Porphyromonas gingivalis antigen preferentially stimulates T cells to express IL-17 but not receptor activator of NF-kappaB ligand in vitro

Although T cells have been implicated in the pathogenesis and are considered to be central to both their progression and control of chronic inflammatory periodontal diseases, the precise contribution of T cells to tissue destruction has not been fully clarified. Recently, interleukin (IL)-17 and receptor activator of Nuclear factor kappaB NF-kappaB ligand (RANKL) have received much attention as a result of their proinflammatory and bone metabolic roles, respectively. We therefore investigated the effect of outer membrane protein (OMP) from Porphyromonas gingivalis (P. gingivalis) on the expression of IL-17 and RANKL in peripheral blood mononuclear cells (PBMCs) and compared these between gingivitis and periodontitis, which are representative of stable and progressive lesions, respectively. The in situ expression of these molecules was also examined. P. gingivalis OMP stimulated PBMCs to express IL-17 at both the mRNA and protein level. Although the mean expression of mRNA was not different between the two groups, the mean level of IL-17 in the culture supernatants was higher in gingivitis patients than in periodontitis patients. However, the frequency of IL-17-positive samples was higher in the periodontitis patients. This stimulatory effect was not evident for RANKL expression in either periodontitis or gingivitis patients. In gingival tissue samples, IL-17 mRNA was detected in gingivitis more frequently than in periodontitis. The expression of RANKL mRNA was much lower than that of IL-17 in terms of both level and frequency. These results suggest that IL-17 but not RANKL may be involved in the pathogenesis of periodontal diseases. However, there may be negative regulatory mechanisms for IL-17 in gingivitis.     

Increased interleukin‐18 in gingival crevicular fluid from periodontitis patients

Introduction: This study aimed to measure the levels of interleukin‐18 (IL‐18) in inflamed shallow sites and inflamed deep sites in patients with periodontitis and to compare the data with results from inflamed shallow sites in patients with gingivitis. A secondary aim was to examine the composition of the subgingival microbiota in the sampled sites. Methods: Gingival crevicular fluid was collected from five gingivitis sites and five periodontitis sites from 18 patients with chronic periodontitis, and from five gingivitis sites from 15 patients with gingivitis. Samples from each site category were pooled and IL‐18 levels were measured using an enzyme‐linked immunosorbent assay. The subgingival microbiota was analyzed by checkerboard DNA–DNA hybridization. Results: All clinical parameters and gingival crevicular fluid volumes were higher in periodontitis sites compared with gingivitis sites from patients with periodontitis and gingivitis. The total amount of IL‐18 was higher in periodontitis sites than gingivitis sites in both periodontitis (P = 0.018) and gingivitis (P = 0.002) patients and was higher in gingivitis sites from periodontitis patients than in those from gingivitis patients (P = 0.015). There were higher levels of Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola (red complex species) in periodontitis sites compared with gingivitis sites in both the periodontitis and gingivitis patients (P < 0.001). Conclusion: Levels of IL‐18 were higher in patients with chronic periodontitis compared with patients with gingivitis, even at sites with similar pocket depths. The presence of similar levels of red complex species in gingivitis sites from periodontitis patients and from gingivitis patients suggested that the higher levels of IL‐18 were not associated with a different microbial challenge.     

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.     

Reduced expression of lipopolysaccharide‐induced CXC chemokine in Porphyromonas gingivalis‐induced experimental periodontitis in matrix metalloproteinase‐8 null mice

