Microhardness Measurements on Tooth and Alveolar Bone in Rodent Oral Disease Models.

The mechanical property, microhardness, is evaluated in dental enamel, dentin, and bone in oral disease models, including dental fluorosis and periodontitis. Micro-CT (µCT) provides 3D imaging information (volume and mineral density) and scanning electron microscopy (SEM) produces microstructure images (enamel prism and bone lacuna-canalicular). Complementarily to structural analysis by µCT and SEM, microhardness is one of the informative parameters to evaluate how structural changes alter mechanical properties. Despite being a useful parameter, studies on microhardness of alveolar bone in oral diseases are limited. To date, divergent microhardness measurement methods have been reported. Since microhardness values vary depending on the sample preparation (polishing and flat surface) and indentation sites, diverse protocols can cause discrepancies among studies. Standardization of the microhardness protocol is essential for consistent and accurate evaluation in oral disease models. In the present study, we demonstrate a standardized protocol for microhardness analysis in tooth and alveolar bone. Specimens used are as follows: for the dental fluorosis model, incisors were collected from mice treated with/without fluoride-containing water for 6 weeks; for ligature-induced periodontal bone resorption (L-PBR) model, alveolar bones with periodontal bone resorption were collected from mice ligated on the maxillary 2nd molar. At 2 weeks after the ligation, the maxilla was collected. Vickers hardness was analyzed in these specimens according to the standardized protocol. The protocol provides detailed materials and methods for resin embedding, serial polishing, and indentation sites for incisors and alveolar. To the best of our knowledge, this is the first standardized microhardness protocol to evaluate the mechanical properties of tooth and alveolar bone in rodent oral disease models.

Extracellular vesicles-Potential link between periodontal disease and diabetic complications.

It has long been suggested that a bidirectional impact exists between periodontitis and diabetes. Periodontitis may affect diabetes glycemic control, insulin resistance, and diabetic complications. Diabetes can worsen periodontitis by delaying wound healing and increasing the chance of infection. Extracellular vesicles (EVs) are heterogeneous particles of membrane-enclosed spherical structure secreted by eukaryotes and prokaryotes and play a key role in a variety of diseases. This review will introduce the biogenesis, release, and biological function of EVs from a microbial and host cell perspective, discuss the functional properties of EVs in the development of periodontitis and diabetes, and explore their role in the pathogenesis and clinical application of these two diseases. Their clinical implication and diagnostic value are also discussed.

The Three-Dimensional In Vitro Cell Culture Models in the Study of Oral Cancer Immune Microenvironment.

The onset and progression of oral cancer are accompanied by a dynamic interaction with the host immune system, and the immune cells within the tumor microenvironment play a pivotal role in the development of the tumor. By exploring the cellular immunity of oral cancer, we can gain insight into the contribution of both tumor cells and immune cells to tumorigenesis. This understanding is crucial for developing effective immunotherapeutic strategies to combat oral cancer. Studies of cancer immunology present unique challenges in terms of modeling due to the extraordinary complexity of the immune system. With its multitude of cellular components, each with distinct subtypes and various activation states, the immune system interacts with cancer cells and other components of the tumor, ultimately shaping the course of the disease. Conventional two-dimensional (2D) culture methods fall short of capturing these intricate cellular interactions. Mouse models enable us to learn about tumor biology in complicated and dynamic physiological systems but have limitations as the murine immune system differs significantly from that of humans. In light of these challenges, three-dimensional (3D) culture systems offer an alternative approach to studying cancer immunology and filling the existing gaps in available models. These 3D culture models provide a means to investigate complex cellular interactions that are difficult to replicate in 2D cultures. The direct study of the interaction between immune cells and cancer cells of human origin offers a more relevant and representative platform compared to mouse models, enabling advancements in our understanding of cancer immunology. This review explores commonly used 3D culture models and highlights their significant contributions to expanding our knowledge of cancer immunology. By harnessing the power of 3D culture systems, we can unlock new insights that pave the way for improved strategies in the battle against oral cancer.

CpG ODN/Mangiferin Dual Delivery through Calcium Alginate Hydrogels Inhibits Immune-Mediated Osteoclastogenesis and Promotes Alveolar Bone Regeneration in Mice.

