Preliminary analysis of the buccal mucosal fungal microbiome in oral lichen planus patients.

OBJECTIVE: To determine the structure and co-occurrence patterns of mucosal fungal community in oral lichen planus (OLP) patients. SUBJECTS AND METHODS: Mucosal swab samples from 20 OLP patients and 10 healthy controls (HCs) were collected and the mucosal mycobiomes were sequenced. The abundance, frequency, and diversity of fungi were analyzed, as well as the inter-genera interactions. The associations between fungal genera and OLP severity were further identified. RESULTS: At the genus level, the relative abundance of unclassified_Trichocomaceae was significantly decreased in the reticular and erosive OLP groups compared to HCs. Meanwhile, significantly lower levels of Pseudozyma were observed in the reticular OLP group compared to HCs. The negative:positive cohesiveness ratio was significantly lower in the OLP group than HCs, indicating a relatively unstable fungal ecological system in the OLP group. In the OLP group, the abundance of unclassified_Nectriaceae was significantly correlated with the reticulation/erythema/ulceration (REU) score. CONCLUSIONS: Compared to HCs, the decreased stability of fungal communities and the decreased abundances of two genera (unclassified_Trichocomaceae and Pseudozyma) on buccal mucosa were identified in OLP patients.

Preliminary analysis of mucosal and salivary bacterial communities in oral lichen planus.

OBJECTIVE: To characterize the bacterial community from different oral niches (buccal mucosa and saliva) in oral lichen planus (OLP) patients. SUBJECTS AND METHODS: This preliminary study analyzed site-specific (mucosa and saliva) microbial landscape of 20 OLP patients and 10 healthy controls. RESULTS: The microbial diversity was similar between OLP patients and healthy controls in both salivary and mucosal communities. However, the topological properties of co-occurrence networks of salivary and mucosal microbiome were different between healthy controls and OLP patients. SparCC analysis inferred three and five keystone taxa in the salivary and mucosal microbial networks of healthy controls, respectively. However, in the salivary and mucosal bacterial networks of OLP patients, only one hub OTU and three OTUs were identified as keystone taxa, respectively. In addition, analysis of community cohesion revealed that mucosal microbial community in OLP patients had lower stability than that in healthy controls. In final, correlation assay showed that the clinical severity of OLP was positively associated with the relative abundance of Rothia in saliva but negatively associated with that of Porphyromonas on mucosa. CONCLUSIONS: The salivary and mucosal bacterial communities of OLP patients differ in terms of composition, the genera associated with OLP severity, and co-occurrence patterns.

Group 1 and 3 innate lymphoid cells are increased in oral lichen planus and oral lichenoid lesions.

OBJECTIVES: Innate lymphoid cells (ILCs) are vital innate immune cells cooperating with T cells. While their phenotypes and functions in oral mucosa kept unclear yet. In the present study, the relative proportions and distribution of different ILC subsets in oral mucosa of oral lichen planus (OLP), oral lichenoid lesions (OLL), and controls were compared. SUBJECTS AND METHODS: Oral mucosal samples were collected from control (n = 29), OLP (n = 20), and OLL (n = 22) donors. ILCs subsets were characterized in single-cell suspensions by flow cytometry. Immunohistochemistry was performed to locate the CD127+ cells in situ. RESULTS: ILCs were present in healthy and increased infiltration in OLP/OLL (p = 0.0092, p = 0.0216). Infiltration of ILC1 increased in OLP/OLL mucosa (p = 0.0225, p = 0.0399), as did the infiltration of ILC3 increase in OLL mucosa (p = 0.0128). The ILC2/ILCs ratio was significantly reduced in OLP and OLL (p = 0.0124, p = 0.0346). CD127+ cells were mainly located closely at the basement membrane. CONCLUSIONS: The results of increased ILC1, decreased ILC2, and increased ILC3 suggested that changes of ILC distributions in oral mucosa may be relevant to persistent inflammation in local tissues, by promoting immune factors and weakening repair capacity.

Association between symptoms of severe periodontitis and post-bronchodilator lung function: results from the China pulmonary health study.

