The Level and Role of Interleukin-17 in Patients of Type 2 Diabetes Mellitus with and without Complications

Background: Type 2 Diabetes Mellitus (T2DM) is the most prevalent metabolic disorder in the world. Recent evidence however suggests that T2DM is not only a disease of metabolism, but also an inflammatory disorder and that inflammation also plays an important role in the pathogenesis of Diabetic complications. Our aim was to study the level of interleukin-17 (IL 17) in Indian populations with T2DM as an inflammatory marker and analyze its role in different diabetic complications. Methods: A total of consecutive 67 patients of T2DM were evaluated for clinical parameters fasting blood glucose (FBG), 2hr-post-prandial blood glucose (PPBG), lipid profile, HbA1c and plasma IL 17. They were divided into three groups—Patients of T2DM without any complications (group A; n = 24), T2DM with acute complications (group B; n = 20), T2DM with chronic complications (group C; n = 23) and compared with 23 healthy controls (group D). Results: Diabetic patients had a higher level of IL 17 as compared to the healthy controls. The level of IL 17 in complicated diabetics was higher than the patients with T2DM without complications. Multiple logistic regression analysis showed positive correlation of IL 17 with Diabetic Retinopathy and Diabetic Neuropathy. IL 17 also showed a positive Pearsons correlation with systolic blood pressure (SBP), diastolic blood pressure (DBP), serum triglycerides (TG), serum total cholesterol (TC), very low density lipoproteins (VLDL), low density lipoproteins (LDL), HbA1c and a negative correlation with HDL. Conclusion: Indian subjects with T2DM with or without complications had higher values of IL 17 as compared to healthy controls. Also diabetic neuropathy and diabetic retinopathy were positively correlated to levels of IL 17. Further levels of IL 17 were positively correlated to hypertension and dyslipidemia indicating a prevaHow to cite this paper: Parhi, A., Das, S., Mahapatra, S., Pradhan, N., Behera, M., Patnaik, B. and Rattan, R. (2019) The Level and Role of Interleukin-17 in Patients of Type 2 Diabetes Mellitus with and without Complications. Journal of Diabetes Mellitus, 9, 176-185. https://doi.org/10.4236/jdm.2019.94017 Received: May 29, 2019 Accepted: November 25, 2019 Published: November 28, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Diabetes Mellitus is a heterogenous chronic metabolic disorder principally characterised by persistent hyperglycemia resulting from defects in insulin secretion, action or both. Recent studies have shown that diabetes is a proinflammatory state [1]. The studies reveal an increased levels of highly sensitive C reactive protein (hs CRP), toll like receptors 2 (TLR 2), TLR 4 and plasminogen activator inhibitor-1 (PAI-1), soluble adhesion molecules, sCD 40 and proinflammatory cytokines interleukin-1β (IL 1β), IL 6, and tumour necrosis factors (TNF-α) in diabetics. Gene profiling has suggested that high glucose exposure to monocytes leads to increased expression of pro-inflammatory cytokines, chemokines and related factors, many of which are regulated by the pro-inflammatory transcription factor nuclear factor κ-B (NF-κB) [2]. In addition to that, Adiponectin, which is anti-inflammatory in nature, is also found to be lower in patients of diabetes mellitus [3]. Its level is also found to be lower in patients of diabetes mellitus with macrovascular and microvascular complications. Diabetes as a proinflammatory state has also been established in our population by Das et al. [4].
Recent studies, suggest that adaptive immune system, especially T lymphocyte, also plays a pivotal role in the pathogenesis of T2DM. One particular type of T lymphocytes named Th17, which is a subset of T helper cells produces IL 17. Recently there has been increasing evidence of the proinflammatory interleukin 17 (IL 17) playing an important role in patients of diabetes mellitus and its complications [5]. IL-17A activates the JAK1, JAK2, PI3K and NF-κB pathways which up-regulate inflammatory gene expression [6]. It also activates MAPK, CEBP cascade which increases proinflammatory gene expression of cytokines like TNFα, IL1, IL6, G-CSF, and MMP which mediate tissue infiltration and destruction. Also recent study described ongoing β-cell destruction by IL1b and TNFα [7]. Also there have been studies regarding increased levels of IL 17 in vitreous humour, and increased Th 17 cells in the renal tissues indicating significant role of IL 17 in the complication of diabetes [8]. Literature in India regarding the role of interleukin 17 in patients of T2DM with and without complications is sparse. Therefore, we decided to evaluate the role of IL 17 in patients of diabetes mellitus with and without complications in our population.

