Suk Ji

Toll-Like Receptor 2 and NALP2 Mediate Induction of Human Beta-Defensins by Fusobacterium nucleatum in Gingival Epithelial Cells

ABSTRACT We previously reported that infection by Fusobacterium nucleatum strongly induced the expression of both human beta-defensin 2 (HBD-2) and HBD-3 by gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptors (PRRs) and regulatory mechanisms involved in the induction of HBD-2 and -3 expression by F. nucleatum in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with live or heat-killed F. nucleatum, and the expression of HBDs and interleukin-8 (IL-8) was examined by real-time reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Live, but not heat-killed, F. nucleatum invaded HOK-16B cells, as seen by confocal microscopy and flow cytometry. Live F. nucleatum induced both HBD-2 and -3 efficiently, whereas heat-killed bacteria induced only HBD-3 at a reduced level. Knockdown of NACHT-LRR- and pyrin domain-containing protein 2 (NALP2), the most abundant intracellular PRR in HOK-16B cells, by RNA interference (RNAi) significantly reduced the induction of HBD-3 but not HBD-2 and IL-8. In addition, knockdown of Toll-like receptor 2 (TLR2) by RNAi reduced the upregulation of HBD-2 and -3 but not IL-8. Heat-killed F. nucleatum was hindered in its ability to activate TLR2 and JNK signaling pathways. Theses data show that TLR2 and NALP2 mediate the induction of HBDs by F. nucleatum in gingival epithelial cells, but thresholds for the two HBD genes are different.

Bacterial invasion and persistence: critical events in the pathogenesis of periodontitis?

Periodontitis is chronic inflammation of the periodontium caused by the hosts inflammatory response to plaque biofilm, which destroys tooth-supporting soft and hard tissues. Periodontitis is a complex disease that involves interactions among three main features - microbial challenge, the host immune response, and environmental and genetic risk factors - in its pathogenesis. Although periodontitis has been regarded as the result of hyperimmune or hyperinflammatory responses to plaque bacteria, recent studies indicate that periodontal pathogens are rather poor activators and/or suppressors of the host immune response. This raises the question of how periodontal pathogens cause inflammation. To resolve this issue, in the present review we propose that bacterial invasion into gingival tissue is a key event in the initiation of periodontitis and that the persistence of these bacteria within host tissue results in chronic inflammation. In support of this hypothesis, we present the ways in which microbial, environmental and genetic risk factors contribute to bacterial invasion. It is hoped that the current model will instigate active discussion and new research to complete the puzzle of this complex disease process.

Gingival fibroblasts from periodontitis patients exhibit inflammatory characteristics in vitro

OBJECTIVE Gingival fibroblasts (GFs) are an important regulatory cell type in the progression of periodontitis. This study aimed to compare the expression levels of genes associated with inflammation, extracellular matrix degradation and bone destruction in GFs isolated from healthy and periodontitis subjects in the absence and presence of Porphyromonas gingivalis. DESIGNS Primary GFs from healthy (n=10) and periodontitis subjects (n=10) were stimulated in vitro with viable P. gingivalis ATCC 49417 and 3 clinical isolates of P. gingivalis with type II fimbriae from one healthy subject (KUMC-H1) and two periodontitis patients (KUMC-P1, -P2). The mRNA expression of proinflammatory cytokines (interleukin (IL)-6, IL-8, IL-1B), anti-inflammatory cytokines (IL-4, IL-10), matrix metalloproteinase (MMP)-1 and 2, tissue inhibitor matrix metalloproteinase (TIMP)-3 and osteoprotegerin (OPG) were assessed using real-time PCR. The levels of IL-6, IL-1β and TIMP-3 protein were measured by an enzyme-linked immunosorbent assay. RESULTS The mRNA expression of IL-6, IL-1B and TIMP-3 was higher in the periodontitis group compared with the healthy group, whereas IL-4 expression was higher in the healthy group both in the absence and presence of the P. gingivalis strains. The expression levels of IL-6, IL-1β and TIMP-3 protein were also higher in the periodontitis group in the absence and/or presence of the P. gingivalis strains. There was inter-strain variability among P. gingivalis strains in the ability to induce expression of the proinflammatory cytokines, MMPs and OPG and in the ability to degrade IL-6 protein. CONCLUSION High expression of proinflammatory cytokines and TIMP-3 and low expression of IL-4 can be a signature of GFs associated with periodontitis.

