Comparative evaluation of serum and gingival crevicular fluid levels of interleukin 21 in periodontally diseased and healthy patients

Introduction

Host inflammatory response is triggered by bacterial biofilm, which is one of the factors contributing to multifactorial disease known as periodontitis (1). A prolonged exposure to bacteria and endotoxins is necessary for the development of gingival inflammation and progressive dysbiotic condition eventually leading to destruction, although it only accounts for 20% of the variation in the clinical manifestation of periodontitis (2). The primary cause of the soft and hard tissue destruction linked to periodontitis is the host immune system’s inflammatory response to the bacterial challenge. As a result, the inflammatory response plays a major role in determining the disease’s destructive nature (3). This includes the active expression of catabolic cytokines and inflammatory mediators like interleukin (IL)-1β and IL-6 through the activation of lymphocytes, fibroblasts, monocytes/macrophages, and other cellular components. Through tissue-derived matrix metalloproteinases, these cytokines and inflammatory mediators can act alone or in combination to promote collagen degradation and alveolar bone resorption (4).

Traditional methods for diagnosing periodontal diseases include clinical and radiographic examinations. These traditional measures provide information only about the existing disease and are incapable of predicting disease progression. As a result, advances in oral and periodontal disease diagnostic research are shifting toward methods for identifying and quantifying periodontal risk using objective measures such as biomarkers. One of these biomarkers that can be detected in several inflammatory conditions is IL-21 (5,6).

Gingival crevicular fluid (GCF) is produced by the blood vessel gingival plexus, which is located next to the epithelial lining of the dentogingival space. Several molecular components originating from the host response are carried by GCF, which is regarded as a major defense against periodontal infection. GCF-present host response mediators can help with both the status of disease activity and the identification of different periodontal diseases (7). Numerous cytokines, including IL-1, IL-6, IL-8, and IL-12, have been thoroughly examined in relation to chronic periodontitis, and research has been done on their role in the breakdown of periodontal tissues (8-11). The research of IL-21, a recently discovered cytokine, has received more attention in recent years.

IL-21, an inflammatory cytokine mostly expressed by activated T-helper (Th)1 and Th17 cells, two distinct proinflammatory lineages, is not expressed by Th2 cells in humans (12,13). It operates by binding to the type I cytokine receptor IL-21R. Because it has been linked to the pathophysiology of inflammatory breakdown in a number of systemic disorders, including rheumatoid arthritis, colitis, and inflammatory bowel disease, IL-21 has recently attracted attention (14-16). Given the critical function that IL-21 plays in inflammation (17), excessive IL-21 production amplifies local inflammation and exacerbates tissue damage and destruction (18).

The levels of IL-21 may rise as a result of periodontal diseases’ propensity for chronic inflammation. Few studies have been conducted on IL-21 in patients with chronic periodontitis (14,19). However, no research has been done to compare GCF and serum IL-21 levels in healthy individuals, gingivitis patients, or periodontitis patients (19). Thus, the current study aims to measure the levels of IL-21 in the blood and GCF of patients with chronic periodontitis, compare them with those of gingivitis and healthy individuals, and assess the usefulness of this information as a diagnostic tool for periodontal disease.

Materials and methods

This cross-sectional study included 45 patients, ages 25 to 60, who were patients at Sri Sai College of Dental Surgery’s Outpatient Department of Periodontics in Vikarabad, Telangana. The Institutional Ethical Committee gave its approval before the trial started. This study included three groups: Group A, or the healthy group, consisted of 15 participants who were 25-60 years old and had a systemically healthy periodontium with probing pocket depth (PPD) of less than 3 mm. Group B, which consisted of 15 individuals aged 25-60 years who were systemically healthy, had gingivitis. They exhibited clinical symptoms of inflammation and had a score of less than 2, using Modified Gingival Index (MGI). Group C, or the periodontitis group, consisted of 15 individuals, aged 25 to 60, who were in general health but had moderate to severe chronic periodontitis. At least four of their teeth had PPDs or clinical attachment loss (CAL) of at least 5 mm, bleeding when probed, and radiographic signs of bone loss. All the parameters, such as plaque index (PI), MGI, Saxton’s bleeding index (BI), PPD, and CAL, were recorded in all the groups.

