LAB strains
KW3110 tested in this study were maintained at Koiwai Dairy Products Co., Ltd. (Saitama, Japan). NRIC1942 and LGG were purchased from collections held at the Culture Collection Center, Tokyo University of Agriculture (Tokyo, Japan). Cultures of LAB strains were grown at 30 °C or 37 °C for 48 h in MRS broth (Oxoid, Hampshire, UK) according to manufacturer’s instructions. Cultured LAB strains were washed twice with sterile distilled water, heat-killed at 70 °C, lyophilized, and suspended in PBS for in vitro studies.
Cell culture and stimulation by heat-killed KW3110
RAW 264.7 murine macrophages were obtained from the American Type Culture Collection (Bethesda, MD, USA). Cells were cultured in DMEM supplemented with 10% FBS, streptomycin (100 μg/ml), and penicillin (100 units/ml) then incubated overnight at 37 °C and 5% CO2. For the experiments, RAW 264.7 cells were seeded and incubated overnight in 24-well plates (2.5 × 105 cells/well), and then treated with heat-killed KW3110 (10 μg/ml) or other LAB (10 μg/ml) for 24 h in confluent state. RAW 264.7 cells were treated with heat-killed KW3110 at a concentration of 1.25, 2.5, 5, 10 μg/ml to verify concentration dependence.
Reagents and antibodies
Cytochalasin D was purchased from Sigma (St. Louis, MO, USA). To block phagocytosis, Cytochalasin D was added 30 min prior to KW3110 stimulation. Gefitinib was obtained from Tocris Bioscience (Bristol, Avon, UK). Anti-mouse Dectin-2 antibodies were from MyBioSource (San Diego, CA, USA).
ELISA
Cell culture supernatants were assayed for mouse IL-10 (BD Biosciences, San Jose, CA, USA), according to manufacturer’s instructions.
RNA extraction and real-time PCR analysis
Total RNAs were extracted using an RNeasy Mini Kit (Qiagen, Venlo, Netherlands), following manufacturer’s recommendation for RNA preparation from bacterial cells, and reverse transcribed into cDNA using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). The cDNA was used as a template in PCR reactions using TB Green premix Ex Taq (Takara Bio, Shiga, Japan). PCR was performed with specific primers listed in Table 1 using a LightCycler 480 (Roche Diagnostics, Tokyo, Japan). The PCR conditions were as follows: 2‑step cycling, 95 °C for 10 s hold; 45 cycles of 95 °C for 5 s and 60 °C for 20 s. Values were then normalized against the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each sample.
Transfection
RAW 264.7 cells were seeded into 12-well plates (3.0 × 105 cells/well) and transfected with 100 nM small interfering RNAs (siRNA) using DharmaFECT 1 Transfection Reagent (Dharmacon, Lafayette, CO, USA) for 24 h before KW3110 treatment. siRNAs were all siGENOME SMARTpool siRNA (Dharmacon, Lafayette, CO, USA). Non-targeting siRNA (siNT, negative control), mouse CLEC7A siRNA (siDectin-1), mouse CLEC4N siRNA (siDectin-2), mouse SYK siRNA (siSyk), mouse SLC15A4 siRNA (siPht-1), and mouse SLC15A3 siRNA (siPht-2) were used in this study.
Flow cytometric analysis
After pre-incubation of RAW 264.7 cells for 24 h, FITC-labeled LAB (10 μg/ml) were added. After 24 h, cells were washed with DMEM and resuspended in 0.2% trypan blue for 5 min to quench the fluorescence of non-ingested, but membrane-associated bacteria. The macrophages were suspended in 4% paraformaldehyde (Wako, Osaka, Japan) for flow cytometry analysis. Flow cytometric analysis was performed on a FACS Canto II (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software. Cell populations were gated by the software program in the scatter diagram (forward-scattered light versus side-scattered light). FITC-positive population was defined as over 103 of intensity. The uptake quantity was determined as Cell Number × MFI.
Microscopic analysis of phagocytosis
After pre-incubation of RAW 264.7 cells for 24 h, FITC-labeled LAB (10 μg/ml) were added. After 24 h, cells were washed with DMEM and suspended in 0.2% trypan blue for 5 min; cells were then washed in and resuspended in PBS. RAW 264.7 cells were observed by confocal laser scanning microscope FV1000 (OLYMPUS, Tokyo, Japan).
Lectin array
The lectin microarray was performed using RayBio Lectin Array 95 (RayBiotech, Inc. Norcross, GA). All LAB species were dissolved at a concentration of 0.2 mg/ml in PBS and used as samples. After their addition to labeling reagent for 1 h, the samples were dialyzed and centrifuged at 1000 rpm for 5 min at 4 °C. Hybridizations were performed on the supernatant according to the manufacturer’s protocol.
Transmission electron microscopy
RAW 264.7 cells were grown for 24 h on 35 mm dishes (5.0 × 105 cells). Cells were washed with PBS and exposed to 1 μg/m1 KW3110 in DMEM for 24 h. After fixation, each cell sample was consigned to Tokai Electron Microscopy Inc. (Aichi, Japan). Cells were washed with PBS buffer and postfixed in 2% osmium tetroxide for 2 h at 4 °C and embedded in resin (Quetol-812) after dehydration in ascending grades of ethanol. Ultrathin sections, 70 nm, were cut and stained with alkaline and lead citrate uranyl acetate. The sections were examined under a TEM (JEM-1400Plus, JEOL Ltd., Tokyo, Japan).
RNA sequencing
Briefly, total RNAs were extracted from RAW 264.7 cells using RNeasy mini columns (Qiagen, Venlo, Netherlands). Library preparation and sequencing were consigned to Takara Bio Inc. Sequencing of each library was performed on a NovaSeq 6000 (illumina, San Diego, California, USA). Sequencing was performed to generate 150 bp paired-end reads.
RNA-seq data analysis
All RNA‐seq data are deposited in the Gene Expression Omnibus database at the National Center for Biotechnology Information. Analysis of the RNA-seq data was consigned to Takara Bio Inc. Preprocessing of the RNA‐seq data was completed using Genedata Profiler Genome. All samples passed quality control criteria. Reads were mapped to the GRCm38 mouse genome using STAR. The category classifications of genes were referred to KEGG36,37,38.
Statistical analysis
Statistical differences were evaluated using the Student’s t-test for comparison of two groups or analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s test for multiple comparisons. p-value < 0.05 was considered significant.
