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. 2018 Oct 2;115(40):E9371-E9380.
doi: 10.1073/pnas.1812744115. Epub 2018 Sep 19.

Chloride Regulates Dynamic NLRP3-dependent ASC Oligomerization and Inflammasome Priming

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Free PMC article

Chloride Regulates Dynamic NLRP3-dependent ASC Oligomerization and Inflammasome Priming

Jack P Green et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The NLRP3 inflammasome is an important regulator of inflammation and immunity. It is a multimolecular platform formed within cells that facilitates the activation of proinflammatory caspases to drive secretion of cytokines such as interleukin-1β (IL-1β). Knowledge of the mechanisms regulating formation of the NLRP3 inflammasome is incomplete. Here we report Cl- channel-dependent formation of dynamic ASC oligomers and inflammasome specks that remain inactive in the absence of K+ efflux. Formed after Cl- efflux exclusively, ASC specks are NLRP3 dependent, reversible, and inactive, although they further prime inflammatory responses, accelerating and enhancing release of IL-1β in response to a K+ efflux-inducing stimulus. NEK7 is a specific K+ sensor and does not associate with NLRP3 under conditions stimulating exclusively Cl- efflux, but does after K+ efflux, activating the complex driving inflammation. Our investigation delivers mechanistic understanding into inflammasome activation and the regulation of inflammatory responses.

