Materials
Thirty different pieces of heart wood from Japanese cedar were collected in August 2020 and March 2021 from Kawakami village, Yoshino, Nara (n = 20), or two other building material production areas, Shikoku and Kyusyu (n = 10) outside the Nara prefecture. Each timber was dried at the felling site and provided in a state ready for use as a building material. We analyzed products that finished the preparation required for building materials. We tested trees that were about 100 years old from Yoshino and about 30–40 years from other areas. These all-timber samples were almost the same diameter, and provided by Kawakami Suppli (Yoshino, Japan) and dried naturally. Each timber sample was chopped and sliced by four-side planing using a molding machine (GMX-5000, Tokiwa Industry, Gifu, Japan) into 50 × 100 × 1–2 mm pieces, and kept at room temperature until use.
Preparation of 37 °C aqueous extracts from Japanese cedar and the measurement of endotoxin levels
The chopped and sliced samples were additionally cut into 5 × 5 × 1–2 mm pieces. One gram of each chipped sample was soaked in 10 ml water and shaken at 200 rpm/min overnight at 37 °C. Each extract was filtered through a 0.45-μm filter (Merck Millipore, Burlington, MA, USA). The extract was used by extracting once from each material. The extracts were stored at − 30 °C until analyzed as aqueous extract. Endotoxin levels in cedar extracts were measured by the ToxinSensor™ Limulus Amebocyte Lysate chromogenic endpoint assay according to the manufacturer’s protocol (GenScript, Piscataway, NJ, USA).
Monocyte isolation and macrophage culture
The study was conducted in accordance with the principles expressed in the Declaration of Helsinki and approved by the Ethics Committee of Nara Medical University (Approval No. 2108). Blood samples were obtained from healthy donors after written informed consent was obtained. Peripheral blood mononuclear cells (PBMC) were obtained by Lymphoprep™ Tube (Abbott Diagnostics Technologies, Oslo, Norway). Monocytes were isolated from PBMC using anti-CD14 magnetic beads (Miltenyi Biotec, Cologne, Germany) based on Kittan et al.’s report [9]. Isolated CD14+ monocytes were cultured at a concentration of 0.5–1.0 × 106 cells/ml in complete RPMI 1640 medium (Fujifilm Wako Pure Chemical Co., Osaka, Japan) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Fujifilm Wako Pure Chemical Co.), supplemented with 20 ng/ml macrophage colony stimulating factor (M-CSF) (PeproTech, Cranbury, NJ, USA). On day 3, fresh complete RPMI 1640 media containing M-CSF (20 ng/ml) was added. On day 7, fully differentiated macrophages were obtained from CD14+ monocytes and harvested and replated in RPMI 1640 medium. After resting overnight, cells were stimulated with or without 1/100 and 1/1000 dilution of extracts from Japanese cedar for 24 h. Sterile pure water was used for the control treatment. After stimulation, we collected the supernatant and cells. The supernatant was collected for the analysis of C–X–C motif chemokine ligand 10 (CXCL10) and IL-12p40 proteins and stored at − 80 °C until used. The cells were collected for mRNAs analysis by quantitative reverse transcriptase–polymerase chain reaction (qRT–PCR). We analyzed six genes, C–C chemokine receptor 7 (CCR7), CXCL10, interleukin 12B (IL12b) which is known as IL-12p40, arachidonate 15-lipoxygenase (ALOX15), folate receptor beta (FOLR2), and mannose receptor C-type 1 (MRC1), by qRT–PCR as described below.