GCF and serum sample collection for IL-21

After instructing the subjects to sit comfortably upright in the dentist chair, the selected test site was air-dried and isolated with cotton rolls. To avoid contamination and obstruction of the microcapillary pipette, supragingival plaque was removed with an ultrasonic scaler while avoiding contact with the marginal gingiva.

The following day, GCF was collected using a calibrated volumetric microcapillary pipette (Sigma Aldrich Chemical Company, USA, Catalog No. p0549) with a black color code. The pipette was used to insert the tip at the entrance of the gingival crevice (unstimulated) and leave it there for 5 to 20 minutes. A 3 μL GCF standard volume was obtained. Adequate GCF was difficult to get from periodontally healthy persons (Fig. 1) and gingivitis subjects; therefore, pooled samples were collected from numerous sites to ensure that each subject received the minimal amount required (3 µL).

When chronic periodontitis is present, gathering GCF takes less time and is less laborious since the site with the highest PPD can provide the necessary amount. The collected samples were wrapped in aluminum foil and kept at 80°C until the assay time. Samples tainted with blood or saliva and those with air bubbles were rejected.

For serum samples, after disinfecting the skin over the antecubital fossa, a 20-gauge needle was used to venipuncture 3 mL of blood into a 5-mL syringe. A sample of collected blood was left to coagulate for approximately half an hour at room temperature (Fig. 2). After separating the serum component, the sample was centrifuged for 10 minutes at 3000 rpm. It was then moved to a polycarbonate container and kept at −80°C until the test was scheduled.

Estimation of interleukin 21 levels

Using a commercially available human IL-21, or IL-21 enzyme-linked immunosorbent assay (ELISA) kit (BioAssay Technology Laboratory affiliated to Shanghai Korain Biotech Co., Ltd., Shanghai, China) (Fig. 3), the levels of GCF and serum IL-21 were determined. Phosphate-buffered saline was used to dilute GCF samples to a level of 100 µL.

Assay principle

An ELISA kit is intended to detect various biomarkers in bodily fluid samples. Human IL-21 antibody was precoated onto the plate. The sample’s IL-21 was introduced, and it binds to the antibodies coated on the wells. After that, biotinylated human IL-21 antibody was added, and it binds to the sample’s IL-21.

Next, streptavidin-horseradish peroxidase (HRP), which binds to the IL-21 antibody that has been biotinylated, was added. During a washing phase, unbound streptavidin-HRP is removed following incubation. After that, the substrate solution was added, and color changed proportionately to the concentration of human IL-21. The addition of an acidic stop solution ends the process, and the absorbance is measured at 450 nm.

Sensitivity

The sensitivity or minimum detectable dose of human IL-21 is reported to be 2.46 ng/L.

Assay procedure

1. Every reagent, standard solution, and sample was made in accordance with the directions. Prior to use, all reagents were brought to room temperature, and the experiment was conducted.
2. After figuring out how many strips were needed for the assay, the strips were put into the frames and used. The leftover strips were kept between 2 and 8°C.
3. The standard well was later filled with 50 μL of standard solution.
4. In addition, 40 μL of the sample and 10 μL of the anti-IL-21 antibody were added to the sample wells. Following a thorough mixing of 50 μL streptavidin-HRP into both the standard and sample wells, the plate was sealed with a sealer and allowed to sit at 37°C for 60 minutes (Fig. 4).

   

5. All of the wells were aspirated, filled to the brim with wash buffer, and automatically cleaned five times. The plate was blotted onto paper towels or other absorbent material.
6. Subsequently, 50 μL of substrate solution A and 50 μL of substrate solution B were applied to each well. The plate was sealed with a fresh sealer and left in the dark at 37°C for 10 minutes.
7. The last step involved adding 50 μL of Stop Solution to each well, which instantly caused the color to shift to yellow (Fig. 5).

   

8. Within 10 minutes of administering the stop solution, each well’s optical density (OD value) was ascertained using a microplate reader set to 450 nm (Tab. 1).

Components	Quantity
Standard solution (1600 ng/L)	0.5 mL ×1
Precoated ELISA plate	12 × 8 well strips ×1
Standard diluent	3 mL ×1
Streptavidin-HRP	6 mL ×1
Stop solution	6 mL ×1
Substrate solution A	6 mL ×1
Substrate solution B	6 mL ×1
Wash buffer concentrate (30×)	20 mL ×1
Biotinylated human IL-21 antibody	1 mL ×1
User instruction	1
Plate sealer	2
Zipper bag	1

ELISA = enzyme-linked immunosorbent assay; HRP = horseradish peroxidase; IL = interleukin; NS = no statistical significance; S = statistical significance (p≤0.05).

