Oral infection with Porphyromonas gingivalis augmented gingival epithelial barrier molecules alteration with aging.

OBJECTIVE: Disruption of the gingival epithelial barrier is often mediated by aging or the pathogen Porphyromonas gingivalis. This study examined the combined effects of aging and P. gingivalis exposure on gingival epithelial barrier molecules. METHODS: In vitro experiments involved treating young- and senescence-induced primary human gingival epithelial progenitor cells (HGEPp) with P. gingivalis lipopolysaccharide (LPS). Transepithelial electrical resistance (TER) and paracellular permeability were measured. In vivo, male C57BL/6J mice aged 10 (young) and 80 (old) weeks were divided into four groups: young, old, young with P. gingivalis (Pg-Young) inoculation, and old with P. gingivalis (Pg-Old) inoculation. P. gingivalis was inoculated orally thrice a week for 5 weeks. The mice were sacrificed 30 days after the last inoculation, and samples were collected for further procedures. The junctional molecules (Claudin-1, Claudin-2, E-cadherin, and Connexin) were analyzed for mRNA expression using qRT-PCR and protein production using western blotting and immunohistochemistry. The alveolar bone loss and inflammatory cytokine levels in gingival tissues were also assessed. RESULTS: LPS-treated senescent cells exhibited a pronounced reduction in TER, increased permeability to albumin protein, significant upregulation of Claudin-1 and Claudin-2, and significant downregulation of E-cadherin and Connexin. Furthermore, the Pg-Old group showed identical results with aging in addition to an increase in alveolar bone loss, significantly higher than that in the other groups. CONCLUSION: In conclusion, the host susceptibility to periodontal pathogens increases with age through changes in the gingival epithelial barrier molecules.

Ingenuity pathway analysis of gingival epithelial cells stimulated with estradiol and progesterone.

OBJECTIVE: Periodontal disease is a risk factor for preterm delivery, and elevated female hormone levels during pregnancy promote hormone-dependent periodontopathogenic bacterial growth and gingivitis. Although the saliva of pregnant women contains female hormones at elevated levels, their effects on the gingiva are poorly understood. Therefore, in this study, we investigated the effects of estradiol and progesterone stimulation on gingival epithelial cells via ingenuity pathway analysis. METHODS: Human gingival epithelial progenitors were cultured in a CnT-Prime medium; 17ß-estradiol (E2) and progesterone (P4) were used as the reagents. Cells treated with dimethyl sulfoxide alone were used as the control group. Cells in the control and experimental groups were incubated for 12 h. RNA was extracted from the cultured cells, RNA-Seq was performed, and pathway analysis was conducted. RESULTS: Differentially expressed genes were detected for 699 (over 2-fold increase) and 348 (decrease) genes in group E2 and for 1448 (increase) and 924 (decrease) genes in group P4 compared with those in the control group (FDR <0.05, n = 4). The z-scores of the pathways suggest that E2 and P4 increased the activity of the wound healing signaling pathway. The activation of this pathway was higher in the E2 and P4 groups than that in the control group. CONCLUSIONS: The results of this study suggest that estradiol and progesterone may affect gingival homeostasis and wound healing.

The effect of Porphyromonas gingivalis on the gut microbiome of mice in relation to aging.

BACKGROUND AND OBJECTIVE: The translocation of oral bacteria, including Porphyromonas gingivalis, to the gut has been shown to alter gut microbiome. However, the effect of P. gingivalis on gut microbiome in relation to aging has not been demonstrated. We hypothesize that P. gingivalis has more detrimental effect on gut environment with increased age. The objective of this study is to investigate the effect of P. gingivalis on gut environment using aged mice. MATERIALS AND METHODS: C57BL/6J mice aged 4 weeks (young) or 76 weeks (old) were divided into four groups: control-young, control-old, P. gingivalis-administered young, and P. gingivalis-administered old. P. gingivalis was orally administered thrice weekly for 5 weeks. At 30 days after the last P. gingivalis administration, 16S rRNA sequencing was performed to study the gut microbiome. The mRNA and protein expression of intestinal junctional barrier molecules and the levels of the inflammatory cytokines IL-1ß and TNF-α in the serum were evaluated. RESULTS: Significant differences in the gut microbiomes between the groups, in terms of taxonomic abundance, bacterial diversity, and predicted metagenome function, were observed. A significant reduction in the alpha diversity and in the abundance of beneficial bacteria, such as Akkermansia and Clostridiaceae, in the P. gingivalis-administered old mice was observed. The mRNA and protein levels of Claudin-1 and Claudin-2 in the intestine were significantly elevated, while E-cadherin was significantly downregulated in the P. gingivalis-administered old mice, as were the serum levels of IL-1ß and TNF-α. CONCLUSION: The effect of P. gingivalis on the gut environment is more pronounced in old mice than in young mice.

Increase in Bifidobacterium is a characteristic of the difference in the salivary microbiota of pregnant and non-pregnant women.

BACKGROUND: The establishment of symbiotic microbiota in pregnant women is important for both the mother and her offspring. Little is known about the salivary symbiotic bacteria in pregnancy, and analysis of composition of microbiome (ANCOM) is useful to detect small differences in the number of bacteria. The aim of this study was to investigate the differences in the salivary bacteria between healthy pregnant and non-pregnant women using ANCOM. METHODS: Unstimulated saliva samples were collected from 35 healthy pregnant women at 35 weeks gestation and 30 healthy non-pregnant women during menstruation. All participants underwent a periodontal examination. Estradiol and progesterone levels were examined by enzyme-linked immunosorbent assay. DNA extracted from the saliva was assessed by 16S ribosomal RNA amplicon sequencing and real-time PCR. RESULTS: Salivary estradiol and progesterone levels were significantly increased in pregnant women. The alpha and beta diversities were higher in pregnant women than in non-pregnant women. The largest effect size difference noted when the microbiota of the pregnant and non-pregnant women were analyzed was that for Bifidobacteriales. Levels of Bifidobacterium dentium, but not of Bifidobacterium adolescentis, were significantly increased in pregnant women, and the levels were significantly correlated with progesterone concentration. CONCLUSION: The results suggest that Bifidobacterium and progesterone levels are elevated in the saliva of healthy pregnant women compared with non-pregnant women.

Gut flora alterations due to lipopolysaccharide derived from Porphyromonas gingivalis.

Gut dysbiosis induces 'leaky gut,' a condition associated with diabetes, NASH, and various auto-immune diseases. Porphyromonas gingivalis is a periodontopathic bacterium which causes periodontal tissue breakdown, and often enters the systemic blood flow. Oral administration of P. gingivalis induced gut dysbiosis in mice model, but no systemic administration of P. gingivalis has been reported thus far. In the present study, we investigated the effect of P. gingivalis-derived lipopolysaccharide (Pg-LPS) on the intestinal flora of our established mouse model. Eight-week-old C57BL/6J mice were intraperitoneally administered Pg-LPS. Three months later, DNA was extracted from stool, and RNA from the small and large intestines. After euthanizing the mice, pathological sections of the intestinal tract were prepared and stained with hematoxylin and eosin (H&E). Tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, and IL-6 expression levels were evaluated using quantitative PCR. 16S rRNA gene PCR amplicon analysis data were acquired using NGS. Microbial diversity and composition were analyzed using Quantitative Insights into Microbial Ecology 2. Furthermore, alterations in microbial function were performed by PICRUSt2. No significant inflammatory changes were observed in the H&E. No significant differences in the mRNA levels of IL-1ß, IL-6, and TNF-α were observed between the groups. Pg-LPS administration decreased the abundance of Allobacterium in the gut. A predictive metagenomic analysis by PICRUSt2 and STAMP showed that 47 pathways increased and 17 pathways decreased after Pg-LPS administration. Systemic application of periodontal pathogens may cause changes in the intestinal flora which may affect the physiological functions of the intestinal tract.

Alterations in the oral microbiome of individuals with a healthy oral environment following COVID-19 vaccination.

BACKGROUND: Several reports suggest that the microbiome of the digestive system affects vaccine efficacy and that the severity of coronavirus disease (COVID-19) is associated with decreased diversity of the oral and/or intestinal microbiome. The present study examined the effects of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine on the oral microbiome. METHODS: Forty healthy Japanese oral healthcare personnel were recruited, and unstimulated saliva was collected before vaccination, after the 1st vaccination, and after the 2nd vaccination. Genomic DNA was extracted from saliva samples, and PCR amplicons of the 16S rRNA gene were analyzed using next-generation sequencing. Microbial diversity and composition were analyzed using Quantitative Insights into Microbial Ecology 2. In addition, alterations in microbial function were assessed using PICRUSt2. RESULTS: SARS-CoV-2 mRNA vaccination significantly increased oral bacterial diversity and significantly decreased the proportion of the genus Bacteroides. CONCLUSIONS: The SARS-CoV-2 mRNA vaccine alters the oral microbiome; accordingly, vaccination might have beneficial effects on oral health.

Metformin accelerates wound healing by Akt phosphorylation of gingival fibroblasts in insulin-resistant prediabetes mice.

BACKGROUND: This study aimed to investigate the effects of metformin on gingival wound healing in insulin-resistant prediabetes. METHODS: C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) for 10 weeks; half of the HFD mice were treated with metformin (HFD+ Met) for the last 2 weeks. Insulin and glucose tolerance tests were performed. The palatal gingiva (2.0 × 0.5 mm) was surgically removed adjacent to the maxillary molars. Post-surgical wound closure was histomorphometrically evaluated for 1 week. The mRNA expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the tissue were quantified by real-time polymerase chain reaction. In vitro, the proliferation and migration of human gingival fibroblasts (HGFs) cultured under high-glucose or control conditions with/without metformin were analyzed. Akt phosphorylation and VEGF expression following the insulin stimulation were evaluated with/without metformin in high-glucose or control media. RESULTS: HFD mice showed significantly higher plasma glucose levels and insulin resistance than ND mice. Gingival wound healing was delayed in HFD group compared with ND group but significantly improved in HFD + MET group. The decreased expression of VEGF and eNOS in HFD group was significantly elevated in the HFD + MET group. The proliferation and migration of HGFs were significantly impaired in high-glucose conditions compared with control; metformin treatment partially attenuated these effects. Metformin treatment significantly recovered the downregulated Akt phosphorylation and VEGF expression in high-glucose conditions. CONCLUSIONS: Metformin improved delayed gingival wound healing in insulin-resistant prediabetes by accelerating HGFs proliferation and migration via Akt phosphorylation in insulin signaling pathway.

Draft Genome Sequence of Neisseria mucosa Strain HSUH001, Isolated from an Aggressive Periodontal Lesion.

We report the draft genome sequence (143 contigs, with a total length of 2,424,805 bp and an N 50 value of 36,066 bp) of a bacterium isolated from an aggressive periodontal lesion in a patient. We assigned strain HSUH001 to Neisseria mucosa through a multilocus sequence analysis.

Evaluation of the Virulence of Aggregatibacter actinomycetemcomitans Through the Analysis of Leukotoxin.

Aggregatibacter actinomycetemcomitans is frequently isolated from localized aggressive periodontitis and periodontitis associated with systemic diseases. A. actinomycetemcomitans produces a leukotoxin, which induces apoptosis in human leukocytes. The leukotoxin expression is dependent on the upstream sequence, likely including the promoter, of the gene encoding leukotoxin; strains with the truncated/short upstream sequence express more leukotoxin than strains with the general/long upstream. This chapter addresses the determination of the type of the leukotoxin promoter by PCR analysis, and detection of the apoptosis in the coculture of human monocyte cell line (THP-1) with A. actinomycetemcomitans by the DNA ladder formation, membrane perturbation, and lactate dehydrogenase release.

Alteration of oral flora in Mongolian patients with Behçet's disease: a multicentre study.

OBJECTIVES: Behçet's disease (BD) is characterised by repeated acute inflammatory attacks with aphthous ulcers of the oral mucosa, uveitis of the eyes, skin symptoms, and genital ulcers. Although its aetiology is still unknown, there is evidence of the involvement of oral bacteria in systemic diseases. Various types of oral bacteria may be involved in the development and progression of BD. The present study investigated alterations in the oral flora of patients with BD in Mongolia. We collected saliva samples from the Mongolian BD group and healthy control (HC) group, and the oral flora were analysed using next-generation sequencer (NGS). METHODS: DNA was extracted from the unstimulated saliva samples from the 47 BD and 48 HC subjects. The DNA was amplified from the V3-V4 region of 16S rRNA using PCR, and the data were acquired using NGS. Based on the obtained data, we analysed the alpha diversity, beta diversity, and bacterial taxonomy of the salivary flora. RESULTS: Beta diversity differed significantly between the BD and HC flora, but no significant differences were observed in alpha diversity. We found that the proportions of three genera - an S24-7 family unknown species, a mitochondria family unknown species, and Akkermansia species associated with IL-10 production - were significantly lower in the BD than in the HC group. CONCLUSIONS: The reduced proportions of the S24-7 family and symbiotic Akkermansia species may be key phenomena in the oral flora of patients with BD.

The periodontopathic bacteria in placenta, saliva and subgingival plaque of threatened preterm labor and preterm low birth weight cases: a longitudinal study in Japanese pregnant women.

OBJECTIVES: This study determined the quantity of periodontopathic bacteria in saliva, subgingival plaque, and placenta on the threatened preterm labor (TPL) and preterm low birth weight (PLBW) subjects in order to identify specific periodontal pathogens with high association to adverse pregnancy outcomes. METHODS: We used real-time PCR with TaqMan probe and ELISA to detect the amount of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, and Prevotella intermedia in subgingival plaque, saliva, and placenta tissue, in addition to serum IgG titers against these bacteria in 28 patients with TPL and 36 healthy pregnant women. RESULTS: Thirteen of 64 births delivered PLBW infants. All 6 periodontopathic bacteria were detected in the placenta samples. The amount of F. nucleatum and detection frequency of T. denticola in placental samples was significantly higher in the TPL group than in the healthy group. Meanwhile, the age, anti-P. gingival IgG in serum, amount of P. gingivalis and T. forsythia in plaque samples, detection frequency of P. intermedia in saliva, and percentage of pocket probing depth ≥ 5 mm were higher in TPL-PLBW births than those in TPL-Healthy delivery (HD) group and/or in H-HD group. Ordinal logistic regression analysis revealed that the presence of F. nucleatum in placental tissues was significantly associated with TPL, while the maternal age was significantly associated with PLBW in TPL. CONCLUSION: Our findings suggested all 6 bacteria may access the placenta. The increased presence of F. nucleatum in placenta might be related to TPL, while advanced maternal age might be associated with PLBW in TPL. CLINICAL RELEVANCE: Periodontal therapy should be applied to reduce the deep periodontal pocket sites and the colonization of periodontal pathogens in high-risk population.

Usefulness of the newly developed artificial denture plaque for practical denture care training.

OBJECTIVES: The aim of this study was to investigate whether the newly developed artificial dental plaque (A-DP) is useful as an educational tool for denture care of dental hygienist that compared it with conventional artificial dental plaque from the viewpoint of practical skills. MATERIAL AND METHODS: The 125 dental hygienist school students and 26 dental hygienists who had clinical experience were subjected a practical training of denture plaque control using the conventional denture plaque (C-DP) and the A-DP. The questionnaires based on the semantic differential method were used to survey whether the A-DP is similar to the real denture plaque (R-DP). Factor analysis by rotation of promax was carried out. RESULTS: In the results of the factor analysis, the two factors could be detected in students and three factors in dental hygienists. The total score of each denture plaque was calculated for each factor, and correlation coefficient was examined. There was significant correlation between the A-DP and the R-DP at the first factors, both students and dental hygienists. C-DP was not similar to R-DP in all factors. CONCLUSIONS: These results suggested that A-DP resembles R-DP better than C-DP. It was concluded that the A-DP was similar to the R-DP and could be a potent educational tool for practical denture care.

The number of microvascular complications is associated with an increased risk for severity of periodontitis in type 2 diabetes patients: Results of a multicenter hospital-based cross-sectional study.

AIMS/INTRODUCTION: To explore the relationships between periodontitis and microvascular complications as well as glycemic control in type 2 diabetes patients. MATERIALS AND METHODS: This multicenter, hospital-based, cross-sectional study included 620 patients with type 2 diabetes. We compared the prevalence and severity of periodontitis between patients with ≥1 microvascular complication and those without microvascular complications. We also compared the prevalence and severity of periodontitis among patients with different degrees of glycemic control. RESULTS: After adjusting for confounding factors, multiple logistic regression analysis showed that the severity of periodontitis was significantly associated with the number of microvascular complications (odds ratio 1.3, 95% confidence interval 1.1-1.6), glycated hemoglobin ≥8.0% (64 mmol/mol; odds ratio 1.6; 95% confidence interval 1.1-2.3), and older age (≥50 years; odds ratio 1.7; 95% confidence interval 1.1-2.6). However, the prevalence of periodontitis was not significantly associated with the number of microvascular complications, but was associated with male sex, high glycated hemoglobin (≥8.0% [64 mmol/mol]), older age (≥40 years), longer duration of diabetes (≥15 years) and fewer teeth (≤25). Furthermore, propensity score matching for age, sex, diabetes duration and glycated hemoglobin showed that the incidence of severe periodontitis was significantly higher among patients with microvascular complications than among those without microvascular complications (P < 0.05). CONCLUSIONS: The number of microvascular complications is a risk factor for more severe periodontitis in patients with type 2 diabetes, whereas poor glycemic control is a risk factor for increased prevalence and severity of periodontitis.

ß2-Glycoprotein I-Dependent Anti-Cardiolipin Antibodies Associated With Periodontitis in Patients With Systemic Lupus Erythematosus.

BACKGROUND: It was reported that patients with systemic lupus erythematosus (SLE) exhibited increased levels of anticardiolipin (anti-CL) antibodies, a class of antiphospholipid antibodies associated with thrombosis. ß2-glycoprotein I (ß2GPI) has been considered as the actual target antigen for anti-CL antibodies. This study investigates the association of periodontal infection with anti-CL antibodies in patients with SLE. METHODS: Fifty-three SLE female patients and 56 healthy female volunteers were recruited in this case-control study. All participants received periodontal examinations. The presence of Porphyromonas gingivalis and Treponema denticola in saliva and plaque samples was detected by polymerase chain reaction. Levels of serum anti-CL and anti-ß2GPI antibodies were examined using enzyme-linked immunosorbent assay. RESULTS: Patients with SLE exhibited more periodontal attachment loss and increased titers of serum anti-CL and anti-ß2GPI antibodies compared with healthy controls. Patients with active SLE who harbored P. gingivalis or P. gingivalis together with T. denticola intraorally exhibited significantly higher anti-CL and anti-ß2GPI antibodies than those without these bacteria. Anti-CL and anti-ß2GPI antibody levels correlated positively with clinical attachment level. Furthermore, increased anti-ß2GPI antibody levels were significantly associated with C-reactive protein and erythrocyte sedimentation rate. CONCLUSIONS: Elevated anti-CL and anti-ß2GPI antibody levels were associated with periodontopathic bacteria and periodontal breakdown in patients with SLE. Periodontitis might be a modifiable risk factor for SLE.

Periodontal treatment with topical antibiotics improves glycemic control in association with elevated serum adiponectin in patients with type 2 diabetes mellitus.

OBJECTIVES: Chronic inflammation of periodontitis aggravates glycemic control in type 2 diabetic patients through aggravation of insulin resistance. Increased or decreased release of various inflammatory mediators, such as high sensitivity C-reactive protein (hs-CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-6 and adipokines, such as adiponectin, leptin, and resistin, are presumed to be responsible for developing and progressing insulin resistance. The purpose of this study was to examine the effects of periodontal treatment on glycemic control, serum inflammatory mediators and adipokines in type 2 diabetes patients with periodontitis. METHODS: Twenty-one type 2 diabetic patients with periodontitis received periodontal treatment with topical antibiotics (intervention group) and 8 patients did not receive periodontal treatment (control group). Periodontal examination, including probing pocket depth (PPD) and bleeding on probing (BOP), and blood sampling were performed at baseline, 2 and 6 months after periodontal treatments. Glycated hemoglobin (HbA1c), hs-CRP, TNF-α, IL-6, adiponectin, leptin, and resistin were analyzed. RESULTS: In the intervention group, improvements of PPD and BOP, decrease in HbA1c and elevation of serum adiponectin were observed, while in the control group, all parameters were not changed. Generalized linear model revealed that changes of serum adiponectin and TNF-α and change of BOP correlated significantly with the reduction of HbA1c at 6 months after periodontal treatments. CONCLUSION: The results demonstrated that periodontal treatment improves periodontal status and glycemic control with elevation of serum adiponectin in type 2 diabetic patients. The results suggest that HbA1c is reduced by amelioration of insulin resistance due to elevated serum adiponectin after periodontal treatments.

Effect of glycemic control on periodontitis in type 2 diabetic patients with periodontal disease.

AIMS/INTRODUCTION: Diabetes mellitus and periodontitis are closely related. A huge number of reports has addressed the effect of periodontal intervention therapy on glycemic control, but no reports have addressed the effect of glycemic intervention therapy on periodontal disease in type 2 diabetic patients. The aim of this study was to examine the effect of improved glycemic control by glycemic intervention therapy on periodontitis in type 2 diabetic patients. MATERIALS AND METHODS: A total of 35 patients underwent intervention therapy to improve glycemic control without periodontal treatment. Glycohemoglobin (HbA1c), high-sensitivity C-reactive protein (hs-CRP), bleeding on probing (BOP), probing pocket depth (PPD) and intraoral community periodontal index (CPI) codes of the World health Organization (WHO) were examined at baseline, and 2 and 6 months after the intervention therapy to improve glycemic control. RESULTS: After the improvement of glycemic control, BOP lesions improved, but deep PPD lesions and WHO CPI codes did not improve. Subanalyses showed that effective glycemic control (average HbA1c reduction 1.8%) improved BOP lesions, but did not affect deep PPD lesions and WHO CPI codes. In addition, high BOP lesions at baseline responded more effectively to glycemic intervention. Further analysis of CPI codes in all individual periodontal sites independent of WHO CPI codes in 35 patients showed that only gingival inflammation without a deep periodontal pocket improved after glycemic intervention. CONCLUSIONS: Effective glycemic control improves BOP lesions in type 2 diabetic patients with periodontitis through ameliorating inflammation at the gingival sites of periodontal tissue. This trial was registered with the University Hospital Medical Information Network (no. UMIN000007670).

The anti-phospholipid antibody-dependent and independent effects of periodontopathic bacteria on threatened preterm labor and preterm birth.

PURPOSE: Periodontal disease is considered to be a risk factor for threatened preterm labor (TPL) and preterm birth (PB), but pathogenic mechanisms have not yet been elucidated. We hypothesized that infection with periodontopathic bacteria may enhance thrombosis through molecular mimicry with TLRVYK peptides on beta-2 glycoprotein I, a target molecule in anti-phospholipid syndrome. This study aimed to examine the effects of periodontitis on TPL and PB. METHODS: Ninety-five pregnant women (47 TPL and 48 healthy subjects) participated. Periodontal clinical parameters and periodontopathic bacteria were examined. Molecular mimicry between TLRVYK peptides and homologous peptides on the periodontopathic bacteria was examined by enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal antibodies specific for the respective peptides (SIRVYK on Aggregatibacter actinomycetemcomitans, TLRIYT on Porphyromonus gingivalis, and TLALYK on Treponema denticola). Serum high-sensitivity C-reactive protein, anti-TLRVYK and anti-SIRVYK IgG antibodies were measured using ELISA. RESULTS: Among the rabbit antibodies specific for the bacterial homologous peptides, only anti-SIRVYK IgG antibody reacted with TLRVYK peptides. Multivariable analysis showed that anti-SIRVYK IgG antibody was significantly associated with diagnosis of TPL. Of 95 births, 14 (14.7 %) delivered preterm. The preterm birth rate was higher in the anti-SIRVYK IgG antibody >median group than in the ≤median group. Of the 47 TPL subjects 13 had PB, and ordinal logistic regression analysis revealed that past smoking, presence of P. gingivalis and anti-SIRVYK IgG antibody were significantly correlated with PB. CONCLUSIONS: Infection with P. gingivalis and the antibody response to SIRVYK might be associated with TPL and PB.

Induction of toll-like receptor expression by Porphyromonas gingivalis.

BACKGROUND: Toll-like receptors (TLRs) play pivotal roles in host immune responses and have been suggested to be involved in the development of many infectious diseases. In this study, the mRNA expression levels of TLR2, TLR4, and TLR9 and their relationship with periodontopathic bacteria in periodontal tissue are examined. Furthermore, the mechanism of TLR induction by Porphyromonas gingivalis is investigated in human gingival fibroblasts (HGFs). METHODS: Gingival tissue and subgingival plaque samples were collected from 19 patients with chronic periodontitis (CP) and 16 control individuals without periodontitis. Gene expression levels in the tissues and in HGFs were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The numbers of periodontopathic bacteria were determined by quantitative real-time PCR. RESULTS: The expression levels of TLR2 and TLR9 were significantly higher in the tissues of patients with CP compared to the tissues of control individuals. The mRNA levels of TLR2 and TLR9, but not TLR4, were positively correlated with the number of P. gingivalis in subgingival plaque. P. gingivalis sonicated extract, P. gingivalis lipopolysaccharide, P. gingivalis DNA, and tumor necrosis factor-α(TNF-α) could significantly upregulate the mRNA expression of TLR2 in HGFs. Furthermore, P. gingivalis-mediated TLR2 expression was suppressed by TNF-α antibody. CONCLUSIONS: This study suggests that P. gingivalis infection induces TLR2 and TLR9 upregulation in patients with CP. P. gingivalis-induced TLR2 expression in HGFs is partially dependent on TNF-α and may lead to sensitization of HGFs to bacterial components encountered in the periodontal microenvironment.

Involvement of Toll-like receptor 2 in apoptosis of Aggregatibacter actinomycetemcomitans-infected THP-1 cells.

BACKGROUND/PURPOSE: Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative bacterium that has been associated with aggressive periodontitis. A actinomycetemcomitans infection induces apoptosis in the human monocytic cell line THP-1, and p38 mitogen-activated protein kinase (p38) activity and tumor necrosis factor-α (TNF-α) production are enhanced by A actinomycetemcomitans infection. However, mechanisms governing the recognition of A actinomycetemcomitans by monocytes during apoptosis have not been elucidated. A actinomycetemcomitans cell wall components stimulate Toll-like receptor (TLR)2 and/or TLR4. The authors examined whether TLR2 and/or TLR4 were involved in the apoptosis of A actinomycetemcomitans-infected THP-1 cells. METHODS: A actinomycetemcomitans-infected THP-1 cells were transferred to six-well culture plates and incubated for 0 to 6 hours. In some experiments, THP-1 cells were incubated with anti-TLR2, anti-TLR4, or isotype control antibody (10 µg/mL) for 30 minutes prior to A actinomycetemcomitans infection. Expression of messenger RNA (mRNA) was examined by reverse transcription-polymerase chain reaction. Intracellular bacteria recovered from A actinomycetemcomitans-infected cells and apoptotic cells were detected by APOPercentage dye (Biocolor Ltd, Northern Ireland, UK) staining. Cellular p38 activity and TNF-α production were assessed with enzyme-linked immunosorbent assay. RESULTS: The expression of TLR2 mRNA was increased by A actinomycetemcomitans infection. Phagocytosis and apoptosis in A actinomycetemcomitans-infected THP-1 cells were inhibited by the addition of anti-TLR2 antibody. Also, anti-TLR2 antibody suppressed the activation of p38 and production of TNF-α in A actinomycetemcomitans-infected THP-1 cells. CONCLUSION: These results suggest that A actinomycetemcomitans induces increased expression of TLR2, leading to phagocytosis and apoptosis of THP-1 cells via p38 activation and TNF-α production.