K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling

doi:10.4049/jimmunol.1402658

. 2015 Apr 15;194(8):3937-52.

doi: 10.4049/jimmunol.1402658. Epub 2015 Mar 11.

K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling

Michael A Katsnelson¹, L Graham Rucker², Hana M Russo¹, George R Dubyak³

Affiliations

¹ Department of Pathology, Case Western Reserve University, Cleveland, OH 44106;
² The Ohio State University College of Medicine, Columbus, OH 43210; and.
³ Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106 george.dubyak@case.edu.

PMID: 25762778
PMCID: PMC4390495
DOI: 10.4049/jimmunol.1402658

K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling

Michael A Katsnelson et al. J Immunol. 2015.

. 2015 Apr 15;194(8):3937-52.

doi: 10.4049/jimmunol.1402658. Epub 2015 Mar 11.

Authors

Michael A Katsnelson¹, L Graham Rucker², Hana M Russo¹, George R Dubyak³

Affiliations

¹ Department of Pathology, Case Western Reserve University, Cleveland, OH 44106;
² The Ohio State University College of Medicine, Columbus, OH 43210; and.
³ Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106 george.dubyak@case.edu.

PMID: 25762778
PMCID: PMC4390495
DOI: 10.4049/jimmunol.1402658

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.

PubMed Disclaimer

Figures

**Figure 1. Nigericin-induced increases in cytosolic [Ca²⁺] in LPS-primed BMDC occur downstream of the NLRP3 inflammasome/ caspase-1/ pyroptotic signaling cascade**
A, Diagram of changes in cation homeostasis occurring in myeloid cells in response to treatment with nigericin and ATP/P2X7. B, IL-1β release from LPS-primed WT, *Nlrp3*^−/−, and *Casp-1/11*^−/− BMDC in response to 10μM nigericin and 5mM ATP was measured by ELISA. Data represent mean of 2 independent experiments. C, LPS-primed WT, *Nlrp3*^−/−, and *Casp-1/11*^−/− BMDC were stimulated with 10μM nigericin or 5mM ATP for 30 min. BMDC were lysed with 10% nitric acid and lysates were analyzed by atomic absorption spectroscopy to measure cellular [K⁺]. Data represent mean of three independent experiments. **D–F**, WT, *Nlrp3*^−/−, or *Casp-1/11*^−/− BMDC were primed with LPS (1μg/ml) for 4h and loaded with 1μg/ml fluo-4 AM for 30 min. Baseline readings were taken for 5 min and 10μM nigericin (NG) or 5mM ATP were added at t=5 min. Cytosolic [Ca²⁺] was determined by measuring fluo-4 fluorescence. Data represent a mean of two independent experiments. **G–I,** LPS-primed WT, *Nlrp3*^−/−, and *Casp-1/11*^−/−BMDC were stimulated with 10μM nigericin or 5mM ATP for 30 min. Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium²⁺. Baseline readings were taken for 5 min and nigericin or ATP were added at t=5 min. Data represent a mean of two independent experiments.

**Figure 2. NLRP3 inflammasome signaling responses to K⁺ efflux agonists are dissociated from influx of extracellular Ca²⁺**
A, LPS-primed BMDC were treated with 10μM nigericin (NG) or 5mM ATP for 30 min in the presence/absence of 1.5mM extracellular [Ca²⁺]. Cytosolic [Ca²⁺] was determined as described in figure 1. Data represent a mean of three independent experiments. **B–E,** LPS-primed BMDC were treated for 30 min with 10μM nigericin or 5mM ATP in the presence/absence of 1.5mM extracellular [Ca²⁺]. B, IL-1β release was determined by ELISA. Data represent a mean of six independent experiments. C, Soluble lysate fraction (Lys) was probed for procaspase-1 and pro-IL-1β, insoluble lysate pellet was crosslinked with DSS and probed for oligomerized ASC, and extracellular medium fraction (ECM) was probed for mature caspase-1 and IL-1β. D, Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium²⁺. Baseline readings were taken for 5 min and nigericin or ATP were added at t=5 min. Data represent mean of four independent experiments. E, BMDC were lysed with 10% nitric acid and lysates were analyzed by atomic absorption spectroscopy to measure cellular [K⁺]. Data represent mean of three independent experiments.