Statistical analysis

Statistical Package for the Social Sciences (SPSS) for Windows (Version 22.0, Released 2013; Armonk, NY: IBM Corp.) was used for providing statistical analysis.

The data was presented as mean ± standard deviation (SD). Using SPSS version 20.0 software, the following statistical tests were used to analyze the parameters.

1. The Kruskal-Wallis test was used to do intergroup analysis for the following variables: serum, GCF IL-21, MGI, PI, PPD, CAL, age, and gender.
2. In group A, the relationship between age and gender and serum and GCF IL-21 levels was assessed. In group B, there was a correlation between the levels of serum and GCF IL-21 and the clinical indices MGI, PI, and BI, as well as age and gender. In group C, the levels of serum and GCF IL-21 were correlated with age, gender, and clinical indices and parameters, such as MGI, PI, BI, PPD, and CAL. The Spearman correlation coefficient test was used to perform all of these group-specific correlations.

Results

Of the 15 individuals in group A, 9 (60%) were male and 6 (40%) were female; of the 15 patients in group B, 8 (55%) were male and 7 (45%) were female; of the 15 patients in group C, 7 (45%) were male and 8 (55%) were female (Tab. 2). According to Table 2, the participants in groups A, B, and C had mean ages of 34.07 ± 2.99, 33.93 ± 2.79, and 34.67 ± 2.35, respectively.

Clinical attachment loss

Mean CAL was 0.00 mm in groups A and B and 4.55 ± 0.864 mm (Fig. 6) in group C. There was a statistically significant difference of CAL values in group C vs. other groups (Tab. 2).

Discussion

Periodontitis is the sixth most common disease in humans, affecting 740 million people worldwide. Periodontitis is a bacterially induced chronic tissue destructive inflammation of the teeth. This periodontal microbiota causes the release of proinflammatory mediators both locally and systemically. As the paradigm of chronic infection in dental pathology, periodontal disease shares several pathogenic pathways with cardiovascular diseases as a result of the low-grade state of systemic inflammation posed by periodontitis (20).

Early diagnosis is essential since disease progression is unpredictable and irreversible. The diagnostic and prognostic efficacy of traditional clinical diagnostic parameters, such as bleeding on probing, PPD, and CAL, is limited (21). Therefore, developments in the diagnosis of periodontal disease are moving in the direction of techniques that allow periodontal risk to be recognized and measured using objective metrics like biomarkers. Diagnostic evidence of the infection/host immune response axis can be obtained through site-specific examination of GCF and subject-based data based on serum assays (6).

Although there is an array of evidence and research about biomarkers for periodontal disease in the literature, appropriate molecular indicators of destruction of both soft and hard tissues that can take the place of clinical gold standards are still missing. However, scientists have been aggressively looking for clear indicators of periodontitis (22).

IL-21 is a pleiotropic cytokine that influences immunological responses. IL-21 promotes the growth of Th17 cells, which contribute significantly to periodontitis. In addition to periodontitis, it has been linked to inflammatory disorders such as rheumatoid arthritis and systemic lupus erythematosus (23). The available information on the connection of IL-21 with chronic periodontitis is limited. This study aims to assess IL-21 levels in serum and GCF of chronic periodontitis patients, correlate them with clinical indicators, and compare them to levels in gingivitis patients and healthy people. IL-21 increases the host’s immunological response and local inflammation. This study suggests that IL-21 plays a significant role in the etiology of periodontal disease (24).

In this investigation, group C had significantly higher mean serum IL-21 concentrations (139.3 ± 113.5 ng/mL) than group A (44.866 ± 8.9 ng/mL), but not group B (87.33 ± 10.1 ng/mL). Lokhande et al (25) found that the periodontitis group had considerably higher serum levels of IL-21 (497.78 ± 297.06) compared to the healthy group (65.34 ± 42.66), which is in accordance with current investigation, where serum levels showed no statistically significant difference between groups B and C.

