Serum IL‐36 cytokines levels in type 2 diabetes mellitus patients and their association with obesity, insulin resistance, and inflammation

Abstract Background The interleukin (IL)‐36 cytokines include IL‐36α, IL‐36β, IL‐36γ, and IL‐36Ra. Little was known about their roles in type 2 diabetes mellitus (T2DM). Methods The study included 40 T2DM patients and 42 healthy control subjects. The anthropometric and biochemical measurements were performed using automatic biochemical analyzer, high‐performance liquid chromatography, and electrochemiluminescence immunoassay. Circulating IL‐36α, IL‐36γ, IL‐36Ra, and IL‐17 levels were determined by enzyme‐linked immunosorbent assay. Results Serum IL‐36α, IL‐36γ, and IL‐17 levels in T2DM patients were significantly higher than those in controls, whereas serum IL‐36Ra levels in T2DM patients were lower. Correlation analysis showed that serum IL‐36α was positively correlated with high sensitivity C‐reactive protein. Serum IL‐36α was negatively correlated with IL‐36Ra. Serum IL‐17 was negatively correlated with low‐density lipoprotein cholesterol. Conclusions This study demonstrated that T2DM patients displayed increased IL‐36α and IL‐36γ expression and decreased IL‐36Ra expression. Moreover, the inflammatory cytokine levels were directly proportional to the inflammation and blood lipid levels. Our results suggest that IL‐36 cytokines may be a new target for the diagnosis or treatment of T2DM.

IL-36Ra has a similar effect to IL-1Ra and inhibits the activation of these pathways by binding to IL-36R. 3 Recent studies found that IL-36 cytokines play a key role in obesity-related metabolic disorders, low-grade inflammation, and IR.
Several studies showed that IL-36 cytokines have effects on psoriasis, arthritis, and systemic lupus erythematosus, but there are few reports on the IL-36 cytokines in T2DM. 4,5 To explore the clinical relevance of IL-36 cytokines in T2DM, we determined the serum IL-36 cytokines concentrations of normal subjects and T2DM patients, and analyzed their relationships with IR, anthropometry, and metabolic parameters. Additionally, 42 healthy individuals with age and gender matching undergoing regular physical examination were recruited as a control group. Exclusion criteria included T1DM, acute or chronic complications, hypertension, or organ failure. The study design was approved by the Ethics Committee of Ningbo University School of Medicine.

| Study design and participants
All subjects gave their informed consent.

| Anthropometric and biochemical measurements
Standard methods for anthropometric measurements, including weight, height, and calculated body mass index (BMI), were applied to all subjects. Appointed nurses used a mercury sphygmomanometer to determine systolic blood pressure (SBP) and diastolic blood pressure (DBP). The subjects were fasted overnight and blood samples collected from 8 to 9 am the next day. The blood samples were kept at room temperature for 2 hours, centrifuged at 1000 g for 20 minutes, and the supernatant was taken aseptically and stored at −80°C.

| Measurements of IL-36 and related cytokines
Enzyme-linked immunosorbent assay was used to determine the serum IL-36α, IL-36γ, IL-36Ra, and IL-17 levels. The kits were purchased from Cusabio Biotech Co., Ltd. The experimental procedures followed the manufacturer's instructions. All samples were assayed in duplicate and random order.

| Characteristics of study participants
*, ****Statistically significant between the T2DM and control subjects.
DBP, or gender distribution between the T2DM patients and the healthy controls (P > .05). Conversely, the BMI and SBP of the T2DM patients were statistically significantly higher than those of the control subjects (P < .05 and P < .0001, respectively).

| The clinical biochemical parameters of the subjects
The GHb, CRP, LDL-C, FBG, FINS, and HOMA-IR levels of patients in the T2DM patients were statistically significantly higher than those of the control subjects (Table 2). Conversely, the HDL-C, TC, and TG contents were not statistically different between the two groups (P > .05).

| Serum IL-36 cytokines and IL-17 levels
We also measured the serum IL-36 cytokines and IL-17 levels. The IL-36α, IL-36γ, and IL-17 levels in the T2DM patients were significantly higher than those in the healthy individuals (P < .0001, Figure 1A,B; P < .001, Figure 1C). Conversely, the serum IL-36Ra level of T2DM patients was significantly lower than of the control subjects (P < .0001, Figure 1D).

| Correlation of serum IL-36 cytokines with inflammatory markers and clinical data
Pearson correlation analysis of serum IL-36 cytokines levels and clinical biochemical parameters in T2DM patients showed that IL-36α was positively correlated with hsCRP (r = .3376, P < .05, Figure 2A).

| D ISCUSS I ON
This study confirmed that T2DM in patients is associated with obesity, inflammation, and IR. In recent years, IL-36α, IL-36β, and IL-36γ up-regulation in many diseases has attracted the interest of numerous researchers. These cytokines play an important role in inflammatory diseases that occur in the skin, joints, blood vessels, heart, and nerves. 7 The current research is focused on IL-36 in psoriasis, 6 inflammatory arthritis, 8 systemic lupus erythematosus, 9 inflammatory bowel disease, including ulcerative colitis and Crohn's disease. 10 However, as far as we know, the role of IL-36 cytokines in T2DM is rarely reported.
In this stduy, we determined the serum IL-36α and IL-36γ levels in T2DM patients and showed that they were significantly higher than those in normal subjects, which indicates that IL-36α and IL-36γ play a role in diabetes progression. Increased plasma hsCRP level is a more sensitive indicator of inflammation, which normally suggests disease progression. 11 We found that hsCRP of T2DM patients was significantly higher than that of normal subjects and that IL-36α was positively correlated with hsCRP, which indicates that IL-36α is related to the inflammation progression of T2DM. The BMI index of T2DM patients in this study group was higher than that of normal subjects, which indicates that T2DM patients had a tendency to be obese. Increased fat tissue is accompanied by inflammatory reactions in obese patients, including the infiltration of macrophages and other immune cells and increased secretion of adipokines, IL-6, MCP-1, TNF-α, and leptin. 12 Inflammatory cytokines interact with the endocrine and immune systems, causing structural changes and dysfunction of pancreatic islet β cells and IR, which ultimately leads to the occurrence of T2DM. 13 However, our data did not show a correlation between IL-36α or IL-36γ and FINS or HOMA-IR.

TA B L E 2 Clinical biochemical parameters
effect. 14 IL-36Ra can inhibit IL-22 and IL-17 production by binding to cell-surface IL-36R. Our results showed reduced IL-36Ra in T2DM patients. Therefore, IL-36Ra as an antagonist for IL-36α, β, and γ may have an antagonistic effect on the inflammatory response of T2DM patients. Our results showed that IL-36α is negatively correlated with IL-36Ra, suggesting that they may play an opposite role in T2DM. Additionally, the IL-17 level was higher in T2DM patients than in normal subjects. However, the IL-17 level was negatively correlated with LDL, which indicates that IL-17 was inversely associated with hyperlipidemia and obesity. We found that there was no correlation between IL-36α and IL-17 levels. Maybe our sample size was small and thus further research is needed. Additionally, this study did not show any correlation between IL-36Ra and HOMA-IR.
In conclusion, our study found that the serum levels of the inflammatory cytokines IL-36α and IL-36γ of T2DM patients were increased, whereas that of IL-36Ra was decreased. Inflammatory