Walter Martins

The broad effects of the functional IL-10 promoter-592 polymorphism : modulation of IL-10, TIMP-3, and OPG expression and their association with periodontal disease outcome

Periodontal diseases are infectious diseases, in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. It occurs through the generation of metalloproteinases and the activation of bone resorption mechanisms. Anti‐inflammatory cytokines such as IL‐10 seem to attenuate periodontal tissue destruction through the induction of tissue inhibitors of metalloproteinases (TIMPs) and the inhibitor of osteoclastogenesis osteoprotegerin (OPG). A high individual variation in levels of IL‐10 mRNA is verified in periodontitis patients, which is possibly determined by genetic polymorphisms. In this study, the IL‐10 promoter ‐592C/A single nucleotide polymorphism (SNP), which is associated with a decrease in IL‐10 production, was analyzed by RFLP in 116 chronic periodontitis (CP) patients and 173 control (C) subjects, and the IL‐10, TIMPs, and OPG mRNA expression levels in diseased gingival tissues were determined by real‐time‐PCR. The IL‐10‐592 SNP CA (P=0.0012/OR=2.4/CI:1.4‐4.1), AA (P=0.0458/OR=2.3/CI:1.1‐4.9), and CA+AA (P=0.0006/OR=2.4/CI:1.4‐3.4) genotypes and the allele A (P=0.0036/OR=1.7/CI:1.2‐2.4) were found to be significantly more prevalent in the CP group when compared with control subjects. Both CA and AA genotypes were associated with lower levels of IL‐10, TIMP‐3, and OPG mRNA expression in diseased periodontal tissues and were also associated with disease severity as mean pocket depth. Taken together, the results presented here demonstrate that IL10‐592 SNP is functional in CP, being associated with lower levels of IL‐10 mRNA expression, which is supposed to consequently decrease the expression of the downstream genes TIMP‐3 and OPG, and influence periodontal disease outcome.

An Interleukin-1β (IL-1β) Single-Nucleotide Polymorphism at Position 3954 and Red Complex Periodontopathogens Independently and Additively Modulate the Levels of IL-1β in Diseased Periodontal Tissues

ABSTRACT Inflammatory cytokines such as interleukin-1β (IL-1β) are involved in the pathogenesis of periodontal diseases. A high individual variation in the levels of IL-1β mRNA has been verified, which is possibly determined by genetic polymorphisms and/or by the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. In this study, we investigated the role of an IL-1β promoter single-nucleotide polymorphism at position 3954 [IL-1β(3954) SNP] and the presence of the periodontopathogens in the determination of the IL-1β levels in the periodontal tissues of nonsmoking chronic periodontitis (CP) patients (n = 117) and control (C) subjects (n = 175) and the possible correlations with the clinical parameters of the disease. IL-1β(3954) SNP was investigated by restriction fragment length polymorphism, while the IL-1β levels and the presence of the periodontopathogens were determined by real-time PCR. Similar frequencies of IL-1β(3954) SNP were found in the C and CP groups, in spite of a trend toward a higher incidence of T alleles in the CP group. The IL-1β(3954) SNP CT and TT genotypes, as well as P. gingivalis, T. forsythia, and T. denticola, were associated with higher IL-1β levels and with higher values of the clinical parameters of disease severity. Concomitant analyses demonstrate that IL-1β(3954) and the red complex periodontopathogens were found to independently and additively modulate the levels of IL-1β in periodontal tissues. Similarly, the concurrent presence of both factors was associated with increased scores of disease severity. IL-1β(3954) genotypes and red complex periodontopathogens, individually and additively, modulate the levels of IL-1β in the diseased tissues of nonsmoking CP patients and, consequently, are potentially involved in the determination of the disease outcome.

Tumor necrosis factor-alpha −308G/A single nucleotide polymorphism and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-α in diseased periodontal tissues

BACKGROUND AND OBJECTIVE Inflammatory cytokines such as tumor necrosis factor-alpha are involved in the pathogenesis of periodontal diseases. A high between-subject variation in the level of tumor necrosis factor-alpha mRNA has been verified, which may be a result of genetic polymorphisms and/or the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola (called the red complex) and Aggregatibacter actinomycetemcomitans. In this study, we investigated the effect of the tumor necrosis factor-alpha (TNFA) -308G/A gene polymorphism and of periodontopathogens on the tumor necrosis factor-alpha levels in the periodontal tissues of nonsmoking patients with chronic periodontitis (n = 127) and in control subjects (n = 177). MATERIAL AND METHODS The TNFA -308G/A single nucleotide polymorphism was investigated using polymerase chain reaction-restriction fragment length polymorphism analysis, whereas the tumor necrosis factor-alpha levels and the periodontopathogen load were determined using real-time polymerase chain reaction. RESULTS No statistically significant differences were found in the frequency of the TNFA -308 single nucleotide polymorphism in control and chronic periodontitis groups, in spite of the higher frequency of the A allele in the chronic periodontitis group. The concomitant analyses of genotypes and periodontopathogens demonstrated that TNFA -308 GA/AA genotypes and the red-complex periodontopathogens were independently associated with increased levels of tumor necrosis factor-alpha in periodontal tissues, and no additive effect was seen when both factors were present. P. gingivalis, T. forsythia and T. denticola counts were positively correlated with the level of tumor necrosis factor-alpha. TNFA -308 genotypes were not associated with the periodontopathogen detection odds or with the bacterial load. CONCLUSION Our results demonstrate that the TNFA -308 A allele and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased tissues of nonsmoking chronic periodontitis patients and consequently are potentially involved in determining the disease outcome.

