Induction of interleukin (IL)-1β, IL-10, IL-8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans

Trindade SC, Olczak T, Gomes‐Filho IS, Moura‐Costa LF, Cerqueira EMM, Galdino‐Neto M, Alves H, Carvalho‐Filho PC, Xavier MT, Meyer R. Induction of interleukin (IL)‐1β, IL‐10, IL‐8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans. J Periodont Res 2012; 47: 27–32. © 2011 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis, an anaerobic gram‐negative bacterium, is associated with chronic periodontitis. This study was undertaken to evaluate the production of interleukin (IL)‐1β, IL‐8 and IL‐10 by human peripheral blood mononuclear cells (PBMC) stimulated with P. gingivalis antigens and to assess the levels of serum immunoglobulin (Ig)G, IgA and IgG subclasses raised against P. gingivalis HmuY protein. Material and Methods: PBMC from patients with chronic periodontitis (CP) and from nonperiodontitis (NP) control subjects were stimulated with P. gingivalis antigens, and the cytokine levels in the culture supernatants were determined by ELISA. The specificity of serum antibodies raised against HmuY was analyzed by Western blotting and by ELISA. Results: Compared with the NP controls, the CP patients produced higher levels of total serum IgG and IgG1 specific for P. gingivalis HmuY. No differences were found between CP and NP groups in the production of IL‐1β and IL‐8 by PBMC stimulated with total P. gingivalis antigens. Only P. gingivalis lipopolysaccharide (LPS) induced higher levels of IL‐10 in the CP group. Higher levels of IL‐1β and IL‐10 were induced by HmuY than by other antigens derived from the wild‐type P. gingivalis strains. In contrast, total antigens derived from the hmuY‐deletion mutant strain induced the production of significantly higher levels of IL‐8 and significantly lower levels of IL‐1β. Conclusion: Our data suggest that P. gingivalis HmuY may be considered an immunogenic protein associated with host–pathogen interactions.     

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.     

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.     

Immunoglobulin G subclass antibody profiles in Porphyromonas gingivalis‐associated aggressive and chronic periodontitis patients

Background/aims: The immunoglobulin G (IgG) antibody response is considered to be protective and beneficial for the control of periodontal lesions. This study analysed IgG subclass antibody levels of Porphyromonas gingivalis in patients with both aggressive periodontitis (AgP) and chronic periodontitis (CP). Methods: Subgingival plaque and peripheral blood samples were collected from patients with localized AgP (n = 13), generalized AgP (n = 28) and generalized CP (n = 27) and from 14 periodontally healthy controls. P. gingivalis was identified in subgingival pockets using a polymerase chain reaction. Simultaneously, serum IgG subclass antibody against P. gingivalis whole cells/P. gingivalis fimbriae were measured using enzyme‐linked immunosorbent assay. Results: P. gingivalis was frequently detected in periodontitis patients. Anti‐P. gingivalis whole cell IgG1 was elevated in all P. gingivalis‐positive patients in the three periodontitis groups. Although increased anti‐P. gingivalis IgG1 was also observed in the bacterium‐positive healthy controls, the level was lower than that found in the three periodontitis groups. Levels of IgG1, IgG2, IgG3 and IgG4 to P. gingivalis did not differ among bacterium‐positive patients in the three periodontitis groups; a significant increase of IgG2 level was not observed in localized AgP. Anti‐fimbriae IgG subclass levels of IgG1, IgG2 and IgG4 did not differ among bacterium‐positive subjects in all groups, while the anti‐fimbriae IgG3 level in generalized CP was significantly higher than that in localized and generalized AgP. Conclusions: P. gingivalis infection elicited an IgG subclass antibody response in both periodontitis patients and healthy subjects, while higher anti‐P. gingivalis IgG1 levels were found in the three periodontitis groups compared with the healthy control group.     

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.     

Coronary heart disease and chronic periodontitis: is polymorphism of interleukin‐6 gene the common risk factor in a Chinese population?

Oral Diseases (2011) 17 , 270–276 Objectives: Coronary heart disease (CHD) and chronic periodontitis (CP) both are multifactorial chronic diseases and related to inflammation. Interleukin‐6 (IL‐6) plays an important role in the pathogenesis of inflammatory diseases. The purpose of the study was to investigate the association among IL‐6 gene polymorphisms, CP and CHD susceptibility in a Chinese population. Material and methods: The investigation was conducted as a case‐control study involving 505 individuals: 113 patients with CHD and CP, 84 patients with CHD, 178 patients with CP and 130 control individuals. The polymorphisms of IL‐6 gene were analyzed by polymerase chain reaction‐restriction fragment length polymorphism. Relationships between the distributions of the genotypes and risk factors were also assessed. Results: Mutations at the loci ‐174 G/C, ‐597 G/A of IL‐6 were rare in a Chinese population. No significant difference for IL‐6‐572C/G polymorphism was detected among moderate CP group, severe CP group and control (P = 0.312 and 0.481), significant differences were found between CHD groups and non‐CHD groups (P ≤ 0.001). After adjustment for CHD risk factors, the G allele resulted in an increased risk (OR = 1.676‐1.856), the GG/CG genotype was nearly two times higher risk compared to CC genotype (OR = 2.010‐2.136). Conclusions: IL‐6‐572C/G polymorphism did not correlate with CP susceptibility, but might be a potential risk factor for CHD in a Chinese population.     

Porphyromonas gingivalis antigen preferentially stimulates T cells to express IL‐17 but not receptor activator of NF‐κB 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 κB NF‐κB 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.     

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).     

Saliva and Serum Levels of B‐Cell Activating Factors and Tumor Necrosis Factor‐α in Patients With Periodontitis

Background: B‐lymphocytes play a central and critical role in the adaptive immune response against invading pathogens. This study evaluates saliva and serum levels of APRIL (a proliferation‐inducing ligand), B‐cell activating factor (BAFF), tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐6, and IL‐10 in patients with chronic periodontitis (CP) or aggressive periodontitis (AgP) and periodontally healthy individuals. Methods: Twenty‐five patients with AgP, 20 patients with CP, and 20 periodontally healthy individuals were included. Smoking status was recorded, and all individuals were divided into non‐smokers and smokers. Saliva and serum samples were collected before clinical periodontal measurements. APRIL, BAFF, TNF‐α, IL‐6, and IL‐10 levels in serum and saliva samples were determined by enzyme‐linked immunosorbent assay. Statistical analysis was performed using multivariate analysis of variance and bivariate correlation. Results: Serum and saliva levels of TNF‐α, APRIL, BAFF, IL‐6, and IL‐10 were similar in CP and AgP groups. Serum levels of TNF‐α, APRIL, and BAFF and saliva levels of BAFF were significantly higher in periodontitis groups than healthy controls (P <0.05). Non‐smokers with CP or AgP had lower levels of saliva TNF‐α and APRIL and serum APRIL and IL‐6 than smokers with CP or AgP (P <0.05). Saliva APRIL and serum TNF‐α and IL‐6 levels were significantly higher in healthy smokers than healthy non‐smokers (P <0.05). Clinical periodontal parameters correlated positively with TNF‐family cytokines and negatively with IL‐10 (P <0.05). Conclusions: Within the limits of this study, it may be suggested that elevated salivary and serum TNF‐α, APRIL, and BAFF in patients with periodontitis may contribute to the dominance of B cells in periodontitis lesions. Moreover, higher levels in healthy smokers than non‐smoking counterparts may play a role in detrimental effects of smoking on periodontal tissues.     

Local and Circulating Levels of Adipocytokines in Obese and Normal Weight Individuals With Chronic Periodontitis

Background: The aim of this study is to evaluate the local and circulating levels of adipocytokines (resistin, adiponectin, leptin, tumor necrosis factor [TNF]‐α, and interleukin [IL]‐6) in individuals who are obese and individuals who are normal weight (NW) with chronic periodontitis (CP). Methods: Periodontal and anthropometric examinations were performed. Based on these measurements, the individuals were divided into one of the following groups: NW non‐periodontitis (NP) (NWNP; n = 20); NWCP (n = 20); obese NP (ONP; n = 18); and obese CP (OCP; n = 20). The levels of adipocytokines were evaluated in the serum and gingival crevicular fluid of shallow and deep sites by enzyme‐linked immunosorbent assay. Results: In serum, resistin levels were higher whereas adiponectin levels were lower in periodontitis than in NP groups (P <0.05). The NWNP group presented the lowest serum leptin levels (P <0.05). The ONP and OCP groups demonstrated higher TNF‐α levels in periodontal sites than the NWNP and NWCP groups (P <0.05). Serum levels of IL‐6 (P = 0.04) and leptin (P = 0.01) were correlated with the OCP group, with odds ratios of 0.99 (95% confidence interval [CI]: –0.01 to –0.00) and 0.99 (95% CI: ?0.00 to ?0.00), respectively. Conclusions: Periodontitis mainly influenced the circulating levels of resistin and adiponectin, whereas both obesity and periodontitis affected the circulating levels of leptin in favor of proinflammation. In addition, obesity upregulated the local levels of TNF‐α.     

Assessment of Interleukin‐6 Receptor Inhibition Therapy on Periodontal Condition in Patients With Rheumatoid Arthritis and Chronic Periodontitis

Background: Overproduction of interleukin (IL)‐6 may play a pathologic role in rheumatoid arthritis (RA) and chronic periodontitis (CP). The present study assesses IL‐6 receptor (IL‐6R) inhibition therapy on the periodontal condition of patients with RA and CP. Methods: The study participants were 28 patients with RA and CP during treatment with IL‐6R inhibitor, and 27 patients with RA and CP during treatment without IL‐6R inhibitor. Periodontal and rheumatologic parameters and serum levels of cytokine and inflammatory markers and immunoglobulin G against periodontopathic bacteria were examined after medication with IL‐6R inhibitor for 20.3 months on average (T1) and again 8 weeks later (T2). Results: No differences were observed between the groups in any parameter values at T1, except for serum IL‐6 levels. The anti–IL‐6R group showed a significantly greater decrease in gingival index, bleeding on probing (BOP), probing depth (PD), clinical attachment level (CAL), and serum levels of IL‐6 and matrix metalloproteinase (MMP)‐3 from T1 to T2 than the control group (P <0.05). A significant correlation was found between changes in serum anticyclic citrullinated peptide levels and those in PD and CAL in the anti–IL‐6R group (P <0.05), whereas both groups exhibited a significant association between changes in serum MMP‐3 levels and those in BOP (P <0.05). Conclusion: Changes in periodontal and serum parameter values were different between the patients with RA and CP during treatment with and without IL‐6R inhibitor.     

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.     

Interleukin-6 gene promoter methylation in rheumatoid arthritis and chronic periodontitis

Background: Methylation status of the cytokine genes may play a role in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis (RA) and chronic periodontitis (CP). This study was undertaken to evaluate whether the DNA methylation profile of the interleukin‐6 (IL‐6) gene promoter was unique to individuals with RA and CP. Methods: The study participants consisted of 30 patients with RA, 30 patients with CP, and 30 age‐, sex‐, and smoking status–balanced healthy controls. Genomic DNA isolated from peripheral blood was modified by sodium bisulfite and analyzed for DNA methylation levels of IL‐6 gene with direct sequencing. Levels of IL‐6 were determined by an enzyme‐linked immunosorbent assay. Results: The region of IL‐6 gene promoter from ?1200 to +27 bp was shown to contain 19 CpG motifs. The methylation levels of the CpG motif at ?74 bp were significantly lower in patients with RA and CP than those in controls (P = 0.0001). Both levels of serum IL‐6 and IL‐6 production by mononuclear cells were significantly different between individuals with and without the methylation at ?74 bp (P = 0.03). The +19 bp motif exhibited differential levels of the methylation among the groups, which was not associated with serum levels of IL‐6. The other 17 CpG motifs exhibited comparable levels of the methylation between the groups. Conclusion: These results suggest that hypomethylated status of a single CpG in the IL‐6 promoter region may lead to increased levels of serum IL‐6, implicating a role in the pathogenesis of RA and CP.     

Salivary Interleukin‐6 and ‐8 in Patients With Oral Cancer and Patients With Chronic Oral Inflammatory Diseases

Background: Previous research has indicated that salivary interleukin (IL)‐6 and IL‐8 are potential biomarkers for oral squamous cell carcinoma (OSCC). However, their levels have been found to be significantly elevated in patients with chronic periodontitis (CP) or oral lichen planus (OLP). The data also showed wide variations in levels among the different studies, and no standardization procedure was ever performed. Therefore, the objective of this study is to determine whether CP or OLP confounds the use of IL‐6 or IL‐8 for OSCC detection. Methods: Saliva samples were collected from five groups: OSCC before treatment (n = 18); CP (n = 21); disease‐active OLP (n = 21); disease‐inactive OLP (n = 20); and healthy controls (n = 21). IL‐6 and IL‐8 concentrations (determined by enzyme‐linked immunosorbent assays) were compared, using total salivary protein–standardized levels to validate the data. The Kruskal–Wallis test (α = 0.05) followed by pairwise Mann–Whitney U (post hoc) tests with Bonferroni adjustments (α = 0.00625) were used for statistical analysis. Results: Salivary IL‐6 levels were significantly higher in patients with OSCC than in patients with CP (P <0.001), disease‐active OLP (P = 0.001), disease‐inactive OLP (P <0.001), and healthy controls (P <0.001). Salivary IL‐8 levels were significantly higher in patients with OSCC than in patients with CP (P <0.001), but only marginally significantly higher than in healthy controls (P = 0.014). Statistical results of standardized IL‐6 and IL‐8 levels were consistent with the non‐standardized levels in all pairs except one. Conclusion: Salivary IL‐6 may be a useful biomarker in the detection of OSCC, unconfounded by CP or OLP.     

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.     

Saliva and Serum Levels of Pentraxin‐3 and Interleukin‐1β in Generalized Aggressive or Chronic Periodontitis

Background: Pentraxin‐3 (PTX3) is a multifactorial protein involved in immunity and inflammation, which is rapidly produced and released by several cell types in response to inflammatory signals. The aim of the present study is to evaluate saliva, serum levels of PTX3, interleukin (IL)‐1β in patients with generalized chronic periodontitis (CP) or aggressive periodontitis (AgP), and periodontally healthy individuals. Methods: A total of 94 participants (25 patients with AgP, 25 patients with CP, and 44 periodontally healthy individuals matched with AgP and CP groups) were recruited. Saliva and serum samples were collected. Clinical periodontal measurements were recorded. PTX3, IL‐1β levels in serum, and saliva samples were determined by enzyme‐linked immunosorbent assay. Data were tested statistically using Kruskal‐Wallis, Mann‐Whitney U, and Spearman ρ rank test. Results: Serum and saliva data were similar in CP and AgP groups. Saliva levels of IL‐1β were significantly higher in the AgP and CP groups than controls (P <0.05). Salivary PTX3 levels were similar in the CP and control groups. Significantly higher salivary concentrations of PTX3 were detected in the AgP group than the control group (P <0.05). Saliva PTX3 levels correlated with plaque index and bleeding on probing in the CP group (P <0.05). Serum and saliva PTX3 levels correlated with those of IL‐1β in the AgP group (P <0.05). Conclusions: It may be suggested that PTX3 is related with periodontal tissue inflammation. Its salivary concentrations may have a diagnostic potential. Additional intervention and follow‐up studies coupling PTX3 concentrations with microbiologic analysis would better clarify its role in periodontal diseases.     

Correlation of Toll‐Like Receptor 4, Interleukin‐18, Transaminases,and Uric Acid in Patients With Chronic Periodontitis and Healthy Adults

Background: Because of the potential association between periodontal disease and inflammation, the purpose of the present study is to examine the level of Toll‐like receptor 4 (TLR‐4), interleukin‐18 (IL‐18), and uric acid as markers of the inflammatory host response in the plasma and saliva of healthy individuals and patients with periodontitis. In addition, routine biochemical parameters such as fasting glucose, insulin, total cholesterol, high‐density lipoprotein (HDL) cholesterol, low‐density lipoprotein (LDL) cholesterol, triglycerides, alanine transaminase (ALT), and aspartate transaminase (AST) were measured. The authors also wanted to check whether patients with chronic periodontitis (CP) exhibit different modulations in salivary and/or plasma concentrations of these parameters compared with clinically healthy individuals. Methods: Saliva and plasma samples were collected from 40 patients with CP and 20 healthy individuals. TLR‐4 and IL‐18 measurements were done using commercially available enzyme‐linked immunosorbent assay kits. Total, HDL, and LDL cholesterol; triglycerides; fasting glucose; AST; and ALT levels were analyzed on a biochemistry analysis system using specific kits. Non‐parametric tests were used for certain parameters in the statistical analyses because the data did not follow Gaussian distribution. Results: Significant differences were observed in plasma and salivary TLR‐4 and IL‐18 levels, along with clinical measurements such as plaque index and probing depth, in patients with CP (P <0.001). The plasma level of TLR‐4 was found to be increased from 0.99 to 3.28 ng/mL in patients with CP. Salivary TLR‐4 levels also showed a slightly higher increase in the diseased state (12.44 to 29.97 ng/mL). A significant increase of ≈46% was recorded in the plasma IL‐18 level. However, salivary IL‐18 levels rose up to >5‐fold in the patients with CP compared with healthy individuals. The level of plasma uric acid was found to be highly significantly increased compared with control individuals. HDL cholesterol and triglyceride also showed significant differences (P <0.02 and P <0.03, respectively). Plasma glucose, total cholesterol, LDL cholesterol, and insulin levels did not show any significant difference. There was only a slight increase in plasma AST and ALT levels between diseased and healthy states (22.55 versus 25.50 IU/L and 12.35 versus 15.95 IU/L, respectively). However, salivary AST and ALT levels showed a ≈6‐fold rise in the patients with CP compared with the healthy individuals. Cross‐correlation analysis in the periodontitis disease group showed a significant association of plasma AST, salivary AST, and salivary ALT with uric acid level. Conclusions: Based on this study, the authors believe that TLR‐4, IL‐18, and uric acid could have a role in the inflammatory pathology of periodontitis. These parameters are suggested to be useful in the prognosis and diagnosis of CP. However, the mechanistic association of these parameters with inflammatory pathology of patients with periodontitis needs to be further elucidated in a higher number of samples.     

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

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