Human gingival fibroblasts release high‐mobility group box‐1 protein through active and passive pathways

Introduction:  The nuclear protein high‐mobility group box‐1 (HMGB1) acts as a late mediator of inflammation when secreted in the extracellular milieu. In this study, we examined the effect of lipopolysaccharides from periodontal pathogens and apoptotic and necrotic cell death on HMGB1 production in human gingival fibroblasts (HGF). Methods:  HGF from healthy periodontal tissue were cultured and stimulated with lipopolysaccharides (LPS) from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Escherichia coli. We also initiated apoptotic and necrotic cell deaths in HGF. The HMGB1 released in the supernatants from stimulated or dying cells was measured. Immunocytochemical staining against HMGB1 was performed in LPS‐stimulated HGF. Results:  A significantly higher amount of HMGB1 was detected from necrotic and apoptotic HGF. LPS from A. actinomycetemcomitans, P. gingivalis, and E. coli significantly induced the production of HMGB1 in a time‐dependent manner. After 6 h of LPS stimulation, HMGB1 was present in the cytoplasm of cells whereas its location was mainly nuclear after 24 h. Conclusions:  LPS from two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis, induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted in the enhancement of HMGB1. Our results suggest that HGF can be a source of HMGB1 by both active secretion and passive release, and that HMGB1 from HGF may contribute to periodontal tissue destruction.     

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.     

Synergistic effects of lipopolysaccharides from periodontopathic bacteria on pro‐inflammatory cytokine production in an ex vivo whole blood model

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been strongly associated with chronic periodontitis. This disease is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. The secretion of high levels of inflammatory cytokines by those cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of whole blood from periodontitis patients following challenges with whole cells of P. gingivalis, T. denticola, and T. forsythia or their lipopolysaccharides (LPS), individually and in combination. Whole blood collected from seven periodontitis patients was stimulated with whole cells or LPS and the production of interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor alpha (TNF‐α) were quantified by enzyme‐linked immunosorbent assays. The mono and mixed challenges with whole bacterial cells or LPS induced the secretion of high amounts of IL‐1β, IL‐6, IL‐8, and TNF‐α by the mixed leukocyte population from periodontitis patients. In addition, P. gingivalis LPS, T. denticola LPS, and T. forsythia LPS acted in synergy to induce high levels of IL‐1β and TNF‐α. This study suggests that P. gingivalis, T. denticola, and T. forsythia may contribute to the immunodestructive host response characteristic of periodontitis through synergistic effects of their LPS on the inflammatory response induced by a mixed population of leukocytes.     

Nicotine and smokeless tobacco effects on gingival and peripheral blood mononuclear cells

Abstract. The pathogenesis of tobacco-related periodontal diseases is not well understood. The purpose of this study was therefore to investigate smokeless tobacco extract (ST) and nicotine effects on prostaglandin E₂ (PGE₂) and interleukin-1β (IL-1β) secretion by peripheral blood mononuclear cells (PBMC, consisting of monocytes and lymphocytes) and gingival mononuclear cells (GMC). Both peripheral blood and gingival tissue adjacent to the alveolar crest were taken from non-smoking adult periodontitis patients. Gingival tissue was treated with collagenase and deoxyribonuclease and GMC and PBMC were isolated by Ficoll-Hypaque centrifugation. GMC and PBMC (100,000 cells/200 μl) were cultured for 24 hours in supplemented RPMI 1640 alone (control), or in supplemented RPMI 1640 containing 1% ST, 100μg/ml nicotine, 1 μg/ml Porphyromonas gingivalis LPS, or 1 μg/ml P. gingivalis LPS and either 100 μg/ml nicotine or 1% ST. Enzyme immunoassays were used to quantity PGE₂ and IL-1β. Treatments were compared by repeated measures ANOVA. 100 μg/ml nicotine (7-fold, p<0.02) and 1% ST (3.5-fold, p<0.004) significantly increased secretion of PGE₂ by PBMC relative to control cultures. 100 μg/ml nicotine and 1% ST, however, had no effect on IL-1β secretion by PBMC. Enhanced PGET secretion also was seen when PBMC were treated with P. gingivalis LPS+100 μg/ml nicotine relative to P. gingivalis LPS alone (p<0.007). In contrast, 100 μg/ml nicotine significantly downregulated IL-1β secretion by GMC relative to medium alone (p<0.008) and had no effect on PGE₂ secretion by GMC, These data indicate that while nicotine and ST can stimulate PBMC to secrete PGE₂, they cannot activate further mononuclear cells extracted from gingiva, possibly due to maximal previous stimulation in the periodontitis lesion.     

