title: NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx.
abstract: Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) is a cytosolic protein that nucleates assembly of inflammasome signaling platforms, which facilitate caspase-1-mediated IL-1β release and other inflammatory responses in myeloid leukocytes. NLRP3 inflammasomes are assembled in response to multiple pathogen- or environmental stress-induced changes in basic cell physiology, including the destabilization of lysosome integrity and activation of K(+)-permeable channels/transporters in the plasma membrane (PM). However, the quantitative relationships between lysosome membrane permeabilization (LMP), induction of increased PM K(+) permeability, and activation of NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester (LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1β release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDCs). Treatment of BMDCs with submillimolar (≤1 mM) LLME induced slower and partial increases in LMP that correlated with robust NLRP3 inflammasome activation and K(+) efflux. In contrast, supramillimolar (≥2 mM) LLME elicited extremely rapid and complete collapse of lysosome integrity that was correlated with suppression of inflammasome signaling. Supramillimolar LLME also induced dominant negative effects on inflammasome activation by the canonical NLRP3 agonist nigericin; this inhibition correlated with an increase in NLRP3 ubiquitination. LMP elicited rapid BMDC death by both inflammasome-dependent pyroptosis and inflammasome-independent necrosis. LMP also triggered Ca(2+) influx, which attenuated LLME-stimulated NLRP3 inflammasome signaling but potentiated LLME-induced necrosis. Taken together, these studies reveal a previously unappreciated signaling network that defines the coupling between LMP, changes in PM cation fluxes, cell death, and NLRP3 inflammasome activation.
authors.affiliation: Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio;
authors.forename: Michael A
authors.initials: MA
authors.lastname: Katsnelson
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio;
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Lozada-Soto
authors.affiliation: Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio;
authors.forename: Hana M
authors.initials: HM
authors.lastname: Russo
authors.affiliation: Department of Pediatrics, Penn State Hershey Children's Hospital, Hershey, Pennsylvania.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Miller
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio; Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio; george.dubyak@case.edu.
authors.forename: George R
authors.initials: GR
authors.lastname: Dubyak
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D053468:CARD Signaling Adaptor Proteins; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D020170:Caspase 1 / Q000172:deficiency / Q000235:genetics; D020169:Caspases / Q000172:deficiency / Q000235:genetics; D053471:Caspases, Initiator; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D058847:Inflammasomes / Q000187:drug effects / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000378:metabolism*; D009336:Necrosis; D009550:Nigericin / Q000494:pharmacology; D010539:Permeability; D011188:Potassium / Q000378:metabolism*; D050053:TRPM Cation Channels / Q000172:deficiency / Q000235:genetics; D054875:Ubiquitination
chemical_list: D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D004151:Dipeptides; C587672:IL1B protein, mouse; D058847:Inflammasomes; D053583:Interleukin-1beta; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C492716:Nlrp3 protein, mouse; C498534:Pycard protein, mouse; D050053:TRPM Cation Channels; C544443:TRPM2 protein, mouse; C045139:leucyl-leucine-methyl ester; C101136:Casp4 protein, mouse; D020169:Caspases; D053471:Caspases, Initiator; D020170:Caspase 1; D009550:Nigericin; D011188:Potassium; D002118:Calcium
keywords: IL-1; caspase-1; cell death; dendritic cell; ion channels