In Vitro Regulation of CCL3 and CXCL12 by Bacterial By-products Is Dependent on Site of Origin of Human Oral Fibroblasts

Introduction

Production of chemokines by tissue resident cells is one of the main mechanisms involved in the inflammatory infiltrate formation during inflammation. The specific ability of fibroblasts from different oral tissues such as gingiva, periodontal ligament, and dental pulp from permanent and deciduous teeth in producing the chemokines CCL3 and CXCL12 under stimulation by bacterial products commonly found in endodontic infections was investigated.

Methods

Cultures of fibroblasts from gingiva and periodontal ligament as well as from dental pulp from permanent and deciduous teeth were established by using an explant technique and stimulated with increasing concentrations of Escherichia coli lipopolysaccharide (EcLPS) and Enterococcus faecalis lipoteichoic acid (EfLTA) for 1, 6, and 24 hours. Supernatants were tested for CCL3 and CXCL12 by enzyme-linked immunosorbent assay.

Results

In general, CCL3 production was induced by EcLPS in the 4 fibroblast subtypes and by EfLTA in fibroblasts from gingiva and periodontal ligament. Constitutive CXCL12 synthesis decreased in all fibroblast subtypes especially under stimulation with EcLPS. Fibroblast from permanent deciduous teeth was the cell type presenting the most expressive reduction in CXCL12 release by both stimuli. On the basis of computational matching of CXCL12 mRNA with the microRNAs miR-141 and miR-200a, their expression was also investigated. Although detected in the fibroblasts, these molecules remained unaltered by bacterial by-product stimulation.

Conclusions

EcLPS and EfLTA induced the production of CCL3 and unbalanced the synthesis of CXCL12 in a manner dependent on the specific tissue origin of fibroblasts.     

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.     

Expression of CD163, interleukin‐10, and interferon‐gamma in oral squamous cell carcinoma: mutual relationships and prognostic implications

Tumor‐associated macrophages (TAMs) and their associated inflammatory cytokines represent the major inflammatory component of the stroma of many tumors and can affect prognosis in the case of neoplasms. The objective of this study was to determine the prognostic significance of CD163⁺ cells, interleukin‐10 (IL‐10), and interferon‐gamma (IFN‐γ) in oral lesions associated with oral squamous cell carcinoma (OSCC). The levels of CD163, IFN‐γ, and IL‐10 in the tissue samples of 240 patients with OSCC and 58 patients with other oral lesions were assessed by immunohistochemistry. Individuals with low IFN‐γ levels, high IL‐10 levels, and low CD163 levels were of special concern with respect to OSCC progression. We found that high levels of CD163, or a combination of low IFN‐γ levels, high IL‐10 levels, and low CD163 levels, were associated with poorer overall survival (OS). CD163⁺ cells provide better predictive power for OS in comparison with traditional markers, such as clinical stage and lymph node metastasis. Therefore, CD163⁺ cells may be effective prognostic predictors of OSCC. IL‐10 may also indicate poor outcomes when IFN‐γ secretion is low and the cells are CD163^?.     

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.     

Cytokines differentially regulate CXCL10 production by interferon-gamma-stimulated or tumor necrosis factor-alpha-stimulated human gingival fibroblasts

Background and Objective: CXC chemokine 10 (CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T‐helper 1 cells. In this study we examined the effects of cytokines on CXCL10 production by human gingival fibroblasts. Material and Methods: Human gingival fibroblasts were exposed to pro‐inflammatory cytokines (interleukin‐1β, tumor necrosis factor‐α), a T‐helper 1 cytokine (interferon‐γ), T‐helper 2 cytokines (interleukin‐4, interleukin‐13), T‐helper 17 cytokines (interleukin‐17A, interleukin‐22) and regulatory T‐cell cytokines (interleukin‐10, transforming growth factor‐β1) for 24 h. CXCL10 production by human gingival fibroblasts was examined by enzyme‐linked immunosorbent assay. Results: Human gingival fibroblasts produced CXCL10 protein upon stimulation with interleukin‐1β, tumor necrosis factor‐α and interferon‐γ. Treatment of human gingival fibroblasts with interferon‐γ in combination with tumor necrosis factor‐α or interleukin‐1β resulted in a synergistic production of CXCL10. However, interleukin‐4 and interleukin‐13 inhibited CXCL10 production by interferon‐γ‐stimulated or tumor necrosis factor‐α‐stimulated‐human gingival fibroblasts. On the other hand, interleukin‐17A and interleukin‐22 enhanced CXCL10 production by human gingival fibroblasts treated with interferon‐γ and inhibited CXCL10 production by tumor necrosis factor‐α‐stimulated human gingival fibroblasts. Furthermore, the anti‐inflammatory cytokine, interleukin‐10, inhibited CXCL10 production by both interferon‐γ‐ and tumor necrosis factor‐α‐stimulated human gingival fibroblasts, but transforming growth factor‐β1 enhanced interferon‐γ‐mediated CXCL10 production by human gingival fibroblasts. Conclusion: These results mean that the balance of cytokines in periodontally diseased tissue may be essential for the control of CXCL10 production by human gingival fibroblasts, and the production of CXCL10 might be important for the regulation of T‐helper 1 cell infiltration in periodontally diseased tissue.     

CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

Objective:

The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS).

Material and Methods:

Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 - 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results.

Results:

Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF.

Conclusion:

These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation.     

Confocal immunolocalization of VE-cadherin- and CXC chemokine-expressing endothelial cells in periapical granulomas

Aim To determine whether endothelial cells (ECs) in periapical granulomas can express vascular endothelial (VE)‐cadherin, CXCL8 and CXCL10 by examining with two‐colour confocal laser scanning microscope. Methodology Periapical lesions were surgically removed from patients with chronic periapical periodontitis (n = 20), and the paraffin‐embedded sections were prepared after being fixed with cold acetone. The 7‐μm‐thick sections were stained with haematoxylin–eosin and then examined pathologically using a light microscope. The lesions diagnosed as periapical granulomas (17 specimens) were analysed further using immunofluorescence and antibodies specific for human VE‐cadherin, CXCL8, and CXCL10. The slides were carefully examined using a confocal laser scanning microscope. The numbers of positive ECs were counted, and the comparison between VE‐cadherin‐positive ECs and CXCL8 or CXCL10 was assessed statistically using one‐way ANOVA followed by a Student–Newman–Keuls test. Results The expression of CXCL8 and CXCL10 by ECs was detected in 60.4 ± 13.4 and 67.2 ± 13.9%, respectively. However, the percentage of VE‐cadherin‐expressing ECs was 40.4 ± 10.5%, which was significantly lower (P < 0.01) than CXCL8 and CXCL10‐expressing ECs. Two‐colour immunofluorescence staining revealed that ECs co‐expressed VE‐cadherin and CXCL8 (37.4 ± 14.1%) or CXCL10 (39.1 ± 13.8%). Conclusions VE‐cadherin expression in ECs was lower than CXCL8 and CXCL10, suggesting that inflamed ECs in periapical granulomas could increase vascular permeability and that leukocyte chemotaxis mediated by ECs might occur. These findings may suggest the possibility that ECs could play a pivotal role in cell recruitment in periapical granulomas.     

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.     

In silico analysis of the competition between Streptococcus sanguinis and Streptococcus mutans in the dental biofilm

During dental caries, the dental biofilm modifies the composition of the hundreds of involved bacterial species. Changing environmental conditions influence competition. A pertinent model to exemplify the complex interplay of the microorganisms in the human dental biofilm is the competition between Streptococcus sanguinis and Streptococcus mutans. It has been reported that children and adults harbor greater numbers of S. sanguinis in the oral cavity, associated with caries‐free teeth. Conversely, S. mutans is predominant in individuals with a high number of carious lesions. Competition between both microorganisms stems from the production of H₂O₂ by S. sanguinis and mutacins, a type of bacteriocins, by S. mutans. There is limited evidence on how S. sanguinis survives its own H₂O₂ levels, or if it has other mechanisms that might aid in the competition against S. mutans, nonetheless. We performed a genomic and metabolic pathway comparison, coupled with a comprehensive literature review, to better understand the competition between these two species. Results indicated that S. sanguinis can outcompete S. mutans by the production of an enzyme capable of metabolizing H₂O₂. S. mutans, however, lacks the enzyme and is susceptible to the peroxide from S. sanguinis. In addition, S. sanguinis can generate energy through gluconeogenesis and seems to have evolved different communication mechanisms, indicating that novel proteins may be responsible for intra‐species communication.     

Interleukin‐1 and interleukin‐8 in nicotine‐ and lipopolysaccharide‐exposed gingival keratinocyte cultures