Association of the invasion ability of Porphyromonas gingivalis with the severity of periodontitis

Porphyromonas gingivalis is one of the well-characterized periodontal pathogens involved in periodontitis. The invasive and proteolytic activities of P. gingivalis clinical isolates have been shown to be associated with heterogenic virulence, as determined in a mouse abscess model. The aims of the present study were to identify a P. gingivalis strain with a low virulence among clinical isolates, based on its invasive ability and cytokine proteolytic activities, and to explore the preferential degradation of a certain cytokine by P. gingivalis. P. gingivalis ATCC 33277, W50, and 10 clinical isolates were used. After incubating bacteria with IL-4, IL-6, IL-10, IL-17A, TNFα, IFNγ, and IL-1α, the amounts of remaining cytokines were determined by ELISA. Invasion ability was measured by a flow cytometric invasion assay. There was inter-strain variability both in the cytokine proteolytic activities and invasion ability. In addition, differential degradation of cytokines by P. gingivalis was observed: while IFNγ and IL-17A were almost completely degraded, inflammatory cytokines TNFα and IL-1α were less susceptible to degradation. Interestingly, the invasion index, but not cytokine proteolytic activities, of P. gingivalis had strong positive correlations with clinical parameters of subjects who harbored the isolates. Therefore, the invasive ability of P. gingivalis is an important virulence factor, and the bacterial invasion step may be a good target for new therapeutics of periodontitis.

Point-of-care diagnosis of periodontitis using saliva: technically feasible but still a challenge

Periodontitis is a chronic inflammation of the periodontium caused by persistent bacterial infection that leads to the breakdown of connective tissue and bone. Because the ability to reconstruct the periodontium is limited after alveolar bone loss, early diagnosis and intervention should be the primary goals of periodontal treatment. However, periodontitis often progresses without noticeable symptoms, and many patients do not seek professional dental care until the periodontal destruction progresses to the point of no return. Furthermore, the current diagnosis of periodontitis depends on time-consuming clinical measurements. Therefore, there is an unmet need for near-patient testing to diagnose periodontitis. Saliva is an optimal biological fluid to serve as a near-patient diagnostic tool for periodontitis. Recent developments in point-of-care (POC) testing indicate that a diagnostic test for periodontitis using saliva is now technically feasible. A number of promising salivary biomarkers associated with periodontitis have been reported. A panel of optimal biomarkers must be carefully selected based on the pathogenesis of periodontitis. The biggest hurdle for the POC diagnosis of periodontitis using saliva may be the process of validation in a large, diverse patient population. Therefore, we propose the organization of an International Consortium for Biomarkers of Periodontitis, which will gather efforts to identify, select, and validate salivary biomarkers for the diagnosis of periodontitis.

Risk assessment for clinical attachment loss of periodontal tissue in Korean adults

PURPOSE The purpose of this study was to assess the prevalence and extent of clinical attachment loss of periodontal tissue and to find out variables related to clinical attachment loss (CAL) in Korean adults older than 40 years of age. MATERIALS AND METHODS Data were collected from 2,519 subjects who were part of a cohort study conducted in Ansan city by Korea University Medical School for Korean Genome project. Age, sex, smoking, drinking, fast glucose, blood pressure, obesity and total cholesterol levels were examined. The oral examination included probing pocket depth, gingival recession and CAL of Ramfords teeth. The severity of periodontitis was classified based on the mean value of CAL. The relationship between each risk factor and the severity of CAL was independently estimated using the chi-square test, the test or one-way ANOVA. Multiple regression analysis was used to determine the significance of each factor in the periodontal disease. RESULTS The prevalences of clinical attachment between 1 and 3 mm, between 3 and < 5 mm, and ≥ 5 mm were 80.27%, 16.75% and < 1%, respectively. Although the univariate analysis showed age, gender, smoking, fasting glucose, blood pressure and total cholesterol levels were significantly related to the severity of CAL, multiple regression analysis indicated that age (P < .0001), gender (P < .0001) and smoking (P < .05) were only significantly related. CONCLUSION Older age, male gender and smoking were significant risk factor for the increase of CAL, and these may be useful indicators of periodontitis high-risk groups.

