Interleukin‐39 is a novel cytokine associated with type 2 diabetes mellitus and positively correlated with body mass index

Abstract Introduction It is suggested that cytokines play a key role in the pathogenesis of type 2 diabetes mellitus (T2DM). Therefore, this study explored two recently discovered cytokines, interleukin (IL)‐37 (anti‐inflammatory) and IL‐39 (pro‐inflammatory), in T2DM due to limited data in this context. Methods Serum IL‐37 and IL‐39 levels were determined in 106 T2DM patients and 109 controls using enzyme‐linked immunosorbent assay kits. Results Serum levels (median and interquartile range) of IL‐37 (79 [47–102] vs. 60 [46–89] ng/L; probability [p] = .04) and IL‐39 (66 [59–69] vs. 31 [19–42] ng/L; p < .001) were significantly elevated in T2DM patients compared to controls. As indicated by the area under the curve (AUC), IL‐39 (AUC = 0.973; p < .001) was more predictable for T2DM than IL‐37 (AUC = 0.582; p = .039). Elevated levels of IL‐39 were significantly associated with T2DM (odds ratio = 1.30; p < .001), while IL‐37 did not show this association. Classification of IL‐37 and IL‐39 levels by demographic and clinical characteristics of patients revealed some significant differences including gender (IL‐39), body mass index (BMI; IL‐37 and IL‐39) and diabetic neuropathy (IL‐39). BMI was positively correlated with IL‐39 (correlation coefficient [r s] = 0.27; p = .005) and glycosylated haemoglobin (r s = 0.31; p = .001), and negatively correlated with age at onset (r s = −0.24; p = .015). Conclusions IL‐37 and IL‐39 levels were elevated in the serum of T2DM patients. Besides, IL‐39 is proposed to be a novel cytokine associated with T2DM and positively correlated with BMI.