Johnson GK, Guthmiller JM, Joly S, Organ CC, Dawson DV. Interleukin‐1 and interleukin‐8 in nicotine‐ and lipopolysaccharide‐exposed gingival keratinocyte cultures. J Periodont Res 2010; 45: 583–588. © 2010 John Wiley & Sons A/S Background and Objective: Tobacco use is associated with increased periodontal destruction in both cigarette smokers and smokeless tobacco users. Gingival keratinocytes are the first cells in contact with microbial and tobacco components and play a key role in the innate immune response to these agents. The objective of this study was to evaluate the effect of nicotine and bacterial lipopolysaccharide (LPS) alone and in combination on gingival keratinocyte production of interleukin‐1α (IL‐1α) and interleukin‐8 (IL‐8). Material and Methods: Gingival keratinocyte cultures were established from 10 healthy, non‐tobacco‐using subjects. The cells were stimulated for 24 h with 1 μm or 1 mm nicotine and/or 10 μg/mL Escherichia coli or Porphyromonas gingivalis LPS. Interleukin‐1α and IL‐8 proteins were quantified using ELISAs. Results: Compared with untreated cultures, 1 mm nicotine stimulated production of IL‐1α (p < 0.001); E. coli and P. gingivalis LPS increased IL‐8 production (p = 0.0014 and p = 0.0232, respectively). A combination of nicotine and LPS produced the highest cytokine quantities. Amounts of IL‐1α and IL‐8 following 1 mm nicotine and LPS exposure were significantly greater than in untreated cultures (p < 0.001). Interleukin‐8 was also responsive to 0.1 μm nicotine combined with E. coli or P. gingivalis LPS compared with control cultures (p < 0.0001 and p = 0.0029, respectively). Both cytokines tended to be elevated following the combined treatment relative to nicotine or LPS treatment alone. Conclusion: These results demonstrate that nicotine and LPS differentially regulate IL‐1 and IL‐8 production by gingival keratinocytes. Combined treatment tended to elevate cytokine production further, which may have implications for the progression of periodontitis in tobacco users.     

Immunomodulation of dendritic cells differentiated in the presence of nicotine with lipopolysaccharide from Porphyromonas gingivalis

Tobacco smoking is a significant risk factor for periodontal diseases. Nicotine, one of the most studied constituents in cigarette smoke, is thought to modify immune responses. Dendritic cells (DCs), which are key mediators between innate and adaptive immunity, stimulate naive T cells to differentiate to effector T‐cell subsets that may be actively involved in the immunopathogenesis of periodontal diseases. In this study, we evaluated the effects of nicotine and lipopolysaccharide (LPS) from Porphyromonas gingivalis, alone and in combination, on the functions of human monocyte‐derived DCs to elucidate the mechanism of tissue destruction of smoking‐associated periodontal diseases. P. gingivalis LPS‐stimulated DCs differentiated with nicotine (NiDCs) induced lower T‐cell proliferation and human leukocyte antigen (HLA)‐DR expression, but elevated expression of programmed cell death ligand 1. Additionally, NiDCs impaired interferon‐γ production but maintained interleukin (IL)‐5 and IL‐10 production in co‐cultured T cells. Furthermore, NiDCs produced lower levels of proinflammatory cytokines compared with DCs differentiated in the absence of nicotine. Interestingly, NiDCs preferentially produced the T helper 2 (Th2)‐type chemokines macrophage chemotactic protein‐1 and macrophage‐derived chemokine. These results suggest that the presence of nicotine during differentiation of DCs modulates the immunoregulatory functions of P. gingivalis LPS‐stimulated DCs.     

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.     

Distribution of carcinoembryonic antigen‐related cellular adhesion molecules in human gingiva

Carcinoembryonic antigen‐related cellular adhesion molecules (CEACAMs) are glycoproteins produced in epithelial, endothelial, lymphoid, and myeloid cells. Carcinoembryonic antigen‐related cellular adhesion molecules mediate cell–cell contact and host–pathogen interactions. The aims of this study were to map the distribution and examine the regulation of CEACAMs in human gingival sites. Quantitative real‐time PCR performed on human gingival biopsies from periodontitis sites revealed mRNA coding for CEACAM1, ‐5, ‐6, and ‐7. Immunohistochemistry showed that CEACAMs were not found in oral gingival epithelium, except for CEACAM5 in periodontitis. Carcinoembryonic antigen‐related cellular adhesion molecules 1, 5, and 6 were present in the oral sulcular epithelium of periodontitis but not in that of healthy gingiva. In junctional epithelium, all three molecules were present in healthy gingiva, but in periodontitis only CEACAM1 and ‐6 were detected. Staining for CEACAM1 and ‐6 was also seen in the inflammatory cell infiltrate in periodontitis. No staining for CEACAM7 was found. Proinflammatory mediators, including lipopolysaccharide (LPS), tumour necrosis factor‐α (TNF‐α)/interleukin‐1β (IL‐1β), and interferon‐γ (IFN‐γ), increased the expression of CEACAM1 and CEACAM6 mRNAs in cultured human oral keratinocytes. CEACAM1 and CEACAM6 mRNAs were also strongly up‐regulated upon stimulation with lysophosphatidic acid. In conclusion, the distribution of different CEACAMs was related to specific sites in the gingiva. This might reflect different functional roles in this tissue.     

