Prediabetes Enhances Periodontal Inflammation Consistent With Activation of Toll‐Like Receptor–Mediated Nuclear Factor‐κB Pathway in Rats

Background: Clinical studies have showed that prediabetes (preDM) is a predisposing factor for periodontitis. However, the pathogenic mechanism involved is unclear. Because it is known that the activation of Toll‐like receptor (TLR)‐mediated nuclear factor‐kappa B (NF‐κB) signaling pathway plays a crucial role in periodontitis, it is hypothesized that preDM enhances periodontal inflammation by activation of the TLR‐mediated NF‐κB pathway. Methods: In this study, a preDM rat model is established by feeding a high‐fat diet (HFD). HFD‐induced rats with preDM (n = 7) and normal chow–fed rats (n = 7) were studied. The animal model was characterized in terms of body weight and the glycemic and insulinemic profiles. The following parameters were assessed to evaluate possible early periodontal alterations and underlying mechanisms: 1) histology analysis of periodontal tissue; and 2) serum and mRNA levels and/or the tissue protein expression of TLRs, myeloid differentiation factor 88 (MyD88), tumor necrosis factor (TNF) receptor–associated factor 6 (TRAF6), NF‐κB, cytokines, advanced glucose ends (AGEs), and free fatty acids (FFAs). Results: Rats with preDM presented higher expression of TLR2 and TLR4 in periodontal tissue in the HFD group compared with the control group. The TLR2 and TLR4 was mostly expressed in gingiva, and TLR4 was expressed in periodontal ligament in rats. Furthermore, the MyD88 and TRAF6 protein levels were significantly increased in gingiva in rats with preDM compared with normal rats. The activity of NF‐κB signals was higher in rats with preDM than in normal rats. Regarding cytokines expression, the TNF‐α protein levels and interleukin‐1β mRNA levels were significantly increased in the HFD group compared with the control group. In the serum, AGEs levels were significantly increased in the rats with preDM. Mean FFAs concentrations were increased in rats with preDM compared with normal rats, but it did not reach statistical significance. Conclusion: In rats with preDM, TLR2 and TLR4 gene and protein levels were higher in periodontal tissue, and the activation of NF‐κB may, through TLRs/MyD88, cause more cytokine secretion, which is associated with the onset or development of periodontal disease.     

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.     

Moesin‐induced signaling in response to lipopolysaccharide in macrophages

Zawawi KH, Kantarci A, Schulze‐Späte U, Fujita T, Batista EL Jr, Amar S, Van Dyke TE. Moesin‐induced signaling in response to lipopolysaccharide in macrophages. J Periodont Res 2010; 45: 589–601.©2010 John Wiley & Sons A/S Background and Objective: Many physiological and pathophysiological conditions are attributable in part to cytoskeletal regulation of cellular responses to signals. Moesin (membrane‐organizing extension spike protein), an ERM (ezrin, radixin and moesin) family member, is involved in lipopolysaccharide (LPS)‐mediated events in mononuclear phagocytes; however, its role in signaling is not fully understood. The aim of this study was to investigate the LPS‐induced moesin signaling pathways in macrophages. Material and Methods: Macrophages were stimulated with 500 ng/mL LPS in macrophage serum‐free medium. For blocking experiments, cells were pre‐incubated with anti‐moesin antibody. Moesin total protein and phosphorylation were studied with western blotting. Moesin mRNA was assessed using quantitative real‐time PCR. To explore binding of moesin to LPS, native polyacrylamide gel electrophoresis (PAGE) gel shift assay was performed. Moesin immunoprecipitation with CD14, MD‐2 and Toll‐like receptor 4 (TLR4) and co‐immunoprecipitation of MyD88–interleukin‐1 receptor‐associated kinase (IRAK) and IRAK–tumor necrosis factor receptor‐activated factor 6 (TRAF6) were analyzed. Phosphorylation of IRAK and activities of MAPK, nuclear factor κB (NF‐κB) and IκBα were studied. Tumor necrosis factor α, interleukin‐1β and interferon β were measured by ELISA. Results: Moesin was identified as part of a protein cluster that facilitates LPS recognition and results in the expression of proinflammatory cytokines. Lipopolysaccharide stimulates moesin expression and phosphorylation by binding directly to the moesin carboxyl‐terminus. Moesin is temporally associated with TLR4 and MD‐2 after LPS stimulation, while CD14 is continuously bound to moesin. Lipopolysaccharide‐induced signaling is transferred downstream to p38, p44/42 MAPK and NF‐κB activation. Blockage of moesin function interrupts the LPS response through an inhibition of MyD88, IRAK and TRAF6, negatively affecting subsequent activation of the MAP kinases (p38 and ERK), NF‐κB activation and translocation to the nucleus. Conclusion: These results suggest an important role for moesin in the innate immune response and TLR4‐mediated pattern recognition in periodontal disease.     

Calcium Phosphate Particles Induce Interleukin‐8 Expression in a Human Gingival Epithelial Cell Line via the Nuclear Factor‐κB Signaling Pathway

Background: Dental calculus is calcified plaque composed primarily of calcium phosphate mineral salts, and there is a clear association between the presence of calculus and the initiation/progression of periodontitis. However, it is still inconclusive whether dental calculus can be a direct causative factor. The authors examined the effect of nano/microsized calcium phosphate particles, which may be generated in the process of early precipitation and/or dissolution of calcium phosphate mineral, on the expression of interleukin (IL)‐8 in human gingival epithelial cells. Methods: Primary human gingival epithelial cells and/or a human gingival carcinoma cell line (Ca9‐22) were stimulated with calcium phosphate particles. Gene and protein levels were assessed by real‐time polymerase chain reaction analysis and enzyme‐linked immunosorbent assay, respectively. The activity of nuclear factor (NF)‐κB signaling was measured by an immunofluorescence assay to evaluate NF‐κB p65 nuclear translocation. Results: The results show that nano/microsized particles stimulate IL‐8 expression in human gingival epithelial cells at gene and protein levels. The activity to induce IL‐8 expression depends on the particle size: particles with a diameter of 200 nm are more effective than those of 40‐nm and 5‐μm diameters. Calcium phosphate particles (diameter 200 nm) stimulated NF‐κB activity. Pretreatment with BMS‐345541, an NF‐κB signaling inhibitor, inhibited the particle‐mediated IL‐8 gene induction, suggesting a requirement for the NF‐κB signaling pathway. Conclusion: These findings suggest that calcium phosphate particles, which may be related to calculus development, may act as a direct causative factor in the pathogenesis of gingival epithelium.     

MAPKs, activator protein-1 and nuclear factor-κB mediate production of interleukin-1β-stimulated cytokines, prostaglandin E₂ and MMP-1 in human periodontal ligament cells

Murayama R, Kobayashi M, Takeshita A, Yasui T, Yamamoto M. MAPKs, activator protein‐1 and nuclear factor‐κB mediate production of interleukin‐1β‐stimulated cytokines, prostaglandin E ₂ and MMP‐1 in human periodontal ligament cells. J Periodont Res 2011; 46: 568–575. © 2011 John Wiley & Sons A/S Background and Objective: Determination of the interleukin‐1 (IL‐1) signaling cascades that lead to the production of various inflammatory mediators and catabolic factors may clarify attractive targets for therapeutic intervention for periodontitis. We comprehensively assessed the involvement of MAPKs, activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB) in IL‐1β‐induced production of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin E₂ (PGE₂) and MMP‐1 in human periodontal ligament cells. Material and Methods: Human periodontal ligament cells were pretreated with an inhibitor for each of the MAPKs or NF‐κB and subsequently treated with IL‐1β. Following treatment, phosphorylation of three types of MAPK (ERK, p38 MAPK and c‐Jun N‐terminal kinase), IκB kinase (IKK) α/β/γ and IκB‐α, as well as the DNA binding activity of AP‐1 and NF‐κB and the production of IL‐6, IL‐8, PGE₂ and MMP‐1, were determined by western blotting, a gel mobility shift assay and ELISA, respectively. Results: The three MAPKs, simultaneously activated by IL‐1β, mediated the subsequent DNA binding of AP‐1 at various magnitudes, while IKKα/β/γ, IκB‐α and NF‐κB were also involved in the IL‐1 signaling cascade. Furthermore, IL‐1β stimulated the production of IL‐6, IL‐8, PGE₂ and MMP‐1 via activation of the three MAPKs and NF‐κB, because inhibitors of these significantly suppressed the IL‐1β‐stimulated production of these factors. Conclusion: Our results strongly suggest that MAPK, AP‐1 and NF‐κB mediate the IL‐1β‐stimulated synthesis of IL‐6, IL‐8, PGE₂ and MMP‐1 in human periodontal ligament cells. Therefore, inhibition of activation of MAPK, AP‐1 and/or NF‐κB may lead to therapeutic effects on progression of periodontitis.     

