Mice

Eight-week-old female BALB/c Cr Slc (BALB/c) mice, obtained from Sankyo Laboratories (Tokyo, Japan), were provided regular mouse feed and water ad libitum. The mice were maintained under specific-pathogen-free conditions on temperature-controlled clean racks with a 12-h light-dark cycle. All animal experiments were performed in accordance with the guidelines of the Bioscience Committee of Nihon University and were approved by the Institutional Animal Care and Use Committee of Nihon University (Approval number: AP11MD016).

Lactobacillus gasseri SBT2055 (LG2055) preparation

LG2055 was provided by the Milk Science Research Institute, Megmilk Snow Brand Co., Ltd. LG2055 was cultured in MRS broth (Difco Laboratories, Detroit, MI, USA) at 37 °C for 18 h and harvested by centrifugation at 10,000 × g for 15 min at 4 °C. The cells were washed twice with sterile PBS (−), resuspended in 25% trehalose solution, and stored at −80 °C until use. The influence on total viable bacterial count by freeze thawing was minimal.

Bacterial strain

Porphyromonas gingivalis strain 381 was cultured anaerobically as described previously37. The bacteria were harvested from brain heart infusion broth (Difco Laboratories) supplemented with hemin (5 mg/mL) and menadione (0.4 mg/mL) and resuspended in 5% carboxymethyl cellulose (CMC) for oral infection.

Experimental design

Mice were randomly divided into three groups (n = 36 per group; Fig. 6). The first and second groups were orally intubated with a 25% trehalose solution or LG2055 suspension (1 × 109 CFU/200 µL/mouse) through a syringe fitted with a ball-type feeding needle once per day for 5 weeks. At 3 weeks after oral intubation was started, the mice were orally infected with live P. gingivalis (1 × 108 CFU/100 μL with 5% CMC/mouse) once per day for 14 days. The third group consisted of sham-infected mice that received CMC without oral intubation. LG2055 and P. gingivalis were administered at specific time intervals.

Figure 6 Experimental procedure. Eight-week-old female BALB/c mice were randomly divided into three groups (n = 36 per group); groups 1 and 2 were orally intubated with 25% trehalose or LG2055 (1 × 109 CFU/200 μL/mouse) throughout the experiment once per day for 5 weeks. After administration for 3 weeks, mice were orally infected with P. gingivalis (1 × 108 CFU/100 μL with 5% CMC/mouse) once per day for 2 weeks. The third group received 5% CMC without oral intubation. Mice were sacrificed 0, 1, 2, and 3 weeks (n = 5 at each time point) after LG2055 administration for a mouse β-defensin-3 assay, and were sacrificed 1 and 30 days (n = 8 at each time point) after P. gingivalis infection for mouse β-defensin-3 assay, GMC cultures, bacterial detection, histological analysis, and alveolar bone loss evaluation. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/bnmrY9. Full size image

Histological analysis of gingival tissue and the measurement of alveolar bone loss

Thirty days after the last infection, mice were sacrificed and the skin of the lower jaw was removed and fixed in 1% paraformaldehyde in PBS for 24 h. The lower jaw was decalcified by incubation in 150 mM EDTA in PBS for 5–7 days at 4 °C and then embedded in paraffin. Four-micrometer-thick serial sections were then prepared and stained with hematoxylin and eosin. Horizontal bone loss around the maxillary molars was assessed using a morphometric method, as described previously38. Briefly, after removing gingival tissue, skulls were immersed overnight in 3% hydrogen peroxide, pulsed for 1 min in bleach, and stained with 1% methylene blue. The distance from the cementoenamel junction to the alveolar bone crest was measured at 14 buccal sites per mouse39. Measurements were made under a dissecting microscope (×20) fitted with a video image marker measurement system standardized to provide measurements in millimeters.

GMC isolation

At 1 and 30 days after the final P. gingivalis infection, mice were sacrificed and gingival tissues from the upper and lower jaws were carefully removed using microsurgical tweezers under a stereomicroscope. Cells from gingival tissues were prepared by gently teasing the tissue through sterile stainless steel screens, followed by an enzymatic dissociation procedure with 0.3 mg/mL of collagenase (Nitta Gelatin Co. Ltd., Osaka, Japan) in RPMI 1640 (Wako Pure Chemical Industries Ltd., Osaka, Japan)40. GMCs were enriched to 60–80% purity through discontinuous Percoll gradients (Pharmacia Fine Chemicals, Uppsala, Sweden) and resuspended in RPMI 1640 supplemented with HEPES buffer (15 mM), L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (Biofill, Victoria, Australia) (complete medium). GMC-enriched populations (2 × 105 cells) were stained with a combination of fluorescence-conjugated or biotinylated monoclonal antibodies, including anti-CD3, -B220, -CD11c, and -CD11b (BD Pharmingen, San Diego, CA, USA). The samples were subjected to FACS analysis to confirm cell purity and phenotype. The GMCs contained mononuclear cells positive for CD3 and B220, as well as monocytes (including macrophages and DCs) with >99% viability (Supplementary Table S1).

