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Cited by 1,039Inflammasome and immune disordersAtherosclerosis and Cardiovascular DiseasesCholesterol and Lipid Metabolism

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Cholesterol Crystals Activate the NLRP3 Inflammasome in Human Macrophages: A Novel Link between Cholesterol Metabolism and Inflammation

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Rajamäki, Kristiina, Lappalainen, Jani, Öörni, Katariina, Välimäki, Elina, Matikainen, Sampsa, Kovanen, Petri T., Eklund, Kari K.·2010·PLoS ONE
DOI 10.1371/journal.pone.0011765PMC PMC2909263
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Abstract

BACKGROUND: Chronic inflammation of the arterial wall is a key element in the pathogenesis of atherosclerosis, yet the factors that trigger and sustain the inflammation remain elusive. Inflammasomes are cytoplasmic caspase-1-activating protein complexes that promote maturation and secretion of the proinflammatory cytokines interleukin(IL)-1beta and IL-18. The most intensively studied inflammasome, NLRP3 inflammasome, is activated by diverse substances, including crystalline and particulate materials. As cholesterol crystals are abundant in atherosclerotic lesions, and IL-1beta has been linked to atherogenesis, we explored the possibility that cholesterol crystals promote inflammation by activating the inflammasome pathway. PRINCIPAL FINDINGS: Here we show that human macrophages avidly phagocytose cholesterol crystals and store the ingested cholesterol as cholesteryl esters. Importantly, cholesterol crystals induced dose-dependent secretion of mature IL-1beta from human monocytes and macrophages. The cholesterol crystal-induced secretion of IL-1beta was caspase-1-dependent, suggesting the involvement of an inflammasome-mediated pathway. Silencing of the NLRP3 receptor, the crucial component in NLRP3 inflammasome, completely abolished crystal-induced IL-1beta secretion, thus identifying NLRP3 inflammasome as the cholesterol crystal-responsive element in macrophages. The crystals were shown to induce leakage of the lysosomal protease cathepsin B into the cytoplasm and inhibition of this enzyme reduced cholesterol crystal-induced IL-1beta secretion, suggesting that NLRP3 inflammasome activation occurred via lysosomal destabilization. CONCLUSIONS: The cholesterol crystal-induced inflammasome activation in macrophages may represent an important link between cholesterol metabolism and inflammation in atherosclerotic lesions.

