title: NLRP3 inflammasome activation and interleukin-1β release in macrophages require calcium but are independent of calcium-activated NADPH oxidases.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  METHODS
  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.
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD, 20852, USA, radab@uga.edu.
authors.forename: Balázs
authors.initials: B
authors.lastname: Rada
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Park
authors.forename: Payel
authors.initials: P
authors.lastname: Sil
authors.forename: Miklós
authors.initials: M
authors.lastname: Geiszt
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Leto
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D000074623:Dual Oxidases; D057809:HEK293 Cells; D006801:Humans; D058847:Inflammasomes; D053583:Interleukin-1beta / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D019255:NADPH Oxidases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D012822:Silicon Dioxide / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D058847:Inflammasomes; D053583:Interleukin-1beta; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; C492716:Nlrp3 protein, mouse; D015759:Ionomycin; D019284:Thapsigargin; D012822:Silicon Dioxide; D000255:Adenosine Triphosphate; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431513:DUOX1 protein, human; C519130:Duox1 protein, mouse; D002118:Calcium