Association of elevated IL-6 with poor glycemic control in periodontitis patients

Introduction

Periodontitis is a common inflammatory condition of the periodontium that affects 20–50% of the world’s population.¹ Its etiology is linked to a specific or group of specific periodontal microorganisms, and it causes bone loss and loss of tooth attachment. Porphyromonas gingivalis, Tannerella forsynthesis, Prevotella intermedia are few periodontal pathogens which are known to invade the cells of periodontium, possess the ability to activate the immune cells such as monocyte and macrophage and hence ensue an increased production of inflammatory mediators like Interleukin-6 (IL-6), Tumor necrosis factor alpha (TNF α) and IL-1 in both systemic and local environment.^2,3

These inflammatory mediators are encountered in high levels in gingival crevicular fluid (GCF), saliva, serum and inflamed gingival tissues in patients with periodontitis.^4,5 Increased IL-6 levels can affect the activities of cells such as leukocytes, osteoclasts and osteoblasts, therefore, affecting the remodeling of tissues, there by responsible for alveolar bone resorption in periodontitis patients.^6–9 Further, they are also accountable for mediating MMPs and other collagenolytic enzymes,^10,11 and hence contributing to additional loss of tooth supporting collagenous structures.^12,13

IL-6 and C-reactive protein are demonstrated as major inflammatory indicators of systemic inflammation. Amidst the diverse inflammatory mediators, IL-6 is of particular importance. Being a multifunctional cytokine, it performs a salient role in proliferation and differentiation of hematopoietic stem cells, T and B cells, stimulate hepatic CRP in liver, also has it influences on nerve cells, hepatocytes, keratinocytes, renal mesangial cells, megakaryocytes and myeloma/plasmacytoma cells to name a few. As a result, IL-6 plays a significant part in how the body reacts to infection, inflammation, and tissue damage.¹⁴

Meanwhile, studies have documented the involvement of deregulation of IL-6 gene expression in the pathogenesis of polyclonal and monoclonal B cells abnormalities, namely rheumatoid arthritis and multiple myeloma. Additionally, researchers have also observed higher circulating levels of IL-6 in obesity, diabetes and cardiovascular diseases amongst others.¹⁵ Furthermore, records do suggest its role in glucose and lipid metabolism, thereby¹⁶ increase in circulating IL-6 levels have shown to aggravate the glycemic status by enhancing the insulin resistance.¹⁷

Literature suggests that, on the establishment of periodontal disease, complications in diabetes control have been recorded. Moreover, disturbance in the homeostasis with increase in severity of micro vascular and macrovascular components have also been described.¹⁸

Type 2 Diabetes, a chronic public health menace, associated with economic burden is a global concern. Studies estimate that more than 80% of world diabetic population may be concentrated in developing countries and 60% and more so in Asian countries.¹⁹ Diabetes might be a potential epidemic in India with an upsurge in cases recorded across the states and the different challenges being reported within its large population.²⁰ According to an epidemiological survey, Nanditha et al. observed that over a period of 10 years (2006-2016) in Tamil Nadu, South India, there was an increase in the prevalence of diabetes and prediabetes within the population studied.²¹ Blas et al. states that 95% of Indian population are affected by periodontitis.²² While the prevalence of 26.2% of mild to moderate and 19% of severe periodontitis, was observed in a systematic review conducted among the Indian adults with highest among urban population. The authors do suggest that nearly half of the Indian population may have some form of periodontal condition.²³ Balaji et al. reported a 42.3% prevalence of periodontal diseases in a population of 1000 adults screened for periodontitis in Tamil Nadu, India, and concluded the existence of a definitive inflammatory burden within the population.²⁴

The alarming escalation in the demographics on diabetes and periodontitis and the speculative molecular relationship necessitates unraveling the association and the possible influencing factors shared between them. Therefore, the present study aims to quantify and compare the GCF levels of IL-6 in periodontitis patients between diabetic and non-diabetic group from South Indian population to determine the association between periodontitis and diabetes. Further, the study also aims to analyze IL-6 levels in patients with poor glycemic control (HBA1c) thereby evaluating its role in the progression periodontal destruction.

