Interleukin-6 and Interleukin-10 Gene Polymorphisms in Patients with Chronic Periodontitis and Response to Treatment after 3 Years.

Abstract

Objective

The aim of this study was to investigate whether genetic susceptibility to chronic periodontitis, conferred by the presence of the IL-6 -572GG genotype or the IL-10 -592A allele, influences the outcomes following a non-surgical periodontal therapy (NSPT)over a long period of time.

Material and methods

Thirty-seven chronic periodontitis patients were divided into two groups according to genotype as susceptible (SCP) and non-susceptible (NSCP). All subjects were clinically evaluated at baseline and 3 years following NSPT. Blood samples were collected at baseline from the individuals who fulfilled the inclusion criteria. All participants received NSPT from a single periodontist who was blind to the genotype status of each patient. A statistical analysis was performed by comparing the variables between groups using the Mann-Whitney U test and between baseline and 3 years for each group using the Wilcoxon test.

Results

The mean age of the population was estimated to be 47.68±8.64 years and it included 51.4% females, 48.6% smokers, and 45.9% alcohol consumers. Following a genetic analysis, 70.3% of patients were homozygous carriers of the IL-6 -572G (IL-6 SCP), and 46.0% of them were carriers of the IL-10 -592A allele (IL-10 SCP). NSPT reduced all studied parameters (probing depth, attachment loss, bleeding on probing, percentage of sites with 4-6mm and ≥7mm pocket depth and attachment loss) to all participants, but the treatment outcome was not associated with the genotype. The SCP and NSCP individuals showed similar clinical parameters at baseline and at 3 years.

Conclusions

Within the limitations of this 3-year prospective cohort study in Caucasians diagnosed with chronic periodontitis, individuals susceptible to periodontal disease as determined by the presence of the IL-6 -572GG genotype or the IL-10 -592A allele showed similar treatment outcome following NSPT.

Introduction

Periodontitis is a non-communicable multifactorial inflammatory disease associated with dysbiotic plaque biofilms resulting in progressive destruction of the tooth-supporting tissues and eventually tooth loss (1). Periodontal disease onset and progression are a result of the interaction between the dysbiosis of the commensal oral microbiota and the host response (2). In the 2017 World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions it was recognized that population subgroups may exhibit distinct disease conditions due to differences with respect to disease susceptibility and exposure (1).

Susceptibility to periodontal disease and response to treatment vary among individuals and depend on different factors. Disease susceptibility depends on environmental and host risk factors, either modifiable such as smoking, diabetes mellitus, obesity and oral hygiene or unmodifiable including genetic predisposition (3). In a study based on 110 pairs of adult twins, 38-82% of the population variance for periodontal clinical parameters may be attributed to heredity of genetic factors highlighting the role of host genetic influences (4). In recent decades, multiple studies have examined the association between gene polymorphisms and the risk of periodontitis development (5).

Polymorphisms in the promoter region of the Interleukin (IL)-6 gene affect the transcription and expression of IL-6 in individuals leading to the up-regulation of IL-6 levels in serum (6) and gingival tissues (7). Similarly, polymorphisms in the promoter region of the IL-10 gene may change the expression of IL-10 in response to inflammatory disease, thus influencing the disease outcome (8, 9). A meta-analyses aimed to determine whether IL-6 and IL-10 polymorphisms confer susceptibility to periodontitis have indicated a significant association between the IL-6 -572 G allele and IL-10 -592 A allele and AA genotype with chronic periodontitis (10-13).

The treatment of periodontitis aims to arrest disease progression by reducing the bacterial load, in order to reduce the risk of tooth loss and to prevent the disease recurrence (14). A reasonable endpoint of non-surgical periodontal treatment should include the absence of clinical signs of gingival inflammation, shallow pocket depth and low levels of plaque accumulation. Studies have demonstrated that periodontal treatment can lead to positive clinical outcomes and a supportive periodontal therapy is effective in maintaining periodontal health and preventing a long-term tooth loss (15-17).

Although periodontal therapy is highly predictable, a patient- and tooth-related factors have been associated with tooth loss and further disease progression during supportive periodontal therapy including age, smoking, systemic conditions and genetics (18, 19). Rapid progression from gingivitis to periodontitis has been detected in 10-15% of the population and 8-12% of patients within a population exhibit a number of sites that do not respond to a routine periodontal treatment (20, 21). Identifying host-related factors such as gene polymorphisms associated with the development and progression of periodontal diseases may lead to early recognition of patients unresponsive to periodontal treatment (22). Although environmental factors have been widely investigated as possible predictors of disease progression in individuals who received periodontal therapy, information concerning genetic factors is scarce.

