Porphyromonas gingivalis Infection-Associated Periodontal Bone Resorption Is Dependent on Receptor Activator of NF-κB Ligand

Rat oral infection with P. gingivalis.All animals were inbred female Rowett rats (Rnu^/+; 2 to 3 month old) maintained under pathogen-free conditions in laminar flow cabinets. Experiments using these animals were approved by the Forsyth Institute's Internal Animal Care and Use Committee (IACUC). In experiment 1, 36 animals were randomly divided into a control group (18 animals) and an infection group (18 animals). For the infection group, rats were orally infected with live Porphyromonas gingivalis bacteria (ATCC 33277). P. gingivalis was grown in Trypticase soy broth (BD Biosciences, San Diego, CA) containing 1% yeast extract, 5 μg/ml hemin, and 2.5 μg/ml menadione as previously described (22). Bacteria number in culture broth was determined by reading absorbance values using a spectrophotometer and comparing values to a curve derived from a standard plate count. The infection was administered by oral gavage using 10⁹ P. gingivalis bacteria per animal per day for four consecutive days (days 0 to 3). Briefly, an equal volume of sterile 2% (wt/vol) low-viscosity carboxymethylcellulose (CMC) was added and mixed thoroughly, and 1 ml of P. gingivalis culture was administered by oral gavage. The uninfected group was used as a control. Animals were sacrificed, and samples (serum, saliva, cervical lymph nodes [LN], and gingivae) were collected at days 7, 14, and 28 (n = 6 for each time point). In experiment 2, 40 animals were randomly distributed into the following five groups (n = 8 per group): group 1, control uninfected; group 2, P. gingivalis infection only; group 3, P. gingivalis infection and gingival injection of anti-RANKL (xRANKL) IgG antibody (1 μg/site); group 4, P. gingivalis infection and gingival injection of human osteoprotegerin fusion protein (OPG-Fc; 0.5 μg/site); or group 5, P. gingivalis infection and gingival injection of a control fusion protein (L6-Fc; 0.5 μg/site). Each rat received three palatal gingival injections (1 μl/site) on the mesial of the first molar and in the papillae between molars on both sides (three sites on each side) of the maxilla using a 28.5-gauge double-beveled MicroFine needle (Becton, Dickinson). The injections for animals in groups 3 to 5 were administered into rat palatal gingival papillae three times (day 5, day 9, and day 14). Experiments were terminated at day 28. A summary of the experimental protocol is shown in Fig. 1.

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Fig 1

Summary of experimental protocol. For experiment (Exp) 1, 36 animals were randomly divided into a control group (18 animals) and an infection group (18 animals), and after four consecutive days of infection, serum, saliva, cervical LN, and gingival samples from each animal were collected at days 7, 14, and 28 (n = 6 for each time point). For experiment 2, 40 animals were randomly divided into five groups (control, infection only, infection plus anti-RANKL IgG, infection plus OPG-Fc, and infection plus L6-Fc; n = 8 for each group). Samples were collected on day 28 after different treatments. For each group, gingival tissues and maxillae were collected from four animals, and another four animals were used for histological analyses. Ab, antibody; P. g., P. gingivalis.

ELISA.Rat serum and saliva samples were collected as previously described (22). Formalin-fixed P. gingivalis bacteria (10⁷ bacteria/well) were coated onto 96-well plates. For the detection of serum IgG antibody to P. gingivalis (n = 6), diluted rat serum (1:500) was applied to the plate, and rabbit (Rb) anti-rat IgG antibody was added, followed by alkaline phosphatase (ALP)-conjugated goat anti-rabbit IgG antibody (Sigma). For the detection of salivary IgA antibody to P. gingivalis (n = 6), diluted rat whole saliva (1:20) was applied to the plate, and mouse anti-rat IgA antibody (Biosource International, Camarillo, CA) was added, followed by ALP-conjugated goat anti-mouse IgG antibody (Sigma). Colorimetric reactions were developed with p-nitrophenyl phosphate (pNPP) substrate and measured at 405 nm using a spectrophotometer (Biotek, Winooski, VT). Enzyme-linked immunosorbent assay (ELISA) measurements were performed, and results are expressed as ELISA units (EU) based on a reference curve provided by dilution of an antibody-containing hyperimmune rat serum collected from rats immunized with P. gingivalis (18). Gingival tissues were homogenized with a Dounce glass homogenizer in phosphate-buffered saline (PBS) with 0.05% Tween 20, phenylmethylsulfonyl fluoride (Sigma), and proteinase inhibitor cocktail (Sigma) as described previously (5). The quantitation of soluble RANKL (sRANKL) in rat gingival homogenates (n = 6) was assayed in triplicate using a murine sRANKL ELISA development kit (Peprotech), and the concentration (pg/ml) of soluble RANKL in each sample was calculated from a standard curve.

T cell proliferation assay.Immune cells from rat cervical lymph nodes (n = 6) were isolated and cultured in 96-well plates (1 × 10⁴ cells/well) in triplicate in the presence of formalin-fixed P. gingivalis (1 × 10⁷ bacteria/well). [³H]thymidine (0.5 μCi/well) was added for the last 16 h of a 4-day culture. Samples were harvested onto glass fiber filters, and radioactivity (cpm) was measured in a liquid scintillation spectrometer (PerkinElmer, Waltham, MA).