Methods

The Institutional Review Board (IRB) and ethical committee of the SRM Dental College, Ramapuram, Chennai (SRMU/M&HS/SRMDC/2011/M. D. S. PG Student/201) approved the procedures for conducting the current study. A sample size of 60 was chosen (for a 95% confidence interval, P = 0.01) based on the statistician’s recommendations. The study recruited periodontology outpatients from SRM Dental College and SRM General Hospital in Ramapuram, Chennai. Participants were informed of the study’s purpose and gave written consent.

Sixty participants within the age group between 30-70 years old with chronic periodontitis (patient has probing depth ≥ 4 mm) with minimum dentition of 24 teeth with or without diabetes mellitus were included. Patients with systemic condition other than diabetes mellitus, pregnant and lactating women, patients with other chronic inflammatory diseases, cancer patients, rheumatoid arthritis, smokers and alcoholics, acute oral conditions, sepsis, excessive obesity and patients who underwent periodontal and antibiotic therapy six months prior to the study were excluded.

Clinical examination was carried out using a mouth mirror, graduated Williams periodontal probe, sickle shaped explorer and CPITN probe. Oral hygiene status was assessed by Plaque Index (PI) and Oral Hygiene Index (OHI). Periodontitis was confirmed with Community Periodontal Index and Treatment Needs (CPITN) index. The chronic periodontitis participants were then grouped into Group 1 comprising of 30 diabetic subjects with positive confirmation of HBA1c reports who were under oral diabetes agents and Group 2 of 30 non-diabetic subjects who served as controls.

Participants selected for the study were seated comfortably in an upright position on the dental chair with proper illumination. The site with the maximum probing depth was selected for sampling. The supragingival plaque was gently removed after being gently dried and carefully isolated with cotton rolls. Crevicular fluid was obtained by placing 1-5 microliter calibrated volumetric micro-capillary pipette (Sigma-Aldrich chemical company, Bangalore, India) at the gingival margin as per Cimsoni method. Samples of GCF if contaminated by blood or saliva were discarded. The samples later were drained into Eppendorf tubes and stored at -70 degree celsius until further analysis.

Later, the samples were assayed for IL-6 concentration using krishgen human IL-6 ELISA kit (Krishgen Corporation, Mumbai, India- Catalog No. – KB1068) as per manufacturer’s instructions in the central research laboratory of Sri Ramachandra Medical College and Research Institute, Chennai, India. Intensity of measurable signal produced by antibody-target complex and substrate solution were read using ELISA Reader.

The data was recorded, tabulated and interpreted for statistical analysis using SPSS, Microsoft Word and Excel programme. Descriptive and inferential statistics such as mean, standard deviation, central tendency, ANOVA, Pearson’s chi-square (χ²) test, student T-TEST were used.

Results

On the demographics front, 27 females and 33 males with a mean age of 49.2 ± 12.7 years comprised the total 60 study participants. While diabetic group included 18 males & 12 females, non-diabetic group consisted of 15 males and 15 females.

Patient with CPITN score of 3 (21 Patients in both the groups) and 4(9 patients in both the groups) with fair (28 diabetic patients with Fair OHI, 29 non-diabetic patients with Fair OHI) to poor (2 diabetic patients with poor OHI, 1 non-diabetic patients with poor OHI) Oral Hygiene Index (OHI), Plaque index (PI) (patient with fair PI were 29 patients in both the groups, patient with poor PI were 1 patients in both the groups) status were employed to confirm chronic periodontitis among the participants and also were considered to evaluate the difference between the diabetic and non-diabetic groups.

In diabetic group with HBA1c values below 6.8% were 17participantsand HBA1c 6.8-7.7% were 6participants and above 7.7% of HBA1c where 7 participants were included in the study.

On evaluating the immunoassay reading, group 1 samples expressed a range of 4.4 Pg/μl to 7.0 Pg/μl of IL-6 levels with a mean value of 5.8 Pg/μl. The IL-6 expression in group 2 comprised of the non-diabetic group ranged from 1.5 Pg/μl to 4.8 Pg/μl with the mean value of 3.2 Pg/μl (Table 1).

On comparing the mean IL-6 values using Student-T test, an increased expression of IL-6 was evident in the diabetic group than the non-diabetic group. Further, the elevated values were statistically significant in diabetic group with p value <0.001 (Table 2).

