The effects of IL-10 gene polymorphism on serum, and gingival crevicular fluid levels of IL-6 and IL-10 in chronic periodontitis

Abstract Objective Anti-inflammatory cytokines play a crucial role in periodontitis by inhibiting synthesis of pro-inflammatory cytokines. The purpose of this study was to evaluate the effect of interleukin-10 (-597) gene polymorphism and genotype distributions on chronic periodontitis (CP) development and IL-6 and IL-10 levels in gingival crevicular fluid (GCF) and serum before and after non-surgical periodontal treatment. Material and Methods The study population consisted of 55 severe generalized CP patients as CP group and 50 healthy individuals as control group. Plaque index, gingival index, probing depth and clinical attachment level were recorded and GCF and blood samples were taken at both the baseline and the sixth week after non-surgical periodontal treatment. PCR-RFLP procedure was used for gene analyses and cytokine levels were measured via ELISA. Results IL-10 genotype distribution was significantly different between CP and control groups (p=0.000, OR:7, 95%CI, 2.83-60.25). Clinical measurements significantly improved in the CP group after periodontal treatment (p<0.05). Periodontal treatment significantly decreased GCF IL-6 and IL-10 levels. No significant difference was found in clinical parameters between IL-10 AA and AC+CC genotypes at both the baseline and the sixth week (p>0.05). Sixth week GCF IL-10 levels were significantly lower in patients carrying IL-10 AC+CC genotype compared to the patients carrying IL-10 AA genotype (p<0.05). Serum IL-6 and IL-10 levels were lower in patients carrying the IL-10 AA genotype compared to patients with IL-10 AC+CC genotype, but the difference was not significant (p>0.05). Conclusion IL-10 AA genotype carriers had lower IL-6 and IL-6/10 levels in serum; however, GCF IL-6/10 levels were similar in both genotypes. Within the limitations of our study, a possible association between IL-10(-597) gene polymorphism and CP might be considered.

The structure of a protein and/or its expression might change by polymorphism of the specific gene, thus, innate and adaptive immunity may be compromised 13,27 . Therefore, gene polymorphisms might have a deterministic importance in disease outcome.
One of the most important factors responsible for periodontal destruction is the upregulation of proinflammatory cytokines. Interleukin-6 (IL-6), as one of these pro-inflammatory cytokines, was reported to be an effective stimulator of osteoclast differentiation and bone resorption 21 . Along with CP, several inflammatory diseases were also associated with elevated levels of IL-6 12,17 . On the other hand, anti-inflammatory cytokines such as IL-10 can restore balance by inhibiting synthesis of pro-inflammatory cytokines (IL-1, IL-6, TNF-alpha) and stimulating protective antibody production 20, 26 . For this reason, IL-10 gene polymorphism might contribute to periodontitis development.
The gene encoding human IL-10 is located on chromosome one (1q31-32). IL-10 three biallelic polymorphisms within the IL-10 promoter region, at positions -1087, -824, and -597 from the transcription initial site, have also been identified 23 . Although decreased IL-10 levels with IL-10 promotor gene polymorphism were reported, studies evaluating the relationship between this single nucleotide polymorphisms (SNPs) and CP reported conflicting results 3,8,11,20,23 .
Several researches on the clinical utility of cytokine genotyping for periodontal disease were conducted, but the genetic knowledge of periodontitis pathogenesis and effects on treatment results is still limited.
Previously, we reported that IL-6 (-174) GG genotype caused an increase in GCF IL-10 levels without affecting periodontal treatment outcomes and we also found that SNPs in IL-6 (-174) and IL-10 (-597) were associated with generalized aggressive periodontitis (GAgP) 18 . However, there is no information regarding the ratio of inflammatory markers (IL-6/IL-10) in different genotype distribution of IL-10 gene in chronic periodontitis. Therefore, the aim of our study was to assess the IL-10 -597 SNPs in CP patients and to evaluate the effect of polymorphisms on non-surgical periodontal therapy and serum and GCF cytokine (IL-6 and IL-10) levels. Secondly, a possible association between IL-10 genotypes and local and systemic inflammatory status presented as the ratio of IL-6/ IL-10 was investigated.

Study population
A total of 105 participants were involved in this study. CP group consisted of 55 chronic periodontitis patients (CP group) and control group consisted of 50 healthy volunteers (HC group). All participants were systemically healthy and never smoked. The results were converted to numeric values by using standard curves.

DNA extraction and genotyping
The study protocol was carried out about our previous study 7 . For DNA extraction and genotyping, 2 mL of blood from the antecubital vein was driven from all participants and placed into biochemical tubes containing sodium EDTA. Tubes were then kept at -80°C until the genetic analysis day. A commercial kit (Invitrogen, Cambrillo, USA) was used for DNA extraction according to the manufacturer's instructions.
IL-10 gene polymorphism was evaluated via PCR-RFLP by analyzing IL-10 gene at position -597. Table 1  Association was determined with odds ratios (ORs) and 95% confidence intervals. A p-value of <0.05 was considered statistically significant.

Clinical parameters
Four participants from the CP group did not want periodontal treatment and were excluded from study.
The CP group consisted of 17 men and 34 women (aged 20 to 46 years; mean age: 33.4±6.0). The healthy control group consisted of 17 men and 33 women (aged 23 to 46 years; mean age: 33±6.5).
There was no significant difference in age and sex distributions in the groups (p>0.05).
Periodontal treatment decreased all clinical parameters in the CP group at the 6 th week compared to the baseline (p<0.05) (

Biochemical parameters
At the 6 th week, IL-6 and IL-10 levels in GCF were lower than baseline in CP group (p<0.05) ( Table 3).
Both baseline and 6 th week levels of IL-6 in GCF of CP group were higher than those of the HC group (p<0.05). However, unlike GCF levels, serum levels of IL-6 and IL-10 in the CP group did not change after periodontal treatment (p>0.05). In the comparison between groups, serum cytokine levels were higher in the CP group than those of the control group (p<0.05).
According to the ratio of IL-6/IL-10, serum and GCF IL-6/IL10 levels were significantly higher in the CP group than those of the control group (p<0.05).
However, baseline and 6 th week values of IL-6/IL-10 ratio in GCF and serum were similar (p>0.05).  We evaluated the changes in clinical and biochemical parameters in the CP group after categorizing patients according to IL-10 genotype distribution (IL-10 AA and AC+CC). Periodontal treatment provided significant improvements in both genotypes (p<0.05) ( Table 4).

CP
However, clinical parameters between IL-10 AA and AC+CC genotypes were similar in both baseline and 6 th week (p>0.05) ( Table 5).
GCF IL-10 levels at the 6 th week in patients carrying IL-10 AC+CC genotype significantly decreased (p<0.05) ( Table 4). In contrast, GCF and serum IL-6 levels in patients carrying the IL-10 AA genotype were not statistically different (p>0.05). Serum IL-10 levels in patients carrying IL-10 AA genotypes were not different from those of the IL-10 AC+CC genotypes at either baseline or 6 th week (p>0.05). The ratios of IL-6/IL-10, especially in serum, were lower in patients with IL-10 AA genotypes compared to IL-10 AC+CC genotypes (p<0.05).
Periodontal disease susceptibility in the CP group was evaluated with logistic regression analysis of IL-10 AA genotypes after adjustment of age and sex.
Genotype diversity was not affected by either age or sex.

Discussion
The complexity of cytokine network, together with gene polymorphisms of these cytokines, aids in uncovering the molecular mechanisms of inflammatory diseases, such as periodontal diseases. In this way, our study evaluated the role of anti-inflammatory (IL-10) and proinflammatory cytokines (IL-6) in chronic periodontitis patients. Correlation of these results with the gene polymorphism of IL-10 was also performed.
As a result, we found that IL-10 AA genotypes were associated with chronic periodontitis and IL-10 AA genotype carriers showed lower IL-6/IL-10 levels in serum.
In this study, patients with the IL-10 AA genotype had a high susceptibility to chronic periodontitis.
Similarly to our results, Sumer, et al. 12 (2007) reported that the IL-10 gene polymorphism at position -597 seems to be associated with severe generalized CP.
Association of SNPs in vascular endothelial growth factor (VEGF) , IL-6, IL-10, IL-1beta, interferon alpha, TNF-alpha genes with immunoregulatory function revealed that C allele of VEGF, an allele of IL-10 and GG genotype of TNF-alpha, was individually associated with chronic periodontitis 10 . Although A alleles were higher in CP groups in this study, our results showed that the allele frequencies did not differ between chronic periodontitis and healthy control groups.
However, Jaradat, et al. 14 IL6  IL10  IL6/IL10  IL6  IL10  IL6/IL10   IL-  In addition, we evaluated whether the IL-10 AA genotypes presented lower levels of IL-10 and IL-6/IL-10 ratio in serum than those of the AC+CC genotype carriers in our study, but this reduction is not statistically significant. On the other hand, GCF IL-10 levels decreased after treatment in IL-10 AC+CC genotype carriers, but did not change in patients with AA genotypes.
In this study, we did not assess the haplotype analyses for this gene locus, which may be considered a shortcoming. Also, another limitation of this study is the low number of subjects included. However, we excluded periodontitis smoker patients because of the effects of smoking on the immune system.

Conclusion
In conclusion, this study demonstrated that IL-10 -597 AA genotype appeared to be a risk factor for chronic periodontitis. Furthermore, genetic polymorphisms in the IL-10 gene might be useful as a marker to diagnose susceptibility to CP. On the other hand, an important finding of this study is that IL-10 AA genotype may tend to reduce IL-6 production or contribute to the inflammatory status, along with decreased GCF IL-10 levels. Nonetheless, further studies are needed to elucidate the mechanisms involved in cytokine gene polymorphisms and their production in periodontal disease.