A meta-analysis that examined the effects of several susceptible genotypes to chronic periodontitis with the periodontal treatment outcome and tooth loss concluded that pocket depth reduction in the first three and six months after a non-surgical periodontal therapy was associated with susceptible genotypes (23). However, the number of available studies was insufficient to draw clear conclusions and further studies are needed (21). In a prospective study with a short follow-up period (45 days following non-surgical periodontal therapy), IL-6 -572 G/C and IL-10 -592 C/A polymorphisms did not influence the treatment outcome of chronic periodontitis (24). Additional methodologically sound studies are needed to contribute to the prediction of periodontal treatment response.

There is still lack of long-term data regarding the treatment outcome and disease susceptibility in patients with chronic periodontitis. To the best of our knowledge, there are currently no published clinical studies that evaluate the effect of gene polymorphisms on the outcome of non-surgical periodontal therapy over a long period of time. Hence, the aim of this study was to investigate whether genetic susceptibility to chronic periodontitis, conferred by the presence of the IL-6 -572GG genotype or the IL-10 -592A allele, influences the clinical outcomes of non-surgical long-term periodontal therapy.

Materials and methods

This prospective cohort study included consecutive chronic periodontitis patients from a private practice limited to Periodontics and Implant Dentistry in Thessaloniki, Greece between September 2014 and June 2015. All eligible patients consented to the study protocols. Out of 68 treated subjects, 67 were re-evaluated 45 days following the treatment and the results of the analysis have been published separately (24). The subjects were encouraged to attend supportive periodontal therapy on a 3- or 4- month recall protocol based on their individual needs. A total of 37 out of 67 re-evaluated at 45 days patients presented at their 3-year follow-up appointment. The remaining 30 subjects moved away from the area, did not attend a number or all of the maintenance appointments, or had a surgical periodontal treatment as part of their treatment plan.

Patients were included in the study on the basis of the following inclusion criteria:

1. Diagnosis of chronic periodontitis based on the 1999 classification system for periodontal disease (25). Two or more non-adjacent sites with interproximal clinical attachment loss (CAL) ≥ 3 mm, probing pocket depth (PPD) ≥ 5 mm and bleeding on probing (BOP).

2. Attendance of supportive periodontal therapy on a 3-4 month recall protocol.

3. Completion of periodontal re-evaluation 3 years following the active periodontal treatment.

4. Systemic health.

5. Age of 30-70 years.

6. Presence of ≥16 teeth at initial appointment.

The exclusion criteria consisted of:

1. Any periodontal treatment within the last 12 months from the study initiation.

2. Poor attendance of follow-up/recall appointments (failure to attend the recommended supportive periodontal therapy 3-4 month recall protocol).

3. Lack of data at the follow-up examination 3 years after active periodontal treatment.

4. Need for periodontal surgery (resective or regenerative) following the non-surgical periodontal therapy.

5. Presence of systemic diseases or severe medical conditions.

6. Need for antibiotic prophylaxis.

7. Systemic antimicrobial therapy within the past 3 months from the study participation.

8. Pregnant or lactating females.

Clinical examination and periodontal therapy

Details of the clinical examination and treatment sequence were described previously (24). In brief, the periodontal status was assessed including PPD, CAL and BOP by an independent, blinded to the genetic analysis and calibrated (intra-examiner agreement=0.88) periodontist (E.D). The clinical examination was performed using a manual periodontal probe (15 UNC probe, Hu-Friedy, Chicago, IL, USA) at six sites per tooth apart from third molars. All patients enrolled in the study were treated according to a comprehensive periodontal treatment plan including case presentation, oral hygiene instructions and non-surgical periodontal therapy under local anesthesia. Hand instruments (Hu-Friedy, Chicago, USA) and ultrasonic scalers (KaVo SONOsoft LUX, Kavo, Germany) were utilized.