Specificity of Antimicrobial Peptide LL‐37 to Neutralize Periodontopathogenic Lipopolysaccharide Activity in Human Oral Fibroblasts

Background: The antimicrobial peptide LL‐37 is known to have a potent lipopolysaccharide (LPS)‐neutralizing activity in various cell types. Because of observed heterogeneity within periodontopathogenic LPS, the authors hypothesized that LL‐37 had specificity to neutralize such LPS activity. The present study, therefore, aims to investigate the LPS‐neutralizing activity of LL‐37 to various periodontopathogenic LPS in interleukin‐8 (IL‐8) production after challenging them in human oral fibroblasts. Methods: Human periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) were cultured from biopsies of periodontal ligament and gingival tissues. After cell confluence in 24‐well plates, LPS (10 μg/mL) from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were added with or without LL‐37 (10 μg/mL). After 18 hours, the supernatant was collected and analyzed in IL‐8 production by enzyme‐linked immunosorbent assay. Results: All periodontopathogenic LPS statistically significantly induced IL‐8 production in both PDLF and GF (P <0.01). After neutralization with LL‐37, both PDLF and GF showed a statistically significant reduction in IL‐8 production compared with LPS‐treated groups without LL‐37 (P <0.01), and the percentage of reduction in IL‐8 production in PDLF appeared to be higher than in GF. In addition, the percentage of reduction in IL‐8 production varied considerably according to each periodontopathogenic LPS. Conclusions: The antimicrobial peptide LL‐37 had an ability to suppress periodontopathogenic LPS‐induced IL‐8 production in both PDLF and GF. Its LPS‐neutralizing activity revealed specificity to periodontopathogenic LPS and seemed to be dependent on the heterogeneity within LPS between different genera.     

Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell‐surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram‐negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), and tumor necrosis factor‐α (TNF‐α) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer‐membrane proteins from P. gingivalis ATCC 53977. Outer‐membrane protein from P. gingivalis enhanced the production of IL‐6 and IL‐8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL‐8 production activity of polysaccharide from P. gingivalis was higher than that of other cell‐surface components. The levels of IL‐6 and IL‐8 released from the P. gingivalis lipopolysaccharide‐treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer‐membrane protein or lipopolysaccharide inhibited the IL‐6 and IL‐8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer‐membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

Whole‐Blood Cultures From Patients With Chronic Periodontitis Respond Differently to Porphyromonas gingivalis but not Escherichia coli Lipopolysaccharide

Background: Porphyromonas gingivalis lipid A heterogeneity modulates cytokine expression in human cells. This study investigates the effects of two lipid A isoforms of P. gingivalis, lipopolysaccharide (LPS)_1435/1449 and LPS₁₆₉₀, on the secretion of proinflammatory and regulatory cytokines in total blood cultures from patients with and without chronic periodontitis (CP). Methods: A cross‐sectional study was conducted in 38 systemically healthy individuals divided in two groups: 1) the CP group (n = 19), in which patients were diagnosed with CP; and 2) the no periodontitis (NP) group (n = 19), which included control patients without CP. Blood samples were collected from all patients, and whole‐blood cell cultures (WBCCs) were stimulated for 48 hours with P. gingivalis LPS_1435/1449 and LPS₁₆₉₀ and Escherichia coli LPS. Unstimulated WBCCs served as negative controls. The secretion of interferon‐γ (IFN‐γ), interleukin‐10 (IL‐10), and transforming growth factor‐β (TGF‐β) was detected in WBCC supernatants by enzyme‐linked immunosorbent assays. Results: E. coli LPS significantly increased the expression of all cytokines in WBCCs from both the NP and CP groups when compared to non‐stimulated cells (control treatment). P. gingivalis LPS preparations increased IFN‐γ levels in the CP group but not in the NP group when compared with controls (P <0.05). P. gingivalis LPS preparations also increased IL‐10 and TGF‐β levels in both CP and NP groups, but P. gingivalis LPS₁₆₉₀ showed a three‐fold increase on IL‐10 production in the NP group (P <0.05) when compared to P. gingivalis LPS_1435/144. Conclusions: These data demonstrate that WBCC cell populations obtained from healthy individuals and patients with CP may differ in the cytokine response to P. gingivalis but not E. coli LPS. This is consistent with the notion that CP alters the systemic WBCC response and that this can be detected by the different P. gingivalis LPS structures.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

