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.     

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.     

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.     

The Proteasome Inhibitor Bortezomib Inhibits Inflammatory Response of Periodontal Ligament Cells and Ameliorates Experimental Periodontitis in Rats

Background: Periodontitis is a chronic inflammatory disease initiated by bacteria and their virulence factors. Bortezomib (BTZ) is the first proteasome inhibitor for clinical treatment of malignancies. Its anticancer activity is accompanied by an anti‐inflammatory effect. However, there are few reports about its anti‐inflammatory effect and underlying mechanism in periodontal disease, especially on human periodontal ligament cells (hPDLCs), which are considered a promising cell‐based therapy for treating periodontitis. Methods: hPDLCs were treated with lipopolysaccharide (LPS) and pretreated with BTZ. mRNA and protein levels of tumor necrosis factor (TNF)‐alpha, interleukin (IL)‐1β, IL‐6, and IL‐8 were determined. The anti‐inflammatory mechanism of BTZ was studied. Further, experimental rat periodontitis was induced with ligature and LPS injection, and simultaneously and locally treated with BTZ (three injections/week). Four weeks after treatment, microcomputed tomography, immunohistochemical, and histopathologic analyses were performed. Results: Bortezomib administration at safe concentrations (≤1 nM) inhibited production of proinflammatory cytokines in LPS‐stimulated hPDLCs via nuclear factor (NF)‐kappa B, p38/extracellular signal‐regulated kinase, and mitogen‐activated protein kinase/activator protein‐1 pathways. Moreover, in the LPS and ligature‐induced periodontitis rat model, BTZ suppressed expression of TNF‐α, IL‐1β, IL‐6, and IL‐8, reduced the ratio of receptor activator of NF‐κB ligand/osteoprotegerin, and prevented alveolar bone absorption. Conclusion: These findings demonstrate the anti‐inflammatory activity of BTZ against periodontal inflammatory response and present BTZ as a promising therapy for periodontal disease.     

Heme oxygenase‐1 mediates nicotine‐ and lipopolysaccharide‐induced expression of cyclooxygenase‐2 and inducible nitric oxide synthase in human periodontal ligament cells

Pi S‐H, Jeong G‐S, Oh H‐W, Kim Y‐S, Pae H‐O, Chung H‐T, Lee S‐K, Kim E‐C. Heme oxygenase‐1 mediates nicotine‐ and lipopolysaccharide‐induced expression of cyclooxygenase‐2 and inducible nitric oxide synthase in human periodontal ligament cells. J Periodont Res 2010; 45: 177–183. © 2010 John Wiley & Sons A/S Background and Objective: Although heme oxygenase‐1 (HO‐1) plays a key role in inflammation, its anti‐inflammatory effects and mechanism of action in periodontitis are still unknown. This study aimed to identify the effects of HO‐1 on the proinflammatory mediators activated by nicotine and lipopolysaccharide (LPS) stimulation in human periodontal ligament (PDL) cells. Material and Methods: The production of nitric oxide (NO) and prostaglandin E₂ (PGE₂) was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2) and HO‐1 proteins was evaluated by Western blot analysis. Results: Lipopolysaccharide and nicotine synergistically induced the production of NO and PGE₂ and increased the protein expression of iNOS, COX‐2 and HO‐1. Treatment with an HO‐1 inhibitor and HO‐1 small interfering RNAs blocked the LPS‐ and nicotine‐stimulated NO and PGE₂ release as well as the expression of iNOS and COX‐2. Conclusion: Our data suggest that the nicotine‐ and LPS‐induced inflammatory effects on PDL cells may act through a novel mechanism involving the action of HO‐1. Thus, HO‐1 may provide a potential therapeutic target for the treatment of periodontal disease associated with smoking and dental plaque.     

Carbon monoxide releasing molecule‐3 inhibits concurrent tumor necrosis factor‐α‐ and interleukin‐1β‐induced expression of adhesion molecules on human gingival fibroblasts

Song H, Zhao H, Qu Y, Sun Q, Zhang F, Du Z, Liang W, Qi Y, Yang P. Carbon monoxide releasing molecule‐3 inhibits concurrent tumor necrosis factor‐α‐ and interleukin‐1β‐induced expression of adhesion molecules on human gingival fibroblasts. J Periodont Res 2011; 46: 48–57. © 2010 John Wiley & Sons A/S Background and Objective: Carbon monoxide releasing molecule‐3 (CORM‐3) is a newly reported compound that has shown anti‐inflammatory effects in a number of cells. In this study, we aimed to investigate the influence of CORM‐3 on concurrent tumor necrosis factor‐α (TNF‐α)‐ and interleukin (IL)‐1β‐induced expression of adhesion molecules on human gingival fibroblasts (HGF). Material and Methods: HGF were cultured from the explants of normal gingival tissues. Cells were costimulated with TNF‐α and IL‐1β in the presence or absence of CORM‐3 for different periods of time. The expression of adhesion molecules, nuclear factor‐kappaB (NF‐κB) and phosphorylated p38 was studied using western blotting. RT‐PCR was applied to check the expression of the adhesion molecules at the mRNA level. The activity of NF‐κB was analysed using a reporter gene assay. Results: CORM‐3 inhibited the up‐regulation of intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule in HGF after costimulation with TNF‐α and IL‐1β, which resulted in the decreased adhesion of peripheral blood mononuclear cells to these cells. Sustained activation of the NF‐κB pathway by costimulation with TNF‐α and IL‐1β was suppressed by CORM‐3, which was reflected by a reduced NF‐κB response element‐dependent luciferase activity and decreased nuclear NF‐κB‐p65 expression. CORM‐3 inhibited MAPK p38 phosphorylation in response to stimulation with proinflammatory cytokines. Conclusion: The results of this study bode well for the application of CORM‐3 as an anti‐inflammatory agent to inhibit NF‐κB activity and to suppress the expression of adhesion molecules on HGF, which suggests a promising potential for CORM‐3 in the treatment of inflammatory periodontal disease.     

Porphyromonas gingivalis antigen preferentially stimulates T cells to express IL‐17 but not receptor activator of NF‐κB ligand in vitro

Although T cells have been implicated in the pathogenesis and are considered to be central to both their progression and control of chronic inflammatory periodontal diseases, the precise contribution of T cells to tissue destruction has not been fully clarified. Recently, interleukin (IL)‐17 and receptor activator of Nuclear factor κB NF‐κB ligand (RANKL) have received much attention as a result of their proinflammatory and bone metabolic roles, respectively. We therefore investigated the effect of outer membrane protein (OMP) from Porphyromonas gingivalis (P. gingivalis) on the expression of IL‐17 and RANKL in peripheral blood mononuclear cells (PBMCs) and compared these between gingivitis and periodontitis, which are representative of stable and progressive lesions, respectively. The in situ expression of these molecules was also examined. P. gingivalis OMP stimulated PBMCs to express IL‐17 at both the mRNA and protein level. Although the mean expression of mRNA was not different between the two groups, the mean level of IL‐17 in the culture supernatants was higher in gingivitis patients than in periodontitis patients. However, the frequency of IL‐17‐positive samples was higher in the periodontitis patients. This stimulatory effect was not evident for RANKL expression in either periodontitis or gingivitis patients. In gingival tissue samples, IL‐17 mRNA was detected in gingivitis more frequently than in periodontitis. The expression of RANKL mRNA was much lower than that of IL‐17 in terms of both level and frequency. These results suggest that IL‐17 but not RANKL may be involved in the pathogenesis of periodontal diseases. However, there may be negative regulatory mechanisms for IL‐17 in gingivitis.     

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.     

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.