Flow cytometry.Mononuclear cells were isolated from rat gingival tissues (n = 4) as described previously (19) and stained with the following monoclonal mouse anti-rat antibodies (AbD Serotec, Oxford, United Kingdom): OX33 (B cells), R73 (T cells), NK-RP1 (natural killer cells), and ED1 (macrophages). After cells were washed with PBS, they were stained with fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse IgG (Jackson ImmunoResearch). For the detection of membrane RANKL expression, cells were also stained with rabbit anti-RANKL IgG followed with phycoerythrin (PE)-conjugated goat anti-rabbit IgG (Molecular probes). FITC-conjugated mouse and rabbit IgG isotype controls (AbD Serotec, Oxford, United Kingdom) were used as negative controls. The number of positively stained cells in the total counted cells (20,000) was analyzed for each sample using an Epics Altra flow cytometer (Beckman Coulter).

TRAP assay.The mouse macrophage/monocyte cell line RAW 264.7 (ATCC TIB-71) was seeded in 96-well plates at a density of 10³ cells/well in 10% fetal bovine serum (FBS) containing Dulbecco's modified Eagle's medium (DMEM). RAW 264.7 cells were cultured for 5 days with 50 ng/ml murine recombinant RANKL (mrRANKL; R&D Systems) in the presence or absence of rabbit (Rb) normal serum IgG or Rb anti-RANKL IgG. In some experiments, RAW 264.7 cells were cultured for 2 days with mrRANKL (50 ng/ml). Then the medium was replaced with fresh medium without mrRANKL, and cells were cultured with rat gingival homogenates (n = 4) (0.25 μg/ml or 2.5 μg/ml) for another 3 days in the presence or absence of anti-RANKL IgG (1 μg/ml, 5 μg/ml, or 10 μg/ml). Cells were stained for tartrate-resistant acid phosphatase (TRAP) using a leukocyte acid phosphatase kit (Sigma) as previously described (1). TRAP-positive(TRAP⁺) cells with three or more nuclei were considered osteoclasts. TRAP⁺ multinuclear cells were counted, and results are expressed as numbers per well in a 96-well plate.

Measurement of alveolar bone resorption.After defleshing of the maxillary jaws, periodontal bone resorption was measured as previously described (1). Briefly, the distances from cemento-enamel junction (CEJ) to the alveolar bone (AvB) crest on the palatal side of each root were measured using a microscope with a reticule eyepiece under ×25 magnification. Recordings were made in the long axis of the root surfaces of all molar teeth. A total of 14 recordings were evaluated in each rat, including three roots of the first molar and both roots of the second and the third molars. Measurements were made by an examiner without prior knowledge of the group designation of the animals, and the recordings were verified by a second examiner. The bone resorption was expressed as total distance between the CEJ and the AvB crest per animal (n = 6 for experiment 1, and n = 4 for experiment 2).

Tissue preparation and histological TRAP staining.The maxillary samples from each group (n = 4 per group) were dissected and fixed in 4% formaldehyde overnight at 4°C. The specimens were then washed consecutively in 5, 10, and 15% glycerol in PBS and decalcified in 10% EDTA–0.1 M Tris for 3 weeks at 4°C. Decalcified samples were cut in half through the midline of the palate, embedded in optimal cutting temperature (OCT) compound (Miles, Elkhart, IN), and frozen at −80°C for 1 h. Serial sections at 6 μm were cut by cryostat sectioning. For TRAP staining, the slides were incubated for 20 to 40 min in the TRAP staining solution at 37°C in the dark until red staining was visible. The slides were then counterstained with hematoxylin. Osteoclasts were identified as multinucleated TRAP-positive cells, lying on the alveolar bone surface. For quantitative image analysis, the number of osteoclasts per millimeter of the alveolar bone surface was measured using an image analysis program (AxioVision; Carl Zeiss MicroImaging Inc., Thornwood, NY).

Quantitative RT-PCR (qRT-PCR).At the termination of experiment 2 (day 28), oral swabs from each rat were resolved in 200 μl of PBS containing protease inhibitor cocktail (Roche). DNA was extracted from each sample as previously described (22) and subjected to real-time PCR (RT-PCR) amplification of the DNA fragment using P. gingivalis 16S rRNA gene-specific primers (5′-GAGTTTGATYMTGGCTCAG-3′ and 5′-TCAGTCGCAGTATGGCAA-3′). A universal 16S rRNA gene primer pair was used to quantitate the total bacteria recovered from each animal (5′-GAGTTTGATYMTGGCTCAG and 5′-AAGGAGGTGWTCCARCC-3′). Amplification reactions were carried out in 25 μl of SYBR green reaction mix (Invitrogen) in an iCycler (Bio-Rad) according to the manufacturer's instructions. The quantity of P. gingivalis DNA or total bacterial DNA from each sample was extrapolated from the DNA standard curve derived from serial dilution of bacteria with known concentrations. P. gingivalis numbers were normalized to the total bacteria count in each sample. Each experiment was carried out in duplicate, and the data were obtained from all animals from each group (n = 8).

Statistical analysis.Results obtained from periodontal bone resorption and quantitation of P. gingivalis DNA are expressed as means ± standard deviations (SD). Data compiled from T cell proliferation assays, from ELISAs for serum IgG, salivary IgA, and sRANKL, from TRAP staining, and from flow cytometry are expressed as means ± standard errors (SE). A Student's t test was used to evaluate significance using the software Excel 2010 (Microsoft). P values of ≤0.05 were considered statistically significant.