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Highlights
· Inflammation is orchestrated by cytokine in initiating and destructive stages of type 1 diabetes mellitus (T1DM).
· The low-production phenotype of transforming growth factor-β1 protect against T1DM susceptibility.
· Cytokine single-nucleotide polymorphisms have a role in regulation of autoimmune diseