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.     

Oral bacterial DNA differ in their ability to induce inflammatory responses in human monocytic cell lines

Background: Deoxyribonucleic acids (DNA) of periodontal pathogens, Porphyromonas gingivalis (Pg) and Tannerella forsythia, stimulate cytokine production in human monocytic cells (THP‐1) through Toll‐like receptor 9 (TLR‐9) and nuclear factor‐κB signaling. Fusobacterium nucleatum (Fn) is one of the most frequently isolated bacteria in periodontally diseased tissues and is reported to synergize with Pg, enhancing the pathogenicity. We investigate inflammatory mediator production in THP‐1 cells challenged with Fn and Streptococcus sanguinis (Ss) DNA, a non‐pathogenic oral bacteria, and further assess whether cytokines triggered by whole pathogens or Pg lipopolysaccharide (LPS) are affected by TLR‐9 signaling inhibitors (chloroquine). Methods: THP‐1 cells were stimulated with Pg‐DNA (100 ng/μL), Fn‐DNA (100 ng/μL), Ss‐DNA (100 ng/μL), Pg‐LPS (10 ng/μL), and heat‐killed whole bacteria (multiplicity of infection, 1:100) for 16 hours with or without chloroquine pretreatment (10 μg/mL). Interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor‐α levels were determined using enzyme‐linked immunosorbent assay. Statistical analyses included analysis of variance with multiple comparisons using Dunnett or Tukey methods and paired t test. A value of P <0.05 was significant. Results: Inflammatory mediator levels were increased in response to all the stimuli with the exception of Ss‐DNA (P <0.05). Chloroquine pretreatment significantly decreased cytokine production from THP‐1 cells with the exception of IL‐6 production triggered by whole Fn and Ss (P <0.05). Conclusions: Differences exist among oral bacterial DNA in inducing immune responses. By altering the conditions in cytosolic compartments, we can interfere with cellular responses triggered by extracellular receptor activation. Thus, alternative treatment approaches targeted to intracellular receptors might be of benefit in controlling periodontal inflammation.     

Azithromycin may inhibit interleukin-8 through suppression of Rac1 and a nuclear factor-kappa B pathway in KB cells stimulated with lipopolysaccharide

Background: Recent studies have shown that the 15‐member macrolide antibiotic azithromycin (AZM) not only has antibacterial activity, but also results in the role of immunomodulator. Interleukin (IL)‐8 is an important inflammatory mediator in periodontal disease. However, there have been no reports on the effects of AZM on IL‐8 production from human oral epithelium. Therefore, we investigated the effects of AZM on IL‐8 production in an oral epithelial cell line. Methods: KB cells were stimulated by Escherichia coli or Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS) with or without AZM. IL‐8 mRNA and protein expression and production in response to LPS were analyzed by quantitative polymerase chain reaction, flow cytometry, and enzyme‐linked immunosorbent assay. The activation of nuclear factor‐kappa B (NF‐κB) and Rac1, which is important for IL‐8 expression, was analyzed by enzyme‐linked immunosorbent assay and Western blotting, respectively. Results: IL‐8 mRNA expression, IL‐8 production, and NF‐κB activation in LPS‐stimulated KB cells were inhibited by the addition of AZM. LPS‐induced Rac1 activation was also suppressed by AZM. Conclusions: This study suggests that AZM inhibits LPS‐induced IL‐8 production in an oral epithelial cell line, in part caused by the suppression of Rac1 and NF‐κB activation. The use of AZM might provide possible benefits in periodontal therapy, with respect to both its antibacterial action and apparent anti‐inflammatory effect.     

High Glucose Concentrations Suppress the Proliferation of Human Periodontal Ligament Stem Cells and Their Differentiation Into Osteoblasts

Background: Diabetes mellitus (DM) is a major risk factor for periodontal disease and affects various cellular functions. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration; however, the effect of hyperglycemia on PDLSCs is unclear. The aim of this study is to investigate whether hyperglycemia affects periodontal tissue regeneration, using human PDLSCs and high‐glucose medium as a model of DM. Methods: PDLSCs were obtained from healthy adult human mandibular third molars. Cell proliferation, osteoblastic differentiation, and proinflammatory cytokine expression were investigated by culturing PDLSCs in media supplemented with four different glucose concentrations representative of control patients (5.5 mM), patients with postprandial or controlled DM (8.0 mM), and patients with uncontrolled DM (12.0 and 24.0 mM). The molecular effects of hyperglycemia on PDLSC physiology were examined with a focus on the nuclear factor (NF)‐(κB signaling pathway. The involvement of NF‐κB was investigated with a specific NF‐κB inhibitor in PDLSCs under hyperglycemic conditions. Results: High glucose levels inhibited PDLSC proliferation and differentiation into osteoblasts but induced NF‐κB activation and subsequent interleukin (IL)‐6 and IL‐8 expression. Treatment with an NF‐κB inhibitor rescued the defects in cell proliferation and osteoblastic differentiation and inhibited the IL‐6 expression caused by the high‐glucose environment. Conclusion: The results of this study demonstrate that hyperglycemia inhibits human PDLSC proliferation and osteoblastic differentiation.     

Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts

Tamai R, Kiyoura Y, Sugiyama A. Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts. J Periodont Res 2011; 46: 13–20. © 2010 John Wiley & Sons A/S Background and Objective: Nitrogen‐containing bisphosphonates (NBPs) are widely used as anti‐bone‐resorptive drugs. However, use of NBPs results in inflammatory side‐effects, including jaw osteomyelitis. In the present study, we examined the effects of alendronate, a typical NBP, on cytokine production by human peripheral blood mononuclear cells (PBMCs) and gingival fibroblasts incubated with lipid A. Methods: The PBMCs and gingival fibroblasts were pretreated with or without alendronate for 24 h. Cells were then incubated in the presence or absence of lipid A for a further 24 h. Levels of secreted human interleukin (IL)‐1β, IL‐6, IL‐8 and monocyte chemoattractant protein‐1 (MCP‐1) in culture supernatants were measured by ELISA. We also examined nuclear factor‐κB (NF‐κB) activation in both types of cells by ELISA. Activation of Smad3 in the cells was assessed by flow cytometry. In addition, we performed an inhibition assay using SIS3, a specific inhibitor for Smad3. Results: Pretreatment of PBMCs with alendronate promoted lipid A‐induced production of IL‐1β and IL‐6, but decreased lipid A‐induced IL‐8 and MCP‐1 production. In human gingival fibroblasts, alendronate pretreatment increased lipid A‐induced production of IL‐6 and IL‐8, and increased NF‐κB activation in gingival fibroblasts but not PBMCs stimulated with lipid A. In contrast, alendronate activated Smad3 in both types of cells. Finally, SIS3 inhibited alendronate‐augmented IL‐6 and IL‐8 production by human gingival fibroblasts but up‐regulated alendronate‐decreased IL‐8 production by PBMCs. Conclusion: These results suggest that alendronate‐mediated changes in cytokine production by gingival fibroblasts occur via regulation of NF‐κB and Smad3 activity.     

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.     

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.     

Rosuvastatin Inhibits Interleukin (IL)‐8 and IL‐6 Production in Human Coronary Artery Endothelial Cells Stimulated With Aggregatibacter actinomycetemcomitans Serotype b

Background: Rosuvastatin exhibits anti‐inflammatory effects and reduces periodontal diseases and atherosclerosis; however, its role in regulating periodontopathogen‐induced endothelial proinflammatory responses remains unclear. The purpose of this study is to determine whether rosuvastatin can reduce the proinflammatory response induced by Aggregatibacter actinomycetemcomitans (Aa) in human coronary artery endothelial cells (HCAECs). Methods: HCAECs were stimulated with purified Aa serotype b lipopolysaccharide (LPS) (Aa‐LPS), heat‐killed (HK) bacteria (Aa‐HK), or live bacteria. Expression of Toll‐like receptors and cellular adhesion molecules were evaluated by fluorometric enzyme‐linked immunosorbent assay. Endothelial cell activation was evaluated by quantifying nuclear factor (NF)‐kappa B‐p65 and cytokine expression levels by quantitative polymerase chain reaction and flow cytometry. Effect of rosuvastatin in expression of the atheroprotective factor Krüppel‐like factor 2 (KLF2) and cytokines were also studied using similar approaches. Results: HCAECs showed increased interleukin (IL)‐6, IL‐8, intercellular adhesion molecule 1, and platelet endothelial cell adhesion molecule 1 expression when stimulated with Aa‐LPS or Aa‐HK. NF‐κB‐p65 activation was induced by all antigens. Aa‐induced IL‐6 and IL‐8 production was inhibited by rosuvastatin, particularly at higher doses. Interestingly, reduced IL‐6 and IL‐8 levels were observed in HCAECs stimulated with Aa in the presence of higher concentrations of rosuvastatin. This anti‐inflammatory effect correlated with a significant increase of rosuvastatin‐induced KLF2. Conclusions: These results suggest Aa‐induced proinflammatory endothelial responses are regulated by rosuvastatin in a mechanism that appears to involve KLF2 activation. Use of rosuvastatin to prevent cardiovascular disease may reduce risk of endothelial activation by bacterial antigens.     

E‐selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide‐binding oligomerization domain‐like receptors and Toll‐like receptors

Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide‐binding oligomerization domain (NOD)‐like receptors (NLRs) and Toll‐like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E‐selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E‐selectin expression. Porphyromonas gingivalis also induced nuclear factor‐κB (NF‐κB) and P38 mitogen‐activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF‐κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF‐κB and P38 MAPK significantly attenuated E‐selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non‐redundant roles in endothelial cell activation following P. gingivalis infection.     

Inflammatory response of uric acid produced by Porphyromonas gingivalis gingipains

Uric acid is a potential metabolite that serves as a danger‐associated molecular pattern (DAMP) and induces inflammatory responses in sterile environments. Porphyromonas gingivalis is a keystone periodontopathogen, and its gingipain proteases play a critical role in the pathogenesis of periodontitis. In this study, we demonstrate that P. gingivalis gingipains play a role in THP‐1 macrophage uric acid production by increasing the expression and activity of xanthine oxidoreductase (XOR). Uric acid sodium salt induces caspase‐1 activation, cell death, and the expression of proinflammatory cytokines, including IL‐1α, IL‐6, and IL‐8, in the human keratinocyte HOK‐16B cell line. Our results suggest that gingipain‐induced uric acid can mediate inflammation in periodontal tissue cells.     

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.