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Originally published online as doi:10.1189/jlb.0308184 on August 25, 2008

Published online before print August 25, 2008
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(Journal of Leukocyte Biology. 2008;84:1565-1573.)
© 2008 by Society for Leukocyte Biology

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

Marcela Claudino*, Ana Paula F. Trombone{dagger}, Cristina R. Cardoso{dagger}, Samuel B. Ferreira, Jr*, Walter Martins, Jr{ddagger}, Gerson F. Assis*, Carlos F. Santos*, Paula C. Trevilatto§, Ana Paula Campanelli*, João S. Silva{dagger} and Gustavo P. Garlet*,1

* Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, São Paulo, Brazil;
{dagger} Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil;
{ddagger} Department of Periodontics, Dentistry School of University of Ribeirão Preto, Ribeirão Preto, Brazil; and
§ School of Dentistry, Pontifical Catholic University of Paraná, Curitiba, Brazil

1 Correspondence: Bauru School of Dentistry (FOB/USP), Department of Biological Sciences, Al. Octávio Pinheiro Brisolla, 9-75, Bauru, SP 17012-901, Brazil. E-mail: garletgp{at}usp.br


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ABSTRACT
 
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.

Key Words: interleukin-10 • genetic polymorphism • single nucleotide polymorphism • cytokine • immunoregulation • osteoprotegerin • periodontal disease


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INTRODUCTION
 
Periodontal diseases are infectious diseases, in which periodontopathogens trigger chronic inflammatory and immune responses that determine the disease outcome. Inflammatory mediators, such as TNF-{alpha} and IL-1β, initiate tissue destruction through the generation of proteases that degrade extracellular matrix, mainly matrix metalloproteinases (MMPs, a family of zinc- and calcium-dependent proteases), and the activation of mechanisms for bone resorption driven by the interaction between RANK (receptor activator for nuclear factor-{kappa}B) and its ligand RANKL (receptor activator for nuclear factor {kappa}B ligand), which leads to the differentiation and activation of osteoclasts [1 , 2 ].

Conversely, anti-inflammatory cytokines, such as IL-10, are widely expressed in diseased periodontal tissues and are associated with lower disease severity [3 , 4 ]. Indeed, IL-10 presents a protective role toward tissue destruction, inhibiting both MMPs and RANK systems [4 , 5 ]. IL-10 characteristically induces the up-regulation of a group of endogenous proteins named tissue inhibitors of metalloproteinases (TIMPs), which are capable of inhibiting almost every member of the MMP family in a nonspecific way [6 , 7 ]. In addition, IL-10 stimulates the production of OPG, a decoy receptor of RANKL, which strongly inhibits bone resorption by preventing RANK-RANKL engagement [8 , 9 ]. In agreement, a positive correlation between the levels of IL-10, TIMPs, and OPG was demonstrated in both human and experimental periodontal diseases [2 , 4 ].

However, studies demonstrate a high individual variation in the levels of IL-10 in diseased periodontal tissues, and such range is thought to be significant in the determination of disease outcome [2 , 10 , 11 ]. Indeed, heritable differences are reported in IL-10 production, and several polymorphic sequences have been identified in the IL-10 gene promoter [12 13 14 ]. Three common single nucleotide polymorphisms (SNPs) in the IL-10 gene promoter (–1082 G/A, –819 C/T, –592 C/A) show strong linkage disequilibrium and form two common haplotypes, designated as [ATA] and [GCC] haplotypes. Therefore, because of the linkage disequilibrium the presence of these haplotypes can be fully determined by the analysis of the –592 C/A polymorphism, in which the occurrence of the A allele indicates the presence of the [ATA] haplotype [15 , 16 ]. The [ATA] haplotype has been associated with decreased synthesis of IL-10 in vitro and in vivo [13 , 17 , 18 ] and is frequently associated with pathological conditions [19 , 20 ]. Regarding periodontal diseases, the IL-10–592 C/A and –819 C/T SNPs were previously associated with chronic periodontitis susceptibility in a Brazilian population [21 ], while the IL10-1082 SNP was associated with the occurrence of severe chronic periodontitis in Swedish Caucasians [18 ]. Conversely, other IL-10 gene SNPs have not been associated with periodontal disease in different populations [22 , 23 ].

