Interleukin-17 levels in gingival crevicular fluid of aggressive periodontitis and chronic periodontitis patients

Abstract

Context:

Interleukin-17 (IL-17) plays an important function in initiation, progression of any inflammatory condition. It is a pro-inflammatory cytokine and considered to be founding member. Periodontitis being an inflammatory condition of periodontium, IL-17 does have a potential role in periodontitis. The current research has shown positive correlation between the IL-17 and severity of periodontitis. Therefore, this research study aimed to evaluate the levels of IL-17 in aggressive periodontitis (AgP), chronic periodontitis (CP), and “healthy gingival sites.”

Materials and Methods:

A total of 45 selected subjects (90 samples) were equally divided into three different groups. Group 1 (Healthy gingiva on clinical examination): Subjects with healthy gingiva and no evidence of periodontal attachment level (PAL), probing pocket depth (PPD) <3 mm papillary bleeding index (PBI) <1. Group 2 (AgP): Subjects having age <35 years with at least six permanent teeth apart from the incisors and first molars with PPD and PAL ≥5 mm. Group 3 (CP): It consist of subjects with clinical features of inflammation of gingiva with minimum six teeth in each jaw, with PPD ≥4 mm, PAL ≥4 mm. IL-17 levels were analyzed by using Gingival crevicular fluid (GCF). It was collected from the sulcus of first maxillary molar sites for quantitative analysis.

Results:

GCF IL-17 levels in AgP were found to be 1.12 ± 0.29, 1.96 ± 1.71 in CP and 0.64 ± 0.23 in healthy gingival sites. Clinical parameters such as (1) plaque index (Turesky et al., modification of Quigley-Hein), (2) PPD, (3) PBI were higher (significantly) in periodontitis group as compared to healthy gingiva. Positive correlation was found to be significant between levels of IL-17 with PAL and PPD in AgP however in cases of CP, positive correlation of IL-17 levels was found only with PAL but not PPD.

Conclusion:

Our study shows that the IL-17 levels were comparatively higher in GCF of AgP, CP in comparison to healthy gingiva, but the GCF IL-17 levels were higher in CP as compared to AgP.

INTRODUCTION

“Periodontitis” is an inflammatory condition which results from the interaction of periodontal pathogens and immune responses from host resulting into destruction of periodontium. These interactions are initiated and controlled by cytokines produced by neutrophils, fibroblasts, macrophages, monocytes, lymphocytes, dendritic cells, and endothelial cells. They (Cytokines) are soluble protein molecules which are considered responsible for the pathogenesis of various inflammatory diseases including periodontitis.[1]

Cytokines regulate physiological as well as pathological tissue reorganization, hematopoiesis, angiogenesis, and wound healing. Cytokines are protein molecules which play crucial role in many of the physiological responses. They are extremely potent, act at picomolar concentration and interact with specific cell receptors. Individual cytokine has multiple cell regulatory actions. The actions and reactions of a cytokine on a cell depends on the type of cell, local concentration and other cell regulatory factors to which it gets exposed.[2,3] The term interleukins (ILs) have replaced the cytokines owing to their role in sharing the information between leukocytes.[4,5]

IL-17 is produced by Th17 and it is a subset of CD4 ⁺ T-cells.[6,7,8] The IL-17 family consists of six members from IL-17A to 17F, IL-17 E is also named as IL-25.[9,10,11,12,13] IL-17 take a leading role in production and release of other proinflammatory cytokines (IL-1, IL-6, IL-8 and Tumor Necrosis Factor-α), matrix-metalloproteinases and osteoclastogenesis related mediators which causes inflammation mediated destruction of periodontium.[12,14]

IL-17 is the most important pro-inflammatory cytokine which has effects on different cell types to stimulate the activation of neutrophils and mediate the proinflammatory reactions.[15] IL-17 is 17–kd protein, released as a dimer and it shares no similarity with rest of the other known proteins (mammalian proteins). Therefore, it comprises specific cytokine family. IL-17 shows its action on receptors present on cell-membrane.[16,17]

Previous studies have shown its (IL-17) role in initiation and progression of chronic periodontitis (CP) therefore suggesting its role in periodontal tissue destruction and bone loss.[18,19,20,21,22] There is abundant scientific data available to correlate IL-17 levels in CP but miniscule scientific evidence is available about its relation with aggressive periodontitis (AgP). Therefore, the present study was undertaken to evaluate and compare the IL-17 levels between AgP, CP and healthy controls (healthy gingiva on clinical examination). It also intends to formally assess the relationship between clinical parameters and levels of IL-17 in healthy controls and patients with periodontitis.

MATERIALS AND METHODS

A total of 45 (90 samples) subjects aged between 20 and 65 years (average = 37.5 years) were categorized into three groups with each group comprising of 15 subjects. The sample size was estimated based on power analyses at a confidence interval of 95%. Group 1 (Healthy gingiva on clinical examination): Subjects with healthy gingiva and no evidence of periodontal attachment level (PAL), probing pocket depth (PPD) <3 mm, papillary bleeding index (PBI) <1.[23] Group 2 (AgP): Subjects <35 years of age and at least six permanent teeth apart from the first molars and incisors with PPD and PAL ≥5 mm.[24] Group 3 (CP): Subjects with clinical features of gingival inflammation, i.e., Bleeding on probing,[25] change in color (redness), consistency, contour[26] and minimum six teeth in each jaw, with PPD ≥4 mm, PAL ≥4 mm.

Exclusion criteria were: (a) patients with systemic diseases which affect the periodontium, (b) pregnant female and lactating mothers, (c) patients receiving periodontal treatment in past 1 year, (d) patients on antibiotic therapy or on any type of medication during the 6 months before the study, and (e) consumption of tobacco products, smokers and alcoholics. The study designed was explained and written approval was obtained from the subjects. The study protocol received the approval from the Institutional Ethical committee (DMIMS [DU]/IEC/2017-2018/6427). Demographic details, clinical information, and others details were recorded beforehand.

Clinical parameters includes (1) PBI by Mühlemann,[27] (2) Plaque Index (PI) given by Turesky et al. modification of Schneider et al.,[28] (3) PPD, (4) PAL. PPD and PAL were measure at four different sites of teeth (buccal, palatal/lingual, mesial and distal aspects).[29] PAL was measured from the cemento-enamel junction to the base of the probable pocket. PPD was measured from the gingival margin to base of the pocket. In all the cases before gingival crevicular fluid (GCF) collection, supragingival and subgingival biofilm was removed with the ultra-sonic scaler. GCF samples for IL-17 were collected from the maxillary first molar sites (in both right and left quadrant), so as to prevent saliva contamination.[17] Sampling sites were isolated with the help of cotton rolls and air dried. GCF samples were collected by placing the tip of microcapillary calibrated volumetric pipettes (Sigma-Aldrich chemical company, United States America) extra-crevicularly (unstimulated) until the consistent volume of 1 ml crevicular fluid was collected and then transferred into tubes (Eppendorf) and immediately stored at −70°C until the laboratory analyses were carried out.

Laboratory procedures

GCF samples were analyzed for IL-17 using commercially available human enzyme-linked immunosorbent assay kit (Diaclone IL-17, France, Catalog No. 850.940.096). Analyses were performed as per the manufacturer’s guidelines. GCF samples and standards were first incubated for 2 h at room temperature. The plates were wash for thrice and 50 ml diluted biotinylated anti-IL-17 was added. Again, it was incubated for 1 h. Plates were washed for thrice and 100 ml of streptavidin-horseradish peroxidase solution was added. It was further incubated for 30 min and later plates were washed. One hundred microliter ready to use Tetra methyl benzidine (TMB) substrate solution was added into wells and incubated in the dark for 15 min, followed by addition of 100 ml of H₂SO_4. Results were calculated using extrapolating optical density values against IL-17 standard concentration by using the standard curve with 450 nm absorbance value serving as a primary wavelength.