The immune system plays an important role in the skeletal system during bone repair and regeneration. The controlled release of biological factors from the immune system could facilitate and optimize the bone remodeling process through the regulation of the activities of bone cells. This study aimed to determine the effect of the controlled delivery of immunomodulatory biologicals on bone regeneration. Immunostimulatory cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) and glucosylxanthone Mangiferin (MAG)-embedded microbeads were incubated with P. gingivalis-challenged splenocytes, or co-cultured with RAW264.7 cells. The effect of CpG ODN/MAG-containing microbeads on bone regeneration was then tested in vivo in a mouse alveolar bone defect model. The results demonstrated that MAG significantly antagonized P. gingivalis proliferation and reduced the live/dead cell ratio. After the addition of CpG ODN + MAG microbeads, anti-inflammatory cytokines IL-10 and IL-4 were upregulated on day 2 but not day 4, whereas pro-inflammatory cytokine IL-1ß responses showed no difference at both timepoints. RANKL production by splenocytes and TRAP+ cell formation of RAW264.7 cells were inhibited by the addition of CpG ODN + MAG microbeads. Alveolar bony defects, filled with CpG ODN + MAG microbeads, showed significantly increased new bone after 4 weeks. In summary, this study evaluated a new hydrogel-based regimen for the local delivery and controlled release of biologicals to repair and regenerate alveolar bony defects. The combined CpG ODN + MAG treatment may promote alveolar bone regeneration through the anti-microbial/anti-inflammatory effects and the inhibition of RANKL-mediated osteoclastogenesis.

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.

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.

Extracellular Release of Citrullinated Vimentin Directly Acts on Osteoclasts to Promote Bone Resorption in a Mouse Model of Periodontitis.

Elevated osteoclast (OC)-mediated bone resorption, a common pathological feature between periodontitis and rheumatoid arthritis (RA), implicates a possible mutually shared pathogenesis. The autoantibody to citrullinated vimentin (CV), a representative biomarker of RA, is reported to promote osteoclastogenesis (OC-genesis). However, its effect on OC-genesis in the context of periodontitis remains to be elucidated. In an in vitro experiment, the addition of exogenous CV upregulated the development of Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear OCs from mouse bone marrow cells and increased the formation of resorption pits. However, Cl-amidine, an irreversible pan-peptidyl arginine deiminase (PAD) inhibitor, suppressed the production and secretion of CV from RANKL-stimulated OC precursors, suggesting that the citrullination of vimentin occurs in OC precursors. On the other hand, the anti-vimentin neutralizing antibody suppressed in vitro Receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced OC-genesis. The CV-induced upregulation of OC-genesis was abrogated by the Protein kinase C (PKC)-δ inhibitor Rottlerin, accompanied by the downmodulation of OC-genesis-related genes, including Osteoclast stimulatory transmembrane protein (OC-STAMP), TRAP and Matrix Metallopeptidase 9 (MMP9) as well as extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP)-kinase phosphorylation. Elevated levels of soluble CV and vimentin-bearing mononuclear cells were found in the bone resorption lesions of periodontitis induced in mice in the absence of an anti-CV antibody. Finally, local injection of anti-vimentin neutralizing antibody suppressed the periodontal bone loss induced in mice. Collectively, these results indicated that the extracellular release of CV promoted OC-genesis and bone resorption in periodontitis.

Porphyromonas gingivalis outer membrane vesicles exacerbate retinal microvascular endothelial cell dysfunction in diabetic retinopathy.

