Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway,but not through the p38 MAPK pathway

Zhang D, Zheng H, Zhao J, Lin L, Li C, Liu J, Pan Y. Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway, but not through the p38 MAPK pathway. J Periodont Res 2011; 46: 31–38. © 2010 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis is a major pathogen in the development and progression of periodontal disease. The aim of this study was to investigate whether endothelial intracellular adhesion molecule‐1 (ICAM‐1), an inflammation biomarker for periodontitis, could be modified by infection with either of two strains of P. gingivalis with different virulence capacities: avirulent ATCC 33277 and virulent W83. Material and Methods: We examined the expression of ICAM‐1, IκBα, phospho‐p38 MAPK and nuclear factor‐kappaB (NF‐κB) p65 in an umbilical vein endothelial cell line (ECV‐304) treated with ATCC 33277 and W83, with or without the NF‐κB antagonist MG132 and/or a specific p38 inhibitor (SB203580), by real‐time PCR, western blotting and immunofluorescence. Results: Both strains could induce ICAM‐1 expression; additionally W83 was able to increase ICAM‐1 expression more significantly than ATCC 33277. In P. gingivalis‐infected endothelial cells, both p38 MAPK and NF‐κB signaling pathways were triggered by a rapid increase of p38 MAPK phosphorylation and a more delayed degradation of IκBα, followed by the nuclear translocation of NF‐κB. It was found that ICAM‐1 production in endothelial cells was abrogated by inhibition of the NF‐κB pathway, but not by inhibition of the p38 MAPK pathway, using the inhibitors of the latter two molecules. Conclusion: The induction of ICAM‐1 by infection of umbilical vein endothelial cells with P. gingivalis might be mediated through the NF‐κB pathway, but not by the p38 MAPK pathway.     

Role of high endothelial postcapillary venules and selected adhesion molecules in periodontal diseases: a review

Periodontitis is accompanied by the proliferation of small blood vessels in the gingival lamina propria. Specialized postcapillary venules, termed periodontal high endothelial‐like venules, are also present, and demonstrate morphological and functional traits similar to those of high endothelial venules (HEVs) in lymphatic organs. The suggested role of HEVs in the pathogenesis of chronic periodontitis involves participation in leukocyte transendothelial migration and therefore proinflammatory effects appear. Recent observations suggest that chronic periodontitis is an independent risk factor for systemic vascular disease and may result in stimulation of the synthesis of acute phase protein by cytokines released by periodontal high endothelial cells (HECs). However, tissue expression of HEV‐linked adhesion molecules has not been evaluated in the gingiva of patients with chronic periodontitis. This is significant in relation to potential therapy targeting expression of the adhesion molecules. In this review, current knowledge of HEV structure and the related expression of four surface adhesion molecules of HECs [CD34, platelet endothelial cell adhesion molecule 1, endoglin and intercellular adhesion molecule 1 (ICAM‐1)], involved in the key steps of the adhesion cascade in periodontal diseases, are discussed. Most studies on the expression of adhesion molecules in the development and progression of periodontal diseases pertain to ICAM‐1 (CD54). Studies by the authors demonstrated quantitatively similar expression of three of four selected surface markers in gingival HEVs of patients with chronic periodontitis and in HEVs of reactive lymph nodes, confirming morphological and functional similarity of HEVs in pathologically altered tissues with those in lymphoid tissues.     

Alcohol Consumption and Periodontitis: Quantification of Periodontal Pathogens and Cytokines

Background: There are few studies on periodontal status related to microbiologic and immunologic profiles among individuals not or occasionally using alcohol and those with alcohol dependence. The aim of this study is to determine the effect of alcohol consumption on the levels of subgingival periodontal pathogens and proinflammatory cytokines (interleukin [IL]‐1β and tumor necrosis factor [TNF]‐α) in the gingival fluid among individuals with and without periodontitis. Methods: This observational analytic study includes 88 volunteers allocated in four groups (n = 22): individuals with alcohol dependence and periodontitis (ADP), individuals with alcohol dependence and without periodontitis (ADNP), individuals not or occasionally using alcohol with periodontitis (NAP), and individuals not or occasionally using alcohol without periodontitis (NANP). Levels of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Eikenella corrodens, and Fusobacterium nucleatum were determined by real‐time polymerase chain reaction on the basis of the subgingival biofilm, and IL‐1β and TNF‐α were quantified by enzyme‐linked immunosorbent assay in gingival fluid samples. Results: Individuals with alcohol dependence showed worse periodontal status and higher levels of P. intermedia, E. corrodens, F. nucleatum, and IL‐1β than non‐users. No significant correlations between TNF‐α and bacterial levels were observed. However, in the ADP group, higher levels of E. corrodens were correlated with higher levels of IL‐1β. Conclusion: A negative influence of alcohol consumption was observed on clinical and microbiologic periodontal parameters, as well as a slight influence on immunologic parameters, signaling the need for additional studies.     

Systemic Inflammatory Biomarkers and Their Association With Periodontal and Diabetes‐Related Factors in the Diabetes and Periodontal Therapy Trial,A Randomized Controlled Trial

Background: The present study evaluates effects of non‐surgical periodontal treatment on serum biomarkers in patients with type 2 diabetes mellitus (t2DM) and chronic periodontitis who participated in the Diabetes and Periodontal Therapy Trial (DPTT); and associations among diabetes markers, serum biomarkers, and periodontal measures in these patients. Methods: DPTT participants randomized to receive immediate or delayed non‐surgical periodontal therapy were evaluated at baseline and 6 months. Serum samples from 475 participants with 6‐month data were analyzed for the following biomarkers: 1) high sensitivity C‐reactive protein; 2) E‐selectin; 3) tumor necrosis factor (TNF)‐α; 4) vascular cell adhesion molecule (VCAM); 5) interleukin (IL)‐6; 6) IL‐8; 7) intercellular adhesion molecule; and 8) IL‐10. Changes in biomarker levels from baseline and correlations among biomarker levels and clinical findings were analyzed. Results: No differences between treatment and control groups were observed for any biomarkers at baseline or 6 months (P >0.05 for all variables). VCAM levels increased by an average (standard deviation) of 17.9 (99.5); ng/mL (P = 0.006) and E‐selectin decreased by 2.33 (16.08) ng/mL (P = 0.03) in the treatment group after 6 months. E‐selectin levels were significantly correlated with DM‐related variables (hemoglobin A1c [HbA1c] and fasting glucose) at baseline and with 6‐month change in both groups; no significant correlations were found among periodontal clinical parameters and serum biomarkers or DM‐related variables. Neither HbA1c or body mass index varied during the study period in either study group. Conclusions: Non‐surgical periodontal therapy and periodontal disease severity were not associated with significant changes in serum biomarkers in DPTT participants during the 6‐month follow‐up. Correlations among changes in E‐selectin, IL‐6, and DM‐related variables suggest that t2DM may be the primary driver of systemic inflammation in these patients.     

Rosuvastatin Inhibits Interleukin (IL)‐8 and IL‐6 Production in Human Coronary Artery Endothelial Cells Stimulated With Aggregatibacter actinomycetemcomitans Serotype b

Background: Rosuvastatin exhibits anti‐inflammatory effects and reduces periodontal diseases and atherosclerosis; however, its role in regulating periodontopathogen‐induced endothelial proinflammatory responses remains unclear. The purpose of this study is to determine whether rosuvastatin can reduce the proinflammatory response induced by Aggregatibacter actinomycetemcomitans (Aa) in human coronary artery endothelial cells (HCAECs). Methods: HCAECs were stimulated with purified Aa serotype b lipopolysaccharide (LPS) (Aa‐LPS), heat‐killed (HK) bacteria (Aa‐HK), or live bacteria. Expression of Toll‐like receptors and cellular adhesion molecules were evaluated by fluorometric enzyme‐linked immunosorbent assay. Endothelial cell activation was evaluated by quantifying nuclear factor (NF)‐kappa B‐p65 and cytokine expression levels by quantitative polymerase chain reaction and flow cytometry. Effect of rosuvastatin in expression of the atheroprotective factor Krüppel‐like factor 2 (KLF2) and cytokines were also studied using similar approaches. Results: HCAECs showed increased interleukin (IL)‐6, IL‐8, intercellular adhesion molecule 1, and platelet endothelial cell adhesion molecule 1 expression when stimulated with Aa‐LPS or Aa‐HK. NF‐κB‐p65 activation was induced by all antigens. Aa‐induced IL‐6 and IL‐8 production was inhibited by rosuvastatin, particularly at higher doses. Interestingly, reduced IL‐6 and IL‐8 levels were observed in HCAECs stimulated with Aa in the presence of higher concentrations of rosuvastatin. This anti‐inflammatory effect correlated with a significant increase of rosuvastatin‐induced KLF2. Conclusions: These results suggest Aa‐induced proinflammatory endothelial responses are regulated by rosuvastatin in a mechanism that appears to involve KLF2 activation. Use of rosuvastatin to prevent cardiovascular disease may reduce risk of endothelial activation by bacterial antigens.     

Role of p38 mitogen-activated protein kinase pathway in Porphyromonas gingivalis lipopolysaccharide-induced VCAM-1 expression in human aortic endothelial cells

Background: Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) has been reported to induce the expression of vascular cell adhesion molecule‐1 (VCAM‐1) in vascular endothelial cells. This finding suggests the potential roles for Pg in the pathogenesis of atherosclerosis. However, the mechanism involved in Pg LPS‐induced VCAM‐1 production in endothelial cells remains unclear. Methods: Quantitative real‐time polymerase chain reaction and Western blotting were used, respectively, to investigate the mRNA expression and protein production of VCAM‐1 in human aortic endothelial cells (HAECs) induced by Pg LPS. The involvement of the p38 mitogen‐activated protein kinase (p38 MAPK) cell signaling pathway in VCAM‐1 expression was investigated by assays with specific inhibitors. Results: Pg LPS–induced expression in HAECs of VCAM‐1 occurred in a dose‐ and time‐dependent manner. In addition, the p38 MAPK inhibitor (SB 203580) significantly attenuated Pg LPS–induced VCAM‐1 expression. Conclusion: Activation of p38 MAPK is at least partially involved in Pg LPS–induced VCAM‐1 expression in HAECs, which may contribute to the acceleration of atherosclerosis.     

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.     

Intracellular Adhesion Molecule‐1 Is Regulated by Porphyromonas gingivalis Through Nucleotide Binding Oligomerization Domain‐Containing Proteins 1 and 2 Molecules in Periodontal Fibroblasts

Background: The mechanism by which Porphyromonas gingivalis regulates intracellular adhesion molecule 1 (ICAM‐1) expression in human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs) is unknown. The aim of this study is to investigate whether nucleotide binding oligomerization domain‐containing protein (NOD) 1 and NOD2 are involved in this process and the clinical significance of ICAM‐1 in periodontitis. Methods: hPDLCs and hGFs were treated with P. gingivalis, l ‐Ala‐γ‐d ‐glutamyl‐mesodiaminopimelic acid (an agonist for NOD1), and muramyl dipeptide (an agonist for NOD2). Alternatively, cells transfected with small interfering RNA targeting NOD1and NOD2 were treated with P. gingivalis. ICAM‐1, NOD1, and NOD2 were detected at mRNA and protein levels. In addition, clinical examinations were performed in 30 healthy controls and 40 patients with chronic periodontitis (CP) before and after treatment, and serum‐soluble ICAM‐1 (sICAM‐1) levels in these individuals were detected by enzyme‐linked immunosorbent assay. Results: This study shows that P. gingivalis caused an increase in ICAM‐1, NOD1, and NOD2 expression in periodontal fibroblasts. There was a linear correlation between ICAM‐1 and NOD1 and NOD2 levels. Activation of NOD1 and NOD2 by the specific agonist led to the upregulation of ICAM‐1, whereas knocking down NOD1 and NOD2 caused a reduction in P. gingivalis–induced ICAM‐1 production. Furthermore, sICAM‐1 levels were higher in patients with CP than in healthy controls and were positively related to the clinical periodontal parameters. After periodontal treatment, sICAM‐1 levels decreased significantly. Conclusions: The present results indicate that sICAM‐1 levels are correlated to the severity of periodontitis. NOD1 and NOD2 mediate P. gingivalis–induced ICAM‐1 production in periodontal fibroblasts. NOD1 and NOD2 could be considered potential targets for periodontal therapy.     

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.     

E‐selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide‐binding oligomerization domain‐like receptors and Toll‐like receptors

Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide‐binding oligomerization domain (NOD)‐like receptors (NLRs) and Toll‐like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E‐selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E‐selectin expression. Porphyromonas gingivalis also induced nuclear factor‐κB (NF‐κB) and P38 mitogen‐activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF‐κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF‐κB and P38 MAPK significantly attenuated E‐selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non‐redundant roles in endothelial cell activation following P. gingivalis infection.     

Oxidized low‐density lipoprotein increases interleukin‐8 production in human gingival epithelial cell line Ca9‐22

Suzuki K, Sakiyama Y, Usui M, Obama T, Kato R, Itabe H, Yamamoto M. Oxidized low‐density lipoprotein increases interleukin‐8 production in human gingival epithelial cell line Ca9‐22. J Periodont Res 2010; 45: 488–495. © 2010 John Wiley & Sons A/S Background and Objective: Recent epidemiological studies have shown a correlation between periodontitis and hyperlipidemia. We have found high levels of oxidized low‐density lipoprotein (OxLDL) in the gingival crevicular fluid of dental patients. In the present study, we tried to examine the possible role of OxLDL in periodontal inflammation in vitro. Material and Methods: Cells of the human gingival epithelial cell line Ca9‐22 were cultured in media containing OxLDL, and the amounts of interleukin‐8 (IL‐8) and prostaglandin E₂ (PGE₂) produced were measured using ELISAs. Results: Production of IL‐8 by Ca9‐22 cells was significantly increased when the cells were treated with OxLDL, but not with native LDL or acetylated LDL. Production of PGE₂ by Ca9‐22 cells was enhanced by co‐incubation with OxLDL and interleukin‐1β (IL‐1β). Scavenger receptor inhibitors, fucoidan and dextran sulfate, inhibited the OxLDL‐induced IL‐8 and PGE₂ production in the presence of IL‐1β. The p³⁸ MAPK inhibitors SB203580 and SB202190 and the ERK inhibitor PD98059 inhibited the OxLDL‐induced IL‐8 production. Among oxidized lipids and chemically modified LDL, 7‐ketocholesterol enhanced IL‐8 production. Conclusion: This is the first report to show that OxLDL enhances IL‐8 production in epithelial cells.     

Two‐dimensional and three‐dimensional models for studying atherosclerosis pathogenesis induced by periodontopathogenic microorganisms

Epidemiological studies have established a clinical association between periodontal disease and atherosclerosis. Bacteremia and endotoxemia episodes in patients with periodontitis appear to link these two diseases by inducing a body‐wide production of cardiovascular markers. The presence of oral bacteria in atherosclerotic lesions in patients with periodontitis suggests that bacteria, or their antigenic components, induce alterations in the endothelium associated with atherosclerosis. Therefore, a causal mechanism explaining the association between both diseases can be constructed using in vitro models. This review presents current experimental approaches based on in vitro cell models used to shed light on the mechanism by which periodontal pathogenic microorganisms, and their antigenic components, induce proatherosclerotic endothelial activity. Monolayer cultures of endothelial vascular or arterial cells have been used to assess periodontal pathogenic bacteria and their antigenic compounds and endothelial activation. However, these models are not capable of reflecting the physiological characteristics of the endothelium inside vascularized tissue. Therefore, the shift from two‐dimensional (2D) cellular models toward three‐dimensional (3D) models of endothelial cells resembling an environment close to the physiological environment of the endothelial cell within the endothelium is useful for evaluating the physiological relevance of results regarding the endothelial dysfunction induced by periodontopathogens that are currently obtained from 2D models. The use of in vitro 3D cellular models can also be relevant to the search for therapeutic agents for chronic inflammatory diseases such as atherosclerosis. Here, we present some strategies for the assembly of 3D cultures with endothelial cells, which is useful for the study of periodontopathogen‐mediated disease.     

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.     

Salivary Colony Stimulating Factor‐1 and Interleukin‐34 in Periodontal Disease

Background: Colony‐stimulating factor (CSF)‐1 and interleukin (IL)‐34 are macrophage growth factors and regulators of osteoclastogenesis. Their potential involvement in periodontal disease is yet unknown. The aim of this study is to explore the presence of CSF‐1 and IL‐34 in whole saliva in relation to periodontal disease. Methods: Protocol validation was assessed in saliva of healthy donors (n = 21) by enzyme‐linked immunosorbent assay. Salivary CSF‐1, IL‐34, and matrix metalloproteinase (MMP)‐8, a biomarker candidate of periodontitis, were determined in 48 patients (29 patients with periodontitis, 12 with gingivitis, and seven healthy patients) and related to the following clinical periodontal parameters: bleeding on probing, probing depth, clinical attachment loss, and plaque index. An additional separate group of patients with gingivitis (n = 21) and some of the patients with periodontitis (n = 11) were subjected to non‐surgical periodontal treatment, whereupon changes in salivary CSF‐1, IL‐34, and MMP‐8 levels were determined and related to periodontal outcome. Results: Patients with periodontitis displayed higher CSF‐1 and MMP‐8 levels in saliva compared with healthy patients, and IL‐34 levels were lower. A higher CSF‐1/IL‐34 ratio was observed in patients with periodontitis compared with healthy patients. There was a positive correlation between CSF‐1 and MMP‐8, which both correlated negatively to IL‐34, in patients with gingivitis and periodontitis. Clinical periodontal parameters correlated positively with CSF‐1, MMP‐8, and with the CSF‐1/IL‐34 ratio, and negatively with IL‐34 in patients with periodontitis. After treatment CSF‐1 and MMP‐8 levels decreased together with observed clinical improvement in patients with gingivitis. Conclusion: CSF‐1 and IL‐34 are present in saliva and seem to have complementary roles in periodontal disease: IL‐34 in steady‐state and CSF‐1 in inflammation.     

