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. 2009 Oct 5;187(1):61-70.
doi: 10.1083/jcb.200903124.

Glyburide inhibits the Cryopyrin/Nalp3 inflammasome

Mohamed Lamkanfi  1 , James L Mueller, Alberto C Vitari, Shahram Misaghi, Anna Fedorova, Kurt Deshayes, Wyne P Lee, Hal M Hoffman, Vishva M Dixit
Affiliations

Glyburide inhibits the Cryopyrin/Nalp3 inflammasome

Mohamed Lamkanfi et al. J Cell Biol. .

Abstract

Inflammasomes activate caspase-1 for processing and secretion of the cytokines interleukin-1beta (IL-1beta) and IL-18. Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by microbial ligands, danger-associated molecular patterns (DAMPs), and crystals. Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of gouty arthritis, Alzheimer's, and silicosis. Therefore, inhibitors of the Nalp3 inflammasome offer considerable therapeutic promise. In this study, we show that the type 2 diabetes drug glyburide prevented activation of the Cryopyrin inflammasome. Glyburide's cyclohexylurea group, which binds to adenosine triphosphatase (ATP)-sensitive K(+) (K(ATP)) channels for insulin secretion, is dispensable for inflammasome inhibition. Macrophages lacking K(ATP) subunits or ATP-binding cassette transporters also activate the Cryopyrin inflammasome normally. Glyburide analogues inhibit ATP- but not hypothermia-induced IL-1beta secretion from human monocytes expressing familial cold-associated autoinflammatory syndrome-associated Cryopyrin mutations, thus suggesting that inhibition occurs upstream of Cryopyrin. Concurrent with the role of Cryopyrin in endotoxemia, glyburide significantly delays lipopolysaccharide-induced lethality in mice. Therefore, glyburide is the first identified compound to prevent Cryopyrin activation and microbial ligand-, DAMP-, and crystal-induced IL-1beta secretion.