Given the important role of IL-10 in periodontitis immunopathogenesis and the possible modulation of its levels by SNPs, in the present study, we investigated the association between the IL-10-592 C/A SNP with the levels of IL-10, TIMPs, and OPG mRNA expression in periodontal tissues, as well as with clinical parameters of periodontal disease severity.


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MATERIAL AND METHODS
 
Study population and clinical examination
Patients and controls, from southeastern Brazil, scheduled for treatment at the Dentistry School of University of Ribeirão Preto (UNAERP), were submitted to anamnesis and to clinical, periodontal and radiographic examination. Before beginning the study, all subjects signed a consent form that was approved by an Institutional Review Board and received supragingival prophylaxis to remove gross calculus and allow probing access. All teeth, with the exception of third molars, were scored by only one experienced clinician (W. M. Jr.), before the genotyping procedures, for bleeding on probing (BOP), probing depth (PD), clinical attachment loss (CAL) at 6 sites per tooth. The patients were categorized according to the classification of the American Academy of Periodontology into control or chronic periodontitis (CP) groups, as described previously [22 , 23 ]. Inclusion criteria comprised partially or fully dentate patients (at least 14 natural teeth, including 10 posterior teeth, excluding third molars), systemically healthy with no evidence of known systemic modifiers of periodontitis such as diabetes, osteoporosis, and rheumatoid arthritis. Exclusion criteria comprised patients who did not give informed consent, patients with a significant medical history, indicating evidence of known systemic modifiers of periodontal disease, as described above; pregnant or lactating females; patients who had taken systemic antibiotic, anti-inflammatory, hormonal or other assisted drug therapy in the last 6 months prior to the study; or those that had been submitted to periodontal therapy in the previous 2 years. Smokers were specifically excluded.

After the diagnostic phase, CP patients (n=116), presenting moderate to advanced PD, that is, at least 1 tooth per sextant with PD >6 mm and CAL >3 mm, and radiographic evidence of extensive bone loss) received basic periodontal therapy, which consisted in oral hygiene instruction, scaling, and root planning. Biopsies of gingival tissue (one sample from each patient, comprising junctional epithelium, gingival crevicular epithelium, and connective gingival tissue) were obtained during surgical therapy of the sites that exhibited no improvement in clinical condition (i.e., persistent bleeding on probing and higher probing depth) 3-4 weeks after the basic periodontal therapy, as described previously [4 ]. The control group (n=173) comprised subjects presenting gingival tissues clinically healthy (low scores of BOP – under 10% of the sites; no sites with PD >3 mm or presenting CAL) scheduled to restorative dentistry procedures. A representative fraction of control group (n=58, 27f/31m, age: 42.82±7.58, 49/74.48% Caucasians and 09/15.48% African-Americans/mulattos, mean probing depth: 2.09±0.29, sampled site probing depth: 2.27±0.64, sampled site attachment loss: 0, mean BOP: 4.77±1.37%, sampled site BOP sites: 0%) was also scheduled to surgical procedures due to prosthetic reasons, and during the surgery, biopsies of gingival tissue were taken (all of the sampled sites showed no BOP and PD <3 mm). The clinical features of the groups are summarized in Table 1 .


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  		Table 1. Clinical Features of Control and Chronic Periodontitis Groups

Analysis of IL10 592 SNP
DNA was extracted from epithelial buccal cells with sequential phenol/chloroform solution and precipitated with salt/ethanol solution [21 ]. For genotyping IL10-592, DNA fragments were amplified as described previously [21 ], by use of primer pairs described in Table 2 . The PCR was performed in a 25-µl reaction mixture containing 10x PCR buffer (Promega Corporation, Madison, WI, USA), MgCl2 (25 mM), dNTPs (10 mM), primers (10 pmol/ml each), Taq polymerase (2 U) and the DNA (200 ng). The standard PCR conditions were 95°C (5 min), and then 40 cycles of 94°C (1 min), 60°C (1 min), and 72°C (1 min), with a final extension step at 72°C for 10 min. To investigate the SNPs, an aliquot of PCR product was subjected to the RFLP (restriction fragment length polymorphism) with Rsa I (Promega Corporation), and the products were resolved and separated on 2% agarose gel stained with 0.5 µg/ml ethidium bromide for quantification of amplicons.


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  		Table 2. Primer Sequences and Reaction Properties

Real-time PCR reactions
Total RNA from gingival tissue biopsies was extracted using the TriZOL reagent (Life Technologies, Grand Island, NY, USA), as described previously [2 ]. cDNA (cDNA) was synthesized using 3 µg of RNA through a reverse transcription reaction (Superscript II, Gibco Life Technologies, Grand Island, NY, USA). Real-time-PCR quantitative mRNA analyses were performed in an ABI Prism 7000 Sequence Detection System using the SYBR-Green fluorescence quantification system (Applied Biosystems, Warrington, UK) for quantitation of amplicons as described previously [2 ]. The sequences of human primers were designed using the PrimerExpress software (Applied Biosystems) using nucleotide sequences present in the GenBank database. The primers sequences, the predicted amplicon sizes, and the annealing and melting temperatures are depicted in Table 2 . The standard PCR conditions were 95°C (10 min), and then 40 cycles of 94°C (1 min), 56°C (1 min), and 72°C (2 min), followed by the standard denaturation curve. PCR conditions for each target were conscientiously optimized with regard to primer concentration, absence of primer-dimer formation, and efficiency of amplification of target genes and housekeeping gene control. SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK), 400 nM specific primers, and 2.5 ng of cDNA were used in each reaction. Negative controls without cDNA and without the primer sets were also performed. Calculations for determining the relative levels of gene expression were made from duplicate measurements of the target gene, with normalization to β-actin in the sample, using the cycle threshold (Ct) method and the 2{Delta}{Delta}ct equation, as described previously [4 ].

Statistical analysis
The significance of the differences in observed frequencies of the study polymorphism in control and CP groups was assessed by Fischer exact test, and the risk associated with individual or combined genotypes was calculated as the odds ratio (OR), with 95% confidence intervals (CI). Analyses of continuous variables between C and CP groups were performed with t test, while possible differences between the genotype subgroups of C and CP groups were evaluated by ANOVA, followed by Bonferroni multiple comparison test. Linear regression analysis was used to test possible correlations between the levels of IL-10, TIMPs, and OPG mRNA expression in CP group. For all the tests used, values of P < 0.05 were considered statistically significant. All statistical tests were performed with the GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA, USA).


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RESULTS
 
IL-10-592 C/A SNP frequency analysis in C and CP groups
The subject sample included in this study was similarly composed by male and female subjects (Table 1) . Regarding ethnic status, Caucasians were prevalent (79.23% in C and 77.58% in CP) compared with African-Americans/mulatto individuals (19.38% in C and 22.41% in CP)in both control and CP groups; however, no further analysis was performed based on such classification in view of the high genetic miscegenation of the Brazilian population [24 ]. The frequency of IL-10-592 polymorphic genotypes CA (P=0.0012, OR: 2.4; 95%; CI: 1.4-4.1), AA (P=0.0458, OR: 2.3; 95%; CI: 1.1-4.9), and CA+AA (P=0.0006, OR: 2. 4; 95%; CI: 1.4-3.9), and of the allele A (P=0.0216, OR: 2.3; 95%; CI: 1. 2-4.6) were found to be significantly higher in CP group when compared with the C group (Table 3 ). The distribution of genotypes in both groups was found to be in Hardy-Weinberg equilibrium.


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  		Table 3. Frequencies of IL10-592 SNP Genotypes in Control and Chronic Periodontitis Groups

Quantitative analysis of IL-10 mRNA expression in periodontal tissues and its relationship with IL-10-592 SNP genotype
To evaluate the putative relevance of IL-10-592 SNP in the modulation of the IL-10 levels in diseased gingival tissues, we next evaluated IL-10 mRNA expression in periodontal tissues from CP and C groups by means of real-time PCR (Fig. 1 ). Our data show the absence or a very weak IL-10 expression in periodontal tissues of control subjects, while a significantly stronger expression was found in the tissues harvested from CP patients. Analyzing the possible association between IL-10 mRNA levels and the IL10-592 SNP genotypes, no differences were found in IL-10 levels between the different genotypes in the C group. However, in the CP group, a significant lower IL-10 mRNA expression was verified in both CA and AA genotypes when compared with CC genotype carriers (CC vs. CA: P<0.001; CC vs. AA: P<0.01; CA vs. AA: P>0,05).