Hernández M, Gamonal J, Salo T, Tervahartiala T, Hukkanen M, Tjäderhane L, Sorsa T. Reduced expression of LIX/CXCL5 in Porphyromonas gingivalis‐induced experimental periodontitis in matrix metalloproteinase‐8 null mice. J Periodont Res 2011; 46: 58–66. © 2010 John Wiley & Sons A/S Background and Objective: Matrix metalloproteinase‐8 (MMP‐8) is a central mediator in chronic periodontitis. Recently developed MMP‐8‐deficient mice show an impaired polymorphonuclear neutrophil response and more severe alveolar bone loss in Porphyromonas gingivalis‐induced experimental periodontitis. The main mediators involved in neutrophil and monocyte/macrophage recruitment and in bone loss include lipopolysaccharide‐induced CXC chemokine (LIX/CXCL5), stromal‐derived factor‐1/CXC chemokine ligand 12 (SDF1/CXCL12) and RANKL. Therefore, the aim of this study was to characterize the expression of LIX/CXCL5, SDF1/CXCL12 and RANKL in Porphyromonas gingivalis‐induced experimental periodontitis in MMP‐8^?/^? (knockout) and wild‐type mice. Material and methods:  MMP‐8 null and WT P. gingivalis‐infected and uninfected mice were included. Histopathological changes were assessed and LIX/CXCL5, SDF1/CXCL12 and RANKL were immunodetected and quantified. Results: Typical histopathological features of chronic periodontitis were seen in P. gingivalis‐infected groups. LIX/CXCL5 expression was restricted to the gingival papilla in all four groups. Significantly lower expression of LIX/CXCL5 was seen in the knockout group compared with the wild‐type infected group (p < 0.05). SDF1/CXCL12 and RANKL expression was mainly localized to the alveolar crest, including inflammatory leukocytes, vascular endothelium, osteoblasts and osteoclasts. Significant increases of SDF1/CXCL12 and RANKL were seen in both knockout and wild‐type P. gingivalis‐infected groups compared with uninfected groups (p < 0.05). Conclusion: RANKL and SDF1/CXCL12 are up‐regulated in P. gingivalis‐induced periodontitis and they appear to be associated with the pathogenesis of the disease. MMP‐8 is associated with a reduced expression of LIX/CXCL5 in the P. gingivalis‐induced experimental periodontitis model.     

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.     

Analysis of YKL‐40 Acute‐Phase Protein and Interleukin‐6 Levels in Periodontal Disease

Background: YKL‐40, a new acute‐phase protein, is shown to be elevated in inflammatory diseases, such as rheumatoid arthritis, type 2 diabetes mellitus, and coronary artery diseases. However, there is no data indicating a relationship between YKL‐40 and periodontal disease. Interleukin‐6 (IL‐6) is the major regulator of acute‐phase protein synthesis and one of the most studied inflammatory markers in periodontal disease. The purpose of the present study is to evaluate YKL‐40 and IL‐6 levels in gingival crevicular fluid (GCF) and serum of patients with periodontal disease and healthy individuals. Methods: Periodontally healthy individuals (n = 15), patients with gingivitis (n = 15), and patients with severe chronic periodontitis (CP) (n = 15) without any systemic disease were included in the study. Clinical measurements were recorded; GCF and blood samples were obtained from each participant. GCF and serum YKL‐40 and IL‐6 levels were analyzed by enzyme‐linked immunosorbent assay. Statistical analysis was performed by parametric and non‐parametric tests. Results: Total amounts of YKL‐40 and IL‐6 in GCF as well as serum YKL‐40 and IL‐6 levels were significantly higher in patients with gingivitis and CP compared with healthy controls (P <0.01). YKL‐40 levels in GCF and serum as well as serum IL‐6 levels were significantly higher in patients with CP compared with patients with gingivitis (P <0.01). Conclusions: YKL‐40 levels in GCF as well as serum YKL‐40 and IL‐6 levels increased from gingivitis to periodontitis. Within the limits of the present study, the YKL‐40 molecule might be a potential novel inflammatory marker of periodontal disease.     

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.     

Blocking Proinflammatory Cytokine Release Modulates Peripheral Blood Mononuclear Cell Response to Porphyromonas gingivalis

Background: Chronic periodontitis (CP) is an inflammatory disease in which cytokines play a major role in the progression of disease. Anti‐inflammatory cytokines (interleukin 4 [IL‐4] and IL‐10) were reported to be absent or reduced in diseased periodontal tissues, suggesting an imbalance between the proinflammatory and anti‐inflammatory mediators. This study tests the hypothesis that there is cellular crosstalk mediated by proinflammatory and anti‐inflammatory cytokines and that blocking proinflammatory cytokine (tumor necrosis factor‐α [TNF‐α] and IL‐1) production will enhance anti‐inflammatory cytokine (IL‐4 and IL‐10) production from peripheral blood mononuclear cells (PBMCs) in response to Porphyromonas gingivalis. Methods: PBMCs were isolated from individuals diagnosed with CP or healthy individuals and cultured for 24 hours. Concanavalin A (ConA) was used as an activator of lymphocyte function. Live and heat‐killed P. gingivalis or lipopolysaccharide from P. gingivalis were used as the bacterial stimulants. TNF‐α and IL‐1 production was neutralized by specific antibodies against TNF‐α and IL‐1α or IL‐β. Culture supernatants were evaluated by enzyme‐linked immunosorbent assay for TNF‐α, IL‐1β, IL‐4, and IL‐10 production. Results: Live P. gingivalis did not result in any significant IL‐10 or IL‐4 release, whereas heat‐killed P. gingivalis led to a significant increase in IL‐10 levels compared with unstimulated or live P. gingivalis–stimulated cells from both healthy individuals or those with CP. Overall, PBMCs from patients with CP produced significantly lower IL‐10 in response to ConA and P. gingivalis, suggesting chronic suppression of the anti‐inflammatory cytokine production. Blocking the proinflammatory cytokine response did not result in any substantial change in IL‐10 or IL‐4 response to live P. gingivalis. Blocking the proinflammatory cytokine response restored IL‐10 production by cells from CP in response to P. gingivalis lipopolysaccharide. Conclusions: These findings suggest that PBMCs from patients with CP have suppressed anti‐inflammatory cytokine production that can, in part, be restored by neutralizing proinflammatory cytokines. Monocytes are an important source of IL‐10 production, and monocyte‐derived IL‐10 might play a regulatory role in the pathogenesis of CP.     

Influence of Periodontal Status and Periodontopathogens on Levels of Oral Human β‐Defensin‐2 in Saliva

Background: Expression patterns of human β‐defensin‐2 (HBD‐2) mRNA or HBD‐2 protein concentration and periodontal diseases have been a focus of scientific research. This study compares the salivary levels of HBD‐2 protein concentration of healthy patients and patients with gingivitis and chronic periodontitis (CP) and correlates these levels with the presence of periodontopathogens. Methods: A total of 89 patients were enrolled in this study: 31 periodontally healthy, 27 with gingivitis, and 31 with CP. Plaque and gingival indices, probing depth, and clinical attachment level were measured. The presence of Campylobacter rectus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Prevotella intermedia was evaluated qualitatively by conventional polymerase chain reaction. HBD‐2 quantification in saliva was performed using an immune enzymatic assay. Frequency of periodontopathogens and HBD‐2 protein concentration was assessed. Association between HBD‐2 protein concentration (≥100 pg/mL) and the simultaneous presence of one to two, three to four, or five to six periodontopathogens was tested. Results: Although periodontally healthy individuals and patients with gingivitis showed similar HBD‐2 levels, the CP group displayed an increased level of HBD‐2. P. gingivalis, P. intermedia, T. forsythia, and T. denticola were more prevalent in CP; however, their mere presence was not related to the increased levels of HBD‐2 (Pearson correlation and multinomial logistic regression model). Conclusions: Salivary HBD‐2 protein concentration was higher in patients with CP compared with healthy individuals or patients with gingivitis. These different protein concentrations were not related to the frequency of periodontopathogens. Clinical inflammatory profile had a higher impact on salivary HBD‐2 levels than bacteria.     

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.     

Monocytic TNFα secretion patterns in IDDM patients with periodontal diseases*

Abstract The aim of the present study was to identify whether monocytic TNFα secretion patterns could serve as a potential phenotypic discriminator for periodontal disease susceptibility within insulin-dependent diabetes mellitus (IDDM) patients. In 32 IDDM individuals the lipopolysaccharide (LPS) stimulated monocytic TNFα secretion dose-response characteristics were analyzed and related to two different periodontal status categories. Diabetics were divided into group A (gingivitis or mild periodontal disease) and group B (moderate to severe periodontal disease). In addition, 17 non-diabetic individuals with various degrees of periodontal disease served as control patients. Diabetics as a group had a significantly higher monocytic TNFα production in response to increasing Porphyromonas gingivalis A 7436 lipopolysaccharide concentrations (0, 0.003, 0.03, 0.3 and 3.0 μg/ml) as compared to non-diabetic patients with gingivitis or adult periodontitis (p <0.05). A significant difference in the dose response was also noted in the level of TNFα secreted as a function of P. gingivalis LPS concentrations between group A and B diabetics, as determined by two-way repeated measurements ANOVA (p <0.05). Furthermore, there was no significant difference in the mean HbA_1C between the two diabetic groups, and the TNFα level was not significantly associated with the HbA_1C level within diabetic patients. These data suggest that the diabetic state results in an upregulated monocytic TNFα secretion phenotype (4.6-fold increase) which, in the presence of Gram-negative bacterial challenge, is associated with a more severe periodontal disease expression. In addition, approximately 40% (10 of 24) IDDM periodontitis patients in group B demonstrated a 62-fold elevation in TNFα secretion relative to non-diabetic gingivitis or periodontitis patients and a 13.5-fold increase relative to IDDM group A (gingivitis or mild periodontitis) patients.     

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.     

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.     

Clinical association between chronic periodontitis and the leukocyte extravasation inhibitors developmental endothelial locus‐1 and pentraxin‐3

This clinical study aimed to determine whether periodontal disease is associated with expression of developmental endothelial locus‐1 (Del‐1) and pentraxin‐3 (PTX‐3), endogenous inhibitors of leukocyte extravasation in humans. Expression of DEL1, PTX3, interleukin‐17A (IL17A), and lymphocyte function‐associated antigen‐1 (LFA1) was determined, using RT‐PCR and melting curve analysis, in biopsies of gingival tissues from 95 patients: 42 with moderate periodontitis; 40 with severe periodontitis; and 13 healthy controls. Relative expression of DEL1 and PTX3 was statistically significantly weaker in patients with periodontitis than in the control subjects. On the contrary, both IL17A and LFA1 showed statistically significant stronger expression in patients with periodontitis than in healthy controls. Correlation analysis, performed using Spearman's test, showed that expression of DEL1 was statistically significantly linked to periodontitis (ρ = ?0.103) and to age (ρ = ?0.134), but not to the gender of the patient, and that expression of PTX3 was significantly correlated with periodontitis (ρ = ?0.354). Expression of neutrophil extravasation inhibitors DEL1 and PTX3 show significant, but weak, association with the clinical manifestation of chronic periodontitis.     

Response of periodontitis and healthy patients in a Porphyromonas gingivalis‐stimulated whole‐blood model

Aim: To investigate the inflammatory responses of periodontitis patients and healthy patients in a whole‐blood model stimulated with Porphyromonas gingivalis. Methods: Whole blood collected from 17 periodontitis patients and six healthy patients was stimulated with Porphyromonas gingivalis cells. The secretion of cytokines and matrix metalloproteinases was quantified by enzyme‐linked immunosorbent assay. An analysis of covariance with the ancova model was used to evaluate the significance of differences in secreted host molecules by whole blood from the periodontitis and healthy groups. Results: Porphyromonas gingivalis induced the secretion of interleukin‐1β, interleukin‐6, interleukin‐8, tumor necrosis factor‐α, monocyte chemoattractant protein‐1, interferon inducible protein‐10 by whole blood from patients in the periodontitis and healthy groups. Matrix metalloproteinase‐8 and ‐9 levels secreted by whole blood also increased following stimulation. No significant differences (P < 0.05) in the amounts of secreted host molecules were observed between periodontitis and healthy patients. Conclusion: This study suggests that Porphyromonas gingivalis can provoke an inflammatory response and promote the progression of periodontitis by inducing the secretion of high levels of cytokines and matrix metalloproteinases by a mixed leukocyte population. However, the whole‐blood model did not reveal any significant differences in the inflammatory response between periodontitis patients (n = 17) and periodontally‐healthy patients (n = 6).     

Identification of gingipain‐specific I‐Ab‐restricted CD4+ T cells following mucosal colonization with Porphyromonas gingivalis in C57BL/6 mice

Chronic periodontitis is associated with Porphyromonas gingivalis infection. Although virulence factors of P. gingivalis are hypothesized to contribute to the pathogenesis of periodontitis, it is unclear whether the local CD4⁺ T‐cell‐mediated response they elicit prevents or contributes to periodontal bone destruction. We hypothesize that major histocompatibility complex class II I‐A^b‐binding peptides existing in Kgp and RgpA are presented to CD4⁺ T cells during P. gingivalis oral colonization. The protein sequences of gingipains RgpA and Kgp, and OMP40 and OMP41 of P. gingivalis were scanned using an I‐A^b‐binding matrix. From this analysis we identified 53 candidate peptides that had the potential to engage the peptide‐binding groove of the I‐A^b molecule of C57BL/6 mice. An ELISpot‐based screen revealed those peptide‐primed effector/memory CD4⁺ T cells that could be re‐stimulated in vitro with P. gingivalis or the peptide itself to produce interleukin‐17A or interferon‐γ. Two immunodominant peptides, Kgp_467–477 (pKgp) and RgpA_1054–1064/Kgp_1074–1084 (pR/Kgp) were identified and engineered to be displayed on I‐A^b molecular tetramers. Peptide pR/Kgp is conserved across all sequenced P. gingivalis strains. C57BL/6 mice were orally inoculated with P. gingivalis strain 53977 and cervical lymph node cells were stained with phycoerythrin‐conjugated pKgp::I‐A^b and pR/Kgp::I‐A^b tetramers. We found that only pR/Kgp::I‐A^b bound with the desired specificity to gingipain‐specific CD4⁺ T cells. The pR/Kgp::I‐A^b tetramer complex will allow the identification of effector/memory CD4⁺ T cells specific for two virulence factors of P. gingivalis strains associated with periodontal disease.     

Porphyromonas gingivalis HmuY‐Induced Production of Interleukin‐6 and IL‐6 Polymorphism in Chronic Periodontitis

Background: In chronic periodontitis (CP), the gene polymorphism of interleukin‐6 (IL‐6) to 174C/G has been associated with the altered production of this cytokine. The aim of this pilot study is to compare the allelic and genotypic frequencies in patients with CP with control individuals without periodontitis (NP) and to measure the production of IL‐6 by whole blood cells stimulated with Porphyromonas gingivalis HmuY protein. Methods: DNA was isolated from peripheral blood cells of 49 patients with CP and 60 control individuals classified as NP, and genotyping was performed by polymerase chain reaction using sequence‐specific primers. Whole blood cells from 29 patients with CP and 30 control individuals were stimulated for 48 hours with HmuY, and IL‐6 levels were measured using enzyme‐linked immunosorbent assay. Results: The proportion of individuals carrying the G allele at position –174 of the IL‐6 gene was higher in the group with CP (85.7%) than in the normal control group (73.3%; P <0.03). P. gingivalis HmuY‐induced production of IL‐6 was higher in the group with CP (P <0.05). Conclusions: Our findings suggest that P. gingivalis HmuY may be associated with increased IL‐6 production during CP. Furthermore, patients with periodontitis and individuals with higher HmuY‐induced production of IL‐6 show a high frequency of the G allele at position –174.