The current study is the first of its type to measure GCF levels of IL-21 in distinct degrees of periodontal inflammation, including gingivitis and periodontitis, when compared to a healthy cohort. In this investigation, groups C and B had considerably higher mean GCF concentrations of IL-21 (66.400 ± 7.82 and 66.400 ± 9.47 ng/mL, respectively) compared to group A (21.933±9.1 ng/mL). There was no significant difference between groups B and C, demonstrating that IL-21 is a generic inflammatory marker.

The current study suggests that IL-21 may play a role in periodontitis by causing inflammation and tissue damage. The current investigation found no significant correlation between illness severity (defined by PPD and CAL) and IL-21 levels in GCF or serum in group C. Dutzan et al (23) found a substantial positive association between GCF levels of IL-21 and PPD and CAL, which contradicts our findings. Lokhande et al (25) found a strong association between PPD and serum IL-21 levels, but no correlation between IL-21 levels in saliva or serum with CAL.

Zhao et al (26) found that nonsurgical periodontal therapy decreased Th17-related cytokines (IL-17, IL-21) in GCF of chronic periodontitis patients. Reduced IL-21 levels suggest a link between periodontal inflammation and disease activity.

Napimoga et al (27) conducted a study on the local levels of IL-21 and IL-21 receptor in gingival tissues of chronic periodontitis and periodontally healthy subjects, as well as their relationship with salivary immunoglobulin A (IgA) levels. The study found that chronic periodontitis patients had considerably greater mean IgA levels and messenger ribonucleic acid (mRNA) levels for IL-21 compared to those who were periodontally healthy, which has consistent results with our study. Elevated IgA levels in the study suggest that IL-21 plays a considerable role in periodontal damage, sometimes even more than periodontal bacteria themselves.

Vahabi et al (28) conducted a study that compared salivary concentrations of IL-17 and IL-18 in patients with chronic periodontitis and healthy individuals. They concluded that an elevated salivary IL-18 level in chronic periodontitis patients has the potential to be a biomarker for periodontal tissue destruction, which was in accordance with our study where we concluded that elevated levels of IL-21 were found in chronic periodontitis individuals.

The current study’s lack of association could be attributed to the small sample size, disease progression, and site specificity. Although clinical parameters indicate cumulative damage, it is impossible to assess the current state of disease activity within tissues. It has been proposed that disease activity is not continuous, but rather consists of active and remission states. If a sample is collected from a remission site that was previously active, the levels may not correlate with clinical criteria. More research is needed with bigger patient samples with different levels of active and inactive areas.

GCF collection in periodontitis locations may not produce comparable amounts due to variations in production and flow rates based on the periodontium’s inflammatory condition. Our study found a strong positive connection between serum levels of IL-21 and BI in group C. Gingival bleeding is an early and clear indicator of gingival irritation. Since IL-21 is an inflammatory biomarker, there may be a positive link between these two traits.

The study found no significant association between age and GCF or serum IL-21 levels across three groups. Age is a variable that requires a bigger sample size for evaluation, and no attributes can be made at this time. Chronic periodontitis is a polymicrobial disease with multiple causes. The host’s susceptibility is determined by interactions between bacteria, the immune system, and the environment, with the host factor playing a significant influence. Estimating host response indicators may aid in monitoring disease progression in advanced gingivitis or early periodontitis patients, as early tissue damage can be challenging to diagnose (29). Estimating host response indicators may aid in assessing disease progression. More cross-sectional and interventional studies with higher sample sizes are needed to determine the efficacy of IL-21 as a biomarker in diagnosis and treatment.

Conclusion

To the best of our knowledge, the current study is the first of its kind to quantify GCF and serum levels of IL-21 in various stages of periodontal inflammation, including gingivitis and periodontitis, when compared to a healthy cohort. Despite the limitations of this study, IL-21 could be a viable biomarker for detecting periodontitis. Further cross-sectional and interventional research with bigger sample sizes are needed to compare the association of IL-21 levels in various types of periodontal disease and consider it as a potential diagnostic biomarker.

DISCLOSURES

Conflict of interest: The authors declare no conflict of interest.

Financial support: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Authors contribution: All authors contributed equally to this manuscript.

Compliance with ethics guidelines: The study protocol is approved by the University Ethics Committee for Human Trials of Sri Sai College of Dental Surgery, Vikarabad, Telangana (Ref no. 615/SSCDS/IRB-E/2017). Written informed consent has been obtained from the participants.

Availability of data and materials: All data generated or analyzed during this study are included in this published article (and its supplementary information files).