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.     

Tumor Necrosis Factor‐α and Interleukin (IL)‐1β, IL‐6, and IL‐8 Impair In Vitro Migration and Induce Apoptosis of Gingival Fibroblasts and Epithelial Cells,Delaying Wound Healing

Background: Multiple factors affect oral mucosal healing, such as the persistence of an inflammatory reaction. The present study evaluates effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β, IL‐6, and IL‐8 on epithelial cells (ECs) and human gingival fibroblasts (GFs) in vitro. Methods: GFs and ECs were seeded in 96‐well plates (1 × 10⁴ cells/well) in plain culture medium (Dulbecco’s modified Eagle’s medium [DMEM]) containing 1% antibiotic/antimycotic solution and 10% fetal bovine serum, and incubated for 24 hours. Both cell lines were exposed for 24 hours to the following cytokines: 1) TNF‐α (100 ng/mL); 2) IL‐1β (1 ng/mL); 3) IL‐6 (10 ng/mL); and 4) IL‐8 (10 ng/mL). All cytokines were diluted in serum‐free DMEM. Control cultures were exposed only to serum‐free DMEM. Effects of exposure to inflammatory cytokines were determined by means of: 1) apoptosis (anexin V); 2) cell migration (wound healing assay); 3) inflammatory cytokine synthesis (enzyme‐linked immunosorbent assay). Data were analyzed by Kruskal–Wallis and Mann–Whitney U tests (α = 0.05). Results: Increased apoptosis rates were noted when cells were exposed to inflammatory cytokines, except ECs exposed to IL‐1β. Cell migration was negatively affected by all inflammatory cytokines for both cell lines. ECs and GFs exposed to IL‐6 and IL‐8 significantly increased synthesis of TNF‐α and IL‐1β. Conclusions: Demonstrated results indicate negative effects of tested inflammatory cytokines on ECs and GFs, inducing apoptosis and impairing cell migration. These results can justify delayed oral mucosa healing in the presence of inflammatory reaction.     

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.     

MyD88 or TRAM Knockdown Regulates Interleukin (IL)‐6, IL‐8, and CXCL12 mRNA Expression in Human Gingival and Periodontal Ligament Fibroblasts

Background: In a previous report, it was shown that Toll‐like receptor (TLR) 2 knockdown modulates interleukin (IL)‐6 and IL‐8 but not the chemokine CXCL12, an important mediator with inflammatory and proangiogenic effects, in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). This study investigates whether knocking down two important TLR adaptor molecules, such as myeloid differentiation protein 88 (MyD88) and TRIF‐related adaptor molecule (TRAM), could affect mRNA expression of IL‐6, IL‐8, and CXCL12 in HGF and HPDLF. Methods: After small interfering (si) RNA‐mediated silencing of MyD88 or TRAM, HGF and HPDLF were stimulated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) or two synthetic ligands of TLR2 (Pam2CSK4 and Pam3CSK4) for 6 hours. IL‐6, IL‐8, and CXCL12 mRNAs were evaluated by quantitative polymerase chain reaction. Results: Knockdown of MyD88 or TRAM partially impaired the IL‐8 mRNA upregulation in both fibroblast subpopulations. Similarly, IL‐6 upregulation was partially prevented by siMyD88 or siTRAM in HGF stimulated with Pg LPS, as well as in both fibroblast subtypes challenged with Pam2CSK4. Conversely, constitutive CXCL12 mRNA levels were upregulated by MyD88 or TRAM knockdown in non‐stimulated cells. Conclusions: These results suggest that TLR adaptor molecules knockdown, such as MyD88 or TRAM, can decrease IL‐6 and IL‐8 mRNA and increase CXCL12 mRNA expression in HGF and HPDLF. This can be an important step for better understanding the mechanisms that control the inflammatory cytokine and chemokine expression, which in turn contributes to periodontal pathogenesis.     

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.     

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.     

Cytokine expression profile in the bone‐anchored hearing system: 12‐week results from a prospective randomized,controlled study

Objective

To study the effect of implanting the percutaneous bone‐anchored hearing system (BAHS) itself and inflammation of the peri‐abutment skin warrant clarification. In this study, we aimed to acquire further insight into the immune responses related to BAHS surgery and peri‐implant skin inflammation.

