Functionality and opposite roles of two interleukin 4 haplotypes in immune cells.

Cytokines expression can be influenced by polymorphisms in their respective coding genes. We associated the CTI/TTD haplotype (Hap-1) and TCI/CCI haplotype (Hap-2) in the IL4 gene formed by the -590, +33 and variable number of tandem repeat polymorphisms with the severity of chronic periodontitis in humans. The functionality of these IL4 haplotypes in the response of immune cells to phorbol 12-myristate 13-acetate (PMA) with Ionomycin and IL-1ß (as inflammatory stimuli) was evaluated. Gene expression (quantitative real-time PCR), profile of secreted cytokines (multiplex) and phenotypic polarization of T cells (flow cytometry) were the outcomes assessed. Green fluorescent protein reporter plasmid constructs containing specific IL4 haplotype were transiently transfected into JM cells to assess the influence of the individual haplotypes on promoter activity. In response to inflammatory stimuli the immune cells from Hap-1 haplotype had increased expression of anti-inflammatory IL4; conversely, the Hap-2 haplotype showed higher levels of pro-inflammatory cytokines. The haplotype CTI proved to be the most important for the regulation of IL4 promoter, regardless of the nature of the inflammatory stimulation; whereas the polymorphism in the promoter region had the least functional effect. In conclusion, IL4 haplotypes studied are functional and trigger opposite immune responses: anti-inflammatory (Hap-1) and pro-inflammatory (Hap-2). In addition, we identified the CTI haplotype as the main responsible for the regulation of IL4 transcriptional activity.

The effect of conventional mechanical periodontal treatment on red complex microorganisms and clinical parameters in Down syndrome periodontitis patients: a pilot study.

Periodontal disease (PD) is induced by a complex microbiota, such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola (together called the red complex), which triggers intense inflammatory reaction. Down syndrome (DS) individuals demonstrate a high prevalence of PD compared with those who are otherwise chromosomally normal (euploids). This pilot study aimed to evaluate the effect of non-surgical periodontal treatment in DS chronic periodontitis patients on clinical and microbiological parameters. Patients with chronic periodontitis, 23 DS and 12 euploids (control group), were submitted to non-surgical mechanical periodontal treatment, followed by maintenance for 45 days. Clinical parameters after periodontal treatment were similar in diseased and healthy sites, independent of the genetic background. Diseased sites of DS and control patients harbored similar levels of P. gingivalis and T. forsythia at baseline, but significantly higher levels of T. denticola were found in DS patients. Increased levels of P. gingivalis at healthy sites were found in DS individuals. Non-surgical periodontal therapy decreased the levels of red complex microorganisms and improved the tested clinical parameters of diseased sites in both groups. However, the levels of red complex bacteria were higher in diseased sites of DS patients after the periodontal treatment. We conclude in this pilot study that, although the mechanical periodontal treatment seemed to be effective in DS subjects over a short-term period, the red complex bacteria levels did not decrease significantly in diseased sites, as occurred in controls. Therefore, for DS patients, it seems that the conventional non-surgical periodontal therapy should be improved by utilizing adjuvants to reduce the presence of periodontopathogens.

TLR2 promoter hypermethylation creates innate immune dysbiosis.

Periodontitis is a common chronic inflammatory disease that is initiated by a complex microbial biofilm that poses significant health and financial burdens globally. Porphyromonas gingivalis is a predominant pathogen that maintains chronic inflammatory periodontitis. Toll-like receptors (TLRs) play an important role in periodontitis by recognizing pathogens and maintaining tissue homeostasis. Deficiencies in TLR expression and downstream signaling may reduce the host's innate defenses against pathogens, leading to bacterial persistence and exacerbated inflammation, which are now being better appreciated in disease pathologies. In the case of periodontitis, gingival epithelial cells form the first line of defense against pathogens. Innate immune dysregulation in these cells relates to severe disease pathology. We recently identified a blunted TLR2 expression in certain gingival epithelial cells expressing diminished cytokine signaling upon P. gingivalis stimulation. Upon detailed analysis of the TLR2 promoter CpG Island, we noted higher CpG methylation in this dysregulated cell type. When these cells were treated with DNA methyltransferase inhibitor, TLR2 mRNA and cytokine expression were significantly increased. If TLR2 expression plasmid was ectopically expressed in dysfunctional cells prior to P. gingivalis stimulation, the cytokine expression was increased, confirming the requirement of TLR2 in the P. gingivalis-mediated inflammatory response. We designed a chronic in vitro infection model to test if P. gingivalis can induce DNA methylation in normal gingival epithelial cells that express higher TLR2 upon agonist stimulation. Chronic treatment of normal epithelial cells with P. gingivalis introduced de novo DNA methylation within the cells. In addition, increased DNA methylation was observed in the gingiva of mice infected with P. gingivalis in a periodontitis oral gavage model. Moreover, tissues obtained from periodontitis patients also exhibited differential TLR2 promoter methylation, as revealed by bisulfite DNA sequencing. Taken together, DNA methylation of TLR2 can modulate host innate defense mechanisms that may confer increased disease susceptibility.