Diabetic retinopathy (DR) is one of the leading causes of blindness. Periodontitis is one of the highest oral incidences and has been closely related to various systemic conditions through Porphyromonas gingivalis (P. gingivalis). P. gingivalis OMVs, derived from P. gingivalis, can cause endothelial dysfunction and potentially affect microvascular diseases. Current epidemiological studies provide limited evidence suggesting that periodontitis is associated with DR. However, there is a lack of basic research elucidating how periodontitis affects the severity of DR. This study aimed to explore the potential of P. gingivalis OMVs to contribute to the pathogenesis of DR and explore how it affect the retinal microvascular endothelium. The results demonstrated that P. gingivalis OMVs accelerated the blood-retinal barrier damage in DR mice. In vitro studies showed that the expression of inflammatory factors in human retinal microvascular endothelial cells (HRMECs) was increased after P. gingivalis OMVs stimulation, and the increased reactive oxygen species production, mitochondrial dysfunction, apoptosis, and altered endothelial permeability were observed in HRMECs under P. gingivalis OMVs stimulation. In addition, we found that protease-activated receptor-2 (PAR-2) regulated OMVs-induced TNF-α, MMP-9 mRNA expression, cell death, and endothelial permeability. Overall, we suggested that P. gingivalis OMVs induced mitochondria-related cell death of HRMECs and accelerated endothelial dysfunction, thus aggravating DR, in which PAR-2 plays a potential role. This study is the first research report to delineate the potential molecular mechanism of P. gingivalis OMVs on DR pathogenesis, which uniquely focused on elucidating the possible impact of periodontal pathogen derivatives on DR progression.

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.

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.

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.

Prevalence, General and Periodontal Risk Factors of Gastroesophageal Reflux Disease in China.

Purpose: There is insufficient information about the prevalence and risk factors of gastroesophageal reflux disease (GERD) in the Chinese adult population. We aimed to assess the prevalence and identify the risk factors of GERD in China. Methods: We collected data from a nationally representative sample (50,991 subjects) of Chinese adults from a large nation-wide cross-sectional survey. GERD was diagnosed by a standardized Chinese-language GERD questionnaire with a score of ≥ 8. The demographic characteristics, comorbidities and periodontal factors of all participants were collected. Results: Fifty-thousands-one-hundred-eighty-three participants were finally included in this study. The overall prevalence of GERD was 5.6% (95% CI, 5.4-5.8%) among the general Chinese population aged 20 years or older. Women, smokers, and people with older age, BMI ≥ 25.0 kg/m2, urban residence, lower education level or comorbidities were more prevalent with GERD (p < 0.001). Symptoms of severe periodontitis (OR = 1.40, 95% CI 1.28-1.52, p < 0.001) and lower frequency of tooth brushing (OR = 2.01, 95% CI 1.76-2.29, p < 0.001) were significantly associated with risk of GERD. Conclusion: Symptom-based GERD is highly prevalent in the Chinese population. Overweight and smoking are major preventable risk factors for GERD. Periodontal factors are novel potential risk factors for GERD and should be given more attention in GERD prevention.

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.

Intralesional and peripheral plasma of oral lichenoid reactions exhibit different cytokine profiles: A preliminary study.

BACKGROUND/PURPOSE: Oral lichenoid reactions (OLRs) are commonly characterized by the infiltration and activation of inflammatory cells at the interface of the oral mucosa. This study aimed to compare the cytokine profiles between intralesional and peripheral plasma from patients with OLRs and elucidate the cytokine profile in the OLR microenvironment. MATERIALS AND METHODS: A total of 26 paired intralesional and peripheral plasma samples were collected from patients with OLRs. A panel of 15 cytokines was measured using a Luminex assay. The reticular, erythema, and ulcerative score was used to evaluate the degree of OLR severity. RESULTS: IL-10 was detected in a fewer number of intralesional samples (19/26) compared to peripheral samples (26/26, p = 0.01). The intralesional plasma exhibited significantly elevated levels of granzyme B (median 108.94 vs. 16.00), TGF-ß1 (mean 30448.92 vs. 10199.04), TGF-ß2 (mean 1659.73 vs. 1308.49), and TGF-ß3 (mean 914.33 vs. 573.13) compared to the peripheral plasma (p = 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively). The levels of intralesional IL-2 (median 2.84 vs. 3.45, p = 0.019) and TNF-α (median 7.66 vs. 10.34, p = 0.048) were significantly lower in the intralesional plasma compared to the peripheral plasma. CONCLUSION: The intralesional concentrations of granzyme B and TGF-ß were elevated, whereas IL-2 and TNF-α were decreased in the OLR microenvironment compared to the peripheral plasma. These findings may contribute to establishing a panel of biomarkers that can be used to monitor the disease activity of OLRs in a large cohort study in future.

