NLRP3 inflammasomes are required for
atherogenesis and activated by cholesterol crystals
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Nature | Letter
Nature Volume: 464, Pages: 1357-1361 Date published: (29 April 2010)
Peter Duewell, Hajime Kono, Katey J. Rayner, Cherilyn M. Sirois, Gregory Vladimer, Franz G. Bauernfeind, George S. Abela, Luigi Franchi, Gabriel Nunez, Max Schnurr, Terje Espevik, Egil Lien, Katherine A. Fitzgerald, Kenneth L. Rock, Kathryn J. Moore, Samuel D. Wright, Veit Hornung & Eicke Latz
"It is likely that cholesterol crystals form as a result of the activity of acid cholesterol ester hydolases, which transform cholesteryl esters supplied by oxidized LDL into free cholesterol......As indicated above, minimally modified LDL can prime cells for the NLRP3 inflammasome activation.....The chronic inflammation in gout, silicosis and asbestosis is thought to derive from the inability of cells to destroy the ingested aggregates, leading to successive rounds of apoptosis and reingestion of the crystalline material23. In the same way, immune cells cannot degrade cholesterol; instead they depend on exporting the cholesterol to high-density lipoprotein (HDL) particles, which carry the cholesterol to the liver for disposal. The success of this or any cellular mechanism in clearing crystals may thus depend on the availability of HDL. A low concentration of HDL in the blood is one of the most prominent risk factors for atherosclerotic disease24, and pharmacological methods of increasing HDL concentration are being actively pursued as treatments."
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 inflammation1. 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) inflammasome2, 3, 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-1α/ß-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.
Cholesterol, an indispensable lipid in vertebrates, is effectively insoluble in aqueous environments, and elaborate molecular mechanisms have evolved that regulate cholesterol synthesis and its transport in fluids4. Cholesterol crystals are recognized as a hallmark of atherosclerotic lesions5 and their appearance assists the histopathological classification of advanced atherosclerotic lesions6. However, crystalline cholesterol is soluble in the organic solvents used in histology, so that the presence of large crystals is identifiable but only indirectly as so-called cholesterol crystal clefts, which delineate the space that was occupied before sample preparation7. The large cholesterol crystal clefts in atherosclerotic plaques were typically observed only in advanced lesions; crystal deposition was therefore thought to arise late in this disease. However, given that atherosclerosis is intimately linked to cholesterol levels, we were interested to determine when and where cholesterol crystals first appear during atherogenesis.
We fed atherosclerosis-prone Apo-E-deficient mice on a high cholesterol diet to induce atherosclerosis8, 9 and used a combination of
laser reflection and fluorescence confocal microscopy3 to identify crystalline materials and immune cells. Many small crystals appeared as early as two weeks after the start of the atherogenic diet within small accumulations of subendothelial immune cells in very early atherosclerotic sinus lesions (Fig. 1a, b and Supplementary Figs 1 and 2). The reflective material was identified by filipin staining as being mostly cholesterol crystals (not shown). Crystal deposition and immune-cell recruitment increased steadily with diet feeding, and the appearance of crystals was correlated with that of macrophages (r2 = 0.99, P<0.001) (Fig. 1a-e). Cholesterol crystals were detected not only in necrotic cores but also in subendothelial areas and found to localize both inside and outside cells (Fig. 1b). In corresponding haematoxylin/eosin-stained sections cholesterol crystal clefts were visible only after 8weeks of diet, and smaller crystals remained invisible (Fig. 1a). As expected, we failed to detect macrophages or accumulation of crystals in the aortic sinus sections in mice
fed on a regular chow diet (Fig. 1a, b, bottom panels). In addition, in human atherosclerotic lesions small crystals were abundant in areas rich in immune cells (Supplementary Figs 3 and 4). These studies establish that crystals emerge at the earliest time points of diet-induced atherogenesis together with the appearance of immune cells in the subendothelial space.
