Investigating the association between interleukin 18 and type 2 diabetes mellitus in the Iraqi population

Extensive research has been conducted for over a decade on the association between obesity, inflammation, and type 2 diabetes mellitus (T2DM). The primary objective of this study is to investigate the correlation between levels of IL-18 and T2DM among different age groups. Ninety blood samples were collected from three groups: old diabetic patients, young diabetic patients and healthy control group. Thirteen blood samples for each group and both sexes had been enrolled in this research. The HbA1c test and interleukin 18 ELISA test were done for all these samples. The results were statistically analyzed by using: One-way ANOVA, Duncan multiple range comparison, chi-square, Pearson correlation test and ROC test. There were significant differences between patients and healthy groups regarding HbA1c and no significant differences between groups in IL18. The results of the ROC test confirmed that there wasn’t any significant relationship between IL18 and T2DM. In conclusion, our study indicates that the impact of IL-18 in the evolution of type 2 diabetes mellitus (T2DM) is not significant and it may not serve as a reliable predictor of T2DM.


Introduction
Type 2 diabetes mellitus (T2DM) is recognized as an endocrine and metabolic disorder with severe effects on human health.It is characterized by chronic hyperglycemia and disorders of carbohydrate, lipid, and protein metabolism due to defects in insulin secretion, insulin action, dysfunction, and organ failure [1,2].Patients with diabetes are predisposed to cardiovascular, cerebrovascular, and peripheral vascular diseases [3].The majority of diagnosed cases of diabetes mellitus (DM) are type 2 diabetes mellitus (T2DM).It is a chronic low-grade inflammation with changing levels of inflammatory markers [4].Cytokines are inflammatory indicators that are extracellular polypeptides or glycoproteins.They are produced by immune cells, primarily T-cells, neutrophils and macrophages as signaling molecules [3].Their concentrations rise in disease situations and cause inflammation-related disorders.Inflammatory cytokines like TNF-α are improperly regulated in T2DM (adult human population and animal models) [6].Interleukins are a diverse group of cytokines that allow leukocytes to communicate with each other by adhering to their specific receptors [7].Interleukin 18 is known as an "IFN-inducing factor", a member of the IL-1 family.It promotes TH1 and TH2 cell responses and increases the toxicity of natural killer (NK) cells [8].IL-18 represents an important factor that regulates innate and adaptive immune responses [9].It's probably one of the elements that participate in the development of autoimmune diseases.It's produced by monocytes, dendritic cells, and activated macrophages [7].Changes in the ratio of IL-18 to IL-18-binding protein impair its function, resulting in disease.In addition, it plays a role in the pathogenesis of autoimmune and chronic inflammatory diseases [10].Similar to IL-6 and TNF, high levels of IL-18 in the blood are indicative of a metabolic syndrome [11].These interleukins play a significant role in the development of numerous metabolic diseases, including obesity, cardiovascular disease and type 2 diabetes [12,13].Therefore, this study aimed to explore the role of IL-18 in T2DM and reveal its relationship with gender, age, and HbA1c, among the Iraqi population.

Design study and subjects
Ninety peripheral venous blood samples had been collected and divided into three study groups (30 blood samples for each group); young T2DM patients, old T2DM patients, and a healthy control group.The ages of these groups were (10-35 y), (40-80 y) and (10-80 y) respectively.Both males and females were included in this study.A random selection of patients from Baghdad's private medical clinics was made, while the healthy control group was chosen from apparently healthy volunteered people.All participants agreed to participate in this research after a full understanding of the purpose and the required tests.Physicians followed the American Diabetes criteria for diagnosis of the patients, fasting blood glucose above 126 mg/dl which equals 8 mmol/l.In the present study, we chose the T2DM patients who had HbA1c ≥ 8 percent.Pregnancy, liver disease, alcoholism, chronic renal disease, thyroid disorders, and any endocrine disease were excluded from our study.

Samples Collection
Five milliliters of peripheral venous blood samples were withdrawn from each participant.They were divided into two tubes according to the research requirements.The first tube was the EDTA tube, which contained 2 ml of blood, stored at -20 Cº till it was used in the HbA1c test.The remaining blood was added into a clot activator gel tube, kept at room temperature 20 to 25 °C for a few minutes, and centrifugated for 15 minutes at 3000 rpm/minute to collect the upper serum of each sample.The serum was stored at -20 to be used in the immunological test for measuring IL-18 levels.

Biochemical test
The HbA1c test was conducted using standard clinical procedures following the manufacturing instructions of the Ichroma TM HbA1c Glycosylated hemoglobin kit (Biotek med, Spain).

Immunological test
It was conducted following the manufacturer's instructions of Human Interleukin 18 (IL-18) ELISA Kit (SunLong Biotech: SL0980Hu -China) to measure the concentration of IL-18 in serum samples, by using an enzyme-linked immunosorbent assay (ELISA) reagent.Duplicate measurements were accomplished on 10% of the serum samples.

Statistical analysis
The data were statistically analyzed with SPSS 26 (SPSS Inc., Chicago, USA).The results were presented as the mean ± SD.The p-value is used to indicate statistical significance.When p≤0.05* or p≤0.0001** the data are considered significant.One-way ANOVA and Chi-square were used to analyze the statistical significance of the mean differences between the three groups.The multiple range comparisons (DMRTs) test developed by Duncan assigned distinct letters to statistically distinct means.A ROC curve (receiver operating characteristic curve) was performed to assess the specificity and sensitivity of gene expression and to determine if it plays a role in the disease.

Ethical approval
This study gained the approval of the local ethical committee of the Genetic Engineering and Biotechnology Institute for postgraduate studies-University of Baghdad-Iraq, Ref: EC/1310-B on 15/5/2022.Formal informed consent was obtained from the participants.

Age distribution result
Ninety blood samples were collected in this study, which was divided into three groups: young T2DM patients, old T2DM patients and healthy control group.The distribution of age categories for each group is illustrated in (Table 1).The current study tried to include a wide range of age categories of the T2DM patients, therefore two patient groups were enrolled (Figure 1).

Sex distribution analysis results
The two genders were included in this study, and the number of participants for each gender was statistically analyzed (Table 2) and (Figure 2).b-The HbA1c level for the old T2DM patient.
c-The HbA1c level for the healthy control group.
The young and old diabetic groups had HbA1c means (9.5103± 0.99) and (10.4867± 1.83) respectively, while the mean (5.0693± 0.35) was recorded for the healthy control group.

Results of IL-18 according to groups
The IL-18 levels were calculated according to the manufacturing instructions of the utilized kit, the results were statically analyzed as in (Table 3) and (Figure 4).

Results of IL-18 according to sex distribution
The results of IL-18 were statically analyzed according to sex distribution among the three tested groups.There were significant differences in the means of IL-18 between males and females among the three tested groups, p-value <0.05, as in (Table 4).The females had higher IL-18 Levels in comparison with males.

Results of IL-18 Receiver Operating Characteristic Curve
Using the Receiver Operating Characteristics (ROC) test, the relationship between IL-18 serum concentration level and T2DM disease has been evaluated.The results revealed that the area under the curve (AUC) was 0.410, which is low compared to the accepted values.Our findings indicate that the hypothesis of persistence correlation between IL-18 and T2DM fails (p-value > 0.05).The optimal cutoff for IL-18 was 13.514 pg/mL, with sensitivity and specificity percentages of 43.3% and 53.3%, respectively (Table 5) and (Figure 5).

Results of correlation analysis between age, HbA1c and IL-18
The Pearson correlation test was conducted to explore the relationship between IL-18 and each age and HbA1c, as illustrated in (Table 6).The correlation analysis between IL-18 and each age and HbA1c revealed the absence of a significant correlation between them, the p-p-value> 0.05 which indicates no significant results.

Discussion
Type two diabetes mellitus is a complex metabolic syndrome that involves insulin resistance and beta-cell dysfunction, leading to glucose buildup and hyperglycemia [14,15].It accounts for 90-95% of diabetic cases, mostly in adults, although it recently has increased in children [16].Numerous studies have emphasized the importance of inflammatory pathways and biomarkers in diabetes and metabolic diseases.
A cytokine is one of these crucial inflammatory markers [17,18].They are frequently found accumulating at sites of inflammation, infection, and trauma; therefore, variations in their concentrations can be used to predict abnormality [19].Interleukins, which are essential cytokine family members were a subject of research for an extended period; researchers have even attempted to correlate their levels with varying glycemic status [20].Previous studies mentioned variations of interleukin roles during inflammation, some of them had a master role, while others had less importance [4].For instance, a previous study on the Egyptian population mentioned that IL-2 (which is an anti-inflammatory cytokine) had a lower level of T2DM than the non-diabetic control subjects.It is considered a dual-natured interleukin that varies depending on the studied population [21].Another study in England indicated no significant association of IL-2 with T2DM, but a significant relationship with T1DM [22].Interleukin-18 (IL-18) is a proinflammatory cytokine that is generated by activated macrophages, epithelial cells, osteoblasts, keratinocytes, and cancer cells, among other cytokines implicated in inflammation.There is still a great deal to learn about IL-18's function [9].A previous study in Pisa mentioned that IL-18 had been recorded by its correlation with T2DM [23].In another Research on diabetic nephropathy, they confirmed that inflammation affects the kidney and that IL-18 has a role in T2DM progress to diabetic nephropathy [24].These results disagreed with ours because the ROC test revealed that IL-18 had no significant correlation with T2DM disease.This finding may be interpreted as population differences [21].The structure and biological activity of a particular cytokine may be altered by cytokine gene polymorphisms, which can result in an increase or reduction in cytokine output.People who inherit SNPs may be susceptible to certain diseases or less resistant to them [25].Various studies conducted on a diverse group of people revealed an extensive diversity of results.Differences in the duration of exposure to hyperglycemia, population size, age, epigenetic factors, or ethnicity might have all contributed to the controversy [26].
Many factors such as a healthy diet and practical exercise were recorded as important factors that may influence the IL-18 level [27] [28].Racial differences may also affect the IL-18 levels among different populations and could interpret the conflict results in data between different studies, such as racial heterogeneity in promoter regions of the IL-18 gene found in the Brazilian population, which recorded conflict data in a comparison with other studies in other countries [29].Another study presented data confirmed that the promoter polymorphisms of the IL-18 gene represent a major determinant of IL-18 production, despite the presence of multiple factors that affect the IL-18 gene expression [30].

Conclusions
The interleukin 18 didn't show a significant correlation with T2DM, females had higher serum levels in comparison with males.Genetic analysis for IL-18 should be conducted to discover the genetic cause behind these results.Other interleukins may be tested to explore their possible correlation with this disease.

Figure 1 .
Figure 1.Bar chart of age group categories.

Figure 2 .
Figure 2. Bar chart of gender distribution for the three groups

Figure 3 .
Figure 3. a-The HbA1c level for the young T2DM patient.

Figure 4 .
Figure 4. Means of the IL-18 among the three groups.
A S, El-Kafoury A A, Haroun M A and Embaby A M 2014 Genetic association between interleukin IL-18-137G/C and IL-18-607 C/A polymorphisms and type 1 diabetes in Egyptian population Iraqi J. Biotechnol.13 47-54 [2] Ridha R H A and Kandala N J 2016 Association of Genetic Polymorphisms in a Sample of Iraqi Patients with Type2 Diabetes Mellitus Iraqi J. Biotechnol.15 78-84 [3] Zhang J, Liu J and Qin X 2018 Advances in early biomarkers of diabetic nephropathy Rev. Assoc.Med.Bras.64 85-92

Table 1 .
Age distribution categories for the three tested groups

Table 2 .
Sex distribution among the three groups

Table 3 .
IL-18 levels for the three tested groups

Table 4 .
IL-18 results according to sex distribution.

Table 5 .
Receiver Operating Characteristic curve data for IL18