Activation of the TREM‐1 pathway in human monocytes by periodontal pathogens and oral commensal bacteria

Periodontitis is a highly prevalent disease caused in part by an aberrant host response to the oral multi‐species biofilm. A balance between the oral bacteria and host immunity is essential for oral health. Imbalances in the oral microbiome lead to an uncontrolled host inflammatory response and subsequent periodontal disease (i.e. gingivitis and periodontitis). TREM‐1 is a signaling receptor present on myeloid cells capable of acting synergistically with other pattern recognition receptors leading to amplification of inflammatory responses. The aim of this study was to investigate the activation of the TREM‐1 pathway in the human monocyte‐like cell line THP‐1 exposed to both oral pathogens and commensals. The relative expression of the genes encoding TREM‐1 and its adapter protein DAP12 were determined by quantitative real‐time polymerase chain reaction. The surface expression of TREM‐1 was determined by flow cytometry. Soluble TREM‐1 and cytokines were measured by enzyme‐linked immunosorbent assay. The results demonstrate that both commensal and pathogenic oral bacteria activate the TREM‐1 pathway, resulting in a proinflammatory TREM‐1 activity‐dependent increase in proinflammatory cytokine production.     

PI3K/Akt mediates expression of TNF‐α mRNA and activation of NF‐κB in calyculin A‐treated primary osteoblasts

Objective: The effect of calyculin A (CA), a serine/threonine protein phosphatase inhibitor, on tumor necrosis factor‐α (TNF‐α) in primary osteoblasts was investigated to determine whether protein phosphatases could affect primary osteoblasts and if so which signaling pathways would be involved. Materials and methods: Primary osteoblasts were prepared from newborn rat calvaria. Cells were treated with 1 nM CA for different time periods. The expressions of TNF‐α and GAPDH mRNA were determined by RT‐PCR. Cell extracts were subjected to SDS‐PAGE and the activation of Akt and NF‐κB were analyzed by western blotting. Results: Calyculin A‐treatment markedly increased the expression of TNF‐α mRNA and enhanced the phosphorylation level of Akt (Ser473) in these cells. Pretreatment with the PI3K inhibitor LY294002 suppressed the increase in TNF‐α mRNA expression and the phosphorylation of Akt in response to CA. Western blot analysis showed that CA stimulated the phosphorylation and nuclear translocation of NF‐κB in primary osteoblasts, and these responses were blocked by pretreatment with LY294002. Conclusion: Calyculin A elicits activation of PI3K/Akt pathway which leads to expression of TNF‐α mRNA and activation of NF‐κB. This NF‐κB activation involves both phosphorylation and nuclear translocation of NF‐κB.     

Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway,but not through the p38 MAPK pathway

Zhang D, Zheng H, Zhao J, Lin L, Li C, Liu J, Pan Y. Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway, but not through the p38 MAPK pathway. J Periodont Res 2011; 46: 31–38. © 2010 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis is a major pathogen in the development and progression of periodontal disease. The aim of this study was to investigate whether endothelial intracellular adhesion molecule‐1 (ICAM‐1), an inflammation biomarker for periodontitis, could be modified by infection with either of two strains of P. gingivalis with different virulence capacities: avirulent ATCC 33277 and virulent W83. Material and Methods: We examined the expression of ICAM‐1, IκBα, phospho‐p38 MAPK and nuclear factor‐kappaB (NF‐κB) p65 in an umbilical vein endothelial cell line (ECV‐304) treated with ATCC 33277 and W83, with or without the NF‐κB antagonist MG132 and/or a specific p38 inhibitor (SB203580), by real‐time PCR, western blotting and immunofluorescence. Results: Both strains could induce ICAM‐1 expression; additionally W83 was able to increase ICAM‐1 expression more significantly than ATCC 33277. In P. gingivalis‐infected endothelial cells, both p38 MAPK and NF‐κB signaling pathways were triggered by a rapid increase of p38 MAPK phosphorylation and a more delayed degradation of IκBα, followed by the nuclear translocation of NF‐κB. It was found that ICAM‐1 production in endothelial cells was abrogated by inhibition of the NF‐κB pathway, but not by inhibition of the p38 MAPK pathway, using the inhibitors of the latter two molecules. Conclusion: The induction of ICAM‐1 by infection of umbilical vein endothelial cells with P. gingivalis might be mediated through the NF‐κB pathway, but not by the p38 MAPK pathway.     

Effects of oral commensal and pathogenic bacteria on human dendritic cells

Background/aims: The oral cavity harbors a diverse and complex microbial community. Bacteria accumulate on both the hard and soft oral tissues in sessile biofilms and engage the host in an intricate cellular dialog, which normally constrains the bacteria to a state of commensal harmony. Dendritic cells (DCs) are likely to balance tolerance and active immunity to commensal microorganisms as part of chronic inflammatory responses. While the role played by DCs in maintaining intestinal homeostasis has been investigated extensively, relatively little is known about DC responses to oral bacteria. Methods: In this study, we pulsed human monocyte‐derived immature DCs (iDCs) with cell wall extracts from pathogenic and commensal gram‐positive or gram‐negative oral bacteria. Results: Although all bacterial extracts tested induced iDCs to mature and produce cytokines/chemokines including interleukin‐12p40, tumor necrosis factor‐α, and monocyte chemoattractant protein‐1 (MCP‐1), the most important factor for programming DCs by oral bacteria was whether they were gram‐positive or gram‐negative, not whether they were commensal or pathogenic. In general, gram‐negative oral bacteria, except for periodontopathic Porphyromonas gingivalis, stimulated DC maturation and cytokine production at lower concentrations than gram‐positive oral bacteria. The threshold of bacteria needed to stimulate chemokine production was 100‐fold to 1000‐fold lower than that needed to induce cytokines. In addition, very low doses of oral commensal bacteria triggered monocytes to migrate toward DC‐derived MCP‐1. Conclusion: Oral commensal and pathogenic bacteria do not differ qualitatively in how they program DCs. DC‐derived MCP‐1 induced in response to oral commensal bacteria may play a role, at least in part, in the maintenance of oral tissue integrity by attracting monocytes.     

Streptococcus cristatus modulates the Fusobacterium nucleatum-induced epithelial interleukin-8 response through the nuclear factor-kappa B pathway

Zhang G, Chen R, Rudney JD.Streptococcus cristatusmodulates theFusobacterium nucleatum‐induced epithelial interleukin‐8 response through the nuclear factor‐kappa B pathway. J Periodont Res 2011; 46: 558–567.©2011 John Wiley & Sons A/S Background and Objective: We previously reported that the interleukin‐8 (IL‐8) response to Fusobacterum nucleatum was attenuated in the presence of Streptococcus cristatus. Here, we further examined the underlying mechanism(s) involved in the modulating effect of S. cristatus by looking specifically at its impact on the nuclear factor‐kappa B (NF‐κB) pathway under the toll‐like receptor (TLR) signaling background. Material and Methods: OKF6/TERT‐2 and KB cells were co‐cultured with F. nucleatum and S. cristatus, either alone or in combination. Secretion of IL‐8 protein was measured by ELISA. The nuclear translocation of NF‐κB was evaluated by confocal microscopy, while DNA‐binding activity was quantified using TransAM? ELISA kits. Western blot analysis was performed to determine whether the anti‐inflammatory effect of S. cristatus is related to the modulation of the NF‐κB inhibitory protein IκB‐α. Results: Incubation with F. nucleatum significantly enhanced the nuclear translocation of NF‐κB. Exposure to S. cristatus alone did not cause detectable NF‐κB translocation and was able to inhibit the F. nucleatum‐induced NF‐κB nuclear translocation. The TransAM assay further confirmed that S. cristatus blocked the nuclear translocation of NF‐κB in response to F. nucleatum stimulation. In contrast to the nearly complete degradation of IκB‐α induced by F. nucleatum alone, the presence of S. cristatus stabilized IκB‐α. Pre‐incubation with TLR2 and TLR4 antibodies, however, did not affect the epithelial response to either species alone or in combination. Conclusion: The mechanism by which S. cristatus attenuates F. nucleatum‐induced proinflammatory responses in oral epithelial cells appears to involve blockade of NF‐κB nuclear translocation at the level of IκB‐α degradation.