**Figure 3. NLRP3 inflammasome signaling responses to lysosomal destabilization are dissociated from influx of extracellular Ca²⁺**
**A–C,** LPS-primed BMDC were stimulated with 1mM LLME for 30 min. A, LPS-primed WT, *Nlrp3*^−/−, and *Casp1*^−/− BMDC were stimulated with LLME and IL-1β release was determined by ELISA. Data represent a mean of two independent experiments. B, LPS-primed WT, *Nlrp3^−/−*, and *Casp1^−/−* BMDC were stimulated with LLME, lysed with 10% nitric acid, and the lysates analyzed by atomic absorption spectroscopy to measure cellular [K⁺]. Data represent mean of three independent experiments C, LPS-primed WT BMDC were stimulated with LLME in the presence/absence of 1.5mM extracellular [Ca²⁺] and IL-1β release was determined by ELISA. Data represent a mean of two independent experiments. **D, F,** Cytosolic [Ca²⁺] was determined as described in figure 1 in WT (panels D, F) *Nlrp3^−/−*, and *Casp1^−/−* (Panel F) BMDC. Baseline readings were taken for 5 min and LLME was added at t=5 min. Data represent a mean of two independent experiments. E, LLME-induced propidium²⁺ influx was measured in LPS-primed BMDC in the presence/absence of 1.5mM extracellular [Ca²⁺]. Baseline readings were taken for 5 min and LLME was added at t=5 min. Data represent mean of two independent experiments..

**Figure 4. NLRP3 inflammasome signaling responses to K⁺ efflux agonists are dissociated from release of thapsigargin-sensitive intracellular Ca²⁺ stores**
A, LPS-primed BMDC were treated with 300nM thapsigargin (TG) at t=0 in the absence of extracellular Ca²⁺. BMDC were washed at t=30 min and 10 min of baseline readings were taken prior to addition of 10μM nigericin or 5mM ATP at t=40 min. Cytosolic [Ca²⁺] was determined as described in figure 1. Data represent a mean of three independent experiments. **B and C,** LPS-primed BMDC were treated with 300nM thapsigargin in the absence of extracellular Ca²⁺. BMDC were then stimulated with 10μM nigericin or 5mM ATP in the absence of extracellular Ca²⁺. B, Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium²⁺. Baseline readings were taken for 5 min and nigericin or ATP were added at t=5 min. Data represent a mean of three independent experiments. C, IL-1β release was determined by ELISA. Data represent a mean of six independent experiments.

**Figure 5. NLRP3 inflammasome signaling responses to K⁺ efflux agonists are dissociated from changes in cytosolic [Ca²⁺] in BMDC primed with TLR2 or TNF receptor agonists**
**A and E,** BMDC were primed with Pam₃CSK₄ (2μg/ml) or TNFα (100ng/ml) for 4h. Baseline fluorescence measurements were taken for 5 min prior to addition of 10μM nigericin or 5mM ATP at t=5 min. Cytosolic [Ca²⁺] was determined as described in figure 1. Data represent a mean of two independent experiments. **B–C and F–G,** Pam₃CSK₄-primed or TNFα-primed BMDC were treated with 10μM nigericin or 5mM ATP for 30 min in the presence or absence of 1.5mM extracellular [Ca²⁺]. **B and F,** Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium²⁺. Baseline readings were taken for 5 min and nigericin or ATP were added at t=5 min. Data represent mean of two independent experiments. **C and G,** IL-1β release from Pam₃CSK₄-primed BMDC or TNFα-primed BMDC was determined by ELISA. Data represent a mean of two independent experiments for each panel. **D and H,** Pam₃CSK₄-primed or TNFα-primed BMDC were pretreated with 300nM thapsigargin (TG) for 30 min in the absence of extracellular Ca²⁺. BMDC were washed and stimulated with 10μM nigericin or 5mM ATP for 30 min in the absence of extracellular Ca²⁺. IL-1β release from Pam₃CSK₄-primed BMDC or TNFα-primed BMDC was determined by ELISA. Data represent a mean of two independent experiments for each panel.

