“Gingival Crevicular Fluid Levels of Interleukin-35 in Periodontal Health, Chronic Periodontitis & the Effect of Scaling & Root Planning on Interleukin-35 Levels in Chronic Periodontitis – A Randomized Controlled Clinical Study”

Introduction

Periodontal diseases are multifactorial diseases, where the periodontopathogens trigger various inflammatory and immune system responses. They are characterized by loss of periodontal connective tissue attachment and irreversible loss of alveolar bone, which simultaneously results in the loss of affected teeth.¹ These periodontal tissues are subjected to infiltration by various immune cells such as neutrophils, macrophages, B cells and T cells and leads to the generation of high concentrations of cytokines and other mediators. Cytokines are soluble proteins that bind to specific receptors present on target cells, which play a major role in triggering and inhibiting intracellular signalling cascades.²

T cells play an important role in regulating the immune responses at the inflammation site by secreting various pro-inflammatory and anti-inflammatory cytokines. T cells which down regulate the immune response are called regulatory T (T_reg) cells. They are classified as natural T_reg (nT_reg) and inducible T_reg (iT_reg) cells, nT_reg cells are CD4+CD25+ T cells that migrate from the thymus to mediate the suppressive role in immune system homeostasis and iT_reg cells are derived from T-effector cells. Based on their cytokine release, iT_reg cells are categorised into type 1 T_reg (Tr1), type 3 T-helper (Th3), and interleukin-35 (IL-35) producing T (iTr35) cells.²

IL-35 was identified in the 2000s and first reported by Dario Vignali and colleagues.³ It is a novel member of IL-12 super-family. They are composed of an 𝛼 chain (p19, p28, or p35) and a 𝛽 chain (p40 or Ebi3). It has IL-27 𝛽 chain Ebi3 (Epstein-Barr virus induced gene 3) and IL-12 𝛼 chain p35.1 IL-35 is unique in its nature in that instead of being expressed primarily by antigenpresenting cells, it is expressed by T_reg cells.³ IL-35 is considered as an effective inhibitory cytokine and is known for its suppressive effect. It is shown to be most effective at high inflammation sites and as a potent activator of T_reg cells.²

Although previous studies have identified the presence of IL-35 in Gingival Crevicular Fluid (GCF) of periodontally healthy and chronic periodontitis patients, the effect of periodontal therapy on the levels of IL-35 has not yet been reported. Hence, the objective of this study was to assess GCF levels of IL-35 in healthy subjects and chronic periodontitis patients and to compare GCF levels of IL-35 before and after scaling and root planing in chronic periodontitis patients.

Materials And Methods

This is a randomised controlled clinical study conducted on individuals reported to Department of Periodontics of the institution. All the selected participants were explained about the need, design of the study and its potential benefits and were made to sign an informed written consent prior to the commencement of the study. The study was approved by the Ethical Committee of the Institution (EC No.: DSCDS/EC/NOV2015/7). This single centre, parallel arm design study was carried out from August 2016 to March 2017. Sample size was estimated using the mean score (Group I: 61.2±8.03 and Group II: 40.9±3.14) of pilot study conducted with 10 subjects. Power of the study was kept at 80% and error probability was fixed at 0.05. Sample size was calculated as 20 subjects per group.

The inclusion criteria for participants were as follows – for periodontally healthy individuals age ranged 25-65 years, gingiva with bleeding on probing scores <20%, no attachment loss and probing pocket depth (PPD) ≤3mm; for chronic periodontitis patients, age ranged 25-65 years, PPD ≥5mm, clinical attachment level (CAL) ≥3 mm and bleeding on probing scores >50%. The exclusion criteria included patients with systemic diseases, use of antiinflammatory drugs and antibiotics in last six months, patients who had undergone periodontal treatment in the last six months, pregnant and lactating mothers, smokers, alcoholics and drug abusers.

Totally, 60 samples were collected from 40 subjects and they were divided into Group 1 [n ═ 20] with periodontally healthy individuals, Group 2A [n ═ 20] with chronic periodontitis patients and Group 2B, comprising Group 2A patients who were evaluated six weeks after scaling and root planing. The study design is as described in Figure 1.

A proforma was created to facilitate systematic and methodological recording of all the observations and information and included a brief case history and periodontal examination. On the day of the first visit, periodontal examination included the clinical measurements of Ainamo and Bay Gingival Bleeding Index⁴ (GBI) which consider only presence or absence of bleeding on gentle probing of gingival sulcus, PPD measured to the nearest millimetre mark, as the distance from the gingival margin to the bottom of periodontal pocket and CAL, measured to the nearest millimetre mark, as the distance from cemento-enamel junction to the base of the pocket.