annually and ranks ninth among other chronic diseases in causing mortality. 1 Relative insulin deficiency and resistance, as well as impaired glucose and lipid metabolism, are hallmarks of T2DM. Thus, hyperglycaemia and dyslipidaemia are common features associated with T2DM that increase the risk of long-term microvascular and macrovascular complications, especially cardiovascular disease and peripheral neuropathy. 2 The underlying mechanisms involved in the pathophysiology of T2DM are complex and not well defined.
However, low-grade systemic inflammation is a characteristic feature of the disease and elevated systemic levels of inflammatory mediators, such as acute phase proteins and cytokines, are well documented in T2DM patients. 3 Cytokines are low molecular weight glycoproteins produced by many types of immune and non-immune cells and secreted into the circulation, where they regulate various aspects of immune responses and inflammatory reactions through their autocrine, paracrine and endocrine actions. 4 Among them cytokines proposed to be involved in the pathogenesis of T2DM are members of the interleukin (IL)-1 and IL-12 cytokine families, 5,6 but some of these cytokines have been neither extensively explored (i.e., IL-37) nor investigated (i.e., IL-39).
IL-37, a member of the IL-1 cytokine family identified in 2000, is an anti-inflammatory cytokine known for its ability to reduce innate inflammation and suppress adaptive immune responses.
Many types of immune and non-immune cells produce IL-37, including monocytes, macrophages, dendritic cells, B cells, plasma cells, natural killer cells and epithelial cells, in response to proinflammatory stimuli. 7 Dysregulated production of IL-37 has been associated with several human inflammatory conditions, such as enterocolitis, Behçet's disease, periodontitis, asthma, SARS-CoV-2 infection and liver inflammation. 8 Regarding T2DM, there is some evidence to suggest that IL-37 may have a role in the pathogenesis.
In this context, IL-37 has been identified as a key anti-inflammatory cytokine that has the potential to attenuate obesity-induced inflammation and insulin resistance. 9 Besides, up-regulated expression of IL-37 has been associated with increased insulin sensitivity in elderly patients with T2DM. 10 A link between IL-37 and a dysregulated immune response in COVID-19 and diabetes has also recently been proposed. 11 IL-39, the latest discovered member of the IL-12 family of cytokines, is heterodimer glycoprotein composed of two covalently linked subunits, IL-23p19 and Epstein-Barr virus-induced gene (Ebi3). 12 In vitro and in vivo studies in mice revealed that IL-23p19 and Ebi3 are secreted by B cells upon stimulation with lipopolysaccharides (LPS). Dendritic cells and macrophages are other immune cells that also express IL-23p19 and Ebi3 mRNA, and their expression levels are up-regulated in the presence of IL-4 (an anti-inflammatory cytokine), whereas LPS inhibits this expression in these cells. B cells and plasma cells of lupus-like mice are also promoted to express high levels of IL-39. 13 In humans, there is some controversy about IL-39 secretion. Ecoeur et al. considered IL-39 to be a theoretical cytokine and its production and/ or biological activity could not be assigned in the human system.
The authors speculated that IL-39 might be an immunoregulatory cytokine only in mice. 14 On the contrary, detectable levels of IL-39 have been demonstrated in human serum. 15,16 Research on the immune functions of IL-39 is still in its preliminary stage. However, pro-inflammatory effects of IL-39 have been suggested in lupuslike mice, and thus this cytokine may contribute to the immunopathogenic mechanisms involved in systemic lupus erythematosus (SLE). 12 Further, significantly elevated levels of IL-39 were found in the serum of patients with acute coronary syndrome and the authors suggested that IL-39 could be considered an indicator of systolic dysfunction. 15 In patients with neuromyelitis optica spectrum disorders, an inflammatory demyelinating autoimmune disorder, up-regulated levels of IL-39 have been demonstrated. 16 IL-39 has also been associated with exacerbation of concanavalin A-induced hepatitis. 17 These studies point to the potential involvement of IL-39 in the pathogenesis of human inflammatory diseases. Since low-grade inflammation is a common feature of T2DM, 3 IL-39 may be a relevant cytokine whose pathogenic role in disease should be understood.
This study planned with the aims to evaluate IL-37 and IL-39 levels in the serum of patients with T2DM in order to assess their biomarker significance and association with disease. In addition, the correlation between IL-37 and IL-39 levels and demographic and clinical characteristics of T2DM was also evaluated.  18 The modified Toronto Clinical Neuropathy Scoring (TCNS) system was followed to assess diabetic sensorimotor polyneuropathy in patients. 19

| Anthropometric and biochemical measurements
Anthropometric measurement included body mass index (BMI), which was calculated by dividing weight (kilogram; kg) by the square of height (meter; m). According to the scheme of the World Health Organization (WHO), T2DM patients were classified into three categories of BMI; normal-weight (18.5-24.9 kg/m 2 ), overweight (25-29.9 kg/m 2 ) and obese (≥ 30 kg/m 2 ). 20 Biochemical measurements included the following parameters: FPG, HbA1c, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol, triglycerides, high-density lipoprotein (HDL) and low-density lipoprotein (LDL). An automated biochemical analyser (Cobas c 311analyzer, Cobas-Roche, Germany) pre-loaded with respective reagents was used to measure the biochemical parameters. A blood sample (5 mL) was collected in a plain tube after 8-10 hours of fasting, and after coagulation for at least 30 minutes at room temperature (20-25°C), the tube was centrifuged (1000 × g, 15 min). Separated serum was frozen at −20°C until assessment.