BACKGROUND: The association between periodontitis and post-bronchodilator lung function is unclear. We aimed to determine the associations between symptoms of severe periodontitis (SSP) and post-bronchodilator lung function in the Chinese population. METHODS: A cross-sectional study (China Pulmonary Health study) was conducted from 2012 to 2015 in a large Chinese nationally representative sample of 49,202 participants aged 20-89 years. Data on demographic characteristics and periodontal symptoms of participants were collected by questionnaire. Participants who had at least one of the two severe symptoms (tooth mobility and natural tooth loss) in the past year were defined to have SSP, which was set as one variable for analyses. Post-bronchodilator lung function data including forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were collected by spirometry. RESULTS: The values of post-FEV1, post-FVC and post-FEV1/FVC of the participants with SSP were all significantly lower than the participants without SSP (all p < 0.001). SSP were significantly associated with post-FEV1/FVC < 0.7 (p < 0.001). In the multiple regression analyses, SSP were still negatively associated with post-FEV1(b = -0.04, 95%CI (-0.05 -0.03), p < 0.001), post-FEV1/FVC (b = -0.45, 95%CI (-0.63, -0.28), p < 0.001) and significantly associated with post-FEV1/FVC < 0.7 (OR = 1.08, 95%CI 1.01-1.16, p = 0.03) after full adjustment for potential confounders. CONCLUSIONS: Our data suggest that SSP were negatively associated with post-bronchodilator lung function in the Chinese population. Longitudinal cohort studies are needed to confirm these associations in the future.

TLR9 Signaling Is Required for the Porphyromonas gingivalis-Induced Activation of IL-10-Expressing B Cells.

Immune cell pattern-recognition receptors such as Toll-like receptors (TLRs) play important roles in the regulation of host responses to periodontal pathogens. Our previous studies have demonstrated that immune regulatory B cells were activated by TLRs and alleviated periodontitis inflammation and bone loss. The purpose of this study is to determine the role of TLR9 signaling in the activation and IL-10 production of the primed-immune B cells in vitro. Wild-type (WT) and TLR9 knockout (TLR9KO) mice (C57BL/6 background, n = 5) were pre-immunized intraperitoneally with 1 × 108 formalin-fixed P. gingivalis and boosted once with 1 × 107 formalin-fixed P. gingivalis. Isolated splenocytes and purified B cells from each mouse were cultured with 1 × 108 formalin-fixed P. gingivalis for 48 h. Immunocytochemistry was performed to detect CD45+ IL-10+ cells. Levels of IL-10 expression and secretion in splenocytes and B cells were detected using qRT-PCR and ELISA, respectively. After stimulation with fixed P. gingivalis, the percentage of CD45+ IL-10+ B cells and the level of IL-10 expression were significantly increased (p < 0.01) in splenocytes and purified B cells isolated from WT mice. However, these changes were not observed in splenocytes and purified B cells from TLR9KO mice when the cells were treated with fixed P. gingivalis. The percentage of CD45+ IL-10+ B cells was significantly reduced in splenocytes and purified B cells from TLR9KO mice compared to those from WT mice when challenged with P. gingivalis. IL-10 expression in B cells from TLR9KO mice was significantly decreased compared to those from WT mice at both the mRNA and protein levels. Additionally, P. gingivalis-induced up-regulation of TNF-α mRNA expressions were consistently observed in B cells from both WT and TLR9KO mice. P. gingivalis-induced B10 activation and IL-10 production during adaptive responses by primed B cells requires TLR9 signaling and can be achieved independent of T-cell help.

The Lysyl Oxidase G473A Polymorphism Exacerbates Oral Cancer Development in Humans and Mice.

Oral cancer is primarily squamous-cell carcinoma with a 5-year survival rate of approximately 50%. Lysyl oxidase (LOX) participates in collagen and elastin maturation. The propeptide of LOX is released as an 18 kDa protein (LOX-PP) in the extracellular environment by procollagen C-proteinases and has tumor-inhibitory properties. A polymorphism in the propeptide region of LOX (rs1800449, G473A) results in a single amino acid substitution of Gln for Arg. Here we investigated the frequency of rs1800449 in OSCC employing TCGA database resources and determined the kinetics and severity of precancerous oral lesion development in wildtype and corresponding knockin mice after exposure to 4-nitroquinoline oxide (4 NQO) in drinking water. Data show that the OSCC is more common in humans carrying the variant compared to the wildtype. Knockin mice are more susceptible to lesion development. The immunohistochemistry of LOX in mouse tissues and in vitro studies point to a negative feedback pathway of wildtype LOX-PP on LOX expression that is deficient in knockin mice. Data further demonstrate modulations of T cell phenotype in knockin mice toward a more tumor-permissive condition. Data provide initial evidence for rs1800449 as an oral cancer susceptibility biomarker and point to opportunities to better understand the functional mechanism of LOX-PP cancer inhibitory activity.