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Figure 1 Macrophages accumulate cholesteryl esters when incubated with cholesterol crystals (CHCs). (A) Primary macrophages and THP-1 macrophages were incubated with 0.1–2 mg/ml CHCs and cholesteryl esters (CE) were measured from cellular lipid extracts by TLC. (B) Cytochalasin D (cytD; 2 µM) was employed to block cytoskeletal movements during a 16 h incubation of primary and THP-1 macrophages with CHCs (0.5 and 1.0 mg/ml, respectively). (C) THP-1 macrophages treated with CHCs ± cytD were stained with fluorophore-conjugated cholera toxin subunit B (cell membrane; red) and Hoechst (nuclei; blue). The cells were imaged using confocal fluorescence microscopy, combined with detection of CHCs by confocal reflection of the 488 nm laser line (green) (panels i, iii). CHCs are indicated by arrows in the bright field panels (ii, iiii). Scale bar 5 µm. Each experiment was performed ≥4 times. The data are means ± s.e.m. ** = p<0.01, compared to the control cells.
Figure 1 Macrophages accumulate cholesteryl esters when incubated with cholesterol crystals (CHCs). (A) Primary macrophages and THP-1 macrophages were incubated with 0.1–2 mg/ml CHCs and cholesteryl esters (CE) were measured from cellular lipid extracts by TLC. (B) Cytochalasin D (cytD; 2 µM) was employed to block cytoskeletal movements during a 16 h incubation of primary and THP-1 macrophages with CHCs (0.5 and 1.0 mg/ml, respectively). (C) THP-1 macrophages treated with CHCs ± cytD were stained with fluorophore-conjugated cholera toxin subunit B (cell membrane; red) and Hoechst (nuclei; blue). The cells were imaged using confocal fluorescence microscopy, combined with detection of CHCs by confocal reflection of the 488 nm laser line (green) (panels i, iii). CHCs are indicated by arrows in the bright field panels (ii, iiii). Scale bar 5 µm. Each experiment was performed ≥4 times. The data are means ± s.e.m. ** = p<0.01, compared to the control cells.CC BY · PubMed Central
Figure 2 Monocytes and macrophages respond to cholesterol crystals (CHCs) by IL-1β secretion. Primary monocytes (A), primary macrophages (B,D,E), and THP-1 macrophages (C) were incubated with CHCs for 16 h, 24 h, and 8 h, respectively. LPS (1 µg/ml) was used as a co-stimulant for the primary cells. Concentrations of cytokines were subsequently determined from cell culture supernatants. The inset in 2C verifies the presence of mature 17 kDa IL-1β in cell culture supernatants of THP-1 macrophages by Western blotting. The data are means ± s.e.m. from ≥4 experiments. * = p<0.05 and ** = p<0.01, compared to untreated cells.
Figure 2 Monocytes and macrophages respond to cholesterol crystals (CHCs) by IL-1β secretion. Primary monocytes (A), primary macrophages (B,D,E), and THP-1 macrophages (C) were incubated with CHCs for 16 h, 24 h, and 8 h, respectively. LPS (1 µg/ml) was used as a co-stimulant for the primary cells. Concentrations of cytokines were subsequently determined from cell culture supernatants. The inset in 2C verifies the presence of mature 17 kDa IL-1β in cell culture supernatants of THP-1 macrophages by Western blotting. The data are means ± s.e.m. from ≥4 experiments. * = p<0.05 and ** = p<0.01, compared to untreated cells.CC BY · PubMed Central
Figure 3 Analysis of inflammasome-related gene expression after exposure of cells to cholesterol crystals (CHCs). Primary macrophages from 3 donors were incubated for 1–9 h with (A) 0.5 mg/ml CHCs and 1 µg/ml LPS, (B) with LPS alone, or (C) with CHCs alone. Untreated control cells were included at each time point. After the incubation, mRNA levels were determined by real-time quantitative RT-PCR. The data are expressed as mean fold changes ± s.e.m. relative to the untreated cells at each time point.
Figure 3 Analysis of inflammasome-related gene expression after exposure of cells to cholesterol crystals (CHCs). Primary macrophages from 3 donors were incubated for 1–9 h with (A) 0.5 mg/ml CHCs and 1 µg/ml LPS, (B) with LPS alone, or (C) with CHCs alone. Untreated control cells were included at each time point. After the incubation, mRNA levels were determined by real-time quantitative RT-PCR. The data are expressed as mean fold changes ± s.e.m. relative to the untreated cells at each time point.CC BY · PubMed Central
Figure 4 Mechanism of cholesterol crystal (CHC)-induced IL-1β secretion. THP-1 macrophages were incubated with CHCs in the absence or presence of caspase-1 inhibitor zYVAD-fmk (25 µM), cytochalasin D (2 µM), KCl (130 mM), or cathepsin B inhibitor CA-074Me (10 µM). After the incubation, cell culture supernatants were analyzed for IL-1β (average response to CHCs 662 pg/ml). The data are means ± s.e.m. from ≥5 experiments. ** = p<0.01 and *** = p<0.001, compared with CHC-treated cells.
Figure 4 Mechanism of cholesterol crystal (CHC)-induced IL-1β secretion. THP-1 macrophages were incubated with CHCs in the absence or presence of caspase-1 inhibitor zYVAD-fmk (25 µM), cytochalasin D (2 µM), KCl (130 mM), or cathepsin B inhibitor CA-074Me (10 µM). After the incubation, cell culture supernatants were analyzed for IL-1β (average response to CHCs 662 pg/ml). The data are means ± s.e.m. from ≥5 experiments. ** = p<0.01 and *** = p<0.001, compared with CHC-treated cells.CC BY · PubMed Central
Figure 5 Cholesterol crystals (CHCs) cause destabilization of lysosomes and leakage of cathepsin B into the cytoplasm. CHC-treated or untreated live THP-1 macrophages were stained with cathepsin B substrate z-Arg-Arg-cresyl violet (panels i, iii) or with acridine orange (panels ii, iiii). The fluorescent cresyl violet group of z-Arg-Arg-cresyl violet is dequenched upon cleavage of one or both of the arginines by cathepsin B. Acridine orange aggregates in the acidic pH of lysosomes, which changes the fluorescence emission of the dye from green to red. The images are representative of 3 experiments.
Figure 5 Cholesterol crystals (CHCs) cause destabilization of lysosomes and leakage of cathepsin B into the cytoplasm. CHC-treated or untreated live THP-1 macrophages were stained with cathepsin B substrate z-Arg-Arg-cresyl violet (panels i, iii) or with acridine orange (panels ii, iiii). The fluorescent cresyl violet group of z-Arg-Arg-cresyl violet is dequenched upon cleavage of one or both of the arginines by cathepsin B. Acridine orange aggregates in the acidic pH of lysosomes, which changes the fluorescence emission of the dye from green to red. The images are representative of 3 experiments.CC BY · PubMed Central
Figure 6 Silencing of NLRP3 attenuates cholesterol crystal (CHC)-induced IL-1β secretion. (A) NLRP3 mRNA levels were reduced by 72% after treatment of THP-1 macrophages with NLRP3-targeted small interfering RNA (siRNA). (B) CHC-induced IL-1β secretion was abolished after treatment of cells with NLRP3 siRNA, whereas treatment of cells with negative control siRNA had no effect. The data are means ± s.e.m. from 5 (A) and 3 (B) experiments. ** = p<0.01.
Figure 6 Silencing of NLRP3 attenuates cholesterol crystal (CHC)-induced IL-1β secretion. (A) NLRP3 mRNA levels were reduced by 72% after treatment of THP-1 macrophages with NLRP3-targeted small interfering RNA (siRNA). (B) CHC-induced IL-1β secretion was abolished after treatment of cells with NLRP3 siRNA, whereas treatment of cells with negative control siRNA had no effect. The data are means ± s.e.m. from 5 (A) and 3 (B) experiments. ** = p<0.01.CC BY · PubMed Central
Figure 7 Proposed mechanism of cholesterol crystal (CHC)-induced inflammasome activation. CHCs are phagocytosed by macrophages, causing lysosomal destabilization and leakage of cathepsin B to cytoplasm, where the enzyme indirectly activates the NLRP3 inflammasome. Lowering of intracellular potassium concentration, stemming from potassium efflux caused by phagocytosed CHCs, is also required for NLRP3 activation.
Figure 7 Proposed mechanism of cholesterol crystal (CHC)-induced inflammasome activation. CHCs are phagocytosed by macrophages, causing lysosomal destabilization and leakage of cathepsin B to cytoplasm, where the enzyme indirectly activates the NLRP3 inflammasome. Lowering of intracellular potassium concentration, stemming from potassium efflux caused by phagocytosed CHCs, is also required for NLRP3 activation.CC BY · PubMed Central

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Authors
Rajamäki, K., Lappalainen, J., Öörni, K., Välimäki, E., Matikainen, S., Kovanen, P. T., & Eklund, K. K.
Affiliations
  • Wihuri Research Institute (FI)
  • Finnish Institute of Occupational Health (FI)
Corresponding author
Rajamäki, Kristiina
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2010-07-23
Volume
5
Issue
7
Pages
e11765
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0011765
PMC
PMC2909263
OpenAlex
W2028545985
Semantic Scholar
fa6cd20e147dd066a3358c77aa3fc9b4dc9ff451
Cited by
1,039
References
60
Citations by year
2026: 65 · 2025: 51 · 2024: 45 · 2023: 77 · 2022: 80 · 2021: 74 · 2020: 72 · 2019: 57 · 2018: 66 · 2017: 77 · 2016: 52 · 2015: 76 · 2014: 75 · 2013: 70 · 2012: 59
FWCI
21.86
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
Jenny ja Antti Wihurin Rahasto
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Inflammasome and immune disorders
  • Life Sciences › Immunology and Microbiology › Immunology › Atherosclerosis and Cardiovascular Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
Keywords
cholesterol crystals, NLRP3 inflammasome, macrophages, atherosclerosis, IL-1β secretion, caspase-1, lysosomal destabilization, cathepsin B, cholesterol esters, cholesterol metabolism, inflammation, monocytes
MeSH terms
NLR Family, Pyrin Domain-Containing 3 Protein, Carrier Proteins, Cathepsin B, Cells, Cultured, Cholesterol, Chromatography, Thin Layer, Cytoplasm, Humans, Inflammation, Lipopolysaccharides, Lysosomes, Macrophages, Monocytes, Phagocytosis, Potassium, Blotting, Western, Cytochalasin D, Microscopy, Confocal, Reverse Transcriptase Polymerase Chain Reaction, Caspase 1, RNA, Small Interfering, Interleukin-1beta, Caspase Inhibitors

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