RPMI1640 media, fetal bovine serum (FBS), penicillin, streptomycin, phosphate buffered saline without calcium and magnesium (PBS (-)), and Hank's balanced salt solution (HBSS) were purchased from Gibco (Carlsbad, CA, USA). Thioglycollate was obtained from Becton Dickinson (Cockeysville, MD, USA). Phorbol myristate acetate (PMA), dimethyl sulfoxide (DMSO), 2',7'-dichlorofluorescin diacetate (DCFH-DA), Escherichia coli lipopolysaccharide (LPS, 0111:B4), sulfanilamide, naphthylethylenediamine dihydrochloride, phosphoric acid (H3PO4), sodium nitrite (NaNO2), boron trifluoride methanol, nonacosanoic acid, and tricosenoic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Murine recombinant interferon (IFN)-γ was purchased from BD Pharmingen (San Diego, CA, USA).
ALDP-deficient mice  backcrossed to C57BL/6J for 10 generations were obtained from the National Institute for Longevity Sciences of the National Center for Geriatrics and Gerontology (Obu City, Aichi, Japan) and males aged 12 to 14 weeks were used for experiments. Male C57BL/6J, wild-type counterparts, were purchased from Charles River Laboratory Inc. (Japan). All animals were housed and maintained in specific pathogen-free condition at a controlled temperature (23 ± 2°C), on a 12 h light/dark cycle, and fed a normal chow diet (Funabashi Farm, Chiba, Japan) and tap water ad libitum. The animal experiments were approved by the Morinaga Milk Industry Animal Research Committee, and the mice were maintained according to the guides for the care and use of laboratory animals of Morinaga Milk Industry Co., Ltd.
To isolate thioglycollate-elicited murine peritoneal exudate macrophages (MPMs), mice were injected intraperitoneally with 2 ml of 4% (wt/vol) thioglycollate solution. Four days later, they were sacrificed by anesthetizing with ether and their abdominal cavities washed out twice with 5 ml of cold PBS (-). The peritoneal lavage fluid was centrifuged at 1200 rpm, 4°C for 10 min. After washing twice, cell pellets were gently resuspended at 1 × 106 cells/ml in RPMI1640 medium with 2 mM L-glutamine, supplemented with 10% endotoxin-free FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. To recover adherent cells, MPMs were incubated for 2 h and washed with PBS(-) twice, and non-adherent cells were removed by aspiration. All culture incubations were performed in a humidified 37°C, 5% CO2 incubator unless otherwise stated. Cell viability was checked using Tripan blue exclusion and the viability was greater than 95%.
Measurements of Plasma Lipid and Glucose Levels
Mice were fasted for 16 h and blood samples were harvested by cardiac puncture after ether anesthesia and collected into EDTA-disodium containing tubes. Samples were centrifuged at 3000 rpm for 15 min to obtain plasma. Plasma lipid such as triglyceride (TG), total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured by the HPLC column method at Skylite Biotech Inc. (Akita, Japan) , and plasma glucose levels were measured by the Glucose CII Test Wako (Wako Pure Chemical Industries, Ltd, Tokyo, Japan).
C26:0 Quantification and Fatty Acid Composition Analyses
MPMs (1.5 × 107 cells) were rinsed with PBS(-) and total lipid was extracted by the Folch method . The extract was then transmethylated with 14% boron trifluoride methanol solution at 90°C for 90 min. Quantification of C22:0 and C26:0 was performed with a gas chromatography-mass spectrometry (GS-MS) system (QP2010, Shimadzu Corporation, Kyoto, Japan) equipped with a fused silica capillary column (Rtx-5 MS, 30 m × 0.25 mm i.d.; 0.25 μm film thickness, Restek, USA) using nonacosanoic acid (C29:0) methyl ester as an internal standard. The mass spectrum acquisitions of C22:0, C26:0, and C29:0 methyl esters were performed in selected-ion monitoring (SIM) mode and the target ions of these fatty acid methyl esters were 354.50, 410.40, and 452.40 m/z, respectively. For fatty acid composition analysis, total lipid extract from MPMs was transmethylated as described above and incubated at 90°C for 60 min. The measurement of fatty acids was performed with a GC-FID system (6890N, Agilent technologies, Tokyo, Japan) equipped with a fused silica capillary column (Omegamax 250, 30 m × 0.25 mm i.d.; 0.25 μm film thickness, Supelco, USA) using tricosenoic acid (C23:0) methyl ester as an internal standard.
The production of NO was determined by Griess reagent solution . MPMs (1.0 × 105 cells/well) were seeded in a 96-well flat-bottom plates and stimulated with various concentrations (1, 10, 100 ng/ml) of LPS plus 2 ng/ml IFN-γ for 24 h for dose-dependent analysis. Control media contained neither LPS nor IFN-γ. For time-dependent analysis, MPMs were stimulated with 10 ng/ml of LPS plus 2 ng/ml IFN-γ and incubated for various times (0, 12, 18, 24 h). After incubation, 50 μl of cell culture supernatant was mixed with 100 μl of Greiss reagent solution (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% H3PO4, respectively) and incubated for 10 min at room temperature. Absorbance of the mixture was measured at 550 nm in a microplate reader (Corona, Ibaraki, Japan) and NO concentration was determined using a serial dilution (1 to 125 μM final concentrations) of NaNO2 in culture medium as a standard. The levels of NO production were normalized to cell viablitiy using CellTiter-Glo Luminescent Cell Viability Assay (Promega, WI, USA).
Real-time Quantitative Reverse Transcription-PCR for iNOS
MPMs (1.0 × 106 cells/well) were seeded in 6-well flat-bottom plates and stimulated with 10 ng/ml LPS plus 2 ng/ml IFN-γ for 18 hours. Then, total RNA was extracted using Trizol (Invitrogen, CA, USA) following the manufacturer's protocol. Total RNA (40 ng/each reaction) was reverse transcribed into cDNA using random primers (High Capacity cDNA Reverse Transcription kit; Applied Biosystems, CA, USA) at 25°C for 10 min, 37°C for 120 min, and 85°C for 5 s. For PCR amplification, 4 μl of reverse transcription reaction was added to a 16 μl reaction mixture containing TaqMan Fast Universal PCR Master Mix (Applied Biosystems, CA, USA) and TaqMan Gene Expression Assays reagents (Applied Biosystems, CA, USA) for iNOS (Mm00440485_m1). The β-actin gene (Mm00607939_s1) was amplified in the same experiment to serve as the reference gene. The reaction was carried out in an Applied Biosystems 7500 Fast Real-Time PCR System at 95°C for 20 s, cycled at 95°C for 3 s and 60°C for 30 s for 40 cycles. The mRNA expression levels were normalized to those of β-actin.
Intracellular Reactive Oxygen Species (ROS) Assay
To measure ROS levels in MPMs, flow cytometric analysis was conducted with DCFH-DA . MPMs (1.0 × 106 cells/well) were seeded in 6-well flat-bottom plates then incubated with 10 μM of DCFH-DA in PBS(-) for 15 min at 37°C. After incubation, MPMs were rinsed with PBS(-) to remove unincorporated DCFH-DA and then stimulated with 0.5 μg/ml of PMA for 20 min at 37°C. To stop reaction, MPMs were rinsed with PBS(-) and gently scraped with a cell scraper. Cell suspensions were transferred to a tube and washed twice with cold PBS(-). Cellular fluorescence was determined using a FACSCanto flow cytometer (BD Biosciences, USA). Intracellular fluorescence was measured at 530/30 nm after excitation of cells at 488 nm with an argon ion laser. MPMs were discerned by the combination of forward-scattered and side-scattered laser light and aggregated and/or fragmented cells were excluded. 10,000 events were recorded for analyses.
Quantification of Pro-inflammatory Cytokine Production
MPMs (1.0 × 105 cells/well) were seeded in 96-well flat-bottom plates and stimulated with 10 ng/ml LPS plus 2 ng/ml of IFN-γ for 24 h. Culture supernatants were collected and cytokines (IL-6, TNF-α and IL-12p70) in media were measured by flow cytometer using the cytometric bead array (CBA) method (Mouse inflammation kit; BD Biosciences, USA) according to the manufacturer's instructions. Data were acquired using a FACS Canto flow cytometer (BD Biosciences, USA) and analyzed using BD cytometric bead array software (BD Biosciences, USA). The levels of pro-inflammatory cytokines production were normalized to cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, WI, USA).
All values are expressed as mean ± SD. Statistical analysis was performed with a two-sided Student's t-test using SAS software (version 9.1.3; SAS Institute, Cary, NC). Differences were considered significant at P < 0.05.