NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals - PubMed (original) (raw)

. 2010 Apr 29;464(7293):1357-61.

doi: 10.1038/nature08938.

Hajime Kono, Katey J Rayner, Cherilyn M Sirois, Gregory Vladimer, Franz G Bauernfeind, George S Abela, Luigi Franchi, Gabriel Nuñez, Max Schnurr, Terje Espevik, Egil Lien, Katherine A Fitzgerald, Kenneth L Rock, Kathryn J Moore, Samuel D Wright, Veit Hornung, Eicke Latz

Affiliations

NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals

Peter Duewell et al. Nature. 2010.

Erratum in

Abstract

The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease.

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Figures

Fig. 1

Fig. 1. Cholesterol crystals appear in early atherosclerotic lesions

a, H&E stain and b, confocal fluorescence and reflection microscopy of adjacent aortic sinus sections of Apo-E-KO mice fed a high cholesterol diet (upper panels) as indicated or a regular diet (lower panel). Areas in white or black boxes were enlarged; crystal reflection signal is color coded green. c, d, e, Quantification of lesion size (c), crystal or macrophage marker MoMa-2 staining amount presented as absolute values (d) or percent of lesions size (e). Representative sections of three mice from each group are shown (a, b). Means and s.e.m. are shown (c, d, e).

Fig. 2

Fig. 2. Cholesterol crystals activate the NLRP3 inflammasome

a, b, Resting or LPS-primed human PBMCs were treated with cholesterol crystals as indicated, MSU crystals (250 µg/ml) or ATP in the presence or absence of the caspase-1 inhibitor zYVAD-fmk (10mM) (b). ELISA and immunoblot (IB) were performed for IL-1β. c, d, IB for caspase-1 in supernatants and cell lysates (c) or ELISA for IL-1β in supernatants (d) from LPS-primed wild-type, NLRP3- or ASC-deficient macrophages stimulated with cholesterol crystals, transfected dsDNA (dAdT), nigericin or ATP. e, IL-18 ELISA of supernatants from LPS-primed wild-type macrophages stimulated with cholesterol crystals or nigericin. Means and s.e.m. of four donors (a, b) or one out of three independent experiments (c, d, e) are shown.

Fig. 3

Fig. 3. Cholesterol crystals activate the NLRP3 inflammasome by inducing lysosomal damage

a, IL-1β ELISA of supernatants from LPS-primed murine macrophages stimulated with cholesterol crystals or nigericin in the presence or absence of cytochalasin D. b, c, Combined confocal fluorescence and reflection microscopy of murine macrophages incubated with DQ ovalbumin alone (b, left) or together with cholesterol crystals (125 µg/ml) (b, right; c) for 2h and plasma membrane was stained with A647-conjugated choleratoxin B (b, c). c, Cells were fixed, permeabilized (0.05% saponin) and internal membranes additionally stained with A555-conjugated choleratoxin B. Nuclei were stained with Hoechst dye. d, Murine macrophages stimulated with cholesterol crystals for 6h, stained with acridine orange and analyzed by FACS. e, f, IL-1β ELISA of supernatants from LPS-primed, murine macrophages stimulated with cholesterol crystals in the presence or absence of bafilomycin A1 (e) or from LPS-primed wild-type, cathepsin B or L deficient macrophages stimulated with cholesterol crystals (f). g, h, Murine macrophages stimulated with 100 µg/ml oxLDL for the indicated times and fluorescent dextran (g, 20h) were imaged by combined confocal fluorescence and reflection microscopy i, Unprimed murine macrophages were incubated with oxidized LDL as indicated for 24 h and IL-1β in supernatants was measured by ELISA. (a, d-f, i) One out of three independent experiments are shown; means and s.d. (a, e, f); Representative images of five (b, c) or two (g, h) independent experiments.

Fig. 4

Fig. 4. The NLRP3 inflammasome mediates crystal-induced peritoneal inflammation and atherosclerosis in vivo

a, C57BL/6 (n=23), B6-129 (n=13) or mice deficient in genes encoding IL-1R (n=11), IL-1α/b (n=11), IL-1α (n=4), IL-1β (n=4), caspase-1 (n=7), ASC (n=15), cathepsin B (n=10), cathepsin L (n=5), NLRP3 (n=10) were peritoneally injected with cholesterol crystals or PBS (C57BL/6 n=14; B6-129 n=4). Peritoneal lavage cells were analyzed for neutrophils after 15h. Data represent means and s.e.m. from pooled groups of mice out of experiments repeated 2–4 times. b–d, Female LDLR-KO mice reconstituted with C57BL/6 (n=7), NLRP3-KO (n=9), ASC-KO (n=8) or IL-1α/b-dKO (n=7) bone marrow were fed a high fat diet for 8 weeks and analyzed for serum IL-18 concentration (b) and average aortic sinus lesion size (c, d). (c) Each dot represents the mean lesion size of serial cross-sections from individual mice. (d) Representative photographs of the aortic sinus stained with Giemsa. Insets show 2-fold magnified portions of the images (black box), arrows point to atherosclerotic lesions.

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