The means and standard deviations for the clinical parameters and IL-17 were calculated for all the groups. Statistical analysis was carried out using various tests such as (1) one-way analysis of variance (ANOVA), (2) Tukey’s test, (3) Pearson’s correlation coefficient test, (4) student unpaired test. If the probability value (p) was >0.05, the difference observed was considered nonsignificant and if it was <0.05, it was considered significant. Correlation between PPD and PAL with IL-17 in AgP and CP were done. SPSS 22.0 version software was used for analysis (IBM, Armonk, NY, USA) and GraphPad Prism 6.0 version (San Diego, California, USA).

RESULTS

The derived illustrative data for IL-17 levels in GCF (pg/ml) in all three groups along with clinical parameters are shown in Table 1. The levels of IL-17 are compared within AgP, CP, and healthy gingiva. The results derived from the ANOVA test indicated that the deviation in IL-17 levels among all the groups were statistically significant (P < 0.004) [Table 1].

Table 1.

Interleukin-17 levels in healthy gingiva, aggressive periodontitis, chronic periodontitis

Group	n	Mean±SD (pg/ml)	SE	95% CI for mean (lower limit–upper limit)	Minimum	Maximum
Healthy gingiva	30	0.64±0.23	0.06	0.51–0.77	0.35	1.20
Aggressive periodontitis	30	1.12±0.29	0.07	0.96–1.29	0.50	1.77
Chronic periodontitis	30	1.96±1.71	0.44	1.01–2.91	0.85	6.40
One-way ANOVA
Source of variation		Sum of squares	Df	Mean square	F	P
Between groups		13.37	2	6.68	6.48	0.004 (S)
Within groups		43.27	42	1.03
Total		56.64	44

SD – Standard deviation; SE – Standard error; CI – Confidence interval; df – Degree of freedom; F – Ratio of two mean square value; S – Significant (P<0.05); n – Number of participants; P – Probability value; ANOVA – Analysis of variance

Multiple comparison between groups were done by Tukey’s test which showed that statistically significant difference was found when IL-17 level in CP cases were compared with healthy gingiva. Higher amount of IL-17 was found in AgP cases as compared with healthy gingiva but was not statistically significant. No statistically significance was found between aggressive and CP [Table 2].

Table 2.

Comparison of interleukin-17 levels in healthy gingiva, aggressive periodontitis, chronic periodontitis

Group	Mean difference (I-J)	SE	P	95% CI (lower limit-upper limit)
Healthy gingiva
Chronic periodontitis	−1.31 (1-3)*	0.37	0.003 (S)	−2.21-−0.41
Aggressive periodontitis	−0.48 (1-2)*	0.37	0.403 (NS)	−1.38-0.41
Chronic periodontitis
Aggressive periodontitis	0.83 (3-2)*	0.37	0.073 (NS)	−0.06-1.73

*1 (healthy gingiva on clinical examination), 2 (aggressive periodontitis), 3 (chronic periodontitis). S – Significant (P<0.05); SE – Standard error; P – Probability value; I−J – Mean difference for these two groups; CI – Confidence interval; NS – Nonsignificant

Clinical parameters such as PI, PBI, and PPD have been found to be statistically significant in AgP, CP as compared to gingival site. Furthermore, significant result for PI, PBI, and PPD were found in CP as compared to AgP [Tables 3–5]. PAL was found to be statistically significant in CP as compared to AgP [Table 6]. Positive correlation was found significantly between IL-17 level with PAL and not with PPD in CP cases [Table 7]. Positive correlation was found between PPD and PAL with IL-17 levels in AgP cases [Table 8].

Table 3.

Comparison of plaque index in healthy gingiva, aggressive periodontitis, chronic periodontitis

Group	n	Mean±SD	Mean difference (I-J)	SE	P	95% CI (lower limit-upper limit)
Healthy gingiva	30	0.23±0.10
Aggressive periodontitis	30	2.08±0.04
Chronic periodontitis	30	2.62±0.60
Healthy gingiva
Chronic periodontitis			−2.39 (1-3)*	0.12	0.0001 (S)	−2.70-−2.08
Aggressive periodontitis			−1.85 (1-2)*	0.12	0.0001 (S)	−2.16-−1.53
Chronic periodontitis
Aggressive periodontitis			0.54 (3-2)*	0.12	0.0001 (S)	0.22-0.85

*1 (healthy gingiva on clinical examination), 2 (aggressive periodontitis), 3 (chronic periodontitis). n – Number of participants; S – Significant (P<0.05); SD – Standard deviation; P – Probability value; I−J – Mean difference for these two groups; CI – Confidence interval; SE – Standard error

Table 5.