Materials and Methods

During surgery and 12 weeks post‐implantation, skin biopsies were obtained. If applicable, additional biopsies were taken during cases of inflammation. The mRNA expression of IL‐1β, IL‐6, IL‐8, TNFα, IL‐17, IL‐10, TGF‐ß, MIP‐1α, MMP‐9, TIMP‐1, COL1α1, VEGF‐A, FGF‐2 TLR‐2, and TLR‐4 was quantified using qRT‐PCR.

Results

Thirty‐five patients agreed to the surgery and 12‐week biopsy. Twenty‐two patients had mRNA of sufficient quality for analysis. Ten were fitted with a BAHS using the minimally invasive Ponto surgery technique. Twelve were fitted with a BAHS using the linear incision technique with soft‐tissue preservation. Five biopsies were obtained during episodes of inflammation. The post‐implantation mRNA expression of IL‐1β (P = .002), IL‐8 (P = .003), MMP9 (P = .005), TIMP‐1 (P = .002), and COL1α1 (P < .001) was significantly up‐regulated. IL‐6 (P = .009) and FGF‐2 (P = .004) mRNA expression was significantly down‐regulated after implantation. Within patients, no difference between post‐implantation mRNA expression (at 12 weeks) and when inflammation was observed. Between patients, the expression of IL‐1β (P = .015) and IL‐17 (P = .02) was higher during cases of inflammation compared with patients who had no inflammation at 12‐week follow‐up.

Conclusions

As part of a randomized, prospective, clinical trial, the present study reports the molecular profile of selected cytokines in the soft tissue around BAHS. Within the limit of this study, the results showed that 12 weeks after BAHS implantation the gene expression of some inflammatory cytokines (IL‐8 and IL‐1β) is still relatively high compared with the baseline, steady‐state, expression. The up‐regulation of anabolic (COL1α1) and tissue‐remodeling (MMP‐9 and TIMP1) genes indicates an ongoing remodeling process after 12 weeks of implantation. The results suggest that IL‐1β, IL‐17, and TNF‐α may be interesting markers associated with inflammation.     

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.     

Focal adhesion kinase activation is required for TNF‐α‐induced production of matrix metalloproteinase‐2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts

Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF‐α) and its effects on interleukin (IL)‐6 and IL‐8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP‐2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real‐time PCR. Tumor necrosis factor alpha dose‐dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF‐α‐induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL‐6, IL‐8, and MMP‐2 in a dose‐dependent manner. Knockdown of FAK significantly suppressed TNF‐α‐induced expression of IL6 and IL8 mRNA and release of IL‐6 and IL‐8 protein in HPDLFs. Similarly, MMP‐2 down‐regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF‐α‐induced IL‐6, IL‐8, and MMP‐2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.     

Short‐Term Effects of Non‐Surgical Periodontal Treatment on the Gingival Crevicular Fluid Cytokine Profiles in Sites With Induced Periodontal Defects: A Study on Dogs With and Without Streptozotocin‐Induced Diabetes

Background: The aim of this study is to assess the short‐term effects of non‐surgical periodontal therapy (NSPT) on the gingival crevicular fluid (GCF) cytokine profile in sites with standardized periodontal bony defects in beagle dogs with and without diabetes. Methods: Four beagle dogs with streptozotocin (STZ)‐induced diabetes and four healthy dogs were included. Fasting blood glucose levels were measured using a glucometer. In all animals, a 3‐walled bony defect was created on the mesial surface of the second premolar and first molar in all quadrants. After 12 weeks, all animals underwent weekly NSPT for 3 weeks. Baseline and post‐NSPT GCF samples were collected, and levels of interleukin (IL)‐1, IL‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α were measured using enzyme‐linked immunosorbent assay. Statistical analyses were performed using a software program, and P values <0.05 were considered statistically significant. Results: Mean fasting blood glucose levels were significantly higher in dogs with induced diabetes than those without diabetes (P <0.01). At baseline, mean IL‐6 (P <0.01) and IL‐8 (P <0.05) levels were higher in dogs with diabetes than those without diabetes. A significant reduction in levels of IL‐1, IL‐1β, IL‐6, IL‐8, and TNF‐α was noted in dogs without diabetes 1 week after NSPT. However, this significant reduction (P <0.05) only appeared 2 weeks after NSPT in dogs with diabetes. Conclusions: NSPT reduces GCF levels of proinflammatory cytokines in dogs with and without STZ‐induced diabetes; however, chronic hyperglycemia seems to retard the effect of NSPT on GCF cytokine concentration.     

