Toll-like receptors (TLRs) play a key role in the innate immune responses to periodontal pathogens in periodontal disease. The present study was performed to determine the roles of TLR2 and TLR4 signaling in alveolar bone resorption, using a Porphyromonas gingivalis -associated ligature-induced periodontitis model in mice. Wild-type (WT), Tlr2 ^−/− , and Tlr4 ^−/− mice (8 to 10 weeks old) in the C57/BL6 background were used. Silk ligatures were applied to the maxillary second molars in the presence or absence of live P. gingivalis infection. Ligatures were removed from the second molars on day 14, and mice were kept for another 2 weeks before sacrifice for final analysis (day 28). On day 14, there were no differences in alveolar bone resorption and gingival RANKL expression between mice treated with ligation plus P. gingivalis infection and mice treated with ligation alone. Gingival interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) expression was increased, whereas IL-10 expression was decreased in WT and Tlr2 ^−/− mice but not in Tlr4 ^−/− mice. On day 28, WT and Tlr4 ^−/− mice treated with ligation plus P. gingivalis infection showed significantly increased bone loss and gingival RANKL expression compared to those treated with ligation alone, whereas such an increase was diminished in Tlr2 ^−/− mice. Gingival TNF-α upregulation and IL-10 downregulation were observed only in WT and Tlr4 ^−/− mice, not in Tlr2 ^−/− mice. In all mice, bone resorption induced by ligation plus P. gingivalis infection was antagonized by local anti-RANKL antibody administration. This study suggests that P. gingivalis exacerbates ligature-induced, RANKL-dependent periodontal bone resorption via differential regulation of TLR2 and TLR4 signaling.

Periodontitis is known to be initiated by periodontal microbiota derived from biofilm formation. The microbial dysbiotic changes in the biofilm trigger the host immune and inflammatory responses that can be both beneficial for the protection of the host from infection, and detrimental to the host, causing tissue destruction. During this process, recognition of Pathogen-Associated Molecular Patterns (PAMPs) by the host Pattern Recognition Receptors (PRRs) such as Toll-like receptors (TLRs) play an essential role in the host–microbe interaction and the subsequent innate as well as adaptive responses. If persistent, the adverse interaction triggered by the host immune response to the microorganisms associated with periodontal biofilms is a direct cause of periodontal inflammation and bone loss. A large number of T and B lymphocytes are infiltrated in the diseased gingival tissues, which can secrete inflammatory mediators and activate the osteolytic pathways, promoting periodontal inflammation and bone resorption. On the other hand, there is evidence showing that immune regulatory T and B cells are present in the diseased tissue and can be induced for the enhancement of their anti-inflammatory effects. Changes and distribution of the T/B lymphocytes phenotype seem to be a key determinant of the periodontal disease outcome, as the functional activities of these cells not only shape up the overall immune response pattern, but may directly regulate the osteoimmunological balance. Therefore, interventional strategies targeting TLR signaling and immune regulatory T/B cells may be a promising approach to rebalance the immune response and alleviate bone loss in periodontal disease. In this review, we will examine the etiological role of TLR signaling and immune cell osteoclastogenic activity in the pathogenesis of periodontitis. More importantly, the protective effects of immune regulatory lymphocytes, particularly the activation and functional role of IL-10 expressing regulatory B cells, will be discussed.

Porphyromonas gingivalis is one of the oral microorganisms associated with human chronic periodontitis. The purpose of this study is to determine the role of the receptor activator of nuclear factor-κB ligand (RANKL) in P. gingivalis infection-associated periodontal bone resorption. Inbred female Rowett rats were infected orally on four consecutive days (days 0 to 3) with 1 × 10 ⁹ P. gingivalis bacteria (strain ATCC 33277). Separate groups of rats also received an injection of anti-RANKL antibody, osteoprotegerin fusion protein (OPG-Fc), or a control fusion protein (L6-Fc) into gingival papillae in addition to P. gingivalis infection. Robust serum IgG and salivary IgA antibody ( P < 0.01) and T cell proliferation ( P < 0.05) responses to P. gingivalis were detected at day 7 and peaked at day 28 in P. gingivalis -infected rats. Both the concentration of soluble RANKL (sRANKL) in rat gingival tissues ( P < 0.01) and periodontal bone resorption ( P < 0.05) were significantly elevated at day 28 in the P. gingivalis -infected group compared to levels in the uninfected group. Correspondingly, RANKL-expressing T and B cells in rat gingival tissues were significantly increased at day 28 in the P. gingivalis -infected group compared to the levels in the uninfected group ( P < 0.01). Injection of anti-RANKL antibody ( P < 0.05) or OPG-Fc ( P < 0.01), but not L6-Fc, into rat gingival papillae after P. gingivalis infection resulted in significantly reduced periodontal bone resorption. This study suggests that P. gingivalis infection-associated periodontal bone resorption is RANKL dependent and is accompanied by increased local infiltration of RANKL-expressing T and B cells.