Keywords: caspase-1; chloride; inflammasome; inflammation; interleukin-1.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Chloride efflux modulates NLRP3 activation and IL-1β processing. (A) IL-1β ELISA, caspase-1, and IL-1β immunoblot of cell lysates (LYS) and supernatants (S/N) from LPS-primed WT and NLRP3 KO BMDMs incubated in a control (145 mM NaCl/5 mM KCl), Cl free (145 mM NaGluconate/5 mM KGluconate), K+ free (150 mM NaCl), or K+ and Cl free (150 mM NaGluconate) solution for 2 h (n = 6). (B) IL-1β ELISA, caspase-1, and IL-1β immunoblot of LYS and S/N from LPS-primed BMDMs pretreated with a vehicle control, DCPIB (10 µM), flufenamic acid (FFA, 100 µM), NPPB (100 µM), or NBC6 (30 µM) and incubated in a K+ and Cl free solution for 2 h (n = 5). (C) Time course of released IL-1β in the supernatant of LPS-primed BMDMs incubated in a control, Cl free, K+ free, or K+ and Cl free solution, determined by IL-1β ELISA. (D) LDH release from LPS-primed WT or NLRP3 KO BMDMs incubated in a control, Cl free, K+ free, or K+ and Cl free solution for 2 h. (E, i) IL-1β ELISA performed on supernatants from LPS-primed iBMDMs pretreated with a vehicle control, DCPIB (10 µM) or NPPB (100 µM) and then stimulated with nigericin (10 µM, 1 h) (n = 4). (E, ii) Intracellular K+ measurements from LPS-primed iBMDM cell lysates pretreated with a vehicle control, DCPIB (10 µM) or NPPB (100 µM) and stimulated with nigericin (10 µM, 30 min) in the presence of Z-VAD-FMK (50 µM) to prevent pyroptosis (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significantly different determined by a one-way ANOVA with Tukey’s (A and D) or Dunett’s (B vs. vehicle control; E, i vs. nigericin; E, ii vs. vehicle) post hoc analysis. Values shown are the mean ± SEM.
Fig. 2.
Fig. 2.
Chloride efflux is sufficient to induce NLRP3-dependent ASC speck formation. (A, i) LPS-primed immortalized BMDMs stably expressing ASC-mCherry (ASC-mCherry iBMDMs) were incubated in a control (145 mM NaCl/5 mM KCl), Cl free (145 mM NaGluconate/5 mM KGluconate), K+ free (150 mM NaCl), or K+ and Cl free (150 mM NaGluconate) solution and ASC speck formation was measured in real time (n = 5). (A, ii) ASC speck formation was analyzed after a 3-h incubation in the respective solutions (n = 5). (A, iii) Representative images of ASC-mCherry iBMDM cells after a 3-h incubation in the respective solutions. (Scale bar, 50 µm, arrows denote ASC speck.) (B, i) LPS-primed ASC-mCherry iBMDMs were incubated in a control, Cl free, K+ free, K+ and Cl free, high K+ and normal Cl (150 mM KCl), or high K+ and Cl free (150 mM KGluconate) solution and ASC speck formation was measured in real time (n = 4). (B, ii) Speck formation was analyzed after a 3-h incubation in the respective solutions (n = 4). (C, i) LPS-primed WT and NLRP3 KO THP-1 macrophages were incubated in a control, Cl free, K+ free, or K+ and Cl free solution for 3 h and immunostained for ASC (n = 4). (C, ii) Representative images of WT and NLRP3 KO THP-1 macrophages incubated for 3 h in the respective solutions and immunostained for ASC. (Scale bar, 50 µm, arrows denote ASC speck.) All experiments were performed in the presence of Z-VAD-FMK (50 µM) to prevent pyroptosis and loss of ASC specks. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significantly different determined by a one-way ANOVA with Tukey’s (A and B) post hoc analysis, or two-way ANOVA with Sidak’s post hoc analysis (C, #P < 0.01 vs. WT in the respective solution). Values shown are the mean ± SEM.
Fig. 3.
Fig. 3.
NLRP3 dependence and caspase-1 recruitment to the chloride-dependent ASC complex. (A) Immunoblot of DSS-crosslinked ASC oligomers in lysates from LPS-primed WT and NLRP3 KO THP-1 macrophages incubated in a control (145 mM NaCl/5 mM KCl), Cl free (145 mM NaGluconate/5 mM KGluconate), K+ free (150 mM NaCl), K+ and Cl free (150 mM NaGluconate) solution, or RPMI ± nigericin (10 µM) for 2 h (n = 3). (B) LPS-primed WT primary BMDMs were incubated in a control, Cl free, K+ free, K+ and Cl free solution, or DMEM ± nigericin (10 µM) in the presence of biotin-VAD-FMK (bVAD, 10 µM) for 2 h (n = 7). Cell supernatants were removed and lysates were pulled down with streptavidin T1 beads and immunoblotted for caspase-1 and ASC. (C) Analysis of ASC speck formation in naïve or LPS-primed ASC-mCherry iBMDMs pretreated with a vehicle control or the NLRP3 inflammasome inhibitor MCC950 (10 µM) and incubated in a Cl free, K+ and Cl free, or high K+ and Cl free solution for 3 h (n = 4). (D) Analysis of ASC speck formation in naïve or LPS-primed ASC-mCherry iBMDMs pretreated with a vehicle control or the chloride channel inhibitors DCPIB (10 µM), flufenamic acid (FFA, 100 µM), or NPPB (100 µM) and incubated in a Cl free or K+ and Cl free solution for 3 h (n = 5). (E) Intracellular K+ measurements from LPS-primed BMDMs incubated in a control, Cl free, K+ free, or K+ and Cl free solution for 2 h, in the presence of Z-VAD-FMK (50 µM) to prevent pyroptosis (n = 4). (F) Intracellular Cl measurements from LPS-primed NLRP3 KO BMDMs incubated in a control, Cl free, K+ free, or K+ and Cl free solution for 2 h (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significantly different determined by a one-way ANOVA with Dunett’s (C and D, vs. vehicle control) post hoc analysis, or a one-sample t test versus hypothetical value of 100% (E and F). Values shown are the mean ± SEM.
Fig. 4.
Fig. 4.
Chloride efflux-induced ASC specks are readily activated by subsequent potassium efflux. (A) LPS-primed BMDMs were incubated in a K+ and Cl free solution (150 mM NaGluconate) and K+ was isosmotically added back with the indicated concentration of KGluconate. Involvement of the NLPR3 inflammasome was assessed by pretreatment with the NLRP3 inhibitor MCC950 (10 μM). Caspase-1 processing and IL-1β release was determined by ELISA and immunoblotting of cell lysates (LYS) and supernatants (S/N) (n = 4). (B) LPS-primed BMDMs were incubated in a control (145 mM NaCl/5 mM KCl) or Cl free (145 mM NaGluconate/5 mM KGluconate) solution for 1 h before stimulation with nigericin (10 µM) for the indicated time (n = 3). Caspase-1 processing and IL-1β release was determined by ELISA and immunoblotting of cell LYS and S/N (n = 3). (C) LPS-primed THP-1 cells were incubated in a Cl free (145 mM NaGluconate/5 mM KGluconate) solution for the indicated time points for up to 120 min, then reperfused with a Cl containing (145 mM NaCl/5 mM KCl) or Cl free solution, and ASC oligomerization was determined by immunoblotting (n = 3). (D) LPS-primed mCherry-ASC iBMDMs were incubated for 120 min in a Cl free solutions before reperfusion with a Cl containing or Cl free solution and ASC speck-containing cells were quantified by immunofluorescence (n = 3). ****P < 0.0001; ns, not significantly different determined by a two-way ANOVA with Sidak’s post hoc analysis. Values shown are the mean ± SEM.
Fig. 5.
Fig. 5.
Chloride efflux-induced ASC specks do not require NLRP3 oligomerization, while active inflammasomes require NEK7-dependent NLRP3 oligomerization. (A) LPS-primed BMDMs were incubated for 30 or 60 min in a control (145 mM NaCl/5 mM KCl), Cl free (145 mM NaGluconate/5 mM KGluconate), K+ free (150 mM NaCl), K+ and Cl free (150 mM NaGluconate) solution, or in DMEM ± ATP (5 mM). Cell lysates were resolved using a blue-native PAGE or SDS/PAGE and immunoblotted for NLRP3 and caspase-1 (n = 3). (B) LPS-primed BMDMs were incubated for 30 or 60 min in a control, Cl free, K+ free, K+ and Cl free solution, or treated with ATP (5 mM) in the control solution. Cell lysates were immunoprecipitated (IP) with NEK7 or IgG antibodies and immunoblotted for NLRP3 and NEK7 (n = 2). (C) LPS-primed BMDMs were incubated for 30 min in a Cl free or K+ and Cl free solution. Cell lysates were resolved by 2D-PAGE and immunoblotted for NLRP3 and NEK7 (n = 2).
Fig. 6.
Fig. 6.
Effects of chloride on LPS-induced inflammasome activation in human PBMCs. (A) Immunoblot of DSS-crosslinked ASC oligomers and IL-1β from human PBMCs incubated in RPMI, PBS, or Cl free PBS ± LPS (1 µg⋅mL−1) for 20 h (n = 5). (B) Immunoblot of ASC oligomers and caspase-1 from PBMCs incubated in PBS or Cl free PBS ± LPS (1 µg⋅mL−1) and the NLRP3 inhibitor MCC950 (5 µM) for 20 h (n = 3). (C) Immunoblot of ASC oligomers and caspase-1 from PBMCs incubated in PBS or Cl free PBS ± LPS (1 µg⋅mL−1) and the chloride channel inhibitor NPPB (100 µM) for 24 h (n = 2). (D) PBMCs were treated as in A ± nigericin (10 µM) added for the last 1 h of treatment (n = 2).

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