Bacterial strains and growth conditions
L. gasseri OLL2809 isolated in our laboratory as described previously [13] was cultured in BD Difco™ Lactobacilli MRS Broth (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 18 h. After fermentation, the bacterial cells were harvested in a refrigerated centrifuge at 10,000×g for 15 min and washed twice with saline solution followed by one wash with distilled water. The cells were then resuspended in distilled water, heated at 75 °C for 1 h, and lyophilized. The lyophilized cells were resuspended in phosphate-buffered saline (PBS; pH 7.4) at a concentration of 200 μg/ml and used for IL-12 production assays.
L. gasseri JCM 1131T, L. plantarum JCM 1149T and L. crispatus JCM 1185T were purchased from the Riken BRC (Wako, Japan). They were cultured, heat treated and lyophilized as described above.
J774.1 cell culture
The murine macrophage cell line J774.1 was purchased from the Riken BRC (Wako, Japan). J774.1 cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI 1640; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Biosera, Nuaille, France), 100 U/mL penicillin (Thermo Fisher Scientific), 100 μg/mL streptomycin (Thermo Fisher Scientific) and 55 μM 2-mercaptoethanol (Thermo Fisher Scientific) in a humidified 5% CO2 incubator at 37 °C.
IL-12 production assay
J774.1 cells were seeded at 5 × 104 cells per 100 μL per well in a 96-well plate and cultivated for 48 h in the absence or presence of 1 μg/ml of the L. gasseri OLL2809. The IL-12 (p40) levels in the culture supernatant were quantified using the mouse IL-12(p40) ELISA set (BD Biosciences, Franklin Lakes, NJ, USA). The same cultivation conditions were used for all experiments, if not otherwise specified.
Fluorescein-4-isothiocyanate (FITC) labelling and confocal microscopy
Lyophilized L. gasseri OLL2809 (10 mg) were resuspended in 1 mL of 50 mM carbonate buffer (pH 9.6) containing 5 μg FITC (Dojindo, Kumamoto, Japan), and incubated at 37 °C for 1 h. After washing twice with PBS (pH 7.4), the cells were used as FITC-labelled L. gasseri OLL2809. J774.1 cells were cultivated with 2 μg/mL FITC-labelled L. gasseri OLL2809 for 24 h. They were then stained with Cell Mask™ Deep Red (Thermo Fisher Scientific) and observed with confocal microscopy (Olympus, Tokyo, Japan).
Flow cytometric analysis
J774.1 cells were cultivated in the presence of 2 μg/mL FITC-labelled L. gasseri OLL2809 for 48 h. They were then harvested and labelled with allophycocyanin (APC) conjugated anti-F4/80 antibody (Bio-Rad, Hercules, CA, USA) and 7-amino-actinomycin D (7-AAD; BD Biosciences). The J774.1 cells were washed 2 twice with PBS (pH 7.4) and 7-AAD-negative, FITC-positive and F4/80-positive cells were counted on the flow cytometer FACSVerse™ (BD Biosciences). The proportion of FITC-positive cells in F4/80-positive cells was defined as phagocytosis index, and that of 7-AAD- positive cells was defined as the dead cell ratio.
Inhibition of phagocytosis, and MYD88 and TLRs 7 and 9 signalling
For inhibition of phagocytosis and Myeloid differentiation factor 88 (MYD88) and TLR 7 and 9 signalling, J774.1 cells were treated with various reagents followed by IL-12 production assays.
For phagocytosis, J774.1 cells were pre-treated with 0, 0.625, 1.25, 2.5 μM cytochalasin D (CyD; Fujifilm Wako Pure Chemical, Osaka, Japan) for 1 h. For MYD88 signalling, J774.1 cells were pre-treated with 150, 200 μM of MYD88 homodimerization inhibitory peptide or the control peptide (Novus Biologicals, Centennial, CO, USA) for 24 h. The culture medium was replenished with fresh media, and then IL-12 production assay was performed. The amino acid sequences of the peptides were as follows;
Control peptide: DRQIKIWFQNRRMKWKK
Inhibitory peptide: DRQIKIWFQNRRMKWKKRDVLPGT
For TLRs 7 and 9, TLR7 antagonist immunoregulatory sequence (IRS)661 [27], and TLR9 antagonist IRS869 [10] as well as the control oligodeoxynucleotide (ODN) [28], were synthesized by Eurofins Genomics (Tokyo, Japan). J774.1 cells were pre-treated with 1 μM of IRS661, or IRS869, or both for 30 min. Nucleotide sequences of ODNs were as follows;
Control: TCCTGCAGGTTAAGT
IRS661 (Antagonist of TLR7): TGCTTGCAAGCTTGCAAGCA
IRS869 (Antagonist of TLR9): TCCTGGAGGGGTTGT
Extraction of RNA and DNA from bacterial strains and transfer to J774.1 cells
Total RNA and genomic DNA were extracted from bacterial strains by using Nucleo Spin® RNA (Takara Bio, Shiga, Japan) or Nucleo Spin® Microbial DNA (Takara Bio). The concentration of the extracted nucleotides was measured by Nano Drop 1000 (Thermo Fisher Scientific). Further, 5 μL of FuGENE® HD (Promega, Fitchburg, WI, USA) was added to 100 μL of Opti-MEM (Thermo Fisher Scientific) containing 2 μg of the extracted nucleotide, and the mixtures were incubated for 10 min at room temperature to form total RNA/FuGENE or genomic DNA/FuGENE complexes. The total RNA/FuGENE or genomic DNA/FuGENE were diluted 20-fold and used to stimulate J774.1 cells for IL-12 production assays.
Statistical analyses
Data are expressed as mean values with standard deviations (SD). Difference between multiple groups was analysed by Tukey-Kramer or Dunnett’s multiple comparison test. For analysis of the two groups in the MYD88 signal inhibition experiment, the Student’s t-test was used. Differences were considered significant when the p-values for the effect were less than 0.05.