Activated T and B lymphocytes in periodontal disease lesions express receptor activator of NF-κB ligand (RANKL), which induces osteoclastic bone resorption. The objective of this study was to evaluate the effects of anti-RANKL antibody on periodontal bone resorptionin vitroandin vivo. Aggregatibacter actinomycetemcomitansouter membrane protein 29 (Omp29) andA. actinomycetemcomitanslipopolysaccharide (LPS) were injected into 3 palatal gingival sites, and Omp29-specific T clone cells were transferred into the tail veins of rats. Rabbit anti-RANKL IgG antibody or F(ab′)₂antibody fragments thereof were injected into the palatal sites in each rat (days −1, 1, and 3). Anti-RANKL IgG antibody significantly inhibited soluble RANKL (sRANKL)-induced osteoclastogenesisin vitro, in a dose-dependent manner, but also gave rise to a rat antibody response to rabbit IgGin vivo, with no significant inhibition of periodontal bone resorption detected. Lower doses (1.5 and 0.15 μg/3 sites) of F(ab′)₂antibody were not immunogenic in the context of the experimental model. Periodontal bone resorption was inhibited significantly by injection of the anti-RANKL F(ab′)₂antibody into gingivae. The sRANKL concentrations for the antibody-treated groups were decreased significantly compared to those for the untreated group. Osteoclasts on the alveolar bone surface were also diminished significantly after antibody injection. Gingival sRANKL concentration and bone loss showed a significant correlation with one another in animals receiving anti-RANKL F(ab′)₂antibody. These results suggest that antibody to RANKL can inhibitA. actinomycetemcomitans-specific T cell-induced periodontal bone resorption by blockade and reduction of tissue sRANKL, providing an immunological approach to ameliorate immune cell-mediated periodontal bone resorption.

Interleukin-10 (IL-10)-producing B cells (B10 cells) play a critical role in the immune system balance by negatively regulating inflammatory responses. This study was conducted to determine the effect of local B10 cell induction on periodontal inflammation and bone loss in ligature-induced experimental periodontitisin vivo. Purified spleen B cells from C57BL/6J mice (8 to 10 weeks old) were cultured with CD40 ligand (CD40L) and the Toll-like receptor 9 (TLR9) agonist cytidine-phosphate-guanosine oligodeoxynucleotide (CpG) to determine effective IL-10 inductionin vitro. Silk ligatures (size 7-0) were tied around the mouse maxillary second molars on day 0, followed by the injection of CD40L and CpG into the palatal gingiva on days 3, 6, and 9. All the mice were sacrificed, and samples were collected on day 14. CD40L and CpG significantly increased the level of IL-10 production by B cellsin vitro, although the frequencies of CD1d^hiCD5⁺and IL-10-producing (IL-10⁺) CD45⁺cells were decreased. IL-10 was predominantly produced by the CD1d^hiCD5⁺subpopulation of B cells.In vivo, both IL-10 mRNA expression and the number of IL-10⁺CD45⁺cells were significantly increased after gingival injection of CD40L and CpG. Periodontal bone loss was significantly decreased and the gingival expression of IL-1β, tumor necrosis factor alpha, and RANKL was significantly reduced. The number of multinucleated tartrate-resistant acid phosphatase-positive cells along the alveolar bone surface was significantly decreased after gingival injection of CD40L and CpG. This study indicates for the first time that the local induction of B10 cell activity could inhibit periodontal inflammation and bone loss.