| Statistical analysis
Categorical variables were given as number and frequency (percentage). Significant differences between frequencies were evaluated using Pearson Chi-square test. Normal distribution of continuous variables was tested using the Kolmogorov-Smirnov and Shapiro-Wilk normality tests. Normally distributed (parametric) variables were given as mean and standard deviation (SD). Significant differences between means were assessed using one-way analysis of variance (ANOVA) test. Non-parametric variables, which did not show a normal distribution, were expressed as median and interquartile range (IQR; 25%-75%).
Significant differences between medians were determined using the Mann-Whitney U test (for comparison of two groups) or Kruskal-Wallis test (for comparison of more than two groups).
Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic performance of IL-37 and IL-

| Power of sample size
The power of the sample size was determined using the G*power software with the following input parameters: number of T2DM patients = 106; number of CTRL = 109; two-tailed α-error p = .05; effect size d = 0.5. The estimated power of sample size (1β error p) was .95. The statistically acceptable power for the sample size is 0.8. 22

| Baseline demographic and clinical characteristics
Baseline demographic and clinical characteristics are given in Table 1.
The mean age of T2DM patients was higher than that of CTRL but the difference was not significant (54 ± 10 vs. 52 ± 8; p = .272).
Further, 90.6% of patients had a family history of DM. Diabetic neuropathy was found in 19.8% of patients and it was mostly mild neuropathy (TCNS score = 6-8). The mean age at onset of T2DM was 46 ± 8 years with disease duration mean of 9.5 ± 5.6 years.

