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.     

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.     

Amphotericin B up-regulates lipid A-induced IL-6 production via caspase-8

Amphotericin B, an antifungal drug used to treat candidiasis, has been reported to induce pro-inflammatory cytokine production in cultured cells. This study investigated the effects of amphotericin B on pro-inflammatory cytokine production in response to lipid A, the bioactive component of lipopolysaccharide (LPS) in the cell walls of Gram-negative bacteria. Amphotericin B alone elicited a slight increase in interleukin (IL)-6 and IL-8 production by human gingival fibroblasts. However, amphotericin B synergistically up-regulated lipid A-induced production of IL-6 and IL-8. While amphotericin B minimally activated nuclear factor (NF)-κB, it synergistically increased lipid A-induced NF-κB activation. Pre-treatment with methyl-β-cyclodextrin (MβCD), a cholesterol-binding agent, reduced IL-6 and IL-8 production in human gingival fibroblasts. Cholesterol-saturated MβCD also reversed cytokine production, suggesting that the synergistic production of cytokines by amphotericin B and lipid A is dependent on cholesterol-rich microdomains. Amphotericin B activated caspase-8. In addition, a caspase-8 inhibitor inhibited IL-6 production by amphotericin B and lipid A. This suggests that caspase-8 is required for the synergistic production of IL-6 by amphotericin B and lipid A. Collectively, our results suggest that periodontal treatment carried out before amphotericin B treatment may protect against lipid A-induced pro-inflammatory cytokine production.     

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.     

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.     

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.     

Effect of bisphosphonates on human gingival fibroblast production of mediators of osteoclastogenesis: RANKL,osteoprotegerin and interleukin‐6

Tipton DA, Seshul BA, Dabbous MKh. Effect of bisphosphonates on human gingival fibroblast production of mediators of osteoclastogenesis: RANKL, osteoprotegerin and interleukin‐6. J Periodont Res 2011; 46: 39–47.© 2010 John Wiley & Sons A/S Background and Objective: Osteonecrosis of the jaw (ONJ) is associated with bisphosphonate (BP) therapy. BPs alter osteoblast production of mediators of osteoclastogenesis, including interleukin (IL)‐6, RANKL and osteoprotegerin (OPG), a RANKL antagonist. This can inhibit bone turnover and lead to necrosis. There is little information on the contribution of gingival fibroblasts, near bone‐resorption sites, to the IL‐6/RANKL/OPG network, the effects of BPs, or fibroblast involvement in ONJ pathogenesis. Therefore, the objective of this study was to determine the effects of alendronate and pamidronate on the constitutive production, or the lipopolysaccharide (LPS)‐ or IL‐1β‐stimulated production, of IL‐6, RANKL and OPG by human gingival fibroblasts. Material and Methods: Human gingival fibroblasts were derived from explants obtained from healthy individuals with noninflamed gingiva. Cytotoxicity was determined by measuring the activity of a mitochondrial enzyme. Fibroblasts were pre‐incubated or not with BPs (0.01 nm–1 μm ), then incubated or not with LPS or IL‐1β. The concentrations of IL‐6, OPG and RANKL were measured using ELISA. Data were analyzed using analysis of variance (ANOVA) and Scheffé’s F procedure. Results: LPS and BPs were not cytotoxic. The cells produced IL‐6, OPG and RANKL, all of which were stimulated by IL‐1β or LPS (p ≤ 0.04). BPs generally increased the production of IL‐6 and OPG (p ≤ 0.04) and decreased the production of RANKL (p ≤ 0.02). BPs generally further increased the production of LPS‐ or IL‐1β‐stimulated IL‐6 (p ≤ 0.04) and had no effect on, or further increased, the production of LPS‐ or IL‐1β‐stimulated OPG (p ≤ 0.04). BPs decreased the production of LPS‐ or IL‐1β‐stimulated RANKL (p ≤ 0.04) and decreased constitutive, LPS‐stimulated and IL‐1β‐stimulated RANKL/OPG ratios (p ≤ 0.02). Conclusion: The action of alendronate and pamidronate on human gingival fibroblasts, through altering the production of RANKL and OPG, appears to contribute to a microenvironment favoring the inhibition of bone resorption and ONJ.     

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.     