**Figure 6. NLRP3 inflammasome signaling responses to K⁺ efflux are dissociated from changes in cytosolic [Ca²⁺] in murine macrophages**
A, Murine BMDM were primed with LPS (1μg/ml) for 4h. Baseline readings were taken for 5 min and 10μM nigericin (NG) or 5mM ATP were added at t=5 min. Cytosolic [Ca²⁺] was determined as described in figure 1. Data represent a mean of two independent experiments. **B and C,** LPS-primed BMDM were treated for 30 min with 10μM nigericin or 5mM ATP in the presence/absence of 1.5mM extracellular [Ca²⁺]. B, Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium²⁺. Baseline readings were taken for 5 min and nigericin or ATP were added at t=5 min. Data represent mean of four independent experiments. C, IL-1β release was determined by ELISA. Data represent a mean of four independent experiments. D, LPS-primed BMDM were treated with 300nM thapsigargin for 30 min in the absence of extracellular Ca²⁺, and then stimulated with 10μM nigericin or 5mM ATP for 30 min in the absence of extracellular Ca²⁺. IL-1β release was determined by ELISA. Data represent a mean of four independent experiments.

**Figure 7. Increased cytosolic [Ca²⁺] induced by Ca²⁺ ionophore or Ca²⁺-mobilizing GPCRs is not a sufficient signal for NLRP3 inflammasome activation**
A, Cytosolic [Ca²⁺] was determined as described in figure 1. Baseline readings were taken for 5 min and 5mM ATP or 3μM ionomycin were added at t=5 min. Data represent a mean of two independent experiments. B, LPS-primed BMDC were treated with 10μM NG, 5mM ATP or 3μM ionomycin for 30 min. IL-1β release was measured by ELISA. Data represent the mean of ten independent experiments. C, LPS-primed BMDC were treated with 30μM R568, 0.1mM ATP, 1μM N-formyl-Met-Leu-Phe (FMLP), or 0.1mM UTP for 30 min. Cytosolic calcium levels were determined as described in figure 1. Baseline readings were taken for 5 min and GPCR agonists were added at t=5 min. Data are representative of two independent experiments. D, LPS-primed BMDC were treated with calcium mobilizing agents for 30 min. IL-1β release was measured by ELISA. Data represent mean of five independent experiments. E, LPS-primed BMDC were treated with 10μM nigericin, 5mM ATP or 3μM ionomycin. Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium²⁺. Baseline readings were taken for 5 min and nigericin, ATP, or ionomycin were added at t=5 min. Data represent a mean of two independent experiments. F, LPS-primed BMDC were treated with 10μM nigericin, 5mM ATP or 3μM ionomycin. Cells were lysed with 10% nitric acid and lysates were analyzed by atomic absorption spectroscopy to measure cellular [K⁺]. Data represent mean of eight independent experiments. G, LPS-primed BMDC were stimulated with 10μM nigericin, 5mM ATP, 3μM ionomycin or 30μM R568 for 30 min. Soluble lysate fraction (Lys) was probed for procaspase-1 and pro-IL-1β, insoluble lysate pellet was crosslinked with DSS and probed for oligomerized ASC, and extracellular medium fraction (ECM) was probed for mature caspase-1 and IL-1β.