Potential association between Fusobacterium nucleatum enrichment on oral mucosal surface and oral lichen planus.

OBJECTIVE: We determined the bacterial community structure of the buccal mucosa in patients with oral lichen planus (OLP) and evaluated the potential association of Fusobacterium nucleatum with OLP. SUBJECTS AND METHODS: We collected buccal mucosal swab samples of patients with OLP (n = 20) and healthy controls (n = 10) and performed 16S rRNA gene sequencing and real-time PCR to determine potentially different bacteria. Damaged and adjacent non-damaged mucosal swab samples of 25 OLP patients were used to detect the amount of F. nucleatum by real-time PCR. RESULTS: Compared with healthy controls, enrichment of Fusobacterium and Granulicatella was more abundant in patients with OLP (p = .0146 and 0.0034). The abundance of Fusobacterium and F. nucleatum was significantly enriched on buccal mucosa of patients with OLP compared with healthy controls (p = .0043 and 0.0235). Compared with adjacent non-damaged buccal mucosa of OLP patients, the amount of F. nucleatum in the damaged mucosa was significantly increased (p = .001). We examined third-level KEGG pathways for bacteria on mucosal surface and found that genes controlling sporulation and ether lipid metabolism were enriched in patients with OLP. CONCLUSIONS: A high amount of F. nucleatum may be associated with OLP. Further studies are required to investigate the precise association of F. nucleatum with OLP.

Toll-Like Receptor Signaling and Immune Regulatory Lymphocytes in Periodontal Disease.

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.

A novel prognostic model for tongue squamous cell carcinoma based on the characteristics of tumour and its microenvironment: iBD score.

AIMS: Tumour budding and invasive depth can predict survival of patients with tongue squamous cell carcinoma (TSCC), while the prognostic value of tumour microenvironment (TME) remains unknown. Here, both characteristics of the tumour and its microenvironment were examined and a novel prognostic model has been proposed. METHODS AND RESULTS: A total of 246 patients with TSCC were included. Using H&E-stained sections, pathological parameters of tumour and the TME were assessed. Inflammatory response (i), tumour budding (B) and invasive depth (D) were combined as iBD score. The association between these variables and the patient survival was determined. Both tumour budding and inflammatory status were independent variables for predicting overall survival (OS) and disease-free survival (DFS) of TSCC patients. Invasive depth was correlated with differentiation, T classification, lymph node metastasis, clinical stage and recurrence (P < 0.05). The novel iBD model was strongly correlated with T classification, lymph node metastasis, clinical stage and recurrence, and showed clear distinction of scores 0, 1 and 2. High iBD score had a strong association with reduced OS and DFS (P < 0.01). CONCLUSIONS: The iBD scoring model is strongly associated with lymph node metastasis and recurrence in TSCC and could be a promising survival predictor for TSCC patients.

OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

Cell fusion-mediated formation of multinuclear osteoclasts (OCs) plays a key role in bone resorption. It is reported that 2 unique OC-specific fusogens [ i.e., OC-stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP)], and permissive fusogen CD9, are involved in OC fusion. In contrast to DC-STAMP-knockout (KO) mice, which show the osteopetrotic phenotype, OC-STAMP-KO mice show no difference in systemic bone mineral density. Nonetheless, according to the ligature-induced periodontitis model, significantly lower level of bone resorption was found in OC-STAMP-KO mice compared to WT mice. Anti-OC-STAMP-neutralizing mAb down-modulated in vitro: 1) the emergence of large multinuclear tartrate-resistant acid phosphatase-positive cells, 2) pit formation, and 3) mRNA and protein expression of CD9, but not DC-STAMP, in receptor activator of NF-κB ligand (RANKL)-stimulated OC precursor cells (OCps). While anti-DC-STAMP-mAb also down-regulated RANKL-induced osteoclastogenesis in vitro, it had no effect on CD9 expression. In our mouse model, systemic administration of anti-OC-STAMP-mAb suppressed the expression of CD9 mRNA, but not DC-STAMP mRNA, in periodontal tissue, along with diminished alveolar bone loss and reduced emergence of CD9+ OCps and tartrate-resistant acid phosphatase-positive multinuclear OCs. The present study demonstrated that OC-STAMP partners CD9 to promote periodontal bone destruction by up-regulation of fusion during osteoclastogenesis, suggesting that anti-OC-STAMP-mAb may lead to the development of a novel therapeutic regimen for periodontitis.-Ishii, T., Ruiz-Torruella, M., Ikeda, A., Shindo, S., Movila, A., Mawardi, H., Albassam, A., Kayal, R. A., Al-Dharrab, A. A., Egashira, K., Wisitrasameewong, W., Yamamoto, K., Mira, A. I., Sueishi, K., Han, X., Taubman, M. A., Miyamoto, T., Kawai, T. OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

Periodontal health: A national cross-sectional study of knowledge, attitudes and practices for the public oral health strategy in China.

AIM: To assess the status of periodontal health knowledge, attitudes and practices (KAP) among Chinese adults. MATERIALS AND METHODS: A cross-sectional study was conducted in a nationally representative sample of adults (N = 50,991) aged 20 years or older from ten provinces, autonomous regions, and municipalities. Percentages of Chinese adults with correct periodontal knowledge, positive periodontal attitudes, and practices were estimated. Multiple logistic regression analyses were used to examine the related factors. RESULTS: Less than 20% of Chinese adults were knowledgeable about periodontal disease. Very few (2.6%) of Chinese adults use dental floss ≥once a day and undergo scaling ≥once a year and visit a dentist (6.4%) in the case of gingival bleeding. Periodontal health KAP was associated with gender, age, body mass index, marital status, place of residence, education level, income, smoking status, and history of periodontal disease. CONCLUSIONS: Periodontal health KAP are generally poor among the Chinese adult population. Community-based health strategies to improve periodontal health KAP need to be implemented. Increasing knowledge of periodontal disease, the cultivation of correct practices in response to gingival bleeding, and the development of good habits concerning the use of dental floss and regular scaling should be public oral health priorities.

miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6.

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1ß, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1ß, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3' untranslated region (UTR) but not TRAF6 3'-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1ß, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.

Scattering center models of backscattering waves by dielectric spheroid objects.

Scattering center models provide a simple and effective way of describing the complex electromagnetic scattering phenomena of targets and have been successfully applied in radar applications. However, the existing models are limited to conducting objects. Numerical results show that scattering centers of dielectric objects are far more complex than conducting objects and most of them are distributed beyond the object. For the lossless and low-loss media, the major scattering contributions to total fields are surface waves and multiple internal reflections rather than the direct reflection. Concise scattering center models for backscattering from dielectric spheroid objects are proposed in this work, which can characterize the backscattered waves by scattering centers with sparse and physical parameters. Good agreement has been demonstrated between the high resolution range profiles simulated by this model with those obtained by Mie series and the full wave numerical method.

Soluble RANKL Cleaved from Activated Lymphocytes by TNF-α-Converting Enzyme Contributes to Osteoclastogenesis in Periodontitis.