Comparison of probing pocket depth in healthy gingiva, aggressive periodontitis, chronic periodontitis

Group	n	Mean±SD	Mean difference (I-J)	SE	P	95% CI (lower limit-upper limit)
Healthy gingiva	30	1.82±0.17
Aggressive periodontitis	30	5.69±0.42
Chronic periodontitis	30	2.51±0.40
Healthy gingiva
Chronic periodontitis			−3.34 (1-3)*	0.16	0.0001 (S)	−3.75-−2.93
Aggressive periodontitis			−3.87 (1-2)*	0.16	0.0001 (S)	−4.28-−3.46
Chronic periodontitis
Aggressive periodontitis			−0.52 (3-2)*	0.16	0.008 (S)	−0.93-−0.12

*1 (healthy gingiva on clinical examination), 2 (aggressive periodontitis), 3 (chronic periodontitis). n – Number of participants; S – Significant (P<0.05); P – Probability value; I−J – Mean difference for these two groups); SD – Standard deviation; SE – Standard error; CI – Confidence interval

Table 6.

Comparison of periodontal attachment level in aggressive periodontitis and chronic periodontitis

	n	Mean±SD	SE	t	P
Chronic periodontitis	30	5.21±0.66	0.17	3.69	0.001 (S)
Aggressive periodontitis	30	5.98±0.44	0.11

n – Number of participants; S – Significant (P<0.05); SD – Standard deviation; P – Probability value; t – Measures the size of the difference (variation) in sample data; SE – Standard error

Table 7.

Correlation between probing pocket depth, periodontal attachment level with interleukin-17 in chronic periodontitis group

	Mean±SD	n	Correlation (r)	P
IL-17	1.96±1.71	30	-	-
PPD	5.16±0.65	30	0.218	0.435 (NS)
PAL	5.21±0.66	30	0.737	0.002 (S)

n – Number of participants; PPD – Probing pocket depth; IL-17 – Interleukin-17; S – Significant (P<0.05); r – Pearson correlation coefficient; SD – Standard deviation; PAL – Periodontal attachment level; P – Probability value; NS – Nonsignificant

Table 8.

Correlation between probing pocket depth, periodontal attachment level with interleukin-17 in aggressive periodontitis group

	Mean±SD	n	Correlation (r)	P
IL-17	1.12±0.29	30	-	-
PPD	5.69±0.42	30	0.593	0.020 (S)
PAL	5.98±0.44	30	0.846	0.0001 (S)

n – Number of participants; S – Significant (P<0.05); IL-17 – Interleukin-17; PPD – Probing pocket depth; r – Pearson correlation coefficient; SD – Standard deviation; P – Probability value; PAL – Periodontal attachment level; ELISA – Enzyme-linked immunosorbent assay

Table 4.

Comparison of papillary bleeding index in healthy gingiva, aggressive periodontitis, chronic periodontitis

Group	n	Mean±SD	Mean difference (I-J)	SE	P	95% CI (lower limit-upper limit)
Healthy gingiva	30	0.13±0.05
Aggressive periodontitis	30	2.57±0.17
Chronic periodontitis	30	2.51±0.40
Healthy gingiva
Chronic periodontitis			−2.37 (1-3)*	0.09	0.0001 (S)	−2.60-−2.15
Aggressive periodontitis			−2.44 (1-2)*	0.09	0.0001 (S)	−2.66-−2.21
Chronic periodontitis
Aggressive periodontitis			−0.06 (3-2)*	0.09	0.790 (NS)	−0.28-0.16

*1 (healthy gingiva on clinical examination), 2 (aggressive periodontitis), 3 (chronic periodontitis). n – Number of participants; S – Significant (P<0.05); P – Probability value; I−J – Mean difference for these two groups); SD – Standard deviation; SE – Standard error; CI – Confidence interval; NS – Nonsignificant