Various crystals that are linked to tissue inflammation, as well as pore-forming toxins or extracellular ATP, can activate IL-1 family cytokines through the triggering of NLRP3 (ref. 10). Of note, cellular priming through nuclear factor (NF)-κB activation leads to the induction of pro-forms of the IL-1 family cytokines and NLRP3 itself, a step that is required for NLRP3 activation, at least in vitro11. To test whether cholesterol crystals could activate the release of IL-1ß, we incubated lipopolysaccharide (LPS)-primed human peripheral blood mononuclear cells (PBMCs) with cholesterol crystals. Cholesterol crystals induced a robust, dose-responsive release of cleaved IL-1ß in a caspase-1-dependent manner (Fig. 2a, b). Cholesterol crystals added to unprimed cells did not release IL-1ß into the supernatant, indicating the absence of any contaminants that would be sufficient for the priming of cells (Fig. 2a)11. IL-1 cytokines are processed by caspase-1, which can be activated by various inflammasomes9. Indeed, as previously observed with other crystals, cholesterol crystals induced caspase-1 cleavage and IL-1ß release in wild-type but not in NLRP3-deficient or ASC-deficient macrophages (Fig. 2c, d). Transfected double-stranded DNA (poly(dA-dT)·poly(dT-dA)), a control activator that induces the AIM-2 inflammasome12, activated caspase-1 and induced IL-1ß release in an ASC-dependent but NLRP3-independent manner, as expected (Fig. 2c, d). In addition, mouse macrophages also produced cleaved IL-18, another IL-1 family member that is processed by inflammasomes (Supplementary Fig. 5a). We also found that chemically pure synthetic cholesterol crystals activated NLRP3, providing further evidence that cholesterol crystals themselves rather than contaminating molecules were the biologically active material (Supplementary Fig. 5b). Priming of cells for NLRP3 activation could be achieved by other pro-inflammatory substances such as cell wall components of Gram-positive bacteria (Supplementary Fig. 5c). Moreover, minimally modified low-density lipoprotein (LDL) also primed cells for NLRP3 activation (Supplementary Fig. 5d)13. Taken together, these data establish that crystalline cholesterol leads to NLRP3 inflammasome activation in human and mouse immune cells.
For further elucidation of the mechanisms involved in cholesterol crystal recognition, we inhibited phagocytosis pharmacologically with cytochalasin D or lantriculin A. We found that these agents inhibited NLRP3 activation by crystals but not by the AIM2 activator poly(dA-dT)·poly(dT-dA) (Fig. 3a and Supplementary Fig. 6a, c, d). To follow the fate of the internalized particles, we analysed macrophages incubated with cholesterol crystals by combined confocal reflection and fluorescence microscopy. Cholesterol crystals induced profound swelling in a fraction of cells (Fig. 3b), as observed for other aggregated materials3, 14. Phagolysosomal membranes contain lipid raft components15, which allowed us to stain the surface of cells with the raft marker choleratoxin B labelled with one fluorescent colour and also to label internal phagolysosomal membranes after cell permeabilization with differently fluorescing choleratoxin B. Indeed, in macrophages that had previously ingested cholesterol crystals, this staining revealed that some cholesterol crystals lacked phagolysosomal membranes and resided in the cytosol of a fraction of cells, thus indirectly indicating crystal-induced phagolysosomal membrane rupture (Fig. 3c). This finding was further supported by crystal-induced translocation of soluble lysosomal markers into the cytosol (see below). In addition, cholesterol crystals dose-responsively led to a loss of lysosomal acridine orange fluorescence, further confirming lysosomal disruption (Supplementary Fig. 6e). These studies suggest that cholesterol-crystal-induced lysosomal damage in macrophages leads to the translocation of phagolysosomal content into the cytosol. In further experiments we found that the inhibition of lysosomal acidification or cathepsin activity blocked the ability of cholesterol crystals to induce IL-1ß secretion (Supplementary Fig. 6f). Similarly, analysis of cells from mice deficient in single cathepsins (B or L) also showed that cholesterol crystals led to a diminished release of IL-1ß in comparison with wild-type cells. However, the dependence of cholesterol-crystal-induced IL-1ß release on single cathepsins was less pronounced at higher doses, suggesting functional redundancy of cathepsin B and L or potentially additional proteases (Fig. 3d). Taken together, these experiments suggest that cholesterol crystals induce translocation of the lysosomal proteolytic contents, which can be sensed by the NLRP3 inflammasome by as yet undefined mechanisms.