In diabetic group, the patients mean IL-6 levels were 6.7 Pg/μl for patients with poor plaque and OHI values while 5.7 Pg/μl for fair plaque and OHI values. No statistical significance was observed on comparison of IL-6 values between patients with poor PI and fair PI values and poor OHI and fair OHI using student T test. Mean IL-6 levels were 5.7 Pg/μl in patients with CPITN score 3 and 5.8 Pg/μl with CPITN score 4 with no statistical significance on comparison.

In non-diabetic group, the patients mean IL-6 levels were 3.9 Pg/μl for patients with poor plaque and OHI values while 3.2 Pg/μl for fair plaque and OHI values. No statistical significance was observed on comparison of IL-6 values between patients with poor PI and fair PI values and poor OHI and fair OHI using student T test. Mean IL-6 levels was 3.1 Pg/μl in patients with CPITN score 3 and 3.2 Pg/μl with CPITN score 4 with no statistical significance on comparison (Table 3).

The analysis was further extended on the HBA₁c values and the expression of IL-6 in the diabetic group. The mean IL-6 values in participants with HBA₁c below 6.8% was 5.1 Pg/μl, HBA1c 6.8-7.7% was 6.3 Pg/μl and above 7.7% of HBA₁c was 6.8 Pg/μl. On intra-group comparison of IL-6 values among the diabetic group with HBA₁c reading above 7.7% and below 6.8% showed a statistically significant increase of IL-6 with the p value < 0.001 (Table 4).

Therefore, the above data is suggestive of an increased expression of IL-6 levels in the GCF of diabetic group when compared to non-diabetic group. Further, a significant raise in the expression of IL-6 values were elicited in participants with poor glycemic control as represented by correlating HBA₁c values with the IL-6 expression.

Discussion

Periodontal disease and diabetes mellitus are both chronic, very common diseases that have a lot in common pathobiologically. Obesity and insulin resistance are two related antecedent states that may be quite important in these dynamics. Recent discoveries and studies have revealed the role of inflammation as a key component in this connection. Diabetes clearly raises the risk of periodontal diseases, and numerous examples of biologically plausible reasons have been found. The effects of periodontal disorders on the glycemic management of diabetes and the processes by which this occurs are less apparent. In a manner comparable to obesity, periodontal disorders may initiate or spread insulin resistance, making glycemic control more difficult.

In diabetes, the high affinity cell surface receptor RAGE on monocytes and macrophages interacts with the synthesis of AGES to cause the release of numerous inflammatory mediators. Diabetes alters how immune cells, such as neutrophils, monocytes, and macrophages, function.²⁵ As poor neutrophil adherence, chemotaxis, and phagocytosis frequently prevent bacteria from being killed in the periodontal pocket, this dramatically increases the risk of periodontal damage.²⁶ Despite the fact that diabetes frequently impairs neutrophil function, the monocyte/macrophage cell line may show up-regulation in response to bacterial antigens. The production of proinflammatory cytokines and mediators such TNF-, IL-6, and IL-1 is dramatically elevated as a result of monocytes’ and macrophages’ hyper reactivity.²⁷

Treatment for periodontitis will therefore lessen not only local inflammation but also the systemic and local availability of these mediators, preventing the periodontium’s cells and their functions from being adversely affected.^28,29 According to Miller,³⁰ between pre-treatment baseline values and 2- to 3-month post-treatment values, periodontal therapy was linked to a 10% drop in HbA1c levels. Consequently, the objective of the current investigation was to compare diabetic patients with chronic periodontitis to non-diabetic patients in terms of the activity of the proinflammatory cytokine IL-6 in GCF. Moreover, in the diabetic group, there was a correlation between IL-6 expression and HBA1C levels.

In the current investigation, patients in group 1 (diabetic periodontitis patients) had statistically higher levels of IL-6 (5.8 pg/l) than patients in group 2 (non-diabetic periodontitis patients; p0.001). These findings are in line with earlier research by Duarte PM et al.³¹ who found that GCF IL-6 levels were significantly higher in diabetic periodontitis patients as a result of long-term poor glycemic control, which increases the formation of AGES, which then couples with RAGE present on monocytes, macrophages, endothelial cells, and other cells, increasing the secretion of various inflammatory cytokines including IL-6.