Cytokine- and β-defensin-specific ELISAs

GMCs (1 × 106/mL) were cultured in complete medium for 3 days and the culture supernatants were collected and subjected to IL-6- and TNF-α-specific ELISAs. We used a mouse IL-6 and TNF-α immunoassay kit (R&D Systems Inc., Minneapolis, MN, USA) to quantify IL-6 and TNF-α in culture supernatants. After removing the culture supernatant, total RNA was extracted from GMCs and subjected to quantitative real-time PCR for IL-6 and TNF-α mRNA. Salivary β-defensin-3 levels were analyzed using a Mouse DEFB3/Beta Defensin 3 ELISA Kit (LSBio, Seattle, WA, USA). Briefly, mouse saliva was collected 1, 2, and 3 weeks after the initial administration of LG2055, and on days 1 and 30 after the last P. gingivalis infection. Since β-defensin has a strong positive electric charge, the saliva samples were pretreated with Tween 20 and dilute HCl to break the bond between β-defensin and components with a negative electric charge (e.g., mucin). Briefly, 0.1 N HCl containing 0.5% Tween 20 was added to the saliva samples at a 1:9 ratio and centrifuged at 15,000 × g for 10 min at 4 °C. The saliva samples were then stored at −20 °C until analysis.

Analysis of gene expression in GMCs, gingival tissue, tongue, and small intestine

Every week from 0 to 3 weeks after LG2055 administration and on days 1 and 30 after the final P. gingivalis infection, total RNA from gingival tissue, tongue, and small intestine samples was extracted using an RNeasy Mini kit and treated with DNase I (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. Aliquots of RNA were then reverse-transcribed with oligo(dT) primers using SuperScript® reverse transcriptase (Invitrogen Corp., Tokyo, Japan) to generate cDNA. Quantitative real-time RT-PCR analyses were performed using a Thermal Cycler Dice real-time PCR system (Takara Bio Inc., Otsu, Japan) in accordance with the manufacturer’s protocol. All reactions were carried out in a total volume of 25 mL, containing 30 ng of reverse-transcribed RNA, 12.5 mL of 2x SYBR Green PCR Master Mix (Takara Bio Inc.), and each primer at 100 nM. Specific primers for IL-6, TNF-α, and GAPDH were supplied by Takara Shuzo (Kyoto, Japan). The specific primer for mbD14 was synthesized as described previously41. The primer sequences were as follows: IL-6 forward (5′-CCACTTCACAAGTCGGAGGCTTA-3′) and reverse (5′-GCAAGTGCATCATCGTGTTCATAC-3′); TNF-α forward (5′-GGAGTAGACAAGGTAC-3′) and reverse (5′-TATGGCCCAGACCCTCACA-3′); mbD14 forward (5′-TCTTGTTCTTGGTGCCTGCT-3′) and reverse (5′-CGACCGCTATTAGAACATCGAC-3′); and GAPDH forward (5′-TGTGTCCGTCGTGGATCTGA-3′) and reverse (5′-TTGCTGTTGAAGTCGCAGGAG-3′). PCR was performed using the following protocol: 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 10 s, and 72 °C for 30 s. The amplification of each gene and melting curve analysis were performed in triplicate. Target mRNA levels were normalized to that of GAPDH mRNA.

Porphyromonas gingivalis- or LG2055-specific 16 S rRNA

Thirty days after the final P. gingivalis infection, DNA was extracted from whole gingival tissues from the upper and lower jaws using a QIAamp DNA Mini Kit (Qiagen). Quantification of P. gingivalis or LG2055 was performed by real-time PCR using P. gingivalis- or LG2055-specific primers based on 16 S rRNA genes. The primer sequences were as follows: P. gingivalis forward (5′-AGGCAGCTTGCCATACTGCG-3′) and reverse (5′-ACTGTTAGCAACTACCGATGT-3′); and LG2055 forward (5′-AGCGACCGAGAAGAGAGAGA-3′) and reverse (5′-TGCTATCGCTTCAAGTGCTT-3′). The number of bacteria per weight of the gingival tissues was calculated.

In vitro culture

Acute monocytic leukemia (THP-1) cells were obtained from the JCRB Cell Bank (Health Science Research Resources Bank, Osaka, Japan) and cultured in RPMI 1640 containing 10% fetal bovine serum (Biofill), 10 mM HEPES, 100 μU/mL of penicillin, and 100 μg/mL of streptomycin (Invitrogen Corp.). Porphyromonas gingivalis antigen was prepared as described previously42. THP-1 cells (1 × 106/well) were pretreated with 0.5 μM PMA for 3 h, and then cultured with P. gingivalis antigen (1000 ng/mL) for 23 h. To assess the anti-inflammatory effects of β-defensin, cells were pre-incubated for 30 min with 0, 0.2, 1.0, and 5.0 µg/mL doses of recombinant human β-defensin-3 (rhBD-3) before adding antigen. The culture supernatants were then collected and subjected to IL-6- and TNF-α-specific ELISAs (R&D Systems Inc.).

Statistical analysis

All results are presented as means ± the standard errors of the mean (SEM), and experimental groups were compared with controls using an unpaired non-parametric Mann-Whitney U test in Statview software.