| DISCUSS ION
The current study examined serum levels of IL-37 and IL-39 in patients with T2DM and age-and gender-matched CTRL. The diagnosis of T2DM was confirmed, with most patients showing levels of FPG and HbA1c exceeding the thresholds established by the ADA criteria. These criteria recommend that FPG of 126 mg/dL or higher and HbA1c of 6.5% or higher be diagnostic thresholds for T2DM. 18 However, we noted that some patients showed normal levels for both tests (about 25%). This was probably due to the use of oral hypoglycaemic drugs, and indicated that these patients had good control of hyperglycaemia, while the others had poor control of hyperglycaemia, even though they were taking oral hypoglycaemic agents. Since the glycaemia status may have an effect on the production of cytokines, 23 the levels of FPG and HbA1c were classified into percentiles to reach a better understanding of IL-37 and IL-39 role in the pathogenesis of T2DM. However, no significant differences were found and IL-37 and IL-39 levels appeared to be unaffected by FPG and HbA1c.
Most of T2DM patients were either overweight or obese (91.5%), and this may link obesity to T2DM aetiology. Consistent with this observation, studies have considered obesity as the most significant risk factor associated with T2DM and weight loss by 5% has been associated with a good improvement of glycaemia control. 24 Furthermore, epidemiological studies indicated that the global increase in obesity prevalence paralleled the increase in T2DM prevalence in a concomitant manner. 25 Besides, circulating cytokine levels have been shown to be influenced by obesity. 26 Therefore, this issue was addressed in the current study in relation to IL-37 and IL-39.
The study also addressed the issue of diabetic neuropathy, one of the most important complications associated with T2DM, as about 50% of T2DM patients eventually develop diabetic neuropathy. 27 In addition, a recent study demonstrated that inflammatory cytokines, particularly tumour necrosis factor (TNF)α, are important predictors of diabetic neuropathy over a period of 5 years. 28 In the current study, about 20% of T2DM patients suffered from diabetic neuropathy, and probably the frequency was higher if we included a larger number of patients. The study also indicated that 90.6% of T2DM patients had a family history of disease (siblings, parents or grandparents). It is generally agreed that T2DM shows a tendency to increase in families and individuals with affected parents and/or sibs are at a greater risk to develop T2DM. This also highlight the importance of genetic predisposition to develop T2DM. 29,30 The main concern of the current study was to understand the found to show an increased expression to counteract and prevent excessive inflammatory reactions. 9 Therefore, the up-regulated serum levels of IL-37 may be due to a feedback mechanism to counter proinflammatory cytokines that exhibit elevated serum levels in T2DM. 3 The current study results were in agreement with those of a recent study, which indicated that serum IL-37 and mRNA levels were upregulated in elderly patients with T2DM (aged 65-95 years). 10  linked to SLE in lupus-prone mice. 33 In patients with acute coronary syndrome, serum levels of IL-39 were also significantly increased and showed a positive correlation with high-sensitivity C-reactive protein (CRP). Its potential has also been suggested as an indicator of cardiac systolic dysfunction. 15 Moreover, IL-39 levels were significantly upregulated in the serum of patients with neuromyelitis optica spectrum disorders compared with CTRL, and were positively correlated with disease severity. 16 Another study indicated that IL-39 was associated with the exacerbation of concanavalin A-induced liver inflammation, and based on this, IL-39 was proposed as a therapeutic target in inflammatory hepatic diseases. 17 Besides, the pathogenic role of IL-39 in acute graft-versus-host disease has been identified and its role in potentiating T-lymphocyte functions has been recognized. 34 However, contradictory results have been reported in patients with Graves' disease and Hashimoto's thyroiditis, and IL-39 showed downregulated levels but was positively correlated with two inflammatory markers, CRP and leukocyte count. 35 Based on these findings and as shown in the results of this study, it may be reasonable to suggest that IL-39 is also a T2DM-associated cytokine and could be considered as a potential biomarker for the disease.
When IL-39 levels were examined in subgroups of T2DM patients, two significant differences were found. Female patients showed elevated levels compared to male patients, and patients with neuropathy showed low levels compared to patients without neuropathy. Although no data are available on sex-related differences in IL-39 levels, experimental in vivo evidence has indicated that inflammatory responses differ between males and females. It has been shown that inflammatory responses to bacterial endotoxins were higher in women than in men, with significantly higher plasma levels of the pro-inflammatory cytokines TNFα and IL-6 and the steroid hormone cortisol. 36 Besides, as in T2DM, there is evidence that females develop low-grade generalized inflammation and elevated levels of inflammatory markers, such as pro-inflammatory cytokines and CRP, during the onset of menstruation when oestrogen levels are lowest. Therefore, inflammation and immunity in women are complicated by sex hormones, especially oestrogen, as well as menopause because pre-menopausal women have higher levels of oestrogen than post-menopausal women. 37 Unfortunately, we did not measure serum levels of oestrogen and other sex hormones in current T2DM patients, and this issue warrants exploration and may aid in a better understanding of IL-39 role in T2DM. With regard to peripheral neuropathy, surprisingly, IL-39 levels were significantly lower in patients with neuropathy than in patients without neuropathy. Because, a recent five-year prospective cohort study indicated that elevated levels of pro-inflammatory cytokines are good predictors of diabetic neuropathy. 28 There is no clear explanation for this difference but the number of patients with diabetic neuropathy (n = 21) may have an effect. In addition, only patients with mild neuropathy were included. Therefore, this issue needs to be explored further by including more patients and covering the full range of TCNS score.
It was interesting to note that IL-39 levels were positively correlated with BMI. BMI may indirectly indicate obesity and the current T2DM patients were either obese or overweight (91.5%). In fact, studies have identified obesity as a complex disease associated with elevated levels of inflammatory markers, which can lead to a chronic, low-grade inflammation with effects on the functions of immune cells, such as macrophages. 24,25,38 Researchers found that adipose tissue in obese subjects was linked to a shift of anti-inflammatory M2 macrophages to pro-inflammatory M1 macrophages, and this was associated with insulin resistance. In this context, the adipose tissue of obese subjects has been found to secrete more pro-inflammatory markers that parallel the development of T2DM. 39  pre-diabetic subjects, as well as newly diagnosed cases, are included.
In conclusion, IL-37 and IL-39 levels were elevated in the serum of T2DM patients. Besides, IL-39 is proposed to be a novel cytokine associated with T2DM and positively correlated with BMI.

ACK N O WLE D G E M ENTS
The authors appreciate the kind assistance and cooperation of the medical staff at the National Diabetes Center (Mustansiriyah University).

FU N D I N G I N FO R M ATI O N
No funds have been received.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no potential financial or nonfinancial conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author upon reasonable request.