The treated patients were re-assessed at 6 weeks following the non-surgical periodontal therapy and clinical examination was completed by the same clinical examiner as carried out at the baseline examination. Supportive periodontal therapy was scheduled on a 3-4 month basis including assessment of PPD, CAL and BOP, re-instruction and re-motivation for effective plaque control, professional tooth cleaning with hand instruments and ultrasonic scalers, and subgingival instrumentation in areas with PPD≥5 mm. The last clinical examination was performed at 3 years following the active periodontal treatment.

Study groups

The recruited individuals were grouped based on the IL-6 -572 G/C and IL-10 -592 C/A polymorphisms: IL-6 SCP: Susceptible to chronic periodontitis conferred by the presence of the IL-6 -572GG genotype; IL-10 SCP: Susceptible to chronic periodontitis conferred by the presence of the IL-10 -592A allele; IL-6 NSCP: Non-susceptible to chronic periodontitis carrying the non-susceptible genotype IL-6 -572C allele; IL-10 NSCP: Non-susceptible to chronic periodontitis carrying the non-susceptible genotype IL-10 -592CC genotype.

Blood samples and genotyping

Blood drops collected from each patient were used to extract genomic DNA using a commercially available genomic DNA isolation kit following the manufacturer’s instructions (QIAamp, DNA mini blood kit, QIAGEN, Germany). Genotyping of IL-6 -572 G/C (rs1800796) and IL-10 -592 C/A (rs1800872) polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism techniques in a final volume of 25 ul using a standard protocol which was described previously (24). The IL-6 and IL-10 polymorphisms were determined by using primers to generate a PCR product which was then digested and separated on polyacrylamide gels stained with silver nitrate (24).

Statistical analysis

The aim of this study was to assess the treatment outcome following non-surgical periodontal therapy between susceptible and non-susceptible individuals to periodontitis. PPD was considered the primary outcome and the sample size calculation was performed on the basis of a minimum difference of 1 mm in the mean full-mouth PPD values of each patient and a standard deviation of 0.5 mm. Aiming to achieve a 95% power of the study, it was determined that 6 individuals per group would be essential. The distribution of the clinical parameters was tested for normality using the Shapiro-Wilk test. The chi-square test was utilized to determine whether the SCP and NSCP groups were composed of similar proportions in regards to gender, smoking status and alcohol consumption while the Mann-Whitney U test was used for age and the number of teeth. To determine the differences between the timepoints (baseline and 3 years) for each group, the Wilcoxon test was used. The Mann-Whitney test was utilized to compare SCP and NSCP groups at baseline and 3 years. A multiple logistic regression analysis was used to identify possible associations between the genotypes, clinical and demographic parameters. To avoid biased results from multiple comparisons, the results were adjusted according to the Bonferroni correction for the 8 variables analyzed. P-value <0.05 was considered statistically significant. The statistical analysis was completed using SPSS v.24.0, IBM, Armonk, NY, USA.

Results

The population investigated in this study comprised a total of 37 Caucasians with a mean age of 47.68± 8.64 and a diagnosis of chronic periodontitis. It was divided into groups of similar age according to their genotype. The demographic characteristics of the study population are presented in Table 1. The IL-6 SCP and NSCP groups demonstrated similar smoking (p = 0.64) and alcohol consumption (p = 0.50) habits with no significant differences in regards to the mean number of teeth present (p = 0.11). Smoking habits remained unchanged throughout the study duration. In the examined population, more male participants were susceptible to periodontal disease than females (p = 0.02). No differences were found between the IL-10 groups with respect to age (p=0.30), gender (p=0.86), alcohol consumption (p=0.90) and number of teeth (p=0.96), whereas non-smokers were more likely to be susceptible to periodontal disease (p=0.03).

Table 1. Demographic characteristics of the study population.

	Interleukin – 6			Interleukin – 10			TOTAL
Parameters	SCP (n=26)	NSCP (n=11)	p-value*	SCP (n=17)	NSCP (n=20)	p-value*	(n=37)
Age (mean ± SD in years)	48.31 ± 9.88	46.18 ± 4.56	0.50	49.29 ± 9.18	46.30 ± 8.12	0.30	47.68 ± 8.64
Gender Male (%) Female (%)	16 (61.5) 10 (38.5)	2 (18.2) 9 (81.8)	0.02	8 (47.1) 9 (52.9)	10 (50) 10 (50)	0.86	18 (48.6) 19 (51.4)
Smoking Yes (%) No (%)	12 (46.2) 14 (53.8)	6 (54.5) 5 (45.5)	0.64	5 (29.4) 12 (70.6)	13 (65) 7 (35)	0.03	18 (48.6) 19 (51.4)
Alcohol consumption Yes (%) No (%)	11 (42.3) 15 (57.7)	6 (54.5) 5 (45.5)	0.50	8 (47.1) 9 (52.9)	9 (45) 11 (55)	0.90	17 (45.9) 20 (54.1)
Number of teeth (mean ± SD)	26.23 ± 1.97	25.00 ± 2.28	0.11	25.88 ± 2.21	25.85 ± 2.08	0.96	25.87 ± 2.11