Strong and persistent microbial and inflammatory stimuli overcome the genetic predisposition to higher matrix metalloproteinase‐1 (MMP‐1) expression: a mechanistic explanation for the lack of association of MMP1‐1607 single‐nucleotide polymorphism genotypes with MMP‐1 expression in chronic periodontitis lesions

AIMS Our objective was to evaluate the association between the MMP1-1607 single-nucleotide polymorphism (SNP), periodontopathogens and inflammatory cytokines with matrix metalloproteinase-1 (MMP-1) mRNA levels in vitro and in vivo. MATERIALS AND METHODS This study investigated the influence of genetic (MMP1-1607 SNP), microbial (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Actinobacillus actinomycetemcomitans) and inflammatory [tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta)] factors on the determination of MMP-1 mRNA levels in periodontal tissues of non-smoker chronic periodontitis (CP, N=178) and control (C, N=190) groups. The effects of single and repeated lipopolysaccharide (LPS) and inflammatory cytokine stimulation of macrophages with distinct MMP1-1607 SNP genotypes were also investigated. RESULTS In healthy tissues, the MMP1-1607 2G allele was associated with higher MMP-1 levels while in CP MMP-1 levels were associated with the presence and load of periodontopathogens, and also with TNF-alpha and IL-1beta expression irrespective of the MMP1-1607 genotype. In vitro data demonstrate that in 2G macrophages low- and intermediate-dose LPS and TNF-alpha+IL-1beta stimulation was associated with increased MMP-1 expression, while strong and repeated stimulation resulted in higher MMP-1 levels irrespective of the MMP1-1607 genotype. CONCLUSION Our data demonstrate a limited role for MMP1-1607 SNP in periodontitis, where the extensive chronic antigenic challenge exposure overcomes the genetic control and plays a major role in the determination of MMP-1 expression.

The use of chronic gingivitis as reference status increases the power and odds of periodontitis genetic studies – a proposal based in the exposure concept and clearer resistance and susceptibility phenotypes definition

AIM Current literature on chronic periodontitis genetics encompasses numerous single nucleotide polymorphisms-focused case-control studies with inconsistent and controversial results, which typically disregards the exposure concept embraced by case-control definition. Herein, we propose a case-control design reappraisal by clear phenotype selection, where chronic gingivitis represents a genetically resistant phenotype/genotype opposing the susceptible cohort. MATERIAL AND METHODS The hypothesis was tested in healthy, chronic periodontitis and gingivitis groups through Real-time PCR-based allelic discrimination of classic variants IL1B-3954, IL6-174, TNFA-308, IL10-592 and TLR4-299. RESULTS Observed allele/genotype frequencies characterize the healthy group with an intermediate genetic profile between periodontitis and gingivitis cohorts. When comparing genotype/allele frequencies in periodontitis versus healthy and periodontitis versus gingivitis scenarios, the number of positive associations (2-4) and the degree of association (p and odds ratio values) were significantly increased by the new approach proposed (periodontitis versus gingivitis), suggesting the association of IL1B-3954, TNFA-308, IL10-592 and TLR4-299 with periodontitis risk. Power study was also significantly improved by the new study design proposed when compared to the traditional approach. CONCLUSIONS The data presented herein support the use of new case-control study design based on the case-control definition and clear resistance/susceptibility phenotypes selection, which can significantly impact the study power and odds of identification of genetic factors involved in PD.