An acute injection of Porphyromonas gingivalis lipopolysaccharide modulates the OPG/RANKL system and interleukin‐6 in an ovariectomized mouse model

Background/aims: In the present study, we attempted to develop a simulated model to explore the causal effects of periodontal pathogens on skeletal homeostasis in postmenopausal osteoporosis. Methods: Fifty‐three female adult ICR mice were randomly assigned to an experimental group (ovariectomized) or a control group. A single injection of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis‐LPS, ATCC 33277) or Escherichia coli lipopolysaccharide (E. coli‐LPS) was administered intraperitoneally 4 weeks after an ovariectomy. Concentrations of interleukin‐6 (IL‐6), osteoprotegerin (OPG), and the receptor activator of nuclear factor‐κB ligand (RANKL) in serum were subsequently analyzed using an enzyme‐linked immunosorbent assay (ELISA). Results: Under stimulation with P. gingivalis‐LPS or E. coli‐LPS, the concentration of OPG rose in both groups. The serum level of RANKL showed a decreasing trend 24 h after the injection in both groups. After injection of P. gingivalis‐LPS in both the experimental and control animals, the OPG : RANKL ratio increased 24 h after the booster (22.26–620.99, P < 0.05). The serum level of IL‐6 in the experimental group significantly increased 1–6 h after administration of E. coli‐LPS and 1–3 h after administration of P. gingivalis‐LPS (P < 0.05). Conclusions: A single booster injection of P. gingivalis‐LPS induced short‐term changes in OPG, RANKL, and IL‐6 serum levels in this ovariectomized mouse model.     

Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis

Takahashi N, Honda T, Domon H, Nakajima T, Tabeta K, Yamazaki K. Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis. J Periodont Res 2010; 45: 512–519. © 2010 John Wiley & Sons A/S Background and Objective: Recent studies have revealed that negative regulatory molecules, including interleukin‐1 receptor‐associated kinase‐M (IRAK‐M), control the overactivation of Toll‐like receptor (TLR) signaling. The role of IRAK‐M in human gingival epithelial cells (HGECs), which express TLRs, remains unclear. The present study examined the role of IRAK‐M on interleukin‐8 and macrophage chemoattractant protein‐1 (MCP‐1) expression in HGECs stimulated with Porphyromonas gingivalis and TLR ligands. Material and Methods: Primary HGECs and an SV40 T‐antigen‐immortalized HGEC line (epi 4) were stimulated with live or heat‐killed P. gingivalis, P. gingivalis lipopolysaccharide or the synthetic lipopeptide PAM₃CSK₄, and subsequent expression of IRAK‐M, interleukin‐8 and MCP‐1 was evaluated at the mRNA and protein levels. The effects of IRAK‐M on interleukin‐8 and MCP‐1 expressions were evaluated by IRAK‐M‐specific RNA interference (RNAi)‐based loss‐of‐function assay. Results: All tested stimulants up‐regulated the expression of IRAK‐M in HGECs. The P. gingivalis lipopolysaccharide or PAM₃CSK₄ increased MCP‐1 expression, whereas live P. gingivalis down‐regulated the MCP‐1 expression in HGECs. However, IRAK‐M RNAi increased the expression of MCP‐1 irrespective of up‐ or down‐regulation mediated by the respective stimulants. Interleukin‐8 gene expression, up‐regulated by all tested stimulants, was further enhanced by IRAK‐M RNAi. In contrast, IRAK‐M RNAi had no effect on the interleukin‐8 protein levels, irrespective of the stimulant, indicating that post‐translational modification, not IRAK‐M, controls interleukin‐8 protein expression. Conclusion: Interleukin‐1 receptor‐associated kinase‐M appeared to have distinct regulatory roles on the interleukin‐8 and MCP‐1 produced by HGECs, further suggesting an important role for interleukin‐8 in the immune reponse to periodontopathic bacteria.     

