice, Inbred C57BL; 03 medical and health sciences; Adenosine Triphosphate; HEK293 Cells; NLR Family, Pyrin Domain-Containing 3 Protein; Animals; Humans; Thapsigargin; Calcium; Carrier Proteins

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