Association of Human T Lymphotropic Virus 1 Amplification of Periodontitis Severity with Altered Cytokine Expression in Response to a Standard Periodontopathogen Infection

BACKGROUND Periodontal diseases (PDs) are infectious diseases in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. Recently, viruses have been implicated in the pathogenesis of PDs. Individuals infected with human T lymphotropic virus 1 (HTLV-1) present with abnormal oral health and a marked increased prevalence of periodontal disease. METHODS In this study, we investigated the patterns of periodontopathogen infection and local inflammatory immune markers in HTLV-1-seropositive individuals with chronic periodontitis (CP/HTLV-1 group) compared with HTLV-1-seronegative individuals with chronic periodontitis (CP group) and periodontally healthy, HTLV-1-seronegative individuals (control group). RESULTS Patients in the CP/HTLV-1 group had significantly higher values of bleeding on probing, mean probing depth, and attachment loss than patients in the CP group. The expression of tumor necrosis factor alpha and interleukin (IL) 4 was found to be similar in the CP and CP/HTLV-1 groups, whereas IL-12 and IL-17 levels trended toward a higher expression in the CP/HTLV-1 group. A significant increase was seen in the levels of IL-1beta and interferon gamma in the CP/HTLV-1 group compared with the CP group, whereas expression of the regulatory T cell marker FOXp3 and IL-10 was significantly decreased in the lesions from the CP/HTLV-1 group. Interestingly, similar frequency and/or load of periodontopathogens (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans) and frequency of viruses (herpes simplex virus 1, human cytomegalovirus, and Epstein-Barr virus) characteristically associated with PDs were found in the CP/HTLV and CP groups. CONCLUSIONS HTLV-1 may play a critical role in the pathogenesis of periodontal disease through the deregulation of the local cytokine network, resulting in an exacerbated response against a standard periodontopathogen infection.

TBX21-1993T/C (rs4794067) polymorphism is associated with increased risk of chronic periodontitis and increased T-bet expression in periodontal lesions, but does not significantly impact the IFN-g transcriptional level or the pattern of periodontophatic bacterial infection

Th1-polarized host response, mediated by IFN-γ, has been associated with increased severity of periodontal disease as well as control of periodontal infection. The functional polymorphism TBX21-1993T/C (rs4794067) increases the transcriptional activity of the TBX21 gene (essential for Th1 polarization) resulting in a predisposition to a Th-1 biased immune response. Thus, we conducted a case-control study, including a population of healthy controls (H, n = 218), chronic periodontitis (CP, n = 197), and chronic gingivitis patients (CG, n = 193), to investigate if genetic variations in TBX21 could impact the development of Th1 responses, and consequently influence the pattern of bacterial infection and periodontitis outcome. We observed that the polymorphic allele T was significantly enriched in the CP patients compared to CG subjects, while the H controls demonstrated and intermediate genotype. Also, investigating the putative functionality TBX21-1993T/C in the modulation of local response, we observed that the transcripts levels of T-bet, but not of IFN-γ, were upregulated in homozygote and heterozygote polymorphic subjects. In addition, TBX21-1993T/C did not influence the pattern of bacterial infection or the clinical parameters of disease severity, being the presence/absence of red complex bacteria the main factor associated with the disease status and the subrogate variable probing depth (PD) in the logistic regression analysis.

CCR5δ32 (rs333) polymorphism is associated with decreased risk of chronic and aggressive periodontitis: A case-control analysis based in disease resistance and susceptibility phenotypes

Abstract Chronic and aggressive periodontitis are infectious diseases characterized by the irreversible destruction of periodontal tissues, which is mediated by the host inflammatory immune response triggered by periodontal infection. The chemokine receptor CCR5 play an important role in disease pathogenesis, contributing to pro‐inflammatory response and osteoclastogenesis. CCR5&Dgr;32 (rs333) is a loss‐of‐function mutation in the CCR5 gene, which can potentially modulate the host response and, consequently periodontitis outcome. Thus, we investigated the effect of the CCR5&Dgr;32 mutation over the risk to suffer periodontitis in a cohort of Brazilian patients (total N = 699), representative of disease susceptibility (chronic periodontitis, N = 197; and aggressive periodontitis, N = 91) or resistance (chronic gingivitis, N = 193) phenotypes, and healthy subjects (N = 218). Additionally, we assayed the influence of CCR5&Dgr;32 in the expression of the biomarkers TNF&agr;, IL‐1&bgr;, IL‐10, IL‐6, IFN‐&ggr; and T‐bet, and key periodontal pathogens P. gingivalis, T. forsythia, and T. denticola. In the association analysis of resistant versus susceptible subjects, CCR5&Dgr;32 mutant allele‐carriers proved significantly protected against chronic (OR 0.49; 95% CI 0.29–0.83; p‐value 0.01) and aggressive (OR 0.46; 95% CI 0.22–0.94; p‐value 0.03) periodontitis. Further, heterozygous subjects exhibited significantly decreased expression of TNF&agr; in periodontal tissues, pointing to a functional effect of the mutation in periodontal tissues during the progression of the disease. Conversely, no significant changes were observed in the presence or quantity of the periodontal pathogens P. gingivalis, T. forsythia, and T. denticola in the subgingival biofilm that could be attributable to the mutant genotype.