Figure 1
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  		Figure 1. Frequency of IL-10-592 SNP and quantitative analysis of IL-10 expression in the different genotypes. DNA was extracted from controls (C, n=173) and chronic periodontitis (CP, n=116) patients, and the IL-10-592 SNP genotype was determined by RFLP. Total RNA was extracted from gingival tissues controls (C n=58, CP n=116), and levels of IL-10 mRNA were measured quantitatively by RealTime PCR SYBR-Green System. The results are presented as the expression of the individual mRNAs, with normalization to β-actin, using the Ct method. All control genotype subgroups were significantly different from the C subjects. *, P < 0.05, statistically significant differences in the CP group.

Quantitative analysis of TIMPs and OPG mRNA expression in periodontal tissues and its relationship with IL-10-592 SNP genotypes and IL-10 expression
We next evaluated whether the different IL-10-592 SNP genotypes could influence the expression of TIMPs and OPG, since the expression of these molecules is characteristically modulated by IL-10 (Fig. 2 ). The expression of TIMP-1, TIMP2, TIMP-3, and OPG mRNA was found to be higher in the diseased tissues than in the healthy ones, in which expression was found to be very weak. In addition, no differences were found in the expression of TIMPs and OPG for the different IL-10-592 SNP genotypes in the tissues of the control subjects. In CP group, no statistically significant differences were found in the expression of TIMP-1 and TIMP-2 in the different IL-10-592 genotype groups, in spite of a trend toward a lower TIMP-1 expression in CA and AA groups. However, our results demonstrate that TIMP-3 expression was significantly lower in CP patients with CA and AA genotype when compared with CC genotype. Similarly, our data also demonstrate that OPG expression was affected by IL-10-592 SNP, since its expression was significantly lower in both CA and AA carriers when compared with patients presenting CC genotype. Interestingly, homozygous subjects to the allele A present a lower expression of IL-10, TIMP-3, and OPG when compared with the heterozygous genotype CA, but such difference was not statistically significant. Positive correlations between the levels of IL-10 mRNA expression and TIMP-1 mRNA expression (P<0.001, r2=0.4891), IL-10, and TIMP-2 mRNA expression (P<0001, r2=0.3630), IL-10, and TIMP-3 mRNA expression (P<0.0001, r2=0.5906), and between IL-10 and OPG mRNA expression (P<0.0001, r2=0.5994) were verified in diseased periodontal tissues, reinforcing the hypothesis that IL-10 levels could influence the expression of other downstream host mediators involved in the immunomodulation of periodontal diseases.


Figure 2
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  		Figure 2. Quantitative expression of TIMPs and OPG in the different IL-10-592 SNP genotypes. DNA was extracted from controls (C, n=58) and chronic periodontitis (CP, n=116) patients and the IL-10-592 SNP genotype was determined by RFLP. Total RNA was extracted from gingival tissues, and levels of TIMPs and OPG mRNA were measured quantitatively by RealTime PCR SYBR-Green System. The results are presented as expression of the individual mRNAs, with normalization to β-actin, using the Ct method. All control genotype subgroups were significantly different from the C subjects. *, P < 0.05, statistically significant differences in the CP group.

IL-10-592 genotypes association with clinical parameters of disease severity
Since IL-10-592 SNP genotypes frequency was found to be associated with the expression of host mediators involved in the immunomodulation of periodontal diseases, we next investigated the possible associations between the IL-10-592 SNP genotypes and clinical parameters of periodontitis severity (Fig. 3 ). Our data demonstrate that CA and AA genotype carriers present significantly higher mean probing depth (a clinical index representative of the overall patient degree of disease severity) values than CC patients. However, no significant differences were found when the parameters only of the site sampled (probing depth and attachment loss) were analyzed in regard to IL-10-592 genotypes.


Figure 3
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  		Figure 3. Clinical parameters of periodontal disease severity in the different IL-10-592 SNP genotypes. Controls (C, n=58) and chronic periodontitis (CP, n=116) patients were subjected to periodontal examination, and the genotype of IL-10-592 SNP was determined by RFLP. All control genotype subgroups were significantly different from the C subjects. *, P < 0.05, statistically significant differences in the CP group.


