The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation - PubMed (original) (raw)

. 2020 Sep 1;32(3):468-478.e7.

doi: 10.1016/j.cmet.2020.07.016. Epub 2020 Aug 12.

Stefano Angiari 1, Svenja Hester 2, Sarah E Corcoran 1, Marah C Runtsch 1, Chris Ling 3, Melanie C Ruzek 4, Peter F Slivka 4, Anne F McGettrick 1, Kathy Banahan 1, Mark M Hughes 1, Alan D Irvine 5, Roman Fischer 2, Luke A J O'Neill 6

Affiliations

The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation

Alexander Hooftman et al. Cell Metab. 2020.

Abstract

The Krebs cycle-derived metabolite itaconate is highly upregulated in inflammatory macrophages and exerts immunomodulatory effects through cysteine modifications on target proteins. The NLRP3 inflammasome, which cleaves IL-1β, IL-18, and gasdermin D, must be tightly regulated to avoid excessive inflammation. Here we provide evidence that itaconate modifies NLRP3 and inhibits inflammasome activation. Itaconate and its derivative, 4-octyl itaconate (4-OI), inhibited NLRP3 inflammasome activation, but not AIM2 or NLRC4. Conversely, NLRP3 activation was increased in itaconate-depleted Irg1-/- macrophages. 4-OI inhibited the interaction between NLRP3 and NEK7, a key step in the activation process, and "dicarboxypropylated" C548 on NLRP3. Furthermore, 4-OI inhibited NLRP3-dependent IL-1β release from PBMCs isolated from cryopyrin-associated periodic syndrome (CAPS) patients, and reduced inflammation in an in vivo model of urate-induced peritonitis. Our results identify itaconate as an endogenous metabolic regulator of the NLRP3 inflammasome and describe a process that may be exploited therapeutically to alleviate inflammation in NLRP3-driven disorders.

Keywords: IL-1β; NEK7; NLRP3; cysteine modification; immunometabolism; inflammasome; itaconate; macrophage; metabolite; pyroptosis.

Copyright © 2020 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

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Graphical abstract

Figure 1

Figure 1

4-OI Blocks NLRP3 Inflammasome Activation (A) Time flow of NLRP3 inflammasome assay with 4-OI in BMDMs. (B and C) LPS (3 h) and ATP (45 min) induced IL-1β release (B, n = 10) and LDH release (C, n = 4 for ATP alone, n = 7 for others, measured as % LDH release of total lysis control) ± 4-OI. (D and E) LPS (3 h) and nigericin (45 min) induced IL-1β release (D, n = 7) and LDH release (E, n = 5 for NS alone, n = 6 for others) ± 4-OI. (F and G) Immunoblot analysis (F) and quantification by densitometry (G, n = 4) of pro- and mature caspase-1 and IL-1β protein in lysates and supernatants of BMDMs treated with LPS (3 h) and nigericin (45 min) ± 4-OI. (H) LPS (3 h) and nigericin (45 min) induced IL-18 release ± 4-OI (n = 5). (I) Percent of all cells positive for ASC specks in LPS- (3 h) and nigericin- (45 min) treated BMDMs ± 4-OI (n = 6). (J) Immunoblot analysis of ASC protein in triton-insoluble pellet and -soluble lysate of LPS- (3 h) and nigericin- (45 min) treated BMDMs ± 4-OI (250 μM). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are mean ± SEM. Blots are representative of a minimum of 3 independent experiments.

Figure 2

Figure 2

4-OI Does Not Block Activation of Other Inflammasomes (A and B) LPS (3 h) and poly(dA:dT) (6 h) induced IL-1β release (A, n = 5) and LDH release (B, n = 5) ± 4-OI. (C and D) Immunoblot analysis (C) and quantification by densitometry (D, n = 3) of pro- and mature caspase-1 and IL-1β protein in lysates and supernatants of BMDMs treated with LPS (3 h) and poly(dA:dT) (6 h) ± 4-OI. (E and F) LPS (3 h) and flagellin (2.5 h) induced IL-1β release (E, n = 8) and LDH release (F, n = 5) ± 4-OI. (G and H) Immunoblot analysis (G) and quantification by densitometry (H, n = 3) of pro- and mature caspase-1 and IL-1β protein in lysates and supernatants of BMDMs treated with LPS (3 h) and flagellin (2.5 h) ± 4-OI. (I and J) LPS priming (3 h) and transfection (18 h) induced IL-1β release (I, n = 5) and LDH release (J, n = 5) ± 4-OI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are mean ± SEM. Blots are representative of a minimum of 3 independent experiments.

Figure 3

Figure 3

Endogenous Itaconate Regulates NLRP3 Activation and 4-OI Dicarboxypropylates C548 on NLRP3 (A and B) LPS (3 h) and nigericin (45 min) induced IL-1β release (A, n = 6) and LDH release (B) in wild-type (WT, n = 7) and _Irg1_−/− (n = 8) BMDMs. (C and D) Immunoblot analysis (C) and quantification by densitometry (D, n = 4) of pro- and mature caspase-1 and IL-1β protein in lysates and supernatants of wild-type (WT) and _Irg1_−/− BMDMs treated with LPS (3 h) and nigericin (45 min). (E) IL-1β release from NLRP3 inflammasome-reconstituted HEK293T cells treated with nigericin (45 min) (n = 3). Immunoblot analysis of pro- and mature IL-1β and IRG1 protein in supernatants and lysates of these cells. (F) Endogenous co-immunoprecipitation of NLRP3 and NEK7 in BMDMs treated with LPS (3 h) and nigericin (45 min) ± 4-OI (250 μM). (G) Tandem mass spectrometry spectrum of C548-containing NLRP3 peptide following 4-OI treatment (250 μM, 24 h). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are mean ± SEM. Blots are representative of a minimum of 3 independent experiments.

Figure 4

Figure 4

4-OI Reduces Inflammation in a Murine In Vivo Model of Peritonitis and Blocks NLRP3 Inflammasome Activation in Healthy Human and CAPS PBMCs (A–C) IL-1β concentration (A), IL-6 concentration (B), and neutrophil number (C) in the peritoneal lavage fluid of mice injected for 6 h with MSU crystals (30 mg/kg) ± 4-OI (50 mg/kg) (n = 3 for PBS groups, n = 8 for MSU groups). (D) LPS or Pam3CSK4 (14 h) and nigericin (2 h) induced IL-1β release (n = 5 for LPS + nigericin, n = 3 for Pam3CSK4 + nigericin) ± 4-OI or 4-O-2-MS (both 250 μM) from healthy human PBMCs. (E and F) Immunoblot analysis (E) and quantification by densitometry (F, n = 3) of pro- and mature IL-1β protein in lysates and supernatants of human PBMCs treated with LPS (14 h) and nigericin (2 h) ± 4-OI (250 μM). (G) LPS (1 h) induced IL-1β release (n = 3) ± 4-OI (250 μM) or MCC950 (500 nM) from PBMCs isolated from CAPS patients. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are mean ± SEM. Blots are representative of a minimum of 3 independent experiments.

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