Materials and Methods
It was a hospital based observational study conducted in the Post Graduate De- , Group C constituted patients with chronic complications (N = 23). Healthy controls were put in Group D (n = 23). Because of financial constraints, we could afford only 1 kit for IL-17 estimation (PicoKine ELISA Kit) which has 96 wells. 6 of the wells were used up for determination of the standard curve and the rest was used for our patients, hence the sample size of 90 patients which were divided into 4 groups. All Type 2 DM patients of both gender of age group more than 30 yr, attending out-patient department or admitted in our wards were included in the study. Patients with Type 1 Diabetes, Gestational Diabetes, Secondary Diabetes, known cases of connective tissue disorder and infections were excluded from the study.
DM was diagnosed according to WHO criteria. Blood glucose estimation was done by glucose oxidase method in venous blood. Glycosylated haemoglobin (HbA1c) estimation was done by ion exchange chromatography method. Blood glucose level estimation was done by glucose oxidase method using a standard kit supplied by Acutex Biochemical Pvt Ltd. Lipid profile, liver function tests (LFT), serum urea, creatinine and electrolytes done by autoanalyzer (TBA120 FR, TOSHIBA) using specific kits. Serum total cholesterol (TC) was estimated using Enzokit supplied by Ranbaxy fine chemicals limited. Triglycerides (TG) was estimated using standard kit by Chemelex SA, Barcelona. Low density lipoprotein (LDL) was estimated using standard kit supplied by Agappe diagnostics ltd. High density lipoprotein (HDL) was estimated using a standard kit supplied by Trans Asia Biochemicals. Very low density lipoproteins (VLDL) was estimated by dividing TG/5. Acute complications of T2DM include hyperglycemic hyperosmolar state (HHS), diabetic ketoacidosis (DKA) and hypoglycaemia. Chronic complications of diabetes include microvascular complications: diabetic nephropathy, diabetic neuropathy and retinopathy as well as macrovascular complications: cerebrovascular accident (CVA), coronary artery disease (CAD) and peripheral artery disease (PAD).
Estimation of IL 17: IL 17 was calculated using 1L 17 PicoKine ELISA Kit manufactured by Boster Biological Technology Co., Ltd. (Catalog no-EKO430) Size-96 wells/kit. It was a Sandwich ELISA kit for Quantitative detection of human IL-17 in cell culture supernates, cell lysates. tissue homogenates, serum and plasma (heparin, EDTA. Sample was kept at −20˚C after proper dilution by diluents buffer.

Preparation and Storage of the Sample
Plasma was collected using EDTA as the anticoagulant. It was centrifuged for 15 Human IL-17 concentration of samples was calculated from the standard curves.
Statistical analysis was done using SPSS statistical package version 21.0. Quantitative variables were described as mean ± standard deviation (SD) unless otherwise indicated qualitative variables were described by percentage. Multiple logistic regression analysis with stepwise additions of variables was performed to assess their association with each of the complication studied. For all statistical tests, P value < 0.05 was considered significant.

Observation
The demographic characteristics, blood pressure values of group A, B, C and D comparing the mean difference between groups are shown in Table 1. Table 2 shows the mean values of different biochemical parameters in the form of FBG, 2 Hr PPBG, Urea, Creatinine. Table 3 shows comparison of plasma level of IL17 (in pg/ml) in different groups (normal value < 10 pg/ml). The highest level of IL-17 is seen in diabetics with chronic complications (group C) with a mean value of 39.727 ± 9.96 pg/ml.
The second highest level is seen in DM with acute complications (group B) with a mean level of 20.3865 ± 6.78761 pg/ml. The mean value of serum IL-17 for control group (group D) is within normal limits. Table 3 also shows the mean levels of IL 17 in males and females among different groups. The mean level of IL 17 of females in each group was more than that of male group. But the control group shows that the mean level is more in case of males as compared to females. Among these, IL17 showed statistically significant association with retinopathy (P-value of 0.017). Diabetic neuropathy shows significant association with IL17 (p-value of 0.033).

Discussion
IL-17, which is a pro-inflammatory cytokine has been studied in development of diabetes. In our study, levels of IL 17 (normal level ≤ 10 pg/ml) was found to be increased in T2DM (mean = 25.06 pg/ml) which is higher than in non-diabetic controls (mean = 5.232 ± 2.867 pg/ml) ( Table 3). These findings are consistent with the studies of Chen et al. which showed increase in level of IL-17 in newly diagnosed diabetes than the healthy controls [9]. The level of IL 17 was even more in the group of patients with acute complications (group B) with a mean  [13]. We too found similar results with a higher mean value of IL 17 among our female patients (Table 3) which shows mean value of IL 17 in females as compared to males across all groups (i.e. among .group A, B, C and D).
In multivariate logistic regression analysis it was found that there is positive association of IL 17 with Diabetic Retinopathy with an odds ratio of 1.309 (P = 0.017) ( Table 4). Our study is at par with Wang et al. who found that IL 17 is an independent risk factor for non proliferative retinopathy and that IL 17 A levels significantly increased the risk of NPDR (odds ratio = 1.22; P < 0.05) [14]. Liu et al. in their study comprising 19 patients with proliferative diabetic retinopathy (PDR) and 20 non diabetic controls found increased IL 17 levels in the sera of patients with PDR as compared to controls. Furthermore, increased expression levels of IL 17 were detected in fibrovascular membranes and in peripheral blood mononuclear cells (PBMC) harvested from patients with PDR [15]. However our results are in disagreement with the results of the study by Nadeem et al.
where it was found that IL 17 has a protective role in Diabetic retinopathy [16].
In multivariate logistic regression analysis we found positive association of IL 17 with Diabetic Neuropathy with a significant P value of 0.033 with odds ratio of 1.647 (Table 5). Sandireddy et al. also suggested that IL 17 can sensitize peripheral receptors causing neuropathic pain in patients of diabetic neuropathy [17]. According to Doupis et al., inflammation and microvascular reactivity are needed for development of diabetic neuropathy and suggested that the chronic state of hyperglycemia culminates to oxidative stress which initiates and amplifies neuroinflammation leading to production of proinflammatory cytokines which are responsible for nerve tissue damage and neuropathies [18].
In multi variate regression analysis, nephropathy did not show any significant association with IL 17 (P = 0.13). A shorter duration of diabetes in patients of Diabetic Nephropathy in our case along with the highly prevalent endemic CKD in our region not due to diabetes explains the failure to attain the significant correlation with nephropathy.
As shown in There was a positive correlation between levels of IL 17 and blood HbA1c with a coefficient(r) value of 0.551 indicating that a higher level of IL 17 is indicative of poor glycaemic control. Studies by Dumanovic et al. showed that there is a significant reduction of HbA1c with decreased IL 17 corroborative with our findings [22]. A small sample size was the primary limitation in this study which was due to financial constraints. A large study with many more variables may shed more light on the relationship between IL-17 and diabetes.

Conclusion
The present study was undertaken to study the levels of IL 17 in patients with T2DM with and without acute and chronic complications. It was found that IL 17 was significantly high in patients of T2DM without any complications as compared to controls. By multivariate logistic regression analysis IL 17 has been found to be an independent marker of severity of degree of neuropathy and retinopathy. Further levels of IL 17 were positively correlated to hypertension and dyslipidemia. We suggest an inflammatory state in diabetes with or without complications. Thus IL 17 can act as a marker of inflammation and complications in patients of diabetes mellitus. However, multicentric studies with larger number of patients should be undertaken for better association.