Porphyromonas gingivalis inhibits M2 activation of macrophages by suppressing α‐ketoglutarate production in mice

Reprograming of metabolic pathways is critical in governing the polarization of macrophages into classical proinflammatory M1 or alternative anti‐inflammatory M2 phenotypes in metabolic diseases, such as diabetes. Porphyromonas gingivalis, a keystone pathogen of periodontitis, causes an imbalance in M1/M2 activation, resulting in a hyperinflammatory environment that promotes the pathogenesis of periodontitis. However, whether P. gingivalis infection modulates metabolic pathways to alter macrophage polarization remains unclear. Bone‐marrow‐derived macrophages (BMDMs) were collected from 6‐week‐old female C57BL/6 mice and stimulated with P. gingivalis, P. gingivalis‐derived LPS or IL‐4. Relative gene expression and protein production were measured by quantitative real‐time PCR, RNA sequencing and western blotting. Colorimetric assays were also performed to assess the amounts of α‐ketoglutarate (α‐KG) and succinate. P. gingivalis or P. gingivalis‐derived LPS‐induced inflammatory responses enhanced M1 macrophages and suppressed M2 macrophages, even in the presence of IL‐4. P. gingivalis inhibited Idh1/2 and Gpt1/2 mRNA expression, and increased Akgdh mRNA expression, thus decreasing the ratio of α‐KG/succinate. Supplementation of cell‐permeable dimethyl‐α‐KG dramatically restored M2 activation during P. gingivalis infection. Our study suggests that P. gingivalis maintains a hyperinflammatory state by suppressing the production of α‐KG by M2 macrophages.     

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.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

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.     

Toll‐like receptors 2 and 5 in human gingival epithelial cells co‐operate with T‐cell cytokine interleukin‐17

Background/aim: Periodontitis begins as the result of perturbation of the gingival epithelial cells caused by subgingival bacteria interacting with the epithelial cells via pattern recognition receptors. Toll‐like receptors (TLRs) have been shown to play an important role in the recognition of periodontal pathogens so we have studied the interaction of TLR ligands with TLR2 and TLR5 for cytokine production in the cultures of gingival epithelial cells. Methods: Immunohistochemistry was used for the localization of TLR2 and TLR5 in tissue specimens. Enzyme‐linked immunosorbent assays were performed to detect the levels of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), released from gingival epithelial cell cultures following stimulation with TLR ligand alone or in combination with IL‐17. Results: Both TLR2 and TLR5 were increased in periodontitis (2128 ± 159 vs. 449 ± 59 and 2456 ± 297 vs. 679 ± 103, respectively, P < 0.001) including gingival epithelial cells that stained strongly. Cultured gingival epithelial cells stimulated with their respective ligands (HKLM, a TLR2 ligand that is also found in Porphyromonas gingivalis, and flagellin, a TLR5 ligand that is also found in Treponema denticola) produced both IL‐1β and TNF‐α. To mimic T‐cell help, IL‐17 was added. This further greatly enhanced TLR ligand‐induced IL‐1β (P < 0.001) and TNF‐α (P < 0.01) production. Conclusions: These findings show how pathogen‐associated molecular patterns, shared by many different periodontopathogenic bacteria, stimulate the resident gingival epithelial cells to inflammatory responses in a TLR‐dependent manner. This stimulation may be particularly strong in periodontitis and when T helper type 17 cells provide T‐cell help in intercellular cooperation.     

Interleukin‐1 β, interleukin‐12 and interleukin‐18 levels in gingival fluid and serum of patients with gingivitis and periodontitis

Introduction: Cytokines are of major importance in periodontal disease progression. Interleukin‐12 (IL‐12) stimulates interferon‐γ production by T helper type 1 (Th1) cells while IL‐18 induces Th1 responses when present with IL‐12 but Th2 responses in the absence of IL‐12. IL‐1β has been correlated with periodontal disease destruction. This study determined the local concentrations of these cytokines in sites of gingivitis and periodontitis. Methods: Gingival crevicular fluid was collected from two sites in each of 10 gingivitis patients and from two gingivitis sites and two periodontitis sites from each of 10 periodontitis patients. Serum samples were also collected. IL‐1β, biologically active IL‐12 p70, the IL‐12 p40 subunit and IL‐18 concentrations were determined by enzyme‐linked immunoabsorbent assay. Results: IL‐1β and IL‐18 concentrations were higher in the gingival crevicular fluid from periodontitis patients than in that from gingivitis patients; IL‐18 concentrations were higher than those of IL‐1β. Very little IL‐12, either p40 or p70, was detected in the gingival crevicular fluid samples. In the serum, very low levels of cytokines were found. The level of serum IL‐12 p40, however, was higher than in the fluid from periodontitis sites of periodontitis patients. Conclusion: The local production of IL‐1β and IL‐18 in the gingival crevicular fluid increased with increasing inflammation and IL‐18 was the predominant cytokine at both gingivitis and periodontitis sites. Very little IL‐12 was detected with levels decreasing with increasing inflammation. These results suggest that there is an association between severity of periodontal disease and levels of IL‐1, IL‐12 and IL‐18.     

