Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease

Accumulated lines of evidence suggest that hyperimmune responses to periodontal bacteria result in the destruction of periodontal connective tissue and alveolar bone. The etiological roles of periodontal bacteria in the onset and progression of periodontal disease (PD) are well documented. However, the mechanism underlying the engagement of periodontal bacteria in RANKL-mediated alveolar bone resorption remains unclear. Therefore, this review article addresses three critical subjects. First, we discuss earlier studies of immune intervention, ultimately leading to the identification of bacteria-reactive lymphocytes as the cellular source of osteoclast-induction factor lymphokine (now called RANKL) in the context of periodontal bone resorption. Next, we consider (1) the effects of periodontal bacteria on RANKL production from a variety of adaptive immune effector cells, as well as fibroblasts, in inflamed periodontal tissue and (2) the bifunctional roles (upregulation vs. downregulation) of LPS produced from periodontal bacteria in a RANKL-induced osteoclast-signal pathway. Future studies in these two areas could lead to new therapeutic approaches for the management of PD by down-modulating RANKL production and/or RANKL-mediated osteoclastogenesis in the context of host immune responses against periodontal pathogenic bacteria

Osteocyte Network; a Negative Regulatory System for Bone Mass Augmented by the Induction of Rankl in Osteoblasts and Sost in Osteocytes at Unloading

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function

Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells

IL-10 expressing regulatory B cells (B10) play a key role in immune system balance by limiting excessive inflammatory responses. Effects of toll-like receptor signaling and co-stimulatory molecules on B10 activity during innate and adaptive immune responses are not fully understood. Objective This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro. Material and Methods Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively. Results P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. Conclusions P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses

GlmS and NagB Regulate Amino Sugar Metabolism in Opposing Directions and Affect Streptococcus mutans Virulence

Streptococcus mutans is a cariogenic pathogen that produces an extracellular polysaccharide (glucan) from dietary sugars, which allows it to establish a reproductive niche and secrete acids that degrade tooth enamel. While two enzymes (GlmS and NagB) are known to be key factors affecting the entrance of amino sugars into glycolysis and cell wall synthesis in several other bacteria, their roles in S. mutans remain unclear. Therefore, we investigated the roles of GlmS and NagB in S. mutans sugar metabolism and determined whether they have an effect on virulence. NagB expression increased in the presence of GlcNAc while GlmS expression decreased, suggesting that the regulation of these enzymes, which functionally oppose one another, is dependent on the concentration of environmental GlcNAc. A glmS-inactivated mutant could not grow in the absence of GlcNAc, while nagB-inactivated mutant growth was decreased in the presence of GlcNAc. Also, nagB inactivation was found to decrease the expression of virulence factors, including cell-surface protein antigen and glucosyltransferase, and to decrease biofilm formation and saliva-induced S. mutans aggregation, while glmS inactivation had the opposite effects on virulence factor expression and bacterial aggregation. Our results suggest that GlmS and NagB function in sugar metabolism in opposing directions, increasing and decreasing S. mutans virulence, respectively

A Hepatocellular Adenoma in a Diet-induced Obese Mouse

A hepatic nodule was noted in a C57BL/6J mouse with diet-induced obesity at 53 weeks of age. Macroscopically, a protruding yellowish white nodule was observed on the visceral surface of the left lateral lobe. Light microscopy demonstrated clear demarcation from the compressed adjacent parenchyma, with loss of the distinct lobular pattern. The proliferating cells of the lesion varied in shape and showed cellular atypia and prominent nucleoli along with vacuoles of various sizes. Some of the cells contained various-sized eosinophilic inclusion bodies in their cytoplasm, and electron microscopy revealed the presence of lipid droplets in the rough endoplasmic reticulum. Eosinophilic inclusions were observed as electron dense granular material in the rough endoplasmic reticulum, with one or a few low density central cores. A diagnosis of hepatocellular adenoma was made based on these findings

Expression levels of novel cytokine IL-32 in periodontitis and its role in the suppression of IL-8 production by human gingival fibroblasts stimulated with Porphyromonas gingivalis

Background:IL-32 was recently found to be elevated in the tissue of rheumatoid arthritis and inflammatory bowel disease. Periodontitis is a chronic inflammatory disease caused by polymicrobial infections that result in soft tissue destruction and alveolar bone loss. Although IL-32 is also thought to be associated with periodontal disease, its expression and possible role in periodontal tissue remain unclear. Therefore, this study investigated the expression patterns of IL-32 in healthy and periodontally diseased gingival tissue. The expression of IL-32 in cultured human gingival fibroblasts (HGF) as well as effects of autocrine IL-32 on IL-8 production from HGF were also examined.Methods:Periodontal tissue was collected from both healthy volunteers and periodontitis patients, and immunofluorescent staining was performed in order to determine the production of IL-32. Using real-time PCR and ELISA, mRNA expression and protein production of IL-32 in HGF, stimulated by Porphyromonas gingivalis (Pg), were also investigated.Results:Contrary to our expectation, the production of IL-32 in the periodontitis patients was significantly lower than in the healthy volunteers. According to immunofluorescent microscopy, positive staining for IL-32 was detected in prickle and basal cell layers in the epithelium as well as fibroblastic cells in connective tissue. Addition of fixed Pg in vitro was found to suppress the otherwise constitutive expression of IL-32 mRNA and protein in HGF. However, recombinant IL-32 in vitro inhibited the expression of IL-8 mRNA by HGF stimulated with Pg. Interestingly, anti-IL-32 neutralizing antibody upregulated the IL-8 mRNA expression in non-stimulated HGF, indicating that constitutive expression of IL-32 in HGF suppressed IL-8 mRNA expression in the absence of bacterial stimulation.Conclusion:These results indicate that IL-32 is constitutively produced by HGF which can be suppressed by Pg and may play a role in the downregulation of inflammatory responses, such as IL-8 production, in periodontal tissue