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DISCUSSION
 
In periodontal diseases, inflammatory mediators trigger tissue destruction through the generation of MMPs and the activation of osteoclasts by RANKL. Conversely, the anti-inflammatory cytokine IL-10 induces the expression of TIMPs and OPG, the respective inhibitors of MMPs and RANKL systems, and therefore, it is thought to attenuate disease severity [3 , 4 ]. Interestingly, periodontitis patients present a high individual variation in the levels of IL-10 mRNA in diseased periodontal tissues, and such range is supposed to be relevant to disease outcome determination [4 , 10 , 11 ]. One hypothesis to explain such variability is the occurrence of genetic polymorphisms in the IL-10 gene promoter, such as IL10-592 SNP, which was found to influence cytokine production in response to inflammatory stimuli [12 13 14 15 16 17 18 ] and was previously associated with CP severity [2 , 4 ]. Because of the strong linkage disequilibrium, the analysis of the –592 C/A polymorphism can determine the occurrence of the common haplotype ATA, which has been associated with decreased synthesis of IL-10 in vitro and in vivo [13 , 16 , 19 , 20 ]. Therefore, in the present study, it was investigated the possible association of IL-10–592 SNP with IL-10, TIMPs, and OPG mRNA levels in the gingival tissues of CP patients and control subjects, and also with clinical parameters of disease severity. The results presented here demonstrate that the frequency of IL-10-592 SNP genotypes in the control group was very similar to that reported for the Brazilian population in previous studies [21 , 25 ]. Furthermore, our data demonstrate that IL10–592 CA, AA, and CA+AA genotypes, and the A allele were found to be more frequent in CP patients than in control subjects, reinforcing their previously described association with the susceptibility to CP [21 ].

However, in spite of the association of IL-10-592 SNP with CP, its putative functionality in periodontal environment remains unknown. Therefore, we next evaluated the tissue levels of IL-10 mRNA in the different IL-10-592 SNP genotype groups. Our results demonstrate that in the CP group, the carriers of CA and AA genotypes presented a significantly lower expression of IL-10 mRNA in periodontal tissues than those with CC genotype [13 , 16 , 19 , 20 ]. In accordance, IL-10-592 SNP was found to modulate quantitatively the IL-10 production in vitro and in vivo [13 , 17 ] and also lead to altered synthesis of IL-10 in response to inflammatory stimuli [18 ]. Concurring, the [ATA] haplotype (determined by the presence of A allele in IL10-592 SNP) [15 , 16 ], has been clearly associated with lower IL-10 production in vitro and in different pathological conditions [13 , 17 , 19 , 20 ]. In accordance, unpublished data from our research group confirm the strong linkage disequilibrium between IL-10–1082, –819 and –592 SNPs in Brazilian subjects, therefore allowing the use of IL-10-592 as a reliable marker of the ATA haplotype in our sample.

In the periodontal environment, IL-10 is thought to be produced by both immune (Th2 lymphocytes and regulatory T cells) and resident cells, probably through a mechanism of negative feedback triggered by microbial and inflammatory mediators [2 , 26 ]. In addition to the attenuation of the inflammatory reaction, IL-10-induced up-regulation of TIMPs and OPG in periodontal environment has been described as an important pathway to control disease progression [4 , 5 ]. In fact, increased TIMPs levels in periodontal tissues are thought to effectively counteract MMPs, and have been associated with the attenuation of disease severity [27 28 29 30 ]. Similarly, the blockade of RANKL by OPG leads to a reduction in the alveolar bone loss in experimental periodontal disease [31 , 32 ] and lower levels of OPG expression were associated with higher disease severity in humans [33 , 34 ].

Therefore, we next investigated whether the IL-10-592 SNP could influence the tissue levels of TIMPs and OPG. Indeed, our data show that carriers of IL-10-592 SNP CA and AA genotypes express significant lower levels of TIMP-3 and OPG in diseased periodontal tissues when compared with patients with the CC genotype. The expression of TIMP-1 was found to be decreased in CA and AA groups, but such reduction is not statistically significant. On the other hand, the expression of TIMP-2 mRNA was not associated with the IL-10-592 SNP. Taken together, these results suggest that IL-10-592 SNP influence IL-10 levels in the diseased periodontium, which consequently could modulate the levels of TIMPs and OPG in these tissues. In accordance with this hypothesis, a correlation was found between the levels of IL-10 with TIMP-1, TIMP-2, TIMP-3, and OPG were found. Concurring, IL-10 modulates the levels of both TIMPs and OPG in vitro [5 , 35 36 37 ] and in vivo [37 ]. In accordance, in both human and experimental periodontitis, the levels of TIMPs and OPG have been associated with IL-10 expression and with the control of disease severity [2 , 4 , 5 , 38 ].