**Figure 8. Suppression of nigericin-stimulated NLRP3 inflammasome signaling by BAPTA can be dissociated from perturbation of Ca²⁺ signaling**
LPS-primed BMDC were loaded with 25μM BAPTA-AM for 45 min per manufacturer’s protocol. Cells were then treated with 10μM nigericin for 30 min in the absence of extracellular Ca²⁺. A, IL-1β release was measured by ELISA. Data represent a mean of three independent experiments. B, Efflux of cellular K⁺ was measured by atomic absorption spectroscopy. Data represent a mean of three independent experiments. C. Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium²⁺. Baseline readings were taken for 5 min and 10μM nigericin was added at t=5 min. Data represent a mean of four independent experiments. D, LPS-primed BMDC were stimulated with 10μM nigericin for 30 min in the presence/absence of BAPTA loading. Soluble lysate fraction (Lys) was probed for procaspase-1 and pro-IL-1β, insoluble lysate pellet was crosslinked with DSS and probed for oligomerized ASC, and extracellular medium fraction (ECM) was probed for mature caspase-1 and IL-1β.

**Figure 9. Suppression of nigericin-stimulated NLRP3 inflammasome signaling by 2-APB can be dissociated from perturbation of Ca²⁺ signaling**
**A–C,** LPS-primed BMDC were incubated with 100μM 2-APB for 20 min prior to treatment with 10μM nigericin for 30 min in the presence of 1.5mM extracellular [Ca²⁺]. A, IL-1β release was measured by ELISA. Data represent a mean of three independent experiments. B, Efflux of cellular K⁺ was measured by atomic absorption spectroscopy. Data represent a mean of three independent experiments. C, Soluble lysate fraction (Lys) was probed for procaspase-1 and pro-IL-1β, insoluble lysate pellet was crosslinked with DSS and probed for oligomerized ASC, and extracellular medium fraction (ECM) was probed for mature caspase-1 and IL-1β. D, LPS-primed BMDC were preincubated with 100μM 2-APB for 20 min in the presence of 1.5mM extracellular [Ca²⁺] and onset of pyroptosis in response to nigericin was assayed by measuring permeability of cells to propidium²⁺. Baseline readings were taken for 5 min and 10μM nigericin was added at t=5 min. E, Cytosolic [Ca²⁺] in LPS-primed BMDC was measured using fluo-4 fluorescence in the presence of 1.5mM extracellular [Ca²⁺]. Baseline readings were taken for 5 min. Cells were treated with 100μM 2-APB at t=5 min (double arrow) and 10μM nigericin (single arrow) at t=15 min. Data are representative of two independent experiments.

See this image and copyright information in PMC

Cited by

Caspase-8 as an Effector and Regulator of NLRP3 Inflammasome Signaling.
Antonopoulos C, Russo HM, El Sanadi C, Martin BN, Li X, Kaiser WJ, Mocarski ES, Dubyak GR. Antonopoulos C, et al. J Biol Chem. 2015 Aug 14;290(33):20167-84. doi: 10.1074/jbc.M115.652321. Epub 2015 Jun 22. J Biol Chem. 2015. PMID: 26100631 Free PMC article.
Unraveling the Role of the NLRP3 Inflammasome in Lymphoma: Implications in Pathogenesis and Therapeutic Strategies.
Stergiou IE, Tsironis C, Papadakos SP, Tsitsilonis OE, Dimopoulos MA, Theocharis S. Stergiou IE, et al. Int J Mol Sci. 2024 Feb 17;25(4):2369. doi: 10.3390/ijms25042369. Int J Mol Sci. 2024. PMID: 38397043 Free PMC article. Review.
Apolipoprotein C3 induces inflammation and organ damage by alternative inflammasome activation.
Zewinger S, Reiser J, Jankowski V, Alansary D, Hahm E, Triem S, Klug M, Schunk SJ, Schmit D, Kramann R, Körbel C, Ampofo E, Laschke MW, Selejan SR, Paschen A, Herter T, Schuster S, Silbernagel G, Sester M, Sester U, Aßmann G, Bals R, Kostner G, Jahnen-Dechent W, Menger MD, Rohrer L, März W, Böhm M, Jankowski J, Kopf M, Latz E, Niemeyer BA, Fliser D, Laufs U, Speer T. Zewinger S, et al. Nat Immunol. 2020 Jan;21(1):30-41. doi: 10.1038/s41590-019-0548-1. Epub 2019 Dec 9. Nat Immunol. 2020. PMID: 31819254
Anti-Inflammatory Therapeutic Mechanisms of Natural Products: Insight from Rosemary Diterpenes, Carnosic Acid and Carnosol.
Habtemariam S. Habtemariam S. Biomedicines. 2023 Feb 13;11(2):545. doi: 10.3390/biomedicines11020545. Biomedicines. 2023. PMID: 36831081 Free PMC article. Review.
The pore-forming subunit Kir6.1 of the K-ATP channel negatively regulates the NLRP3 inflammasome to control insulin resistance by interacting with NLRP3.
Du RH, Lu M, Wang C, Ding JH, Wu G, Hu G. Du RH, et al. Exp Mol Med. 2019 Aug 6;51(8):1-13. doi: 10.1038/s12276-019-0291-6. Exp Mol Med. 2019. PMID: 31387986 Free PMC article.