Glycemic fluctuation exacerbates inflammation and bone loss and alters microbiota profile around implants in diabetic mice with experimental peri-implantitis.

BACKGROUND: The impact of glycemic fluctuation under diabetic condition on peri-implantitis in diabetic patients remains unclear. We hypothesized that glycemic fluctuation has greater adverse effect on experimental peri-implantitis, compared with sustained high blood glucose in diabetes. RESULTS: Maxillary left first and second molars of diabetic db/db mice were extracted and were replaced with one dental implant in the healed edentulous space. Glycemic control or fluctuation were managed by constant or interrupted oral administration of rosiglitazone to these mice. Meanwhile, experimental peri-implantitis was induced by ligation around implants. After 14 weeks, inflammatory responses, and peri-implant bone loss, together with oral microbiota profile were analyzed. Diabetic mice with glycemic fluctuation showed greater peri-implant bone loss, inflammatory cell infiltration, and osteoclastogenesis, compared with mice with sustained hyperglycemia. Compared to sustained hyperglycemia, glycemic fluctuation led to further increase in IL-1ß, TNFα, RANKL, TLR2/4, IRAK1, and TRAF6 mRNA expression in peri-implant gingival tissues. Both rosiglitazone-induced glycemic control and glycemic fluctuation caused microbiota profile change in diabetic mice compared to that in uncontrolled hyperglycemic mice. CONCLUSIONS: This study suggests that glycemic fluctuation may aggravate peri-implantitis inflammation and bone loss, which may be associated with a shift in peri-implant microbial profile towards dysbiotic changes and the activation of TLR2/4-IRAK1-TRAF6 signaling.

MicroRNA-146a regulates the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis lipopolysaccharide.

OBJECTIVES: This study aimed to investigate the effects of microRNA-146a (miR-146a) on the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis (P.gingivalis) lipopolysaccharide (LPS). METHODS: Lymphocytes were harvested from mouse spleen and cultured in vitro. The cells were treated with P. gingivalis LPS, miR-146a mimic, or miR-146a inhibitor. Scramble RNA served as the negative control of mimic and inhibitor. The production of inflammatory cytokines was detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Compared with non-LPS-stimulated group, P. gingivalis LPS could increase the levels of interleukin (IL)-1ß, IL-6, receptor activator NF-κB ligand (RANKL), and IL-10 (P<0.05) and decrease the mRNA level of osteoprotectin (OPG) (P<0.05). However, it did not significantly change the secretion of OPG. Compared with the negative control group, miR-146a mimic upregulated the levels of IL-10 and OPG (P<0.05), downregulated IL-1ß, IL-6, and RANKL (P<0.05). Meanwhile, miR-146a inhibitor had a reverse effect on these cytokines (P<0.05) in P.gingivalis LPS-treated-lymphocytes. CONCLUSIONS: MiR-146a can provide a suitable microenvironment for bone formation by preventing the inflammatory effects of P.gingivalis LPS through the inhibition of IL-1ß, IL-6, and RNAKL, thereby enhancing IL-10 and OPG.

Local promotion of B10 function alleviates experimental periodontitis bone loss through antagonizing RANKL-expressing neutrophils.

