title: K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling.
abstract: Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1-mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic K(+) concentration versus increased cytosolic Ca(2+) concentration ([Ca(2+)]) remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca(2+)] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K(+) efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester. The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca(2+)] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent Leu-Leu-O-methyl ester. Second, agonists for three Ca(2+)-mobilizing G protein-coupled receptors (formyl peptide receptor, P2Y2 purinergic receptor, and calcium-sensing receptor) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared with the K(+) efflux agonists. Third, the intracellular Ca(2+) buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate, widely used reagents for disruption of Ca(2+)-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca(2+) homeostasis. The results indicate that the ability of K(+) efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca(2+)] as a necessary or sufficient signal.
authors.affiliation: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106;
authors.forename: Michael A
authors.initials: MA
authors.lastname: Katsnelson
authors.affiliation: The Ohio State University College of Medicine, Columbus, OH 43210; and.
authors.forename: L Graham
authors.initials: LG
authors.lastname: Rucker
authors.affiliation: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106;
authors.forename: Hana M
authors.initials: HM
authors.lastname: Russo
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106 george.dubyak@case.edu.
authors.forename: George R
authors.initials: GR
authors.lastname: Dubyak
mesh_terms: D000255:Adenosine Triphosphate / Q000276:immunology; D000818:Animals; D001896:Boron Compounds; D020013:Calcium Signaling / Q000187:drug effects / Q000276:immunology*; D002352:Carrier Proteins / Q000276:immunology*; D002614:Chelating Agents / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology; D058847:Inflammasomes / Q000276:immunology*; D053583:Interleukin-1beta / Q000276:immunology; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D009550:Nigericin / Q000494:pharmacology; D011188:Potassium / Q000276:immunology*; D058486:Receptors, Purinergic P2X7 / Q000276:immunology
chemical_list: D001896:Boron Compounds; D002352:Carrier Proteins; D002614:Chelating Agents; D004151:Dipeptides; C587672:IL1B protein, mouse; D007166:Immunosuppressive Agents; D058847:Inflammasomes; D053583:Interleukin-1beta; D007476:Ionophores; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C492716:Nlrp3 protein, mouse; D058486:Receptors, Purinergic P2X7; C045139:leucyl-leucine-methyl ester; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; C109986:2-aminoethoxydiphenyl borate; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D009550:Nigericin; D011188:Potassium