Clinical outcomes of periodontal therapy are not influenced by the ATC/TTC haplotype in the IL8 gene.

BACKGROUND AND OBJECTIVE: Although chronic periodontitis (CP) is a multifactorial condition, few studies have investigated the potential association of gene variants with the outcome of periodontal therapy. In a previous study, we reported that variants in the interleukin-8 (IL8) gene were associated with CP in a Brazilian population. The aim of this nonrandomized study was to investigate whether genetic susceptibility to CP, conferred by the presence of the IL8 ATC/TTC haplotype, influences the clinical outcomes of nonsurgical periodontal therapy and the IL-8 protein levels in the gingival crevicular fluid. MATERIAL AND METHODS: Forty-one individuals were grouped according to the presence (susceptible to CP; n = 21) or absence (not susceptible to CP; n = 20) of the IL8 ATC/TTC haplotype. These individuals received nonsurgical periodontal therapy from one periodontist, who was blinded to the genetic status of each patient, and follow up continued for 45 d. The clinical parameters and gingival crevicular fluid samples were collected at baseline and on day 45. The IL-8 levels were determined by an ELISA. The data were subjected to the Mann-Whitney U-test, Wilcoxon and Spearman tests and to multiple logistic-regression analysis. RESULTS: No significant differences between patients with or without the IL8 ATC/TTC haplotype were found for the outcome of nonsurgical periodontal therapy and IL-8 levels. The multiple logistic-regression analysis did not show a statistically significant association between the IL8 haplotype and the variables studied. CONCLUSION: In this longitudinal clinical study, we observed that neither the outcome of nonsurgical periodontal therapy nor the IL-8 levels were influenced by the IL8 ATC/TTC CP-susceptibility haplotype. Additional studies of CP patients from other ethnic populations are necessary to confirm these results.

Periodontopathogens levels and clinical response to periodontal therapy in individuals with the interleukin-4 haplotype associated with susceptibility to chronic periodontitis.

Periodontitis is an inflammatory disease that results from an interaction between dental biofilm agents and the host immune-inflammatory response. Periodontopathogenic organisms, such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, as well as the host's susceptibility, represented by the host's genetic makeup, are the key factors that influence this complex disease. Recently, we identified haplotypes in the IL4 gene that were associated with chronic periodontitis (CP). This study aimed to evaluate whether subjects with different IL4 haplotypes (TCI/CCI and TTD/CTI) would be differentially colonized by periodontopathogens and whether they would respond differently to non-surgical periodontal therapy. Thirty-nine patients carrying the IL4 haplotype of genetic susceptibility to CP (IL4+) or protection against CP (IL4-) were evaluated. Those groups were further subdivided into individuals with CP (CP IL4+ or CP IL4-) and those that were periodontally healthy (H) (H IL4+ or H IL4-). CP patients were submitted to non-surgical periodontal therapy. Clinical and microbiological analyses were performed considering the data at baseline and 45 and 90 days after periodontal therapy. Periodontopathogens levels were evaluated by absolute quantitative polymerase chain reaction (qPCR). The baseline data revealed that the total levels of periodontopathogens were higher in the CP IL4+ than in the CP IL4- groups. Clinical analyses revealed that the periodontal therapy was equally effective, independent of the subject's IL4 genetic load. The TCI/CCI IL4 haplotype, previously associated with genetic susceptibility to CP, was also associated with increased levels of periodontopathogenic bacteria, but this genetic background did not influence the response to non-surgical periodontal treatment.

Association between IL8 haplotypes and pathogen levels in chronic periodontitis.

Chronic periodontitis (CP) is considered to be a multifactorial disease influenced by microbial and genetic factors. The aim of the present study was to investigate whether the genetic susceptibility to CP in individuals with the IL8 ATC/TTC haplotype is associated with subgingival levels of periodontopathogens. Sixty-five individuals, grouped according to the presence (n = 28) or absence (n = 37) of the IL8 haplotype, were evaluated. After clinical periodontal evaluation, each group was subdivided according to the presence (CP) or absence (H) of periodontitis. Four subgingival samples were obtained from CP and two samples per subject from H patients. The levels and proportions of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were analyzed using quantitative real-time polymerase chain reaction (q-PCR). No differences were found in the proportion of periodontopathogenic bacteria between groups with the presence or absence of the IL8 haplotype. However, in the CP groups, the levels of periodontopathogens were significantly higher in the individuals without the IL8 haplotype than in the individuals with the IL8 haplotype. These results suggest that periodontal destruction may occur in patients who are considered to be genetically susceptible to CP with a lower microbial challenge because of the presence of the IL8 ATC/TTC haplotype than in patients without this haplotype.