Finally, possible association between the genotypes and clinical parameters of disease severity was analyzed. Interestingly, no correlations were found between the genotypes and the parameters from which the biopsy were taken. In fact, when only the sampled sites were considered, the clinical records may be also under a strong/stronger influence of the local factors such as anatomic variations (i.e., enamel pearls or furcation defects), traumatic occlusion and restorations with subgingival or overhanging margins [39 ], which directly influence the disease scores in these specific sites, and consequently can make difficult or impair the association with factors that can influence overall disease severity, such as genetic polymorphisms. In fact, all of the previous studies [21 22 23 , 40 ] have correlated the different genotypes with the mean values of probing depth and attachment levels, and in the view of the evaluation of IL-10 expression in situ, this study seems to be the first to try a correlation between the sampled sites with the genotypes. However, patients with CA and AA genotypes were found to present significantly higher values of the mean probing depth than carriers of the CC genotype. In accordance, the mean probing depth is a clinical index representative of the overall patient degree of disease severity, where the influence of individual local factors is thought to be minimized, and therefore, their associations with IL-10-592 SNP suggest that this genetic variation could be relevant to the determination of disease severity. In accordance with our findings, recent studies demonstrate that the ATA haplotype—which is known as a "low interleukin-10 producer" and can be determined by the presence of IL-10-592 SNP due the strong linkage disequilibrium—is a putative risk indicator for the progression of periodontal disease [41 , 42 ].

Another interesting point is that IL-10-592 SNP is not correlated with the % of bleeding on probing sites, and inflammatory parameter indicative of gingivitis (a clinical variation of the periodontal disease considered of low severity, since it is reversible and not associated with bone loss). However, the possible individual causes for the susceptibility to gingivitis are still poorly known. A series of studies identified high- and low-responders, which respectively exhibited high and low gingival inflammatory responses to similar levels of plaque accumulation [43 ], and further studies demonstrate small, but significant, variations in the frequency of some SNP (IL-1RN, IL-1B-511, IL-10 ATA haplotype) that could account for the determination of gingivitis susceptibility [44 , 45 ]. However, IL-10 ATA haplotype was found to be associated with the gingivitis susceptibility only in children [45 ], which hinder deeper comparisons with our data. Furthermore, several polymorphisms previously associated with periodontitis susceptibility (including the IL-6-174, TNFA-308, IL-1A+4845, and IL-1B+3953 SNPs) were not associated with gingivitis susceptibility or with clinical indicators of gingival inflammation, suggesting that the genetic factors underlying the susceptibility of gingivitis and periodontitis may differ [44 , 46 47 48 ].

The putative risk associated with IL-10-592 SNP was investigated by means of the odds ratio calculation, which resulted in a risk of 1.7 for the A allele and 2.4 for the CA+AA genotypes. However, it is important to consider that due to the complex etiology of periodontal disease, the result of such analysis must be carefully interpreted. The association of IL-10-592 SNP with the susceptibility to the development of periodontal disease is also reinforced by the fact that smokers were specifically excluded of our sample. Smoke is an important risk factor to the development of periodontitis, but the inclusion of smokers in the genetic association study samples comprises an additional confusing factor that hinders the proper interpretation of the results [43 , 49 ]. Accordingly, a recent study demonstrates that smoking represents an additional factor involved in periodontal disease progression that could mask the effects of IL-10 SNPs on periodontitis outcome [42 ]. However, the exclusion of the smokers from the sample may result in a biased populational attributable risk, which could overestimate the risk associated with IL-10-592. It also must be considered that the clinical analyses in our study were performed by an experienced clinician; however, in future studies, it would be very interesting if two or more independent blinded clinicians could rate the disease severity parameters to assure clinical objectivity. On the other hand, unlike in the majority of diseases, in which the distinction between resistant and susceptible subjects is relatively simple, genetic studies of subjects with periodontal disease comprise periodontally healthy subjects, which not necessarily implies a genetic resistance to the disease development, but may only reflect the control of the disease etiologic factors by proper oral hygiene [50 , 51 ]. Therefore, the presence of susceptible subjects, but with a proper control of the microbial etiological factors, could result in an underestimated risk of a given SNP in association studies. It is also important to consider that the variations in the risk attributable to IL-10 SNPs, as well as the absence of association between IL-10 gene SNPs and periodontal disease described in some studies [22 , 23 , 40 ], are possibly due to different criteria to select diseased and control groups, or due to the genotypic frequency differences in IL-10 SNPs in ethnically different populations. Nevertheless, despite the fact that the population living in southeastern region of Brazil is predominantly of European ancestry, previous studies demonstrate a high miscegenation of races leading to a genetic heterogeneity in this population, and therefore, the analysis of genetic polymorphisms in Brazilian individuals represents important information [24 , 52 ].