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Figures

Figure 1.
Figure 1.
Glyburide inhibits LPS+ATP-induced caspase-1 activation, secretion of IL-1β and IL-18, and macrophage cell death. (A–E) LPS-primed BMDMs were treated with glyburide, glipizide, or DMSO for 15 min before 5 mM ATP was added for 30 min. Cell extracts were immunoblotted for caspase-1 (A), and culture supernatants were analyzed for secreted IL-1β (B), IL-18 (C), IL-6 (D), and TNF (E). Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. (F) BMDMs were incubated with 200 µM glyburide, 200 µM glipizide, 200 µM DMSO, or 50 µM calmidazolium for 2 h before brightfield photographs were taken. (G) LPS-primed BMDMs were treated with 200 µM glyburide, glipizide, or DMSO for 15 min followed by 5 mM ATP for the indicated durations. Membrane damage was measured using Live/Dead assay. Bars, 20 µm. (H) BMDMs were left untreated (CTRL), stimulated with 10 µg/ml LPS for 3 h, treated with 5 mM ATP for 1 h, or treated with LPS and ATP. Membrane damage was measured using Live/Dead assay. (I) LPS-primed BMDMs from wild-type (WT), P2X7−/−, Cryopyrin−/−, and caspase-1−/− mice were treated with 5 mM ATP for the indicated durations. Membrane damage was measured with Live/Dead assay. Cytokine and cell death data represent the mean ± SD of triplicate samples from a single experiment, and all results are representative of at least three independent experiments.
Figure 2.
Figure 2.
Glyburide's cyclohexylurea moiety is dispensable for inflammasome inhibition. (A) Chemical structure of glyburide, glipizide, and glyburide-derived analogues G1–G4. (B–G) LPS-primed BMDMs were treated with glyburide, glipizide, or analogues G1–G4 for 15 min before 5 mM ATP was added for 30 min. Cell extracts were immunoblotted for caspase-1 (B and E), and culture supernatants were analyzed for secreted IL-1β (C and F) and IL-6 (D and G). Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. Cytokine data represent the mean ± SD of triplicate samples from a single experiment, and all results are representative of at least three independent experiments.
Figure 3.
Figure 3.
Glyburide inhibits PAMP-, DAMP-, and crystal-induced activation of the Cryopyrin inflammasome. (A) LPS-primed BMDMs were incubated with 200 µM glyburide, glipizide, or DMSO for 15 min before 20 µM nigericin was added for 30 min. Cell extracts were immunoblotted for caspase-1. (B) BMDMs were stimulated with the indicated PAMPs for 3 h, incubated with 200 µM glyburide for 15 min, and stimulated with 20 µM nigericin for 30 min. Cell extracts were immunoblotted for caspase-1. (C and D) LPS-primed BMDMs were incubated with 200 µM glyburide or DMSO for 15 min before 500 µg/ml silica or1 mM of the lysosomotrophic peptide H-LL-OMe was added for 3 h. Cell extracts were immunoblotted for caspase-1 (C), and culture supernatants were analyzed for secreted IL-1β (D). (E) LPS-primed BMDMs were left untreated or incubated with 200 µM glyburide or 10 µM KN-62 for 15 min. 2 µM YoPro-1 was subsequently added, and its uptake was visualized before and after a 5-min ATP pulse. Bars, 50 µm. (F) BMDMs were left untreated (CTRL), transfected with DOTAP alone for 4 h, stimulated with 10 µg/ml LPS for 3 h and subsequently with 5 mM ATP (LPS+ATP), or transfected with DOTAP and 30 µg/ml LPS for 4 h (DOTAP+LPS) in the presence or absence of 200 µM glyburide, glipizide, or DMSO. Cell extracts were immunoblotted for caspase-1. (G) Overview and mapping of inflammasome-activating stimuli and upstream signaling pathways. Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. Cytokine data represent the mean ± SD of triplicate samples from a single experiment, and all results are representative of at least three independent experiments.
Figure 4.
Figure 4.
KATP channels and ABC transporters are dispensable for activation of the Cryopyrin inflammasome. (A–E) BMDMs from wild-type (WT) and SUR2−/− (A and B), Kir6.1−/− and Kir6.2−/− (C and D), or abca1−/−, abcg1−/−, and abca1−/−/abcg1−/− (abca1/g1−/−) mice (E) were left untreated (CTRL), stimulated with 10 µg/ml LPS for 3 h, treated with 5 mM ATP or 20 µM nigericin for 30 min, or stimulated with LPS and treated with ATP (LPS+ATP) or nigericin (LPS+nigericin). Cell extracts were immunoblotted for caspase-1 (A, C, and E), and culture supernatants were analyzed for secreted IL-1β (B and D). (F–H), LPS-primed BMDMs from abca1−/−/abcg1−/− (F), Kir6.1−/− (G), and Kir6.2−/− (H) mice were treated with 200 µM glyburide or DMSO for 15 min before 5 mM ATP or 20 µM nigericin was added for an additional 30 min. Cell extracts were immunoblotted for caspase-1. Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. Cytokine data represent the mean ± SD of triplicate samples from a single experiment, and all results are representative of three independent experiments.
Figure 5.
Figure 5.
Glyburide inhibits the inflammasome upstream of Cryopyrin. (A and B) BMDMs from C57BL/6 (A) or BALB/c mice (B) were stimulated with 10 µg/ml LPS for 3 h and treated with 5 mM ATP for 30 min (LPS+ATP), infected with S. typhimurium for 4 h, or treated with 10 µg/ml anthrax lethal toxin (PA+LF) for 4 h in the presence or absence of 200 µM glyburide. Cell extracts were immunoblotted for caspase-1. Black arrowheads indicate procaspase-1, and white arrowheads mark the p20 subunit. (C) In vitro enzymatic activity of 1 IU mouse caspase-1 incubated with glyburide, glipizide, or the caspase-1 inhibitor Ac-YVAD-cmk. Data represent the mean ± SD of triplicate samples from one out of three independent experiments. (D and E) ATPase activity of purified recombinant Cryopyrin and control eluates in the presence of 100 µM glyburide, compound G1, or glipizide (D). (E) ATPase activity quantified by phosphoimaging. Data represent the mean ± SD of triplicate samples from one out of three independent experiments. (F) Adherent monocytes from FCAS patients (n = 3) were stimulated with 100 ng/ml LPS (4 h) and treated with 5 mM ATP (30 min) in the presence of vehicle controls or 100 µM compound G1 or glipizide before IL-1β levels in culture supernatants were determined. Data represent the mean ± SD of triplicate samples. (G) Adherent monocytes from FCAS patients (n = 3) were cultured at 32°C for 12 h in the presence of vehicle controls or 100 µM compound G1 or glipizide. IL-1β levels were determined in culture supernatants. Data represent the mean ± SD of triplicate samples. (H) Protection against LPS-induced lethality in C57BL/6 mice (n = 10) injected intraperitoneally bid daily with 500 mg/kg glyburide or formulation vehicle. Results are representative of two independent experiments.
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