It has previously been demonstrated that oxidized LDL, a major lipid species deposited in vessels, has the potential to damage lysosomal membranes16. We found that macrophages incubated with oxidized LDL internalized this material and nucleated crystals in large, swollen, phagolysosomal compartments (Fig. 3e); in some cells these compartments ruptured with translocation of the fluorescent marker dye into the cytosol (Fig. 3e, arrows). A time-course analysis revealed that small crystals appeared as early as 1h after incubation with oxidized LDL (not shown), and larger crystals were visible after longer incubation times (Fig. 3f). It is likely that cholesterol crystals form as a result of the activity of acid cholesterol ester hydolases, which transform cholesteryl esters supplied by oxidized LDL into free cholesterol. As indicated above, minimally modified LDL can prime cells for the NLRP3 inflammasome activation (Supplementary Fig. 5d). Recent evidence suggests that this priming proceeds through the activation of a TLR4/6 homodimer and CD36 (ref. 13). This, together with the propensity of minimally modified LDL to form crystals and to rupture lysosomal membranes, suggests that these LDL species could be sufficient to provide both signals 1 and 2 needed to activate IL-1ß release from cells. Indeed, after 24h of incubation we observed a spontaneous release of IL-1ß in the absence of further stimulation of NLRP3 inflammasomes (Fig. 3g).
In mouse atherosclerotic lesions we identified not only macrophages and dendritic cells but also neutrophils accumulated within the intima space (see Supplementary Fig. 2). IL-1ß has a key function in the recruitment of neutrophils, and the IL-1-dependent intraperitoneal accumulation of neutrophils has frequently been used as an in vivo assay for inflammasome activation and IL-1 production2, 17, 18. Using this acute inflammation model we found that cholesterol crystals induced a robust induction of neutrophil influx into the peritoneum (Fig. 4a). Neutrophil influx into the peritoneum after deposition of cholesterol crystals was markedly decreased in mice lacking IL-1 or the IL-1 receptor (IL-1R), indicating that IL-1 production is indeed induced and essential for cholesterol-crystal-induced inflammation in vivo. Moreover, mice lacking NLRP3 inflammasome components or cathepsins B or L also recruited significantly fewer neutrophils into the peritoneum than wild-type mice after injection of cholesterol crystals. However, the decrease in neutrophilic influx observed after deposition of cholesterol crystals was more pronounced in mice lacking IL-1-related genes than in mice lacking NLRP3-inflammasome-related genes (Fig. 4a), presumably because of the contribution of IL-1α signalling and/or caspase-1-independent processing of IL-1ß (ref. 19) in vivo. In any case, these data confirm that cholesterol crystals trigger NLRP3 inflammasome-dependent IL-1 production in vivo.
To test whether the NLRP3 inflammasome is involved in the chronic inflammation that underlies atherogenesis in vessel walls, we tested whether the absence of NLRP3, ASC or IL-1 cytokines might modulate atherosclerosis development in LDLR-deficient mice20, a model for familial hypercholesterolaemia. We reconstituted lethally irradiated LDLR-deficient mice with bone marrow from wild-type or NLRP3-deficient, ASC-deficient or IL-1α/ß-deficient mice and subjected these mice to eight weeks of a high-cholesterol diet. In these bone marrow chimaeras, the LDLR-deficiency radioresistant parenchyma causes the animals to become hypercholesterolaemic when placed on a high-fat diet, whereas their bone marrow-derived macrophages and other leukocytes lack the NLRP3-inflammasome or IL-1 pathway components needed to respond to cholesterol crystals. No significant differences in blood cholesterol levels were observed between the different groups (wild type, 893±144mgdl-1; ASC-/-, 781±114mgdl-1; Nlrp3-/-,753±132mgdl-1; Il1a-/-/b-/-, 832±98mgdl-1). However, mice reconstituted with NLRP3-deficient, ASC-deficient or IL-1α/b-deficient bone marrow showed significantly lower plasma levels of IL-18, an IL-1 family cytokine whose secretion is dependent on inflammasomes and a biomarker known to be elevated in atherosclerosis21 (Fig. 4b). Additionally, mice whose bone marrow-derived cells lacked NLRP3 inflammasome components or IL-1 cytokines were markedly resistant to the development of atherosclerosis (Fig. 4c, d). The lesional area in the aortae of these mice was decreased on average by 69% in comparison with chimaeric LDLR-deficient mice that had wild-type bone marrow. These data demonstrate that activation of the NLRP3 inflammasome by bone marrow-derived cells is a major contributor to diet-induced atherosclerosis in mice. However, the contribution of NLRP3 inflammasome activation in parenchymal cells to the development of atherosclerosis cannot be assessed with this disease model and remains to be examined in mice that are doubly deficient in both LDLR and inflammasome components.