Culture of mouse embryonic fibroblast (MEF) cells
Mouse embryonic fibroblast (MEF) cells, commonly used to test the immunological response through Toll-like receptors (TLRs), expressed high levels of mRNA for TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9 [10]. To test the effect of LPS in aqueous substances isolated from Yoshino cedar on M1 skewing of macrophages, we used TLR4-expressing MEF cells (wild-type, WT) and TLR4-knockout MEF cells (KO). Animal experiments performed in this study were approved by The Animal Care and Use Committee at Nara Medical University (Approval No. 12718), and all experiments were performed following the policy of the Care and Use of Laboratory Animals, Nara Medical University. MEF cells from WT C57BL/6 mice (CLEA Japan, Tokyo, Japan) were harvested as previously described [11]. Briefly, a pregnant female mouse was euthanized at Embryo E14.5 or 15.5, the abdomen was swabbed with 70% ethanol and cut open, the uterus was removed, and embryos were placed in a 10-cm petri dish (Corning, Corning, NY, USA) containing 10 ml sterile PBS on ice. The embryos were minced with fresh, sterile razor blades into small fragments. Then, 5 ml of Trypsin–EDTA (0.25%) (Fujifilm Wako Pure Chemical Co.) was added and transferred to a 50-ml conical tube (Corning) after pipetting up and down several times with a 5-ml pipette to disaggregate the tissue. The tissue was placed into the tube in a 37 °C water bath with shaking for 1 h. Next, 5 ml of Dulbecco’s modified Eagle medium (DMEM) (Fujifilm Wako Pure Chemical Co.) was added, and spun down at 1200 ×g for 5 min at room temperature. MEF cells were expanded in a 100-mm dish containing 10 ml DMEM complete culture medium, then stored in liquid nitrogen until used. MEF cells from TLR4 KO mice were purchased from Oriental Bio Service Co. (Kyoto, Japan). MEF cells from WT and TLR4 KO mice were cultured in complete DMEM medium overnight and then stimulated with or without 1/100 and 1/1000 dilutions of Japanese cedar extracts for 24 h. Sterile pure water was used for the control treatment. After stimulation, we collected supernatants and cells. The supernatant was collected for the analysis of CXCL10 and IL-12p40 protein and stored at − 80 °C until used. The cells were collected for mRNAs analysis by qRT–PCR, and for TLR4 signaling analysis by western blotting using cell lysates. We analyzed five genes, Il1b, Il6, tumor necrosis factor (Tnf), Cxcl10, and Il12b, by qRT–PCR and two proteins, phospho-nuclear factor-kappa B (p-NF-κB) p65, and β-actin, by western blotting using the WES system (Protein Simple Japan Co., Ltd., San Jose, CA, USA).
RNA extraction and qRT–PCR
RNAs from human macrophages and MEF were isolated using an RNeasy mini kit (Qiagen, Germantown, MD, USA). Total RNA was extracted and then 1 μg of total RNA was reverse-transcribed to cDNA with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). qRT–PCR analysis was performed using StepOne with TaqMan PCR master mix (Applied Biosystems, Foster City, CA, USA) on the default setting. The quantification of genes of interest was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as the fold increase over the negative control for each treatment at each time point as previously described [12].
Protein analysis
All protein analyses in supernatants were performed using a commercial ELISA kit. Human CXCL10 was analyzed using a Human CXCL10/IP-10 Immunoassay Quantikine ELISA (Cat No. DIP100, R&D Systems, Inc., Minneapolis, MN, USA). Human IL-12p40 was analyzed using a Human IL-12/IL-23(P40) ELISA MAX TM Standard set (Cat No. 430701, BioLegend, San Diego, CA, USA). Mouse CXCL10 was analyzed using a Mouse CXCL10/IP-10/CRG-2 Duoset ELISA (Cat No. DY466-05, R&D Systems Inc.). Mouse IL-12p40 analysis was performed by ELISA MAX™ Deluxe Set Mouse IL-12/IL-23(P40) (Cat No. 431604, BioLegend). Each detailed procedure was performed according to the manufacturer’s instructions.
TLR4 signaling analysis by western blotting
To test the association of LPS in aqueous substances derived from Yoshino cedar on M1 skewing of macrophages through the TLR4 pathway, we detected the phosphorylation of NF-κB by western blotting using the WES system (Protein Simple Japan Co., Ltd.). Cells were lysed with RIPA buffer (50 mmol/L Tris–HCl (pH 7.6), 150 mmol/L NaCl, 1% nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% sodium deoxysulfate, ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail (Takara Bio Inc., Shiga, Japan)).
Cell lysates were sonicated and centrifuged at 20,000 ×g for 15 min at 4 °C. The supernatants of cell lysates were collected and stored at − 80 °C until used. Protein concentrations were determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). We analyzed two proteins, p-NF-κB p65 (Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033, Cell Signaling Technology, Inc. Danvers, MA, USA) and β-actin (Monoclonal Anti-β-Actin, Clone AC-15, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) according to the manufacturer’s instructions.
Statistical analysis
Data are presented as the mean ± standard error of the mean (SEM) and are representative of at least two independent experiments. Statistical analyses of endotoxin levels and in vitro gene expressions were performed by the Mann–Whitney U test using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Differences in the in vitro gene expression of MEF were analyzed by one-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism 7 (GraphPad Software). P values < 0.05 were considered statistically significant.