Elevated MicroRNA‐128 in Periodontitis Mitigates Tumor Necrosis Factor‐α Response via p38 Signaling Pathway in Macrophages

Background: Periodontitis is a chronic inflammatory disease resulting from an inflammatory response to subgingival plaque bacteria, including Porphyromonas gingivalis. MicroRNA (miRNA) is a current focus in regulating the inflammatory processes. In this study, the inflammatory miRNA expression in gingival tissues of patients with periodontitis and of healthy individuals is compared, and its role in regulating the inflammatory response is examined. Methods: Gingival tissues from patients with periodontitis and healthy individuals were collected for miRNA microarray. THP‐1 and CA9‐22 cells were challenged with P. gingivalis, and miRNA expression was determined by real‐time polymerase chain reaction. Target genes for miRNA were predicted using TargetScanHuman database, and miRNA gene expressions were reviewed using public databases. For the functional study, THP‐1 cells were transfected with a miRNA‐128 mimic, and target gene expression was compared with THP‐1 cells challenged with P. gingivalis. For the tolerance test, THP‐1 cells transfected with miRNA‐128 mimic were treated with phorbol 12‐myristate 13‐acetate (PMA) or paraformaldehyde (PFA)‐fixed Escherichia coli. Tumor necrosis factor (TNF)‐α production was determined by enzyme‐linked immunosorbent assay, and mitogen‐activated protein kinase (MAPK) protein phosphorylation was determined by Western blot. Results: Gingival tissues from patients with periodontitis showed increased expression of miRNA‐128, miRNA‐34a, and miRNA‐381 and decreased expression of miRNA‐15b, miRNA‐211, miRNA‐372, and miRNA‐656. THP‐1 cells and CA9‐22 cells challenged with P. gingivalis showed increased miRNA‐128 expression. Among the predicted miRNA‐128 target genes, several genes that are involved in MAPK signaling pathway showed similar gene expression pattern between P. gingivalis challenge and miRNA‐128 mimic transfection. In THP‐1 cells transfected with miRNA‐128 mimic, TNF‐α production was lower, and phosphorylation of p38 was inhibited when challenged with PMA or PFA‐fixed E. coli. Conclusion: miRNA‐128 may be involved in mitigating the inflammatory response induced by P. gingivalis in periodontitis.     

A Sialidase‐Deficient Porphyromonas gingivalis Mutant Strain Induces Less Interleukin‐1β and Tumor Necrosis Factor‐α in Epi4 Cells Than W83 Strain Through Regulation of c‐Jun N‐Terminal Kinase Pathway

Background: Porphyromonas gingivalis is one of the major periodontal pathogens. In a previous study, a mouse abscess model showed that sialidase deficiency of P. gingivalis weakened its virulence, but the mechanism behind this observation remains unknown. Methods: A sialidase‐deficient mutant strain (△PG0352) and a complemented strain (com△PG0352) were constructed. Epi4 cells were stimulated by wild‐type strain P. gingivalis W83, △PG0352, or com△PG0352. Real‐time polymerase chain reaction was carried out to detect expression of virulent genes in P. gingivalis and interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α in epi4 cells. Activities of sialidase, gingipains, and lipopolysaccharide (LPS) were compared among the different P. gingivalis strains. Levels of IL‐1β and TNF‐α in the epi4 cells supernatant were detected by enzyme‐linked immunosorbent assay and levels of p38, extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase (JNK), and phospho‐c‐Jun were detected by western blotting. Results: Compared with P. gingivalis W83 and com△PG0352, activities of Kgp and Rgp gingipains and amount of LPS decreased in △PG0352, whereas there were no differences in LPS activity among these three strains. Level of phospho‐JNK was lower in epi4 cells stimulated by △PG0352. △pG0352 induced less IL‐1β and TNF‐α and more IL‐8 in epi4 cells; differences in IL‐1β and TNF‐α could not be detected after JNK blocking. Conclusion: A sialidase‐deficient P. gingivalis mutant strain induces less IL‐1β and TNF‐α in epi4 cells than W83 strain through regulation of JNK pathway.