Evaluation of MicroRNA‐146a and Its Targets in Gingival Tissues of Patients With Chronic Periodontitis

Background: MicroRNAs (miRNAs) are a group of small non‐coding RNAs that play an important role in the regulation of gene expression. miRNA‐146a (miR‐146a), a member of the miR‐146 family, is involved in the control of inflammation. Periodontitis is a set of chronic inflammatory disorders of the tissues surrounding the teeth that lead to the breakdown of alveolar bone and tooth loss. In this study, expression levels of miR‐146a and its targets, including tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, and IL‐6, are evaluated in human patients with chronic periodontitis (CP). Methods: The study population consisted of 10 healthy controls and 20 individuals with CP. For each participant, clinical parameters including probing depth and clinical attachment level were measured, and a gingival tissue sample was collected. Levels of miR‐146a, TNF‐α, IL‐1β, and IL‐6 were quantified using real‐time polymerase chain reaction. Results: Levels of miR‐146a were significantly higher in patients with CP (P <0.001). There was a positive correlation between levels of miR‐146a and clinical parameters (P <0.05). Elevated miR‐146a was accompanied by a significant reduction in TNF‐α and IL‐6 (P <0.001). Conclusions: Patients with CP had higher levels of miR‐146a than healthy individuals, accompanied by reduced levels of TNF‐α and IL‐6. A positive relationship between miR‐146a levels and clinical parameters suggests a pathophysiologic role of miR‐146a in CP.     

The Effects of a Novel Botanical Agent on Lipopolysaccharide‐Induced Alveolar Bone Loss in Rats

Background: The development of host‐modulatory agents with low risk of adverse effects has been needed to treat periodontitis, a chronic inflammatory disease. A botanical mixture of extracts from two natural substances, Panax notoginseng and Rehmannia glutinosa Libosch, was developed as a novel botanical agent synthesized with anti‐inflammatory effect. The aim of this study is to evaluate the effects of the botanical mixture on the release of inflammatory cytokines and its inhibitory effect on lipopolysaccharide (LPS)‐induced alveolar bone loss (ABL) in a rat model. Methods: Cytotoxicity was assessed by 3‐(4,5‐dimethylthiazol‐2yl)‐5(3‐carboxymethoxyphenol)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium assay using human gingival fibroblast (hGF) and human periodontal ligament (hPDL) cells. Human acute monocytic leukemia cell line and hGF cells were cultured to assay tumor necrosis factor (TNF)‐α and interleukin (IL)‐6, respectively. Microcomputed tomography analysis and immunofluoresence analysis were performed to evaluate the efficacy of the botanical mixture to inhibit the destruction of alveolar bone and connective tissue in a rat model. Results: The botanical mixture is cytotoxic at concentrations exceeding 2.5 mg/mL (P <0.05). Based on the results from cytotoxicity assay, it can be determined that the pharmacologic ranges of the botanical mixture to be used in all subsequent in vitro and in vivo experiments. The botanical mixture reduced the release of TNF‐α and IL‐6 from human monocytic cells and hGF cells in a dose‐dependent manner (P <0.05). The administration of the botanical mixture significantly reduced the alveolar bone loss in a rat model (P <0.05). In groups treated with the botanical mixture, matrix metalloproteinase (MMP)‐9 was detected along the alveolar bone crest (ABC), but not around the gingival connective tissue, while in the group with LPS‐induced ABL, pronounced expression of MMP‐9 around the ABC, periodontal ligament, and gingival connective tissue was found. Conclusions: The botanical mixture showed a potential adjunctive effect in the treatment of periodontitis. However, the present findings are obtained in vitro and in a rat model, so further clinical study is needed for its clinical application.