* Mann-Whitney tests were used to compare age and number of teeth between SCP and NSCP groups. Chi-square tests were utilized for comparisons between SCP and NSCP groups with respect to gender, smoking, alcohol consumption.

Abbreviations: SCP: Susceptible, NSCP: Non-susceptible

Clinical outcomes of periodontal treatment and IL-6 genotype

The clinical parameters of the IL-6 SCP and NSCP patients at baseline and 3 years following periodontal therapy are shown in Table 2. All clinical variables of the SCP and NSCP groups were similar at baseline irrespective of the genotype (p>0.05) and all decreased significantly three years following a non-surgical periodontal treatment in both groups (SCP and NSCP) (p<0.05) except for the percentage of sites with CAL≥7 mm in the NSCP group (p=0.097). In addition, no significant differences were detected in any of the clinical parameters between the groups after 3 years of treatment (p>0.05).

Table 2. Clinical parameters of the IL-6 SCP and NSCP patients at baseline and 3 years following periodontal therapy.

Clinical parameters	SCP (n=26)	NSCP (n=11)	p-value**
Full-mouth PPD (mm) Baseline 3 years p-value*	3.57 (2.21-4.98) 2.39 (2.00-3.71) <0.001	3.37 (2.68-4.19) 2.21 (2.00-3.21) 0.003	0.32 0.29
Full-mouth BOP (%) Baseline 3 years p-value*	100.00 (21.43-100.00) 8.63 (0-100.00) <0.001	100.00 (10.87-100) 6.52 (0.67-62.50) 0.003	1.00 0.56
Full-mouth CAL (mm) Baseline 3 years p-value*	3.67 (2.21-5.18) 2.66 (2.00-5.64) 0.003	3.55 (2.83-4.31) 2.37 (2.00-5.24) 0.021	0.49 0.15
Percentage of sites with PPD 4-6 mm (%) Baseline 3 years p-value *	26.88 (1.92-49.31) 4.92 (0-34.67) <0.001	30.36 (8.67-45.65) 2.17 (0-27.38) 0.003	0.92 0.84
Percentage of sites with PPD ≥7mm (%) Baseline 3 years p-value*	4.63 (0-23.81) 0 (0-8.64) <0.001	1.45 (0-9.62) 0 (0-2.00) 0.017	0.19 0.52
Percentage of sites with CAL 4-6 mm (%) Baseline 3 years p-value*	29.15 (1.92-52.50) 8.33 (0-87.04) 0.046	35.71 (10.0-46.38) 2.50 (0-78.21) 0.033	0.79 0.35
Percentage of sites with CAL ≥7 mm (%) Baseline 3 years p-value*	4.63 (0-25.00) 0.30 (0-24.07) 0.003	1.67 (0-10.67) 0 (0-15.38) 0.097	0.37 0.22

*Wilcoxon test: comparison between baseline and 3 years.

**Mann-Whitney test: comparison between SCP and NSCP groups.

Abbreviations:

PPD: Probing pocket depth

CAL: Clinical attachment loss

BOP: Bleeding on probing

SCP: Susceptible

NSCP: Non-susceptible

Clinical outcomes of periodontal treatment and IL-10 genotype

The clinical parameters of the IL-10 SCP and NSCP patients at baseline and 3 years following periodontal therapy are shown in Table 3. No significant differences between the SCP and NSCP groups were detected at baseline (p>0.05), while a significant clinical improvement was observed at 3 years (p<0.05) apart from the percentage of sites with CAL 4-6 mm (p=0.057) and the percentage of sites with CAL≥7 mm (p=0.167) in the NSCP group. Individuals SCP to chronic periodontitis exhibited statistically significant reductions in PPD, CAL, BOP, percentage of sites with PPD=4-6 mm, percentage of sites with PPD≥7 mm, percentage of sites with CAL=4-6 mm, and percentage of sites with CAL≥7 mm at 3 years following the treatment (p<0.03). In comparisons between groups, neither SCP nor NSCP patients showed any significant difference with respect to the examined parameters (p>0.05).