Increased bacterial invasion and differential expression of tight-junction proteins, growth factors, and growth factor receptors in periodontal lesions.

BACKGROUND Many pathogens are known to modulate epithelial physical barriers, particularly tight-junction (TJ) proteins, to enter host cells and/or tissues. Growth factors have been implicated in the regulation of TJ proteins. The aim of this study is to determine differences in the levels of TJ proteins, growth factors, and their receptors in relation to bacterial invasion in diseased gingival tissues obtained from patients with periodontitis. METHODS The presence of bacteria and expression of junctional adhesion molecule (JAM)-A, occludin, epidermal growth factor (EGF), keratinocyte growth factor (KGF), insulin-like growth factor-I (IGF-I), EGF receptor, KGF receptor, and IGF-1 receptor (IGF-1R) were evaluated in gingival tissues from healthy (n = 10) and diseased (n = 10) sites in patients with periodontitis by in situ hybridization and immunohistochemistry. RESULTS The bacterial invasion of gingival tissue was increased in periodontal lesions compared with healthy sites. Although the levels of JAM-A and occludin were not significantly different between the healthy and diseased sites, aberrant cytoplasmic expression of JAM-A and occluding was often observed in the lesions. In addition, more leukocytes expressing JAM-A or occludin were observed within the disease-associated epithelia. Compared with the healthy sites, the differential expression of KGF, IGF-I, and IGF-1R was observed in the periodontal lesions. The levels of TJ proteins showed positive correlations with those of growth factors. CONCLUSION The aberrant expression of growth factors and TJ proteins may contribute to increased bacterial invasion and disease progression in periodontal lesions.

Innate immune response to oral bacteria and the immune evasive characteristics of periodontal pathogens

Periodontitis is a chronic inflammation of periodontal tissue caused by subgingival plaque-associated bacteria. Periodontitis has long been understood to be the result of an excessive host response to plaque bacteria. In addition, periodontal pathogens have been regarded as the causative agents that induce a hyperinflammatory response from the host. In this brief review, host-microbe interaction of nonperiodontopathic versus periodontopathic bacteria with innate immune components encountered in the gingival sulcus will be described. In particular, we will describe the susceptibility of these microbes to antimicrobial peptides (AMPs) and phagocytosis by neutrophils, the induction of tissue-destructive mediators from neutrophils, the induction of AMPs and interleukin (IL)-8 from gingival epithelial cells, and the pattern recognition receptors that mediate the regulation of AMPs and IL-8 in gingival epithelial cells. This review indicates that true periodontal pathogens are poor activators/suppressors of a host immune response, and they evade host defense mechanisms.

Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling

Background: Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Results: JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. Conclusion: ODAM mediates JE attachment to healthy teeth. Significance: We investigate ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontal disease. Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin β3- and β6-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.

Relationship between tooth loss and carotid intima-media thickness in Korean adults

PURPOSE The aim of this study was to examine the relationship between tooth loss and sub-clinical atherosclerosis in Korean adults. MATERIALS AND METHODS The subjects were part of a cohort study conducted in Ansan city by the Korea University medical school as part of the Korean Genome project. 749 subjects over than 40 years old were evaluated. After taking panoramic radiography, the amount of tooth loss was calculated. The intima-media thickness (IMT) was assessed by using ultrasonography at the common carotid artery. Traditional cardiovascular risk factors for atherosclerosis were also evaluated. The relationship between tooth loss and the IMT was evaluated using ANOVA with Scheffes multiple comparison method in univariate analysis. Multiple regression analysis was also performed to determine the significance between the IMT and tooth loss. RESULTS With age, tooth loss increased, but there was no significant increase in other traditional cardiovascular risk factors. Univariate analysis revealed the IMT to be positively related with the amount of tooth loss. Regression analysis of the IMT in the anterior and posterior tooth loss revealed that only the posterior tooth loss was significantly related with the IMT at all sites of the common carotid artery (right far wall, P = .015; left far wall, P = .008; right near wall, P < .001; left near wall, P = .001). CONCLUSION This study verified the positive relationship between the increased tooth loss at the posterior area and the accumulation of atheroma in arteries.