BACKGROUND: Persistent host immune responses initiated by oral bacteria protect host against infection but may also elicit the process of sustained periodontal inflammation and subsequent alveolar bone loss. Interleukin-10 (IL-10), an anti-inflammatory cytokine, can downregulate pro-inflammatory cytokine and inhibit neutrophil migration in inflammation. IL-10-expressing regulatory B cells (B10) is termed by negatively regulating immune response through IL-10 and are mainly restricted in CD19+ CD1dhi CD5+ B cells in mice. Our current study was aimed to explore the effect of locally transferred CD19+ CD1dhi CD5+ B cells on inflammation and alveolar bone loss in an experimental periodontitis mouse model. METHODS: Ligation plus P. gingivalis (Pg) infection was used to induce periodontitis in a mouse model. CD19+ CD1dhi CD5+ B cells were sorted by flow cytometry and transferred into the gingivae immediately on the fifth day after ligation. All the mice were sacrificed on day 14 after ligation. RESULTS: H&E staining showed that inflammatory cell infiltration was significantly reduced by the CD19+ CD1dhi CD5+ B cells. Toluidine blue staining showed that the CD19+ CD1dhi CD5+ B cells alleviated alveolar bone loss in the ligature/Pg-induced periodontitis in mice. Immunohistochemical staining showed Receptor Activator of NF-KappaB Ligand (RANKL), Interleukin-1ß(IL-1ß) and Interleukin-17 (IL-17) were decreased after the CD19+ CD1dhi CD5+ B cell transfer. Immunofluorescent staining showed that IL-10 was increased while the number of Ly6G+ neutrophil and its RANKL production were decreased in gingival tissue. CONCLUSIONS: These results indicated that locally transferred CD19+ CD1dhi CD5+ B cells may alleviate alveolar bone loss through inhibiting pro-inflammatory cytokine expression and RANKL-expressing neutrophils in the periodontitis mouse model.

In-vivo imaging revealed antigen-directed gingival B10 infiltration in experimental periodontitis.

Our previous study demonstrated that IL-10 secreting B (B10) cells alleviate inflammation and bone loss in experimental periodontitis. The purpose of this study is to determine whether antigen-specificity is required for the local infiltration of B10 cells. Experimental periodontitis was induced in the recipient mice by placement of silk ligature with or without the presence of live Porphyromonas gingivalis (P. gingivalis). Donor mice were pre-immunized by intraperitoneal (IP) injection of formalin-fixed P. gingivalis, or PBS as non-immunized control. Spleen B cells were purified and treated with LPS and CpG for 48 h to expand the B10 population in vitro. Fluorescence-labelled B10 cells were transferred into the recipient mice by tail vein injection and were tracked on day 0, 3, 5 and 10 using IVIS Spectrum in vivo imaging system. The number of B10 cells and P. gingivalis-binding B cells were significantly increased after in vitro treatment of LPS and CpG. On day 5, the fluorescence intensity in gingival tissues was the highest in mice transferred with B10 cells from pre-immunized donor mice. Gingival expression of IL-6, TNF-α, RANKL/OPG ratio and periodontal bone loss in recipient mice were significantly reduced, and the expression of IL-10 and the number of CD19+ B cells were significantly increased after pre-immunized B10 cell transfer in the presence of antigen, compared to those with non-immunized B10 cell transfer or no antigen presence. This study suggests that antigen specificity dictate the local infiltration of B10 cells into periodontal tissue and these antigen-specific B10 cells promote anti-inflammatory responses.

Saliva Microbiome Changes in Patients With Periodontitis With and Without Chronic Obstructive Pulmonary Disease.

Objective: The oral microbiota plays a key part in the initial colonization by pathogens and the chronic inflammatory reaction of the host. We measured variations in the salivary microbiota and evaluated their potential associations with periodontitis and chronic obstructive pulmonary disease (COPD). Methods: We investigated the salivary microbiota of patients with COPD and periodontitis (n = 21) compared with that in patients with periodontitis alone (n = 36) and with healthy controls (HCs; n = 14), using pyrosequencing of polymerase chain reaction-amplified 16s rRNA genes. Results: Bacterial richness and diversity were significantly higher in patients suffering from COPD, and the bacterial family Lachnospiraceae was observed frequently only among patients with COPD and periodontitis. Veillonella, Rothia, Actinomyces, and Fusobacterium were the core bacterial genera that showed significant differences among patients with coincident COPD and periodontitis, patients with periodontitis alone, and HCs (p < 0.05). Veillonella, Rothia, and Actinomyces were observed much more frequently in patients with COPD and periodontitis, compared with that in HCs. All tested populations were divided into subgroups based on sex, smoking, or periodontitis index. In the subgroup with a bleeding index >2, Rothia was significantly different in periodontitis with and without COPD groups compared with HCs. In the subgroup with a plaque index >2.5, Rothia and Veillonella showed significant differences in periodontitis with and without COPD groups compared with HCs. Conclusion: Variations in salivary microbiota may be associated with COPD and periodontitis.

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.