Evidence Linking the Role of Placental Expressions of Peroxisome Proliferator‐Activated Receptor‐γ and Nuclear Factor‐Kappa B in the Pathogenesis of Preeclampsia Associated With Periodontitis

Background: Peroxisome proliferator‐activated receptor (PPAR)‐γ activation leads to suppression of production of a broad range of proinflammatory molecules. It plays a role in differentiation of trophoblasts and helps in normal placentation and formation of vascular exchange interface. Activation of nuclear factor‐kappa (NF‐κ) B triggers proinflammatory molecules inducing abnormal placentation and premature labor. This study aims to explore expression of PPAR‐γ and NF‐κB in placentas of women with periodontitis‐associated preeclampsia compared with that in normotensive pregnant women. Methods: Fifty pregnant women were included. Twenty‐five were controls (normotensive pregnant women) and 25 were pregnant women with preeclampsia, including those with gestational hypertension. Demographic data, pregnancy characteristics, and periodontal parameters were recorded, including: 1) plaque index; 2) gingival index; 3) bleeding on probing (BOP); 4) probing depth; and 5) attachment loss (AL). Placental tissue samples were collected from both groups and analyzed to quantify expression of PPAR‐γ and NF‐κB using real‐time polymerase chain reaction. Results: BOP and AL were significantly higher in pregnant women with preeclampsia compared with normotensive pregnant women (P <0.05). Expression of PPAR‐γ was downregulated in patients with preeclampsia compared with that of healthy normotensive patients, which was statistically significant (P <0.05), whereas NF‐κB was significantly activated (P <0.05) in pregnant women with preeclampsia compared with normotensive pregnant women. Conclusions: Higher periodontal disease prevalence is found among pregnant women with preeclampsia, with increased percentage of sites with BOP and greater AL. This study provides novel information on host response to systemic inflammation induced by periodontal pathogens through mechanisms involving downregulation of PPAR‐γ and increased activation of NF‐κB.     

Retinoic acid‐inducible gene‐I is induced in gingival fibroblasts by lipopolysaccharide or poly IC: possible roles in interleukin‐1β, ‐6 and ‐8 expression

Retinoic acid‐inducible gene‐I (RIG‐I) is a member of the DExH family of proteins, and little is known of its biological function in the oral region. We previously reported that interleukin 1β (IL‐1β) induced RIG‐I expression in gingival fibroblasts. In this study, we studied the mechanism of RIG‐I expression induced by lipopolysaccharide (LPS) or double‐stranded RNA (dsRNA) in gingival fibroblasts. We also addressed the role of RIG‐I in the expression of IL‐1β, IL‐6 and IL‐8 in gingival fibroblasts stimulated with LPS or dsRNA. We stimulated cultured human gingival fibroblasts with LPS or dsRNA, and examined the expression of RIG‐I mRNA and protein. The effect of cycloheximide, a protein synthesis inhibitor, on RIG‐I induction by these stimuli was examined. The expression of IL‐1β, IL‐6 and IL‐8 in gingival fibroblasts transfected with RIG‐I cDNA stimulated with LPS or dsRNA was examined. LPS or dsRNA induced the expression of mRNA and protein for RIG‐I in concentration‐ and time‐dependent manners. We also examined the localization of RIG‐I, and found that it was expressed in cytoplasm. Cycloheximide did not suppress the LPS or dsRNA‐induced RIG‐I expression. Introduction of RIG‐I cDNA into gingival fibroblasts resulted in enhanced expression of IL‐1β, IL‐6 and IL‐8; moreover, overexpression of RIG‐I stimulated with LPS or dsRNA synergistically increased expression of IL‐1β, IL‐6 and IL‐8. RIG‐I may have important roles in the innate immune response in the regulation of IL‐1β, IL‐6 and IL‐8 expression in gingival fibroblasts in response to LPS and dsRNA.     

Modulation of Matrix Metalloproteinase and Cytokine Production by Licorice Isolates Licoricidin and Licorisoflavan A: Potential Therapeutic Approach for Periodontitis

Background: Inflammatory cytokines and matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in response to periodontopathogens play a major role in the tissue destruction observed in periodontitis, which is a disease that affects tooth‐supporting structures. In the present study, we investigate the effects of licorice‐derived licoricidin (LC) and licorisoflavan A (LIA) on the secretion of various cytokines and MMPs by human monocyte‐derived macrophages stimulated with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS). Methods: Macrophages were treated with non‐toxic concentrations of LC or LIA before being stimulated with A. actinomycetemcomitans LPS. The secretion of cytokines and MMPs and the activation of nuclear factor‐kappa B (NF‐κB) p65 and activator protein (AP)‐1 were assessed by enzyme‐linked immunosorbent assays. Results: LC and LIA inhibited the secretion of interleukin (IL)‐6 and chemokine (C‐C motif) ligand 5 in a concentration‐dependent manner but did not affect the secretion of IL‐8 by LPS‐stimulated macrophages. LC and LIA also inhibited the secretion of MMP‐7, ‐8, and ‐9 by macrophages. The suppression of cytokine and MMP secretion by LC and LIA was associated with the reduced activation of NF‐κB p65 but not that of AP‐1. Conclusion: The present study suggests that LC and LIA have potential for the development of novel host‐modulating strategies for the treatment of cytokine and/or MMP‐mediated disorders such as periodontitis.