There was a statistically significant link between the level of IL-6 and the HBA1C value in the current study’s diabetic patients (good control = 5.18, fair control = 6.38, poor control = 6.89). Those with poorly managed diabetes who had an increased IL-6 level had a p value of less than 0.001. The findings support Débora C. Rodrigues et al. investigation’s which found that persistent AGES build up led to monocyte/macrophage activation and enhanced IL-6 secretion³² and thus elevated IL-6 levels.

According to a study by Mark C. Genovese et al., IL-6 Receptor blockage with sarilumab was linked to a decrease in HbA1c in people with rheumatoid arthritis and diabetes. Hence, these findings imply that IL-6 significantly contributes to the deterioration of glycemic control.³³ Clinical and metabolic outcomes following conventional periodontal therapy were examined between patients with and without type 2 diabetes mellitus by Faria-Almeida R et al. After receiving basic non-surgical periodontal treatment, both patient groups displayed a clinical improvement. After periodontal therapy, the diabetic patients had better metabolic control (lower HBA1c) at 3 and 6 months.³⁴ So, in the future, inhibiting the IL-6 receptor combined with periodontal therapy can lower IL-6 activity and thereby minimise the inflammatory reactions in both illnesses, as well as the complications associated with treating patients with diabetes and periodontitis. According to a study by B Kurtis et al., the expression of IL-6 in healthy subjects was 0.62 Pg/μl and 1.31 Pg/μl in adult periodontitis. However, when compared to the results of the current study, it was found that the non-diabetic group with periodontitis had an increased expression of IL-6 (3.24 Pg/μl).¹⁷

Reverse transcription polymerase chain reaction (RT-PCR) and ELISA tests, as well as several investigations by many other authors, revealed that individuals with periodontitis had higher levels of IL-6 mRNA and protein expression.^35,36 Hence, the results of the current study’s IL-6 levels in 30 patients with periodontitis who did not have diabetes, as measured by ELISA, were consistent with findings from earlier research that suggested periodontitis might spread insulin resistance by upregulating IL-6 expression. As a result of their reciprocal interaction, diabetes and periodontitis both have an impact on interlukin-6 levels.^37–39 IL-6 levels in GCF of teeth with periodontitis may be impacted by periodontal treatment, such as periodontal scaling and root planing, according to a study by Hua Xi Kou et al. from 2001.⁴⁰ Also, in a study conducted in 2003 by Nishimura F ET AL, serum levels were evaluated both before and after scaling and root planning. According to the findings, there was a shift in blood IL-6 levels between pre- and post-treatment.⁴¹ As a result, periodontal therapy may lower inflammatory mediators like IL-6 in both the blood and the GCF, according to the findings of the Nishimura F ET AL study and the Hua Xi Kou et al study. In order to reduce the additional systemic inflammatory burden and potentially avert diabetes, cardiovascular disease, and other systemic morbidities, Mattson JS et al. 2001 advises that efforts to battle diabetes sequelae, particularly periodontitis and gingivitis.²⁸

In terms of sex-related differences in the prevalence of periodontitis and diabetes, the current study’s data show that men had higher rates of both conditions than women did, which is consistent with findings from studies by Anna Nordström et al. on the prevalence of diabetes and Ashish Jain et al. on sex-related differences in the prevalence of periodontitis.^42,43

This study’s findings on PI, CPITN, and OHI scores were in agreement with a study by Roberto Del Giudice et al.⁴⁴ on their favourable correlation with IL-6 levels. By comparing the levels of IL-6 in GCF between diabetic subjects and non-diabetic (healthy subjects) without periodontitis as well as between the levels of IL-6 in GCF before and after periodontal management, more research is needed to understand the function and potential mechanism of IL-6 in the pathogenesis of diabetes and periodontitis. The study’s limitation is the relatively small sample size, which reduces the statistical analysis’s power.

Conclusion

The results of the present study concluded that the IL-6 activity was enhanced more in diabetic subjects than non-diabetic subjects with periodontitis. Among the diabetic group the subjects with good diabetic control have shown less activity of IL-6 in GCF than poor controlled diabetic subjects. So, diabetes may play a major role in the activity of proinflammatory cytokine IL-6 in the progression of periodontitis.