DISCUSSION

Inflammation is a complex reaction in which the host defense mechanism aims to neutralize its initiation and progression thus attempting to restore the normal biologic tissue architecture and functions. A pathological event is a phase of inflammation invoked by the response of PMNs to bacteria. Scientific evidence has shown that IL-17 is one of key mediators for the response of T-cells to the presence of bacteria.[30]

An array of studies reported in the scientific literature put forward that IL-17 plays a significant role for the initiation and progression of periodontitis and also increase levels of IL-17 have been found in CP.[17,18,31,32]

Vernal et al., have provided the scientific confirmation regarding the presence of IL-17 in gingival cells from biopsies collected from CP subjects. Raised GCF IL-17 levels in samples obtained from deeper part of the pockets in CP subjects were estimated compared to the healthy control.[17] Interestingly, Cardoso et al.[18] found increased expression of Th-17 cells in areas of gingival inflammation with CP patients. However, the report regarding the expression of IL 17 in the literature lacks consistency. For example, a study conducted in CP patients of Indian population did not find any expression of IL 17 in the GCF samples.[33]

Findings of our research showed that level of IL-17 was found to be statistically significant in CP as compared to healthy gingival sites. Direct correlation was found between IL-17 with CAL and not PPD in CP. Results of our study with respect to level of IL-17 in CP is found to be in accordance with other studies.[21,22,34]

In our study, GCF collection was done using microcapillary pipettes, whereas many of the studies use filter paper strips and Periotron 6000/8000, which can result into nonspecific adulation of the analyte to filter paper fibers ensuing incorrect reduction in the detectable levels of IL-17, thus underestimate the correlation of IL-17 levels with periodontal disease progression.[21] It might be predictably possible that amount of GCF collected from different methods may lead to difference in the estimation of levels of IL-17 in different studies.

AgP is a condition which leads to significant rapid periodontal destruction at an early age. Apart from microbiological aspect other etiological factors includes defect in leukocytes function and presence of genetic factors.[35] The prevalence and occurrence of AgP are lower than CP, but destruction is more massive compared to CP. However, very few studies are available evaluating GCF IL-17 levels in GCF of AgP cases.[24,36,37]

In 2012, Ay et al. conducted the study to evaluate the IL-17 levels in sulcular fluid of AgP cases and also in healthy group. Result of this study showed the concentration of GCF IL-17 was reduced (significantly) in the generalized AgP than in the healthy group, despite AgP is severe form of periodontitis.[36] Shaker and Ghallab observed in his study that AgP group had significantly reduced concentrations of IL-17 than CP and control.[24] Sadeghi et al. evaluated IL-17 levels in GCF in a chronic and AgP cases. They found that the GCF IL-17 concentration was raised (significantly) in healthy controls as compared to AgP and CP.[37]

Result of our study indicates that the IL-17 levels in AgP had been found to be raised but not significantly as compared to periodontally healthy gingiva. In addition, an IL-17 level in AgP is found to be less as compared to CP cases although not statistically significant. Despite that AgP is more severe form of periodontitis than CP. Our study has also found positive correlation between IL-17 with CAL and PPD in AgP.

Interlukin-17 stimulates the release of cytokines which particularly attract neutrophils to the inflammatory site. However, AgP known to have divergent PMN function shows defect in neutrophils chemotactic response. It might possible that despite stimulation of IL-17, inflammation may remain unnoticed in AgP due to lack of chemotactic response. Study conducted in a Vermin model reveal the significance of IL-17 in mobilization of neutrophil in the control of the any bacterial infection.[38] This could be a possible reason why IL-17 levels are found less in AgP as compared to CP. However, multicentric studies and research on signaling pathway of IL-17 are required to validate this possible reason in case of AgP.

CONCLUSION

Present findings of our study indicated higher level of IL-17 in periodontitis patients compared to the individual with healthy gingiva, highest among CP group as compared to AgP. However, further multicentric randomized controlled trials are needed to clarify the generalized and/or specific role of IL-17 in AgP.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.