Host immune responses play a key role in promoting bone resorption in periodontitis via receptor activator of NF-κB ligand (RANKL)-dependent osteoclastogenesis. Both membrane-bound RANKL (mRANKL) expressed on lymphocytes and soluble RANKL (sRANKL) are found in periodontal lesions. However, the underlying mechanism and cellular source of sRANKL release and its biological role in periodontitis are unclear. TNF-α-converting enzyme (TACE) is reported to cleave the following: 1) precursor TNF-α with release of mature, soluble TNF-α and 2) mRANKL with release of sRANKL. Both soluble TNF-α and sRANKL are found in the periodontitis lesion, leading to the hypothesis that TACE expressed on lymphocytes is engaged in RANKL shedding and that the resulting sRANKL induces osteoclastogenesis. In the current study, upon stimulating PBLs with mitogens in vitro, RANKL expression, sRANKL secretion, and TACE expression were all upregulated. Among the four putative mRANKL sheddases examined in neutralization assays, TACE was the only functional sheddase able to cleave mRANKL expressed on PBL. Moreover, PBL culture supernatant stimulated with mitogens in the presence of anti-TACE Ab or anti-RANKL Ab showed a marked reduction of osteoclastogenesis from osteoclast precursors, indicating that TACE-mediated sRANKL may possess sufficient osteoclastogenic activity. According to double-color confocal microscopy, B cells expressed a more pronounced level of RANKL and TACE expression than T cells or monocytes in periodontally diseased gingiva. Conditioned medium of patients' gingival lymphocyte culture increased in vitro osteoclastogenic activity, which was suppressed by the addition of anti-TACE Ab and anti-RANKL Ab. Therefore, TACE-mediated cleavage of sRANKL from activated lymphocytes, especially B cells, can promote osteoclastogenesis in periodontitis.

B10 Cells Alleviate Periodontal Bone Loss in Experimental Periodontitis.

B10 cells can regulate inflammatory responses in innate immunity. Toll-like receptors (TLRs) play an important role in B cell-mediated immune responses in periodontal disease. This study aimed to determine the effects of TLR-activated B10 cells on periodontal bone loss in experimental periodontitis. Spleen B cells isolated from C57BL/6J mice were cultured with Porphyromonas gingivalis lipopolysaccharide (LPS) and cytosine-phospho-guanine (CpG) oligodeoxynucleotides for 48 h. B10-enriched CD1dhi CD5+ B cells were sorted by flow cytometry and were adoptively transferred to recipient mice through tail vein injection. At the same time, P. gingivalis-soaked ligatures were placed subgingivally around the maxillary second molars and remained there for 2 weeks before the mice were euthanized. Interleukin-10 (IL-10) production and the percentage of CD1dhi CD5+ B cells were significantly increased with treatment with P. gingivalis LPS plus CpG compared to those in mice treated with P. gingivalis LPS or CpG alone. Mice with CD1dhi CD5+ B cell transfer demonstrated reduced periodontal bone loss compared to the no-transfer group and the group with CD1dlo CD5- B cell transfer. Gingival IL-10 mRNA expression was significantly increased, whereas expressions of receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG), tumor necrosis factor alpha (TNF-α), and IL-1ß were significantly inhibited in the CD1dhi CD5+ B cell transfer group. The percentages of CD19+ IL-10+ cells, CD19+ CD1dhi CD5+ cells, and P. gingivalis-binding CD19+ cells were significantly higher in recovered mononuclear cells from gingival tissues of the CD1dhi CD5+ B cell transfer group than in tissues of the no-transfer group and the CD1dlo CD5- B cell transfer group. This study indicated that the adoptive transfer of B10 cells alleviated periodontal inflammation and bone loss in experimental periodontitis in mice.

Local Induction of B Cell Interleukin-10 Competency Alleviates Inflammation and Bone Loss in Ligature-Induced Experimental Periodontitis in Mice.

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 periodontitis in 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 induction in 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 cells in vitro, although the frequencies of CD1dhi CD5+ and IL-10-producing (IL-10+) CD45+ cells were decreased. IL-10 was predominantly produced by the CD1dhi CD5+ 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-1ß, 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.

IL-21/anti-Tim1/CD40 ligand promotes B10 activity in vitro and alleviates bone loss in experimental periodontitis in vivo.

IL-10-expressing regulatory B cells (B10) play an essential role in immune system balance by suppressing excessive inflammatory responses. In this study, we investigated induction of B 10 cell's IL-10 competency in vitro and its effect on ligature-induced experimental periodontitis in vivo. Spleen B cells were isolated from C57BL/6J mice and cultured for 48h under the following conditions: control, CD40L, IL-21, anti-Tim1, CD40L+IL-21, CD40L+anti-Tim1, CD40L+IL-21+anti-Tim1. Silk ligatures were tied around both maxillary second molars of C57BL/6J mice for two weeks. Optimized combination of CD40L, IL-21 and anti-Tim1 and vehicle were injected into contralateral side of palatal gingiva on days 3, 6 and 9. The palatal gingival tissues and maxillary bone were collected on day 14 to determine expressions of IL-10 and periodontal bone resorption respectively. Our results demonstrated that IL-10 expressions of cultured spleen B cells were significantly increased in the presence of CD40L, IL-21 and anti-Tim1 combination when compared with control groups. Gingival IL-10 mRNA and protein expressions were significantly increased after injection of CD40L, IL-21 and anti-Tim1 combination, when compared to the control side. The gingival RANKL expression and periodontal bone loss were significantly decreased on the combination treatment side, as compared to the control side. These results suggest that combination of IL-21, anti-Tim1 and CD40L treatment induced B10 cell's IL-10 competency in vitro and inhibited periodontal bone loss in ligature-induced experimental periodontitis.

Phosphoglycerol dihydroceramide, a distinctive ceramide produced by Porphyromonas gingivalis, promotes RANKL-induced osteoclastogenesis by acting on non-muscle myosin II-A (Myh9), an osteoclast cell fusion regulatory factor.

Among several virulence factors produced by the periodontal pathogen Porphyromonas gingivalis (Pg), a recently identified novel class of dihydroceramide lipids that contains a long acyl-chain has the potential to play a pathogenic role in periodontitis because of its higher level of tissue penetration compared to other lipid classes produced by Pg. However, the possible impact of Pg ceramides on osteoclastogenesis is largely unknown. In the present study, we report that the phosphoglycerol dihydroceramide (PGDHC) isolated from Pg enhanced osteoclastogenesis in vitro and in vivo. Using RAW264.7 cells, in vitro assays indicated that PGDHC can promote RANKL-induced osteoclastogenesis by generating remarkably larger TRAP+ multinuclear osteoclasts compared to Pg LPS in a TLR2/4-independent manner. According to fluorescent confocal microscopy, co-localization of non-muscle myosin II-A (Myh9) and PGDHC was observed in the cytoplasm of osteoclasts, indicating the membrane-permeability of PGDHC. Loss- and gain-of-function assays using RNAi-based Myh9 gene silencing, as well as overexpression of the Myh9 gene, in RAW264.7 cells showed that interaction of PGDHC with Myh9 enhances RANKL-induced osteoclastogenesis. It was also demonstrated that PGDHC can upregulate the expression of dendritic cell-specific transmembrane protein (DC-STAMP), an important osteoclast fusogen, through signaling that involves Rac1, suggesting that interaction of PGDHC with Myh9 can elicit the cell signal that promotes osteoclast cell fusion. Taken together, our data indicated that PGDHC is a Pg-derived, cell-permeable ceramide that possesses a unique property of promoting osteoclastogenesis via interaction with Myh9 which, in turn, activates a Rac1/DC-STAMP pathway for upregulation of osteoclast cell fusion.

Porphyromonas gingivalis exacerbates ligature-induced, RANKL-dependent alveolar bone resorption via differential regulation of Toll-like receptor 2 (TLR2) and TLR4.

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-1ß (IL-1ß) 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.

Activation of Toll­like receptor 9 inhibits lipopolysaccharide­induced receptor activator of nuclear factor kappa­ B ligand expression in rat B lymphocytes.

B lymphocytes express multiple TLRs that regulate their cytokine production.We investigated the effect of TLR4 and TLR9 activation on receptor activator of NF­kB ligand (RANKL) expression by rat spleen B cells. Splenocytes or purified spleen B cells from Rowett rats were cultured with TLR4 ligand Escherichia coli LPS and/or TLR9 ligand CpG­oligodeoxynucleotide (CpG­ODN) for 2 days. RANKL mRNA expression and the percentage of RANKL­positive B cells were increased in rat splenocytes challenged by E. coli LPS alone. The increases were less pronounced when cells were treated with both CpG­ODN and E. coli LPS. Microarray analysis showed that expressions of multiple cyclin­dependent kinase (CDK) pathway­related genes were up­regulated only in cells treated with both E. coli LPS and CpG-ODN. This study suggests that CpG­ODN inhibits LPS­induced RANKL expression in rat B cells via regulation of the CDK pathway.