On the subsequent day of clinical parameter recording, the sites were well isolated and GCF was collected by placing the microcapillary pipette (Sigma Aldrich, St.Louis) at the entrance of the gingival sulcus by gently touching the gingival margin (Figure 2). In the chronic periodontitis group, only one site per subject, showing the greatest CAL and signs of inflammation; whereas in the healthy group, multiple sites (3-5 sites per subject) with an absence of inflammation were sampled, to ensure the collection of an adequate amount of GCF. For Group 2B patients, GCF samples were collected from the same site which showed greatest CAL and signs of inflammation at the baseline level. A standardized volume of 3 μL was collected. Pipette contaminated with saliva and blood was discarded. Samples were transferred to air-tight plastic vials containing phosphate buffered saline and 0.5% bovine serum albumin as transport medium and stored at -800 C till subjected to laboratory analysis using ELISA (Figure 3). The test kit [KINESIS Dx, catalogue number K12-0043] utilizes a double-antibody sandwich ELISA for quantitative detection of level of IL-35 in GCF samples (Figure 4). 

The results were averaged for each continuous parameter. Data were first examined for normality by Kolmogorov Smirnov test and Shapiro Wilk test. Student paired t-test and Chi square test were used to detect differences in frequencies of age and gender between the groups. Intergroup comparison of clinical and biochemical parameters were done by using one way ANOVA test. To find the significantly different means between the two groups, the post hoc test of Tukey’s test was used. Possible correlation between IL-35 levels in GCF and clinical parameters were tested by the Pearson correlation test. In all the above analyses, the level of significance was set at α = 0.05, i.e., P<0.05 was considered to be statistically significant. Statistical analyses were performed using the SPSS software, version 10.5.

Results

In the present study, the selected patients were divided into three different groups classified in accordance with the selection criteria. Demographic and clinical parameters were recorded. A total of 60 samples were collected and biochemically analysed for IL-35 levels.

The mean age for Group 1 and Group 2 was 26.25 and 38.15 years, respectively. The difference in age between the two groups was statistically significant (P<0.001). The χ² value for gender was 8.640 and the difference between the two groups was statistically significant (P=0.003).

All clinical and biochemical parameters were normally distributed except CAL and GBI values for Group 2A, which were not normally distributed (Table 1).

The difference in PPD scores between the groups was highly statistically significant (Table 2). Pair wise comparison also showed statistically significant difference in mean PPD score between group 1 and group 2A, and between group 1 and group 2B (Table 4). The difference in CAL scores between the groups was highly statistically significant (Table 3). On pair wise comparison between the groups, the difference in scores between Group 1 and Group 2A was highly statistically significant, while between Group 1 and Group 2B was not significant (Table 5). The difference in GBI scores between the groups was highly statistically significant (Table 3). On pair wise comparison between all the three groups, the difference in scores was highly statistically significant (Table 5).

The mean IL-35 levels in GCF for Group 1, Group 2A and Group 2B were 530.70, 626.98 and 528.98 (ng/L) respectively. The difference in levels between the groups was highly statistically significant (Table 2). On pair wise comparison between the groups, the difference between Group 1 and Group 2A was statistically significant, while between Group 1 and Group 2B was not significant (Table 4). The difference in PPD and IL-35 scores between Group 2A and Group 2B was highly statistically significant (Table 6). The difference in CAL and GBI scores between Group 2A and Group 2B was highly statistically significant (Table 7). A significant reduction in all clinical and biochemical parameters was noted in Group 2B. The difference in IL-35 levels between Group 2A and Group 2B was highly statistically significant. A weak positive correlation was found between IL-35 and PPD values in Group 2A (R value: 0.233, P value: 0.324) and Group 2B (R value: 0.298, P value: 0.202), while a weak negative correlation was found in Group 1 (R value: -0.200, P value: 0.399) but the correlation was not statistically significant (Graphs 1, 4, 7). A negligible correlation was found between IL35 and CAL values in Group 1 (R value: -0.158, P value: 0.505), and Group 2B (R value: -0.117, P value: 0.623), while a weak negative correlation was found in Group 2A (R value: -0.209, P value: 0.376), and the correlation was not statistically significant (Graphs 2, 5, 8). A moderate negative correlation was found between IL-35 levels and GBI scores in Group 2A (R value: -0.332, P value: 0.153), while a negligible correlation was found in Group 1 (R value: -0.199, P value: 0.401), and Group 2B (R value: -0.192, P value: 0.417), (Graphs 3, 6, 9).

Discussion

IL-35 is a newly identified and youngest member of IL12 cytokine family, produced by T_reg cells (a component of the immune system that supresses inflammation). IL-35 expression is extremely minimal in unactivated T cells. These cells need to be activated, by binding to their T-cell receptor or following inflammation, for the induction of IL-35. This suggests that IL-35 may not be a constitutive marker of T_reg cells, but associated with the activity of Treg cells in peripheral tissues³ .