Characterization and immunostimulatory activity of extracellular vesicles from Filifactor alocis

Filifactor alocis, a gram‐positive, obligate anaerobic rod, is an emerging periodontal pathogen that is frequently isolated from patients with periodontitis, peri‐implantitis, and apical periodontitis. Recent studies have shown that extracellular vesicles (EVs) from gram‐negative periodontal pathogens, so‐called outer membrane vesicles (OMVs), harbor various effector molecules responsible for inducing host inflammatory responses. However, there are no reports of EVs from F. alocis. In this study, we purified and characterized the protein profiles of EVs from F. alocis and investigated their immunostimulatory activity on human monocytic THP‐1 and human oral keratinocyte HOK‐16B cell lines. Highly pure EVs were obtained from F. alocis using density gradient ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy showed that F. alocis EVs were between 50 and 270 nm in diameter. Proteome analysis identified 28 proteins, including lipoproteins, autolysins, F. alocis complement inhibitor (FACIN), transporter‐related proteins, metabolism‐related proteins, and ribosomal proteins. Human cytokine array analysis showed that F. alocis EVs remarkably induced the expression of CCL1, CCL2, MIP‐1, CCL5, CXCL1, CXCL10, ICAM‐1, IL‐1β, IL‐1ra, IL‐6, IL‐8, MIF, SerpinE, and TNF‐α in THP‐1 cells and CXCL1, G‐CSF, GM‐CSF, IL‐6, and IL‐8 in HOK‐16B cells. The immunostimulatory activity of F. alocis EVs was similar to that of the whole bacterial cells. Our findings provide new insight into the role of EVs from gram‐positive oral bacteria in periodontal diseases.     

Increased Expression of Interleukin (IL)‐35 and IL‐17, But Not IL‐27, in Gingival Tissues With Chronic Periodontitis

Background: Interleukin (IL)‐35 plays an important role in immune regulation through the suppression of effector T‐cell populations, including T‐helper 17 (Th17) cells. Although Th17 cells and IL‐17 are involved in the pathogenesis of periodontitis, the level of IL‐35 in inflamed periodontal tissues is unclear. Here, IL‐35, IL‐17, and IL‐27 production/expression in gingival crevicular fluid (GCF) and human gingival tissue were investigated. Methods: GCF samples were collected from buccal (mesial, center, and distal) sites of teeth from patients with chronic periodontitis (CP) and healthy controls and were analyzed by enzyme‐linked immunosorbent assay for IL‐35 (periodontitis, n = 36; healthy, n = 30) and IL‐17 (periodontitis, n = 16; healthy, n = 13). Gingival tissue, including sulcus/pocket epithelium and underlying connective tissue, was collected from an additional 10 healthy participants and 10 patients with CP and were analyzed by quantitative polymerase chain reaction (qPCR) for Epstein Barr virus‐induced gene 3 (EBI3), IL12A, and IL17A. IL27p28 was also tested by qPCR. Results: IL‐35 and IL‐17 were significantly higher in GCF from patients with periodontitis than healthy participants (P <0.01, P <0.05, respectively). In both healthy participants and those with periodontitis, positive correlations were found among IL‐35 and probing depth and clinical attachment level (CAL) as well as between IL‐17 and CAL. EBI3, IL12A (components of IL‐35), and IL17A messenger RNA expression levels were significantly higher in inflamed gingival tissue than in healthy control tissues (P <0.05). IL27p28 was not detected in any sample, suggesting that IL‐27 is not produced in large quantities in periodontal tissue. Conclusion: IL‐35 and IL‐17, but not IL‐27, may play important roles in the pathogenesis of periodontitis.