The molecules that incite inflammation in atherosclerotic lesions have presented a long-standing puzzle. Although the lesions are absolutely dependent on cholesterol, this abundant, naturally occurring molecule has been viewed as inert. Here we show that the crystalline form of cholesterol can induce inflammation. The magnitude of the inflammatory response and the mechanism of NLRP3 activation seem identical to that of crystalline uric acid, silica and asbestos2, 3, 22. All these crystals are known to provoke clinically important inflammation as seen in gout, silicosis and asbestosis, respectively.
The chronic inflammation in gout, silicosis and asbestosis is thought to derive from the inability of cells to destroy the ingested aggregates, leading to successive rounds of apoptosis and reingestion of the crystalline material23. In the same way, immune cells cannot degrade cholesterol; instead they depend on exporting the cholesterol to high-density lipoprotein (HDL) particles, which carry the cholesterol to the liver for disposal. The success of this or any cellular mechanism in clearing crystals may thus depend on the availability of HDL. A low concentration of HDL in the blood is one of the most prominent risk factors for atherosclerotic disease24, and pharmacological methods of increasing HDL concentration are being actively pursued as treatments.
Even though cholesterol cannot be degraded by peripheral cells, it may be transformed to cholesteryl ester by the cellular enzyme acylcoenzyme A:cholesterol acyltransferase (ACAT). Cholesteryl esters form droplets rather than crystals and are considered to be a storage form of cholesterol4. On the assumption that decreased cholesterol storage would be beneficial for decreasing atherosclerosis, ACAT inhibitors were tested in large clinical trials. Studies with two such inhibitors did not show a decrease but rather an increase in the size of the coronary atheroma25, 26. This apparent paradox may be reconciled by our findings that the crystalline form of cholesterol, which would be expected to be increased in concentration after inhibition of ACAT, may be crucial in driving arterial inflammation. Indeed, mouse studies of ACAT deficiency show enhanced atherogenesis with abundant cholesterol crystals27. On the basis of our findings, therapeutic strategies that would reduce cholesterol crystals or block the inflammasome pathway would be predicted to have clinical benefit by decreasing the initiation or progression of atherosclerosis. In this context our findings also indicate novel molecular targets for the development of therapeutics to treat this disease.
Mice were provided as follows: Nlrp3-/- and ASC-/- by Millennium Pharmaceuticals; Casp1-/- by R. Flavell; Ctsb-/- by T. Reinheckel; Ctsl-/- by H. Ploegh; and Il1a-/-, Il1b-/- and Il1a-/-b-/- by Y. Iwakura. B6-129 (mixed background), C57BL/6, Il1r1-/-, Apoe-/- and Ldlr-/- mice were purchased from Jackson Laboratories. Animal experiments were approved by the University of Massachusetts and Massachusetts General Hospital Animal Care and Use Committees.
Bafilomycin A1, cytochalasin D and zYVAD-fmk were from Calbiochem. ATP, acridine orange and poly(dA-dT)·poly(dT-dA) sodium salt were from Sigma-Aldrich, and ultra-pure LPS was purchased from InvivoGen. Nigericin, Hoechst dye, DQ ovalbumin and fluorescent choleratoxin B were purchased from Invitrogen. MSU
crystals were prepared as described17.
Cholesterol crystal preparation
Tissue-culture grade or synthetic cholesterol was purchased from Sigma, solubilized in hot acetone and crystallized by cooling. After six cycles of recrystallization, the final crystallization was performed in the presence of 10% endotoxin-free water to obtain hydrated cholesterol crystals. Cholesterol crystals were analysed for purity by electron impact gas chromatography-mass spectrometry and thin-layer chromatography with the use of silica gel and hexane-ethyl acetate (80:20) solvent. Crystal size was varied with a microtube tissue grinder. Fluorescent cholesterol was prepared by the addition of DiD or DiI dye (Invitrogen) in PBS.