Interestingly, multiple regression analyses demonstrate that IL-10 expression is age-, race- and sex-independent, being only associated with IL-10-592 SNP (data not shown). Conversely, previous studies demonstrated that IL-10 haplotype ATA was associated with periodontal disease only in females [21 ], possibly because of the IL-10-819 SNP location in a DNA motif forming a putative estrogen-responsive element [53 ]. However, these studies comprised relatively small samples composed predominantly of females, which could result in biased conclusions. Furthermore, the estrogen deficiency was demonstrated to increase the severity of established periodontal disease and not to influence their onset [54 ]. In addition, recent studies with higher experimental n demonstrate that the IL-10 haplotype ATA is equally distributed between both genders [55 ] and can be associated with periodontal disease in both males and females [41 , 42 ]. However, it is reasonable to suppose that estrogen variations and IL-10 SNP could present an additive role over periodontitis severity, but further studies are required in order to investigate such a hypothesis.

Interestingly, although some IL-10 promoter polymorphisms have been frequently associated with the susceptibility to different forms of periodontitis in diverse populations [18 , 21 , 45 ], no associations were found to date between SNPs in OPG and TIMP genes and the occurrence of periodontal diseases [56 57 58 ]. Taken together with our results, these studies may suggest that polymorphisms in genes that codify mediators involved in the upstream positions of inflammatory-immune response pathways (such as IL-10), which modulate a broad range of factors, may be relevant to periodontitis outcome [59 , 60 ]. Conversely, polymorphisms in downstream genes (such as TIMPs and OPG), whose products present a narrow action in the pathways involved in periodontal tissue destruction, would play a minor role (or even do not play a significant role) in the development of periodontal diseases. However, as only a few studies to date have evaluated the association of a small number of TIMPs and OPG SNPs, with the susceptibility to periodontitis, the possible influence of functional polymorphisms in these genes on the development of periodontal disease cannot be disregarded. It is also important to consider that periodontitis is a complex trait disease, in which several immunoregulatory cytokines are involved [48 , 61 ]. Furthermore, specific virulent periodontopathogens can also interfere in the course of immune response in the periodontal environment modulating the local cytokine milieu [50 , 62 ]. Indeed, even in experimental periodontitis in knockout mice, in which a given cytokine is completely absent, the disease severity is usually found to be partially, but not completely, increased/decreased [38 , 63 ]. Therefore, it is reasonable to expect that SNPs, even those proven to be functional, such as IL-10-592, potentially present a significant, but not a major role in disease progression. It also must be considered that IL-10-592 SNP may be in linkage disequilibrium with other SNPs that can influence IL-10, TIMPs, and OPG levels, and therefore they could also contribute to the resistance/susceptibility to chronic periodontitis. However, it must be kept in mind that variations in mRNA levels do not necessarily mean variations in the protein levels, and further studies are required to confirm the possible influence of IL10-592 SNP over IL-10, TIMPs, and OPG levels.

Our results demonstrate that the A allele (in both heterozygous and homozygous genotypes CA and AA) of IL-10-592 SNP was more prevalent in the CP group. In addition, our data show that IL-10-592 SNP is functional, being associated with lower levels of IL-10, TIMP-3, and OPG mRNA expression in diseased periodontal tissues, and also with higher mean probing depth in CP patients. Therefore, polymorphisms in genes that codify mediators of upstream positions of inflammatory-immune pathways, such as IL10-592 SNP, seem to consequently modulate a broader range of factors involved in immunoregulation of periodontal disease, and consequently, to be an important factor in the determination of disease severity. The first steps in solving the puzzle of the pathological mechanisms of periodontal disease have been achieved, but further studies are required in order to understand the role of genetic polymorphisms in the immunopathogenesis of this disease. Such knowledge might allow the development of diagnostic, preventive and therapeutic strategies in order to improve the clinical management of periodontal diseases.


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ACKNOWLEDGEMENTS
 
This study was supported by scholarships and grants from Fundação de Amparo à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico.

Received March 14, 2008; revised July 24, 2008; accepted July 29, 2008.


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