Experimental animals
Adult male C57BL/6J mice (10 weeks old) (Japan SLC Inc., Shizuoka, Japan), CCR2-deficient mice on the C57BL/6J background (10 weeks old) (CLEA Japan Inc., Tokyo, Japan), TLR4-deficient mice on the C57BL/6J background (10 weeks old) (Oriental Bio Service Inc., Kyoto, Japan) and CX3CR1-GFP/CCR2-RFP mice on the C57BL/6J background (10 weeks old) (gifted by Dr. R. Yamasaki) were used in this study.31 All mice were maintained on a 12-hour light/dark cycle and fed either a normal laboratory diet or a high-fat high-cholesterol diet and water ad libitum. The study protocol was reviewed and approved by the Committee on the Ethics of Animal Experiments, Kyushu University Faculty of Medicine, which is compatible with the ARRIVE guidelines. All the experiments were conducted following the American Physiological Society guidelines and the NIH Guide for the Care and Use of Laboratory Animals, 8th edition.
Measurement of plasma lipid levels
Plasma was collected for cholesterol, oxysterol, and triglyceride measurements. Total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides were measured using enzymatic assay kits (FUJIFILM Wako Pure Chemical Co., Osaka, Japan). Lipids extracted plasma were saponified at room temperature overnight in the dark. Unsaponified lipids were applied to a Sep-Pak Vac silica cartridge (Waters Corporation, Milford, MA) to separate oxysterols and sterols.47 Oxysterols, including 7-KC, were converted into trimethylsilyl ethers in a mixture of trimethylchlorosilane, 1,1,1,3,3,3-hexamethyldisilazane, and dried pyridine (1 : 3 : 9, v : v : v) for 30 minutes at room temperature. Oxysterols were measured by gas chromatography-mass spectrometry using a GCMS-QP2010 system (Shimadzu Co., Kyoto, Japan) equipped with an SPB-1–fused silica capillary column of 60 m × 0.25 mm and 0.25-µm phase thickness (Supelco Inc., Bellefonte, PA).
Mouse myocardial IR injury model
Mice were fed a normal laboratory diet (CRF-1), WD, or 7KWD for 3 weeks, and then surgery causing myocardial IR was performed. The WD contained 40% fat, 1% cholesterol and 0.5% cholic acid, and the 7KWD contained 40% fat, 0.9% cholesterol, 0.1% 7-ketocholesterol and 0.5% cholic acid. All these diets were purchased from ORIENTAL YEAST CO., Ltd. (Tokyo, Japan), and supplemental 7-KC was purchased from Sigma–Aldrich (St. Louis, MO). A murine model of myocardial IR injury was established as previously described.48,49 Mice were kept warm on a heating pad and anesthetized with 1.5% isoflurane in 2 L/min oxygen. Subsequently, the mice were intubated and ventilated with a respirator. The heart was exposed by a left thoracotomy and pericardiotomy. The left anterior descending artery (LAD) was temporarily ligated with an 8 − 0 nylon suture and silicon tube (1 mm OD) for 30 minutes, and then the silicon tube was removed to restart the blood flow.
Preparation of primary macrophages
As previously described, murine peritoneal and BMDMs were prepared for in vitro assays.50 Peritoneal macrophages were collected as described below. Brewer’s thioglycolate medium (BD Diagnostic Systems, Sparks, MD) was injected into the murine peritoneal cavity 3 days before macrophage collection. Ice-cold phosphate-buffered saline (PBS) was injected into the peritoneal cavity, and cells were harvested. Red blood cells were lysed with ammonium-chloride-potassium lysis buffer (Thermo Fisher Scientific Inc., Cleveland, OH). Then, the cells were washed with PBS, resuspended in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma–Aldrich) and 1% penicillin/streptomycin and plated in culture plates. One hour later, the nonadherent cells were removed, and the remaining adherent cells were cultured further in fresh medium. Bone marrow-derived macrophages were harvested from the tibiae and femurs of donor mice. After red blood cell lysis and washing, the cells were resuspended and cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL M-CSF (R&D Systems, Minneapolis, MN).
siRNA transfection
ON-TARGETplus Non-targeting Control Pool or ON-TARGETplus SMARTPool siRNAs targeting mouse Nr1h3, Nr1h2, Eif2ak3, or Ero1l were purchased from Horizon Discovery Ltd. (Cambridge, UK). All siRNAs were packaged in lipid nanoparticles (LNPs) and transfected into cells. Cultured cells were transfected with siRNA for 24 hours and then treated with cholesterol or 7-KC 12 hours after transfection was completed. siRNA-loaded LNPs were formulated as previously described.51 Briefly, 400 µL of lipid solution containing 1.875 mM ssPalmO-Phe (Product# COATSOME® SS-OP, NOF CORPORATION, Tokyo, Japan), 450 µM polyethylene glycol-dimyristoylglycerol (Product# SUNBRIGHT® GM-020, NOF CORPORATION), and 1.875 mM cholesterol (Sigma–Aldrich) in ethanol was prepared. Then, 10 µg of siRNA in 124 µL of 20 mM malic acid buffer (pH 3.0) was gradually added to the lipid solution under vigorous mixing. The mixed solution was diluted with 1 mL of malic acid buffer and then mixed vigorously with 3 mL of PBS. This diluted solution was subjected to ultrafiltration with Amicon Ultra-4 (Merck Millipore, Billerica, MA) two or more times. The encapsulation efficiency and recovery ratio of siRNA in this LNP solution were measured with a RiboGreen assay (Thermo Fisher Scientific Inc.) as previously described.52
Quantification of myocardial infarction
Twenty-four hours after reperfusion, mice were anesthetized with 1.5% isoflurane in 2 L/min oxygen and intubated. After ligating the LAD again, 2% Evans blue dye (Sigma–Aldrich) was injected into the inferior vena cava to dye the nonischemic area of the heart. Hearts were harvested and washed by saline perfusion. The harvested hearts were frozen in liquid nitrogen and cut into sequential cross-sections at a thickness of 1 mm. The sections were incubated with 1.5% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma–Aldrich) for 20 minutes at 37°C. After dyeing with TTC, the sections were placed in 10% formaldehyde overnight. Then, the sections were photographed under a microscope. The infarcted area (TTC negative/Evans blue negative, white), the noninfarcted area within the area at risk (AAR) (TTC positive/Evans blue negative, red), the nonischemic area (TTC positive/Evans blue positive, purple), and the AAR (Evans blue negative, red and white) were quantified using ImageJ software (version 1.51h).
Immunohistochemistry
Paraffin sections of the heart were prepared for immunohistochemistry. Twenty-four hours after reperfusion, hearts were harvested, fixed in 10% formaldehyde for 24 hours, and then embedded in paraffin. Serial cross-sections of the heart cut at a thickness of 5 µm at the midventricular level were used for analysis. After blocking with 2.5% goat serum (ImmPRESS, Goat Anti-Rat IgG Polymer Kit, Peroxidase, Vector Laboratories, Burlingame, CA), the sections were subjected to immunostaining for macrophages using an anti-mouse Mac-3 antibody (BD Pharmingen, San Diego, CA). After an overnight incubation with the primary antibody at 4°C, the sections were washed 3 times with PBS and incubated with peroxidase-conjugated anti-rat IgG at room temperature for 30 minutes. Then, the sections were washed 3 times with PBS and incubated with a peroxidase substrate (ImmPACT, AMEC Red Peroxidase Substrate Kit, Vector Laboratories) for 10 minutes. After counterstaining with hematoxylin, the sections were imaged with a stereomicroscope. Mac-3-positive cells were counted using ImageJ software (version 1.51h).
Flow cytometry
Twenty-four hours after reperfusion, hearts were harvested, cut into small pieces, and digested with a cocktail of 450 U/mL collagenase type I, 125 U/mL collagenase type XI, 60 U/mL DNase I, and 60 U/mL hyaluronidase (Sigma–Aldrich) in PBS containing 20 mM HEPES at 37°C for 40 minutes. The cell suspensions were washed with PBS, and Fc receptors were blocked with anti-CD16/32 antibodies (BD Pharmingen). After blocking, the cell suspensions were incubated with a cocktail of antibodies against leukocytes (anti-CD45-PE-Cy7), myeloid cells (anti-CD11b-APC-Cy7), B cells (anti-B220-PE), T cells (anti-CD90.2-PE), NK cells (anti-CD49b-PE, NK1.1-PE), granulocytes (anti-Ly-6G-PE), monocyte subsets (anti-Ly-6C-FITC), and macrophages (anti-F4/80-Alexa Fluor 647) (BD Pharmingen) for 1 hour at 4°C. 7-AAD (BD Pharmingen) was applied to exclude nonviable cells just before starting the analysis. The stained cells were analyzed with a Gallios flow cytometer (Beckman Coulter Inc., CA). Monocytes/macrophages were identified as CD45 + CD11b + Lineage (B220/CD90.2/CD49b/NK1.1/Ly-6G)- Ly-6Chigh/low cells.
Quantitative real-time PCR
RNA was extracted from cultured macrophages with an Illustra RNA Spin Mini Kit (GE Healthcare, Chicago, IL), and cDNA was synthesized with a PrimeScript RT Reagent Kit (TAKARA BIO Inc., Shiga, Japan) according to the manufacturer’s instructions. Real-time PCR was performed with TB Green Premix Ex Taq II (TAKARA BIO Inc.) and a StepOnePlus Real-time PCR System (Applied Biosystems, Waltham, MA). The primer sequences are listed in Table S2 in the Data Supplement.
Evaluation of CX3CR1 and CCR2 expression
Bone marrow cells harvested from CX3CR1-GFP CCR2-RFP mice were cultured for 5 days and then pretreated with 10 µM cholesterol or 7-KC for 24 hours before stimulation with 10 ng/mL lipopolysaccharide (LPS) and 10 ng/mL interferon-γ (IFN-γ) for 24 hours to induce differentiation into inflammatory macrophages. After nuclear counterstaining (Hoechst 33342, Thermo Fisher Scientific Inc.), CX3CR1-positive cells (GFP-positive) and CCR2-positive cells (RFP-positive) were observed under a confocal laser scanning microscope (FV1000, Olympus, Tokyo, Japan).
Chemotaxis assay
The chemotactic activity of macrophages was measured in a 96-well chemotaxis chamber with a 5-µm pore-sized membrane (ChemoTx, Neuro Probe Inc., Gaithersburg, MD) according to the manufacturer’s instructions. MCP-1 in RPMI-1640 medium was added to the lower chambers, and peritoneal macrophages harvested from wild-type (WT) mice fed the WD or 7KWD for 3 weeks (1x107 cells per mL) were placed in the upper chambers. After a 4-hour incubation at 37°C, the upper surface of the membrane was washed with PBS, and 20 mM ethylenediaminetetraacetic acid (EDTA) was applied. After a 20-minute incubation at 4°C, the added EDTA was removed and centrifuged. The migrated cells in the lower chambers were counted.
RNA sequencing
Peritoneal macrophages were stimulated with 10 µM cholesterol or 7-KC for 24 hours, and then RNA was extracted with ISOGEN (Nippon Gene, Tokyo, Japan). RNA-Seq was performed using an Illumina NextSeq 500 (Illumina Inc., Tokyo, Japan). Among all detected genes, differentially expressed transcripts between the cholesterol group and the 7-KC group were chosen according to the stated criteria (fold change > 2 or < 0.5 and FDR < 0.05). The obtained data were analyzed using STRING software (version 11.5) and iDEP software (version 0.94).
NF-κB activity assay
NF-κB activity was evaluated by measuring p65 translocation into the nucleus using a TransAM NF-κB p65 Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. Nuclear extracts were extracted from cultured peritoneal macrophages, and protein concentrations were determined with a Bradford-based assay (Pierce Coomassie Plus Assay Kit, Thermo Fisher Scientific Inc.). The nuclear extracts were added to a 96-well plate coated with an oligonucleotide containing the NF-κB consensus sequence (5’-GGGACTTTTCC-3’) and incubated for 1 hour at room temperature. An anti-NF-κB antibody was added to each well, and the plate was incubated for 1 hour at room temperature. After the plate was incubated for 1 hour with a horseradish peroxidase-conjugated secondary antibody, specific binding was detected with a microplate spectrophotometer based on the absorption at 450 nm with a reference wavelength of 655 nm.
Measurement of mitochondrial ROS
Mitochondrial superoxide was observed and quantified using the mitochondrial superoxide indicator MitoSOX Red (Thermo Fisher Scientific Inc.). After peritoneal macrophages were stimulated with 10 µM 7-KC for 24 hours, the cells were incubated with a 2.5 µM MitoSOX solution. After nuclear counterstaining, the fluorescence intensity of MitoSOX was quantified using an FV1000.
Western blotting
Total protein was extracted from peritoneal macrophages using Cell Lysis Buffer (Cell Signaling Technology Inc., Danvers, MA) according to the manufacturer’s instructions. Protein concentrations were determined with a BCA assay (Pierce BCA Protein Assay Kit, Thermo Fish Scientific Inc.). Proteins were separated on 4–20% gradient SDS/polyacrylamide gels and blotted onto PVDF membranes. The following antibodies were used as primary antibodies: anti-PERK (1:2000), anti-BiP (1:2000), anti-CHOP (1:2000) (Cell Signaling Technology Inc.), and anti-β-actin (1:10000, Sigma–Aldrich). Anti-Rabbit IgG, HRP-linked whole Ab donkey (GE Healthcare) was used as secondary antibody (1:5000). Chemiluminescence images were acquire with an Image Quant LAS-4010 (GE Healthcare). Densitometric analyses were performed using an Image Quant TL (GE Healthcare).
Statistical analysis
Data are expressed as the mean ± SEM. Statistical analyses of the differences between two groups were performed using an unpaired t test, and analyses of the differences among three or more groups were performed by one-way or two-way ANOVA, followed by Tukey’s post-hoc multiple comparison test using Prism Software version 8.0 (GraphPad Software, San Diego, CA). Statistical analyses of flow cytometry data were performed using the chi-square test. P values less than 0.05 were considered statistically significant.