Table 3. Clinical parameters of the IL-10 SCP and NSCP patients at baseline and 3 years following periodontal therapy.

Clinical parameters	SCP (n=17)	NSCP (n=20)	p-value**
Full-mouth PPD (mm) Baseline 3 years p-value*	3.18 (2.66-4.98) 2.31 (2.00-3.71) <0.001	3.55 (2.21-4.44) 2.50 (2.00-3.23) <0.001	0.35 0.49
Full-mouth BOP (%) Baseline 3 years p-value*	100.00 (10.87-100.00) 7.50 (0-100.00) 0.002	100.00 (35.26-100.00) 11.26 (0-62.50) <0.001	0.48 0.89
Full-mouth CAL (mm) Baseline 3 years p-value*	3.55 (2.84-5.18) 2.38 (2.00-4.54) 0.002	3.58 (2.21-5.02) 2.66 (2.00-5.64) 0.023	0.63 0.59
Percentage of sites with PPD 4-6 mm (%) Baseline 3 years p-value*	23.81 (8.67-47.44) 4.76 (1.28-34.67) <0.001	29.77 (1.92-49.31) 3.21 (0-27.38) <0.001	0.25 0.80
Percentage of sites with PPD ≥7mm (%) Baseline 3 years p-value*	2.56 (0-23.81) 0 (0-3.33) 0.001	3.13 (0-18.75) 0 (0-8.64) 0.003	0.66 0.70
Percentage of sites with CAL 4-6 mm (%) Baseline 3 years p-value*	29.17 (9.88-49.36) 8.33 (0-87.04) 0.028	33.65 (1.92-52.50) 5.06 (0-78.21) 0.057	0.77 0.76
Percentage of sites with CAL ≥7 mm (%) Baseline 3 years p-value*	4.49 (0-25.00) 0 (0-7.74) 0.001	4.46 (0-22.22) 0 (0-24.07) 0.167	0.50 0.99

*Wilcoxon test: comparison between baseline and 3 years.

**Mann-Whitney test: comparison between SCP and NSCP groups.

Abbreviations:

PPD: Probing pocket depth

CAL: Clinical attachment loss

BOP: Bleeding on probing

SCP: Susceptible

NSCP: Non-susceptible

A subgroup analysis was also performed to compare the clinical parameters at baseline and 3 years following a non-surgical periodontal therapy between individuals susceptible (n=13) to chronic periodontitis carrying both the susceptible genotypes for IL-6 (IL-6 GG) and IL-10 (IL-10 CA or IL-10 AA) with those non-susceptible (n=24) carrying one or all three non-susceptible genotypes IL-6 GC, IL-6 CC, IL-10 CC. Both subgroups showed similar age (p=0.50) and gender (p=0.25) distribution. Following the treatment, significant reductions were observed for the susceptible group in regards to BOP (p=0.001), PPD (p=0.001), CAL (p=0.013), PPD=4-6 mm (p=0.001), PPD≥7 mm (p=0.001) and CAL≥7 mm (p=0.01). Sites with CAL=4-6 mm demonstrated no significant differences between baseline and 3 years in the combined IL-6 and IL-10 susceptible subgroup (p=0.13). In the IL-6 and IL-10 non-susceptible combined subgroup, all examined clinical parameters showed significant reductions (p<0.04). SCP and NSCP subgroups showed similar clinical variables at baseline and 3 years (p>0.05).

Multiple logistic regression analysis

Multiple logistic regression analysis for the association between the IL-6 -572 G/C and IL-10 -592 C/A genotypes and the demographic and clinical variables at baseline and 3 years is shown in Table 4. At baseline, IL-6 genotype was significantly associated with gender (p=0.01) and the number of missing teeth (p=0.05), while IL-10 genotype and smoking status showed a significant association (p=0.04). In addition, there was a significant association between susceptibility to chronic periodontitis (as determined by the presence of the IL-6 -572GG genotype) with gender at 3 years (p=0.01). However, after the Bonferroni adjustment for multiple comparisons, these findings lost statistical significance (p>0.05). After 3 years of periodontal treatment, there was no association between any of the variables tested (p>0.05).

Table 4. Multiple logistic regression analysis for the association between the IL-6 -572 G/C and IL-10 -592 C/A genotypes and the demographic and clinical variables at baseline and 3 years.

	Parameters	OR	95%Cl	p-value*	Adjusted p-value (Bonferroni correction)
IL-6	Baseline Age Gender Smoking Alcohol consumption Number of teeth Full-mouth BOP Full-mouth PPD Full-mouth CAL	1.12 52.09 1.09 0.42 1.79 0.97 0.10 35.69	0.97-1.29 2.41-1125.37 0.09-13.01 0.04-4.78 1.00-3.22 0.93-1.02 0.01-104.32 0.07-18052.25	0.12 0.01 0.95 0.49 0.05 0.21 0.52 0.26	0.99 0.10 7.56 3.86 0.41 1.66 4.17 2.08
	3 years Age Gender Smoking Alcohol consumption Number of teeth Full-mouth BOP Full-mouth PPD Full-mouth CAL	1.09 26.86 1.39 0.35 1.32 0.99 0.28 1.94	0.96-1.25 2.00-360.16 0.18-10.96 0.05-2.38 0.82-2.11 0.95-1.03 0.01-6.11 0.59-6.38	0.19 0.01 0.76 0.29 0.26 0.63 0.42 0.28	1.56 0.10 6.04 2.28 2.06 5.04 3.34 2.22
IL-10	Baseline Age Gender Smoking Alcohol consumption Number of teeth Full-mouth BOP Full-mouth PPD Full-mouth CAL	1.05 1.07 0.18 2.07 1.08 0.98 0.46 3.37	0.95-1.15 0.20-5.78 0.03-0.93 0.36-11.89 0.73-1.59 0.95-1.01 0.01-109.19 0.02-541.77	0.33 0.94 0.04 0.42 0.70 0.16 0.78 0.64	2.63 7.49 0.33 3.33 5.58 1.30 6.25 5.11
	3 years Age Gender Smoking Alcohol consumption Number of teeth Full-mouth BOP Full-mouth PPD Full-mouth CAL	1.05 0.61 0.18 2.1 0.80 1.01 1.58 0.63	0.95-1.15 0.11-3.50 0.30-1.05 0.42-10.64 0.51-1.26 0.98-1.05 0.13-19.75 0.25-1.60	0.34 0.57 0.06 0.37 0.34 0.48 0.72 0.33	2.75 4.59 0.45 2.95 2.68 3.82 5.78 2.62

*p-values in bold denote statistical significance (p<0.05).

Abbreviations:

PPD: Probing pocket depth

CAL: Clinical attachment loss

BOP: Bleeding on probing

Discussion

Current evidence suggests that the treatment outcome following non-surgical periodontal treatment may vary between patients, teeth as well as treated sites within individuals (18, 19). Each individual’s genetic background may explain the variation in treatment response. Since IL-6 -572 and IL-10 -592 gene polymorphisms have been significantly associated with chronic periodontitis (10-13) and since gene variant carriage could possibly affect the response to periodontal therapy (23), we investigated, in this study, the influence of genetic susceptibility to chronic periodontitis on periodontal treatment outcome over a three-year period of time.

In contrast with our hypothesis that patients susceptible to chronic periodontitis as determined by the presence of the IL-6 -572GG genotype or the IL-10 -592A allele would have significantly worse response to non-surgical periodontal treatment than non-susceptible individuals, a similar treatment outcome was observed for both included groups. More specifically, susceptible and non-susceptible to chronic periodontitis patients showed a statistically significant improvement in the examined clinical parameters at 45 days (as shown in our previous publication, 24) which has been maintained up to 3 years following the initial treatment (as shown in the present investigation). However, this significant clinical periodontal improvement was irrespective of a patient’s genetic background.

To the best of our knowledge, this is the first study that evaluated the association between gene polymorphisms and non-surgical periodontal treatment over a long term period. Previous investigations included individuals that were followed-up for a period of time between 45 days to 6 months following a non-surgical periodontal therapy (23). Our findings may differ with previous studies due to the longer period of observation. Long-term monitoring and supportive periodontal treatment are of paramount importance to achieve long-term success of periodontal treatment and to minimize the risk of tooth loss (26). In addition, long-term studies can confirm or reject an association that has been shown in relatively short-term studies. However, it is not always possible to maintain the initial sample size and preserve an equal distribution of the sample size between groups when performing longitudinal studies. The loss of recruited patients during the follow-up time can be considered as inherent limitation of our investigation. Although differences in gender distribution between IL-6 SCP and NSCP groups were observed, gender demonstrated no significant effects on treatment outcome.

Furthermore, the majority of previous studies that aimed to examine the effect of susceptibility to chronic periodontitis on the treatment outcome following non-surgical periodontal therapy have shown similar results (23). Matrix metalloproteinase (MMP)-1, MMP-13, IL-1, IL-4, IL-6, IL-8, mannose-binding lectin (MBL) and monocyte chemoattractant protein (MCP)-1 gene polymorphisms have been analyzed (23, 27). These short-term studies demonstrated no significant differences between genetic polymorphisms of the IL-8, MMP-13, MMP-1, IL-1, IL4, IL-8, MBL genes and response to non-surgical periodontal treatment (23). On the contrary, in a prospective longitudinal study by D’ Aiuto et al., susceptible patients carriers of the C allele of the IL-6 −174 polymorphism with generalized severe periodontitis showed a worse periodontal treatment outcome compared to non-susceptible individuals (28). However, the inclusion of various ethnic populations and the differences in the examined variables may explain the contrasting findings. More specifically, Caucasian, African/Caribbean and Asian patients were included in the same analysis rather than a specific ethnic group and the effect of genotype on periodontal treatment outcome was determined based on the decrease in the number of pockets rather than the evaluation of any continuous periodontal parameters. In addition, a Taiwanese study demonstrated that MCP-1 -2518 A/G genotype was significantly associated with the treatment outcome of non-surgical periodontal treatment as determined by evaluation of the probing depth, gingival index and bleeding index (27). However, this gene polymorphism influenced the treatment outcome only in patients with a diagnosis of aggressive periodontitis rather than in patients with chronic periodontitis.

Although there is evidence supporting that patient-related factors influence the periodontal treatment outcome and the risk of tooth loss (18, 19), genetic susceptibility to chronic periodontitis, conferred by the presence of the IL-6 -572GG genotype or the IL-10 -592A allele, was not associated with a worse treatment outcome in this study. Other factors, such as smoking, age and oral hygiene, may be stronger factors for a poorer treatment outcome than genetic factors (29). Additionally, systemic medical conditions such as diabetes mellitus may affect periodontal healing and tissue homeostasis following periodontal therapy (20). In order to minimize the potential confounding factors, systemically healthy patients were only recruited. Moreover, the lack of association between smoking status and susceptibility to chronic periodontitis after adjusting for multiple comparisons may be due to different smoking habits (light, moderate, heavy smoking) of the included population.

IL-6 is a pleotropic cytokine that promotes the evolution of chronic inflammation and bone resorption through complex interactions (30, 31). Since IL-6 polymorphisms are associated with an increased inflammatory response primarily in the presence of periodontal pathogens, IL-6 gene may play an important role in the pathogenesis of periodontitis (6, 7). IL-10 is considered an anti-inflammatory cytokine that suppresses immune and inflammatory responses which can protect against bone resorption (32). Polymorphisms in the IL-10 promoter -592 region are associated with a decrease in IL-10 production, which may result in the development of periodontal diseases (9). Although both polymorphisms are associated with an increased inflammatory response and therefore play an important role in disease onset and progression, their effect may not influence the healing process after periodontal treatment. IL-6 and IL-10, as shown in this study, may have no effect on the resolution of periodontal inflammation.

Future studies encompassing larger samples with different characteristics including smoking habits, various bacterial strains and systemic conditions from different populations and ethnicities are required to further examine the possible effect of gene polymorphisms on periodontal treatment outcome over a long period of time. In addition, further studies should include a greater number of patients with a generalized form of chronic periodontitis (>30% of sites) to determine whether the patients with a greater extent of periodontitis demonstrate significant differences with respect to treatment outcome. Due to the inclusion criteria of this study (interproximal CAL ≥ 3 mm, PPD ≥ 5 mm and BOP in two or more non-adjacent sites), the individuals with localized and generalized periodontitis were included in the analysis.