Clinical correlations with Porphyromonas gingivalis antibody responses in patients with early rheumatoid arthritis

Introduction: Prior studies have demonstrated an increased frequency of antibodies to Porphyromonas gingivalis (Pg), a leading agent of periodontal disease, in rheumatoid arthritis (RA) patients. However, these patients generally had long-standing disease, and clinical associations with these antibodies were inconsistent. Our goal was to examine Pg antibody responses and their clinical associations in patients with early RA prior to and after disease-modifying antirheumatic drug (DMARD) therapy. Methods: Serum samples from 50 DMARD-naïve RA patients were tested using an enzyme-linked immunosorbent assay with whole-Pg sonicate. For comparison, serum samples were tested from patients with late RA, patients with other connective tissue diseases (CTDs), age-similar healthy hospital personnel and blood bank donors. Pg antibody responses in early RA patients were correlated with standard RA biomarkers, measures of disease activity and function. Results: At the time of enrollment, 17 (34%) of the 50 patients with early RA had positive immunoglobulin G (IgG) antibody responses to Pg, as did 13 (30%) of the 43 patients with late RA. RA patients had significantly higher Pg antibody responses than healthy hospital personnel and blood bank donors (P < 0.0001). Additionally, RA patients tended to have higher Pg antibody reactivity than patients with other CTDs (P = 0.1), and CTD patients tended to have higher Pg responses than healthy participants (P = 0.07). Compared with Pg antibody-negative patients, early RA patients with positive Pg responses more often had anti-cyclic citrullinated peptide (anti-CCP) antibody reactivity, their anti-CCP levels were significantly higher (P = 0.03) and the levels of anti-Pg antibodies correlated directly with anti-CCP levels (P < 0.01). Furthermore, at the time of study entry, the Pg-positive antibody group had greater rheumatoid factor values (P = 0.04) and higher inflammatory markers (erythrocyte sedimentation rate, or ESR) (P = 0.05), and they tended to have higher disease activity scores (Disease Activity Score based on 28-joint count (DAS28)-ESR and Clinical Disease Activity Index) and more functional impairment (Health Assessment Questionnaire). In Pg-positive patients, greater disease activity was still apparent after 12 months of DMARD therapy. Conclusions: A subset of early RA patients had positive Pg antibody responses. The responses correlated with anti-CCP antibody reactivity and to a lesser degree with ESR values. There was a trend toward greater disease activity in Pg-positive patients, and this trend remained after 12 months of DMARD therapy. These findings are consistent with a role for Pg in disease pathogenesis in a subset of RA patients

Bcl2 Deficiency Activates FoxO through Akt Inactivation and Accelerates Osteoblast Differentiation

Osteoblast apoptosis plays an important role in bone development and maintenance, and is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging. Although Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis, the physiological significance of Bcl2 in osteoblast differentiation has not been fully elucidated. To investigate this, we examined Bcl2-deficient (Bcl2(-/-)) mice. In Bcl2(-/-) mice, bromodeoxyuridine (BrdU)-positive osteoblasts were reduced in number, while terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive osteoblasts were increased. Unexpectedly, osteoblast differentiation was accelerated in Bcl2(-/-) mice as shown by the early appearance of osteocalcin-positive osteoblasts. Osteoblast differentiation was also accelerated in vitro when primary osteoblasts were seeded at a high concentration to minimize the reduction of the cell density by apoptosis during culture. FoxO transcription factors, whose activities are negatively regulated through the phosphorylation by Akt, play important roles in multiple cell events, including proliferation, death, differentiation, longevity, and stress response. Expressions of FasL, Gadd45a, and Bim, which are regulated by FoxOs, were upregulated; the expression and activity of FoxOs were enhanced; and the phosphorylation of Akt and that of FoxO1 and FoxO3a by Akt were reduced in Bcl2(-/-) calvariae. Further, the levels of p53 mRNA and protein were increased, and the expression of p53-target genes, Pten and Igfbp3 whose proteins inhibit Akt activation, was upregulated in Bcl2(-/-) calvariae. However, Pten but not Igfbp3 was upregulated in Bcl2(-/-) primary osteoblasts, and p53 induced Pten but not Igfbp3 in vitro. Silencing of either FoxO1 or FoxO3a inhibited and constitutively-active FoxO3a enhanced osteoblast differentiation. These findings suggest that Bcl2 deficiency induces and activates FoxOs through Akt inactivation, at least in part, by upregulating Pten expression through p53 in osteoblasts, and that the enhanced expression and activities of FoxOs may be one of the causes of accelerated osteoblast differentiation in Bcl2(-/-) mice

Overexpression of Bcl2 in Osteoblasts Inhibits Osteoblast Differentiation and Induces Osteocyte Apoptosis

Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis