DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines

Toll‐like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP‐1). THP‐1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA‐initiated cytokine production was determined either by blocking TLR9 signaling in THP‐1 cells with chloroquine or by measuring IL‐8 production and nuclear factor‐κB (NF‐κB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL‐1β, IL‐6, and TNF‐α) was increased significantly in bDNA‐stimulated cells compared with controls. Chloroquine treatment of THP‐1 cells decreased cytokine production, suggesting that TLR9‐mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9‐transfected cells demonstrated significantly increased IL‐8 production (P < 0.001) and NF‐κB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF‐κB genes in response to periodontal bDNA in THP‐1 cells, suggesting that cytokine induction is through NF‐κB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.     

Porphyromonas gingivalis Capsule‐Mediated Coaggregation as a Virulence Factor in Mixed Infection With Fusobacterium nucleatum

Background: Porphyromonas gingivalis (Pg) capsule enables evasion from phagocytosis, invasion of keratinocytes, and bacterial survival. In mixed infection, the capsule also participates in coaggregation, which may lead to characteristic virulence not present in the monoinfection. The aim of this study is to evaluate the role of Pg capsule as a virulence factor in coaggregated mixed infection with Fusobacterium nucleatum (Fn). Methods: Mixed infections containing Fn and non‐capsulated or capsulated strains of Pg were compared with the same infection with lactose as coaggregation inhibitor. Murine experimental periodontitis was used to assess disease severity. Primary polymorphonuclear leukocytes and keratinocytes were used to examine phagocytosis and bacterial invasion, respectively. Results: Mixed infection with capsulated Pg augmented alveolar bone loss compared with that of mixed infection with non‐capsulated Pg. Addition of lactose led to attenuation of bone loss in the capsulated mixed infection and to intensification of bone loss in the non‐capsulated mixed infection. In the latter mixed infection, Fn evaded phagocytosis, whereas in the capsulated mixed infection Pg displayed a greater capacity for invasion of keratinocytes. Conclusions: Pg capsule was found to serve as a unique virulence factor in mixed infection with Fn. Capsule‐dependent coaggregation led to augmented invasion of Pg and may be responsible for the severity of disease after mixed infection with Fn.     

Periodontal and Systemic Responses in Various Mice Models of Experimental Periodontitis: Respective Roles of Inflammation Duration and Porphyromonas gingivalis Infection

Background: The great variability of periodontal and systemic responses to experimental periodontitis reflects the inherent pathogenic complexity of mice models and could limit the resulting interpretations and their extension to human diseases. This study compared the effect of Porphyromonas gingivalis (Pg) infection and experimental periodontitis duration at local and systemic levels in various models. Methods: Periodontitis was induced in C57BL/6J mice by ligatures previously incubated with Pg (LIGPG group) or not (LIG group) or by oral gavage (GAV) with Pg ATCC 33277. Blood samples were taken, and mice were euthanized at different times. Periodontal tissue destruction, osteoclast number, and inflammation were assessed by histomorphometry, tartrate‐resistant acid phosphatase histoenzymology, and cathepsin B (CATB) and matrix metalloproteinase 9 (MMP9) immunochemistry. Serum levels of interleukin‐6 (IL‐6) and IL‐1β were measured using enzyme‐linked immunosorbent assay bioplex methods. Results: Periodontal tissue destruction and osteoclast numbers were significantly elevated in LIGPG models compared to LIG and GAV models. They increased with time with the exception of osteoclast numbers in the LIG model. CATB and MMP9 expression was related to bone destruction processes and Pg infection. The highest serum levels of IL‐6 and IL‐1β were observed in the LIGPG group. A decrease of IL‐6 and an increase of IL‐1β serum level were observed with time in LIGPG group contrary to LIG group. Conclusions: These data indicate that Pg infection worsened periodontal tissue destruction through specific pathogenic pathways and modified systemic response to periodontal inflammation. Furthermore, the blood cytokine response to ligature models showed their relevance for evaluating the systemic impact of periodontal disease.     

Peptide 19 of Porphyromonas gingivalis Heat Shock Protein Is a Potent Inducer of Low‐Density Lipoprotein Oxidation

Background: Although periodontal pathogens show a strong association with development of atherosclerosis, little is known about how a microorganism contributes to disease onset and progression. Oxidation of low‐density lipoprotein (LDL) is a major risk factor of atherogenesis. The principal objective of this study is to evaluate the ability of peptide 19 (Pep19) of Porphyromonas gingivalis (Pg) heat shock protein (HSP) as a potent inducer of LDL oxidation, and a secondary objective is to compare this ability with that of Pep19 from different bacteria. Methods: HSP60, Pep14, and Pep19 from Pg and THP‐1 monocytes were cultured, and the extent of LDL oxidation induced by each peptide was evaluated by an assay for thiobarbituric acid‐reactive substances (TBARS). Pep19 and HSP60 from Chlamydia pneumoniae and Mycobacterium tuberculosis were also cultured with THP‐1 monocytes and evaluated by the TBARS assay. After incubation of macrophages with LDL and peptides from Pg, Oil Red O staining was performed for examination of foam cells, macrophages that took up the oxidized LDL. Results: Monocyte‐mediated native‐LDL oxidation under the influence of Pep19 or HSP60 from Pg was significantly stronger than oxidation induced by the counterpart Pep19 or HSP60 from C. pneumoniae or M. tuberculosis. Pep19 from Pg HSP60 showed a stronger ability to induce LDL oxidation than did Pep14 from Pg HSP60. Conclusion: These results suggest Pep19 from Pg HSP60 has a distinct ability to induce native‐LDL oxidation as a plausible mechanism by which this peptide may drive epitope spreading to the neoantigen, i.e., oxidized LDL, in the pathogenesis of atherosclerosis.     

Role of p38 mitogen-activated protein kinase pathway in Porphyromonas gingivalis lipopolysaccharide-induced VCAM-1 expression in human aortic endothelial cells

Background: Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) has been reported to induce the expression of vascular cell adhesion molecule‐1 (VCAM‐1) in vascular endothelial cells. This finding suggests the potential roles for Pg in the pathogenesis of atherosclerosis. However, the mechanism involved in Pg LPS‐induced VCAM‐1 production in endothelial cells remains unclear. Methods: Quantitative real‐time polymerase chain reaction and Western blotting were used, respectively, to investigate the mRNA expression and protein production of VCAM‐1 in human aortic endothelial cells (HAECs) induced by Pg LPS. The involvement of the p38 mitogen‐activated protein kinase (p38 MAPK) cell signaling pathway in VCAM‐1 expression was investigated by assays with specific inhibitors. Results: Pg LPS–induced expression in HAECs of VCAM‐1 occurred in a dose‐ and time‐dependent manner. In addition, the p38 MAPK inhibitor (SB 203580) significantly attenuated Pg LPS–induced VCAM‐1 expression. Conclusion: Activation of p38 MAPK is at least partially involved in Pg LPS–induced VCAM‐1 expression in HAECs, which may contribute to the acceleration of atherosclerosis.     

Microbial Characteristics of Peri‐Implantitis: A Case‐Control Study

Background: The aim of this case‐control study is to compare oral microbiologic characteristics of patients with healthy peri‐implant conditions and patients with peri‐implantitis and to explore the influence of various patient‐ and implant‐related factors on microbiologic characteristics. Methods: Peri‐implant submucosal microbial samples were collected from 85 patients with peri‐implantitis (cases) and from 69 patients with only implants with healthy peri‐implant conditions (controls). Samples were analyzed using culturing techniques. Multivariable logistic regression was used to explore the association of disease status and various patient‐ and implant‐related factors (sex, patient age, smoking, number of remaining teeth, percentage of teeth with bone loss, implant function time, implant surface, and presence of plaque) with microbiologic characteristics. Results: Peri‐implant disease status was significantly associated with the submucosal presence of Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythia (Tf), and Fusobacterium nucleatum (Fn). The association with disease status was most obvious for Pi (odds ratio [OR]: 15.1; 95% confidence interval [CI]: 5.1 to 45.3) and Tf (OR: 13.3; 95% CI: 5.4 to 32.5). The prevalence of Aggregatibacter actinomycetemcomitans and Staphylococcus species was very low. Conclusions: The periodontal pathogens Pg, Pi, Tf, and Fn are associated with peri‐implantitis. A. actinomycetemcomitans and Staphylococcus species do not seem to play an important role in peri‐implantitis.     

Interferon‐Gamma and Fas Are Involved in Porphyromonas gingivalis–Induced Apoptosis of Human Extravillous Trophoblast‐Derived HTR8/SVneo Cells via Extracellular Signal‐Regulated Kinase 1/2 Pathway

Background: A number of studies recently revealed a link between periodontal disease and preterm birth (PTB). PTB can be induced by dental infection with Porphyromonas gingivalis (Pg), a periodontopathic bacterium. This study aims to investigate responses of human extravillous trophoblast‐derived HTR8/SVneo cells to Pg infection. Methods: Cell apoptosis, cell viability, protein expression, and cytokine production in HTR8 cells were measured via: 1) flow cytometry, 2) CCK‐8 assay, 3) western blot, and 4) enzyme‐linked immunosorbent assay methods, respectively. Results: Pg decreased cell viability and increased cell apoptosis, active caspase‐3 and Fas expression, and interferon‐gamma (IFN‐γ) secretion in HTR8 cells. Extracellular signal‐regulated kinase (ERK) 1/2 inhibitor U0126 and FasL neutralizing antibody NOK1 that blocks FasL/Fas interaction both significantly suppressed Pg‐induced apoptosis. U0126 also inhibited IFN‐γ secretion and Fas expression close to control levels. Moreover, treatment with recombinant IFN‐γ also significantly decreased number of viable HTR8 cells and increased Fas expression, suggesting IFN‐γ may play an important role in Pg‐induced apoptosis of HTR8 cells, at least partially through regulation of Fas expression. Conclusions: To the best of the authors’ knowledge, this is the first study to demonstrate Pg induces IFN‐γ secretion, Fas expression, and apoptosis in human extravillous trophoblast‐derived HTR8/SVneo cells in an ERK1/2‐dependent manner, and IFN‐γ (explored by recombinant IFN‐γ) and Fas are involved in Pg‐induced apoptosis. The finding that Pg infection abnormally regulates inflammation and apoptosis of human trophoblasts may give new insights into the possible link of PTB with maternal periodontal disease and periodontal pathogens.     

Epithelial Cells Secrete Interferon‐γ Which Suppresses Expression of Receptor Activator of Nuclear Factor Kappa‐B Ligand in Human Mandibular Osteoblast‐Like Cells

Background: Prostaglandin (PG)E₂ accumulates in inflamed periodontal tissue and induces receptor activator of nuclear factor kappa‐B ligand (RANKL)‐RANK‐osteoprotegerin (OPG) signaling associated with bone resorption. Although oral epithelial cells maintain tissue homeostasis, the role of these cells in RANKL regulation remains unknown. Methods: To mimic an inflamed condition, RANKL upregulation in human mandibular osteoblast‐like cells (HMOBs) were stimulated with PGE₂. Effect of recombinant human interferon (IFN)‐γ or epithelial‐derived IFN‐γ in constitutively released or Porphyromonas gingivalis lipopolysaccharide (PgLPS)‐stimulated epithelial supernatant was investigated in HMOBs. Some HMOBs were pretreated with an anti‐IFN‐γ antibody before PGE₂ stimulation. THP‐1 human monocytes and HMOBs were cocultured in a transwell system to investigate RANKL‐driven THP‐1 osteoclastic activity. Results: PGE₂ significantly increased RANKL messenger RNA (mRNA) and protein in HMOBs in a dose‐dependent manner, while OPG protein remained similar to baseline. Epithelial cells constitutively released IFN‐γ, which was substantially increased by PgLPS. HMOBs treated with epithelial supernatant or recombinant IFN‐γ, concurrently with PGE₂ stimulation, reduced RANKL, but not OPG, expression. In contrast, anti‐IFN‐γ antibody reversed the effect of epithelial mediators on RANKL expression. When cocultured with THP‐1, RANKL released by PGE₂‐stimulated HMOBs is adequate to drive THP‐1 differentiation as osteoclastogenic gene expression and bone resorption pit are increased. However, recombinant IFN‐γ, or IFN‐γ derived from oral epithelial cells, suppressed RANKL expression at both the mRNA and protein level, resulting in decreased THP‐1‐derived osteoclastic activity. Conclusion: Oral epithelial cells interact with HMOBs by releasing IFN‐γ to regulate RANKL expression and contribute to osteoclastogenesis.     

Major surface protein complex of Treponema denticola induces the production of tumor necrosis factor α, interleukin‐1β, interleukin‐6 and matrix metalloproteinase 9 by primary human peripheral blood monocytes

Gaibani P, Caroli F, Nucci C, Sambri V. Major surface protein complex of Treponema denticola induces the production of tumor necrosis factor α, interleukin‐1β, interleukin‐6 and matrix metalloproteinase 9 by primary human peripheral blood monocytes. J Periodont Res 2010; 45: 361–366. © 2010 John Wiley & Sons A/S Background and Objective: Treponema denticola is a micro‐organism that is involved in the pathogenesis of periodontitis. Major surface protein complex (MSPc), which is expressed on the envelope of this treponeme, plays a key role in the interaction between T. denticola and gingival cells. The peptidoglycan extracted from T. denticola induces the production of a large variety of inflammatory mediators by macrophage‐like cells, suggesting that individual components of T. denticola cells induce the inflammatory response during periodontal disease. This study was designed to demonstrate that MSPc of T. denticola stimulates release of proinflammatory mediators in primary human monocytes. Material and Methods: Primary human monocytes were separated from the blood of healthy donors and incubated for up to 24 h with varying concentrations of MSPc. The production of tumor necrosis factor α (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and matrix metalloproteinase 9 (MMP‐9) was measured at different time points with commercially available enzyme‐linked immunosorbent assays. Results: T. denticola MSPc induced the synthesis of TNF‐α, IL‐1β, IL‐6 and MMP‐9 in a dose‐ and time‐dependent manner. Similar patterns of TNF‐α, IL‐1β and IL‐6 release were observed when cells were stimulated with 100 and 1000 ng/mL of MSPc. The production of MMP‐9 was significant only when cells were treated with 1000 ng/mL of MSPc. Conclusion: These results indicate that T. denticola MSPc, at concentrations ranging from 100 ng/mL to 1.0 μg/mL, activates a proinflammatory response in primary human monocytes.     

Aggregatibacter actinomycetemcomitans‐Induced AIM2 Inflammasome Activation Is Suppressed by Xylitol in Differentiated THP‐1 Macrophages

Background: Aggressive periodontitis is characterized by rapid destruction of periodontal tissue caused by Aggregatibacter actinomycetemcomitans. Interleukin (IL)‐1β is a proinflammatory cytokine, and its production is tightly regulated by inflammasome activation. Xylitol, an anticaries agent, is anti‐inflammatory, but its effect on inflammasome activation has not been researched. This study investigates the effect of xylitol on inflammasome activation induced by A. actinomycetemcomitans. Methods: The differentiated THP‐1 macrophages were stimulated by A. actinomycetemcomitans with or without xylitol and the expressions of IL‐1β and inflammasome components were detected by real time PCR, ELISA, confocal microscopy and Immunoblot analysis. The effects of xylitol on the adhesion and invasion of A. actinomycetemcomitans to cells were measured by viable cell count. Results : A. actinomycetemcomitans increased pro IL‐1β synthesis and IL‐1β secretion in a multiplicity of infection‐ and time‐dependent manner. A. actinomycetemcomitans also stimulated caspase‐1 activation. Among inflammasome components, apoptosis‐associated speck‐like protein containing a CARD (ASC) and absent in melanoma 2 (AIM2) proteins were upregulated by A. actinomycetemcomitans infection. When cells were pretreated with xylitol, proIL‐1β and IL‐1β production by A. actinomycetemcomitans infection was significantly decreased. Xylitol also inhibited ASC and AIM2 proteins and formation of ASC puncta. Furthermore, xylitol suppressed internalization of A. actinomycetemcomitans into differentiated THP‐1 macrophages without affecting viability of A. actinomycetemcomitans within cells. Conclusions: A. actinomycetemcomitans induced IL‐1β production and AIM2 inflammasome activation. Xylitol inhibited these effects, possibly by suppressing internalization of A. actinomycetemcomitans into cells. Thus, this study proposes a mechanism for IL‐1β production via inflammasome activation and discusses a possible use for xylitol in periodontal inflammation caused by A. actinomycetemcomitans.     

Porphyromonas gingivalis Lipopolysaccharide Upregulates Insulin Secretion From Pancreatic β Cell Line MIN6

Background: A close association between periodontitis and diabetes has been demonstrated in human cross‐sectional studies, but an exact relationship between periodontitis and prediabetes has not been established. Previous studies using animal model systems consistently have shown that hyperinsulinemia occurs in animals with periodontitis compared to animals with healthy periodontium (while maintaining normoglycemia). Because bacterial lipopolysaccharide (LPS) plays an important role in the pathogenesis of periodontitis, we hypothesized that LPS may stimulate insulin secretion through a direct effect on β cell function. To test this hypothesis, pancreatic β cell line MIN6 cells were used to determine the effect of Porphyromonas gingivalis (Pg) LPS on insulin secretion. Furthermore, expression of genes altered by Pg LPS in innate immunity and insulin‐signaling pathways was determined. Methods: MIN6 cells were grown in medium with glucose concentration of normoglycemia (5.5 mM). Pg LPS was added to each well at final concentrations of 50, 200, and 500 ng/mL. Insulin secretion was measured using enzyme‐linked immunosorbent assay. Gene expression levels altered by Pg LPS were determined by polymerase chain reaction (PCR) array for mouse innate and adaptive immunity response and mouse insulin‐signaling pathways, and results were confirmed for specific genes of interest by quantitative PCR. Results: Pg LPS stimulated insulin secretion in the normoglycemic condition by ≈1.5‐ to 3.0‐fold depending on the concentration of LPS. Pg LPS treatment altered the expression of several genes involved in innate and adaptive immune response and insulin‐signaling pathway. Pg LPS upregulated the expression of the immune response–related genes cluster of differentiation 8a (Cd8a), Cd14, and intercellular adhesion molecule‐1 (Icam1) by about two‐fold. LPS also increased the expression of two insulin signaling–related genes, glucose‐6‐phosphatase catalytic subunit (G6pc) and insulin‐like 3 (Insl3), by three‐ to four‐fold. Conclusions: We have demonstrated for the first time that Pg LPS stimulates insulin secretion by pancreatic β cell line MIN cells. Pg LPS may have significant implications on the development of β cell compensation and insulin resistance in prediabetes in individuals with periodontitis.     

The Influence of Glycated Hemoglobin on the Cross Susceptibility Between Type 1 Diabetes Mellitus and Periodontal Disease

Background: Periodontal disease is a major complication of type 1 diabetes mellitus (T1DM). The aim of the present study is to investigate the relationship between glycated hemoglobin and circulating levels of interleukin (IL)‐6, IL‐8, and C‐X‐C motif chemokine ligand 5 (CXCL5) in non‐smoking patients suffering from T1DM, with and without periodontitis. In addition, to determine the effect of advanced glycation end products (AGE) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) on IL‐6, IL‐8, and CXCL5 expression by THP‐1 monocytes and OKF6/TERT‐2 cells. Methods: There were 104 participants in the study: 19 healthy volunteers, 23 patients with periodontitis, 28 patients with T1DM, and 34 patients with T1DM and periodontitis. Levels of blood glucose/glycated hemoglobin (International Federation of Clinical Chemistry [IFCC]) were determined by high‐performance liquid chromatography. Levels of IL‐6, IL‐8, and CXCL5 in plasma were determined by enzyme‐linked immunosorbent assay (ELISA). In vitro stimulation of OKF6/TERT‐2 cells and THP‐1 monocytes was performed with combinations of AGE and P. gingivalis LPS. Changes in expression of IL‐6, IL‐8, and CXCL5 were monitored by ELISA and real‐time polymerase chain reaction. Results: Patients with diabetes and periodontitis had higher plasma levels of IL‐8 than patients with periodontitis alone. Plasma levels of IL‐8 correlated significantly with IFCC units, clinical probing depth, and attachment loss. AGE and LPS, alone or in combination, stimulated IL‐6, IL‐8, and CXCL5 expression in both OKF6/TERT‐2 cells and THP‐1 monocytes. Conclusions: Elevated plasma levels of IL‐8 potentially contribute to the cross‐susceptibility between periodontitis and T1DM. P. gingivalis LPS and AGE in combination caused significantly greater expression of IL‐6, IL‐8, and CXCL5 from THP‐1 monocytes and OKF6/TERT‐2 cells than LPS alone.     

Interleukin‐1α stimulation in monocytes by periodontal bacteria: antagonistic effects of Porphyromonas gingivalis

Periodontal pathogenic bacteria are associated with elevated levels of interleukin‐1α (IL‐1α) but it is unclear if all species can induce cytokine production equally. Porphyromonas gingivalis may be able antagonize IL‐1α induced by other species through the activity of its proteases or lipopolysaccharide (LPS). Monomac‐6 cells and primary human monocytes were treated with culture supernatants from Porphyromonas gingivalis, Fusobacterium nucleatum, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Prevotella intermedius, Veillonella atypical and Prevotella nigrescens. IL‐1α protein levels were measured after 6 h of incubation. In addition, monocytes were co‐stimulated with supernatants from P. gingivalis and other bacteria. The role of P. gingivalis proteases was tested using Arg‐X and Lys‐X mutant strains. The role of LPS was investigated using purified P. gingivalis LPS and polymixin depletion. All species tested induced significant IL‐1α production, but P. gingivalis was the weakest. Co‐stimulation of monocytes with P. gingivalis antagonized the ability of other bacterial species to induce IL‐1α production. This effect was at its greatest with C. rectus (resulting in a 70% reduction). Gingipain mutant strains and chemical inhibition of protease activity did not reduce antagonistic activity. However, 100 ng/ml of P. gingivalis LPS can reproduce the antagonistic activity of P. gingivalis culture supernatants. Periodontitis‐associated bacterial species stimulate IL‐1α production by monocytes. P. gingivalis can antagonize this effect, and its LPS appears to be the crucial component. This study highlights the importance of mixed infections in the pathogenesis of periodontal disease because reduction of pro‐inflammatory cytokine levels may impair the ability of the host to tackle infection.