Clinical and immunological effects of azithromycin in Behçet''s disease

Background: The aim of this study was to evaluate the effects of azithromycin on mucocutaneous manifestations, oral health and immune response in Behçet's disease (BD). Methods: Eight BD patients with active mucocutaneous symptoms were treated with azithromycin for 4 weeks. Oral health, clinical manifestations and in vitro interleukin (IL)‐12, interferon (IFN)‐γ, IL‐10 and monocyte chemotactic protein (MCP)‐1 responses were evaluated before and after treatment. Results: The number of folliculitic lesions, healing time of oral ulcers and scores of plaque indexes (PLIs) were lower after azithromycin treatment (P < 0.05). Scores of PLIs correlated positively with the healing time of oral ulcers (P = 0.02). Although a trend towards increased stimulated IL‐10 responses with azithromycin was observed, no statistically significant difference was found. Stimulated and unstimulated MCP‐1, IFN‐γ and IL‐12 responses were similar before and after treatment (P > 0.05). Conclusion: Azithromycin was observed to be effective in decreasing folliculitic lesions and fastening the healing time of oral ulcers in BD.     

Association of CXCL12 gene promoter methylation with periodontitis in patients with diabetes mellitus type 2

ObjectivesCXCL12 is widely expressed, constitutive chemokine involved in tissue repair and regeneration, while the extent of its expression is important in various chronic inflammatory conditions. Involvement of DNA methylation in CXCL12 gene suppression (CXCL12) has been shown in malignancy and some autoimmune diseases. The aim of this study was to investigate whether the alterations in DNA methylation of CXCL12 are also involved in progression of periodontitis in combination with diabetes, as these chronic inflammatory conditions are strongly interrelated.DesignStudy included 72 subjects divided in three groups: healthy control (C, n = 21), periodontitis (P, n = 29) and diabetes/periodontitis group (D/P, n = 22). DNA extracted from epithelial cells obtained by sterile cotton swabs from buccal mucosa was subjected to methylation specific polymerase chain reaction (MSP) to obtain DNA methylation pattern of CXCL12 promoter.ResultsCXCL12 promoter was predominantly unmethylated in all groups. However, increase in the frequency of the methylated form and increase in percent of methylation of CXCL12 promoter in periodontitis and diabetes/periodontitis group compared to control group were found, although without statistical significance. However, statistically significant increase in Tm of MSP products in diabetes/periodontitis group was observed. Correlation analysis revealed statistically significant relationship between the extent of DNA methylation of the CXCL12 promoter and periodontal parameters, as well as between DNA methylation of CXCL12 and glycosylated hemoglobin.ConclusionPresented results suggest that chronic inflammation contributes to the change of CXCL12 DNA methylation in buccal cells and that DNA methylation profile of CXCL12 promoter plays important role in development and progression of periodontal disease.     

MyD88 is essential for alveolar bone loss induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide in mice

Aggregatibacter actinomycetemcomitans is a Gram‐negative bacteria highly associated with localized aggressive periodontitis. The recognition of microbial factors, such as lipopolysaccharide from A. actinomycetemcomitans (_AaLPS), in the oral environment is made mainly by surface receptors known as Toll‐like receptors (TLR). TLR4 is the major LPS receptor. This interaction leads to the production of inflammatory cytokines by myeloid differentiation primary‐response protein 88 (MyD88) ‐dependent and ‐independent pathways, which may involve the adaptor Toll/interleukin‐1 receptor‐domain‐containing adaptor inducing interferon‐β (TRIF). The aim of this study was to assess the involvement of MyD88 in alveolar bone loss induced by _AaLPS in mice. C57BL6/J wild‐type (WT) mice, MyD88, TRIF or TRIF/MyD88 knockout mice received 10 injections of _AaLPS strain FDC Y4 (5 μg in 3 μl), in the palatal gingival tissue of the right first molar, every 48 h. Phosphate‐buffered saline was injected in the opposite side and used as control. Animals were sacrificed 24 h after the 10th injection and the maxillae were removed for macroscopic and biochemical analyses. The injections of _AaLPS induced significant alveolar bone loss in WT mice. In the absence of MyD88 or TRIF/MyD88 no bone loss induced by _AaLPS was observed. In contrast, responses in TRIF^?/? mice were similar to those in WT mice. Diminished bone loss in the absence of MyD88 was associated with fewer TRAP‐positive cells and increased expression of osteoblast markers, RUNX2 and osteopontin. There was also reduced tumor necrosis factor‐α production in MyD88^?/? mice. There was less osteoclast differentiation of hematopoietic bone marrow cells from MyD88^?/? mice after _AaLPS stimulation. Hence, the signaling through MyD88 is pivotal for _AaLPS‐induced osteoclast formation and alveolar bone loss.