ELISA and western blotting
ELISA measurements of IL-1ß (Becton Dickinson) and IL-18 (MBL International) were made in accordance with the respective manufacturer's directions. Experiments for caspase-1 western blot analysis were performed in serum-free DMEM medium. After stimulations, cells were lysed by the addition of a 10xlysis buffer (10% Nonidet P40 in Tris-buffered saline (10mM Tris-HCl, pH7.5, 150mM NaCl) and protease inhibitors), and post-nuclear lysates were separated by 4-20% reducing SDS-PAGE. Anti-mouse caspase-1 polyclonal antibody was provided by P. Vandenabeele. Anti-human cleaved IL-1ß (Cell Signaling) from human PBMCs was analysed in serum-free supernatants as above without cell lysis.
Apoe-/- mice maintained in a pathogen-free facility were fed with a Western-type diet (Teklad Adjusted Calories 88137; 21% (w/w) fat, 0.15% (w/w) cholesterol, 19.5% (w/w) casein; no sodium cholate) starting at 8 weeks of age; this continued for 2, 4, 8 or 12 weeks (three mice in each group). Mice were killed and hearts were collected as described28. Hearts were sectioned serially at the origins of the aortic valve leaflets, and every third section (5µm thick) was stained with haematoxylin/eosin and imaged by light microscopy. Adjacent sections were fixed in 4% paraformaldehyde, blocked and permeabilized (10% goat serum and 0.5% saponin in PBS) and stained for 1h at 37°C with fluorescent primary antibodies against macrophages (MoMa-2; Serotec), dendritic cells (CD11c; Becton Dickinson) or neutrophils (anti-Neutrophil; Serotec) for imaging by confocal microscopy.
Human atherosclerotic lesions were obtained directly after autopsy, serially sectioned at 2-3-mm intervals, and frozen sections (5mm thick) were prepared as above. Parallel sections were stained with Masson's trichrome stain. Tissues were prepared for microscopy as above. Macrophages were stained with anti-CD68 (Serotec); smooth muscle cells were revealed with fluorescent phalloidin (Invitrogen). Human and mouse samples were counterstained with Hoechst dye to reveal nuclei. The atherosclerotic lesions were imaged on a Leica SP2 AOBS confocal microscope where immunofluoroscence staining was revealed by standard confocal techniques, and crystals were observed with laser reflection using enhanced transmittance of the acousto-optical beam splitter as described3. Laser reflection and fluorescence emission occurs at the same confocal plane in this setup. The mean lesion area, amount of crystal deposition and monocyte marker presence were quantified from three digitally captured sections per mouse (Adobe Photoshop CS4 Extended). For quantification of the crystal mass and macrophages present, the sum of positive pixels (laser reflection and fluorescence, respectively) was determined and the area was calculated from the pixel size.
Confocal microscopy of mouse macrophages was performed as described3. DQ ovalbumin fluoresces only on proteolytic processing, marking phagolysosomal compartments in macrophages.
Acridine orange lysosomal damage assay
This assay was performed by flow cytometry as described3.
Bone marrow transplantation and atherosclerosis model
Eight-week-old female Ldlr-/- mice were lethally irradiated (11Gy). Bone marrow was prepared from femurs and tibiae of C57BL/6, Nlrp3-/-, ASC-/- and Il1a-/-Il1b-/- donor mice, and T cells were depleted with complement (Pel-Freez Biologicals) and anti-Thy1 monoclonal antibody (M5/49.4.1; American Type Culture Collection). Irradiated recipient mice were reconstituted with 3.5x106 bone marrow cells administered into the tail vein. After four weeks, mice were fed with a Western-type diet (Teklad Adjusted Calories 88137; 21% (w/w) fat, 0.15% (w/w) cholesterol, 19.5% (w/w) casein; no sodium cholate) for eight weeks. Mice were killed and perfused intracardially with formalin. Hearts were embedded in OTC (Optimal Cutting Temperature) (Richard-Allan Scientific) medium, frozen, and serially sectioned through the aorta from the origins of the aortic valve leaflets; every single section (10µm thick) throughout the aortic sinus (800µm) was collected. Quantification of average lesion area was performed from 12 sections stained with haematoxylin/eosin or Giemsa from each mouse by two independent investigators, with virtually identical results. Serum cholesterol levels were determined by enzymatic assay (Wako Diagnostics), and serum IL-18 was measured by SearchLight protein array technology (Aushon Biosystems).
The significance of differences between groups was evaluated by one-way analysis of variance (ANOVA) with Dunnett's post-comparison test for multiple groups to control group, or by Student's t-test for two groups. R2 was calculated from the Pearson correlation coefficient. Analyses were performed with Prism (GraphPad Software, Inc.).