Potential markers associated with the severity and susceptibility of periodontal disease is now a primary concern of research and has been receiving considerable attention. GCF, saliva and plasma are the primary quantifiers to evaluate their role in pathogenesis of periodontal diseases. GCF is a serum exudate found in the gingival sulcus, and traverses from the microcirculation across inflamed periodontal tissues and carries biological molecular markers gathered from the surrounding site. As GCF flow is increased in case of inflammation, it offers an easy and non-invasive mode of collection from the sites.²

The present study aimed to assess GCF levels of IL35 in periodontally healthy adults as well as those with chronic periodontitis and also to compare GCF levels of IL-35 in chronic periodontitis patients, before and after scaling and root planing. The findings of our study demonstrated a statistically significant difference in all clinical parameters like GBI, PPD and CAL on comparing all the three groups. IL-35 levels were higher in chronic periodontitis patients than healthy individuals. The difference in IL-35 levels between Group 1 and Group 2A was statistically significant, while between Group 1 and Group 2B was not significant. Similar findings were reported by Kalburgi et al1 where they evaluated levels of IL-35 mRNA in gingival tissues of healthy subjects, chronic periodontitis and aggressive periodontitis (AGP) patients. The level of IL-35 mRNA was higher in chronic periodontitis group as compared to AGP and healthy group. Another study done by Mitanni et al,⁵ where they compared IL-35 levels in chronic periodontitis patients and healthy subjects found that GCF concentrations of IL-35 were higher in chronic periodontitis. Periodontal clinical parameters showed significant positive correlation with IL-35 levels.

Okada et al⁶ investigated the effect of IL-35 on Th 17 cells derived from periodontally healthy and chronic periodontitis patients. The proportion of IL-17+CD4+ slightly increased in chronic periodontitis patients compared with healthy individuals; however, there were no significant differences in the percentage. IL17mRNA expression was significantly increased in Th17 cells and the induction was significantly inhibited by addition of rIL-35 (1 ng/mL). Our results are consistent with their interpretations, where they suggested that IL-35 could directly suppress the pro-inflammatory activity of IL17 and might play an important role in suppressing inflammatory responses of periodontium.

Conflicting results were reported by Kaustubh et al,⁷ where they compared GCF levels of IL-35 in periodontally healthy individuals and individuals with gingivitis. Clinical parameters were significantly higher in gingivitis patients but GCF levels were higher in healthy individuals. Another study done by Koseglu et al2 evaluated the levels of IL-35 in plasma, saliva and GCF in chronic periodontitis, gingivitis, and healthy individuals. All clinical parameters were significantly higher in chronic periodontitis patients, but GCF concentrations of IL-35 were significantly higher in healthy individuals. They concluded that increased levels of IL-35 could have an important role in inhibiting periodontal inflammation and maintaining periodontal health. In our study, there was significant reduction in IL-35 levels and clinical parameters when comparing Group 2A and Group 2B. On comparing Group 1 and Group 2B, the GBI scores were statistically significant. However, while comparing Group 2A and Group 2B, there was a significant reduction in gingival inflammation which directly correlates with the reduction in IL-35 levels. The elevated level of IL35 in Group 2A suggests their increased expression in activated T cells following inflammation. Subsequent to their induction, IL-35 may initiate their inhibitory actions on inflammatory conditions. As a result of reduction in inflammatory responses following SRP, the induction of IL-35 by T cells in peripheral tissues also can be limited. The presence of IL-35 in healthy subjects suggests their frequent interaction with the host mechanisms to maintain periodontal health. Our results suggest the suppressive role of IL-35 in periodontal inflammation and their protective role in maintaining periodontal health.

The limitations of the present study can be the discrepancy in IL-35 levels at the time of sample collection; the time elapsed between the collection and storage of samples and the smaller sample size. Even though our data supports the role of IL-35 as a promising biomarker in periodontal diseases, further long-term trials with a larger sample size are necessary to validate and quantify its levels in periodontally healthy and diseased conditions.

Conclusion

Within the study limitations and due to minimal data available for comparative evaluation of IL-35 levels in periodontal diseases, the present study concludes that higher GCF levels of IL-35 in chronic periodontitis patients may reveal the severity of periodontal disease and the role of IL-35 in suppressing the inflammation. The significant reduction of IL-35 levels in chronic periodontitis patients 6-8 weeks after scaling and root planing and their presence in periodontally healthy subjects suggests the role of IL-35 in controlling inflammation and their protective role in maintaining periodontal health.

Conflict of Interest

None.