See all "Cited by" articles

References

1. Warren J. Williams Hematology. 8. McGraw Hill Companies; 2010. The Inflammatory Response. Access Medicine. Web. 7 August 2014.
1. Schroder K, Tschopp J. The Inflammasomes. Cell. 2010;140(6):821–832. - PubMed
1. Gross O, Thomas CJ, Guarda G, Tschopp J. The inflammasome: an integrated view. Immunological Reviews. 2011;243(1):136–151. - PubMed
1. Lu A, Magupalli V, Ruan J, Yin Q, Atianand M, Vos M, Schröder G, Fitzgerald K, Wu H, Egelman E. Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell. 2014;156(6):1193–1206. - PMC - PubMed
1. Cai X, Chen J, Xu H, Liu S, Jiang Q, Halfmann R, Chen Z. Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell. 2014;156(6):1207–1222. - PMC - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- H1 Connect
- scite Smart Citations
Medical
- MedlinePlus Health Information
Miscellaneous
- NCI CPTAC Assay Portal

[1] Warren J. Williams Hematology. 8. McGraw Hill Companies; 2010. The Inflammatory Response. Access Medicine. Web. 7 August 2014.

[2] Warren J. Williams Hematology. 8. McGraw Hill Companies; 2010. The Inflammatory Response. Access Medicine. Web. 7 August 2014.

[3] Schroder K, Tschopp J. The Inflammasomes. Cell. 2010;140(6):821–832. - PubMed

[4] Schroder K, Tschopp J. The Inflammasomes. Cell. 2010;140(6):821–832. - PubMed

[5] Gross O, Thomas CJ, Guarda G, Tschopp J. The inflammasome: an integrated view. Immunological Reviews. 2011;243(1):136–151. - PubMed

[6] Gross O, Thomas CJ, Guarda G, Tschopp J. The inflammasome: an integrated view. Immunological Reviews. 2011;243(1):136–151. - PubMed

[7] Lu A, Magupalli V, Ruan J, Yin Q, Atianand M, Vos M, Schröder G, Fitzgerald K, Wu H, Egelman E. Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell. 2014;156(6):1193–1206. - PMC - PubMed

[8] Lu A, Magupalli V, Ruan J, Yin Q, Atianand M, Vos M, Schröder G, Fitzgerald K, Wu H, Egelman E. Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell. 2014;156(6):1193–1206. - PMC - PubMed

[9] Cai X, Chen J, Xu H, Liu S, Jiang Q, Halfmann R, Chen Z. Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell. 2014;156(6):1207–1222. - PMC - PubMed

[10] Cai X, Chen J, Xu H, Liu S, Jiang Q, Halfmann R, Chen Z. Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell. 2014;156(6):1207–1222. - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling

Affiliations

K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling

Authors

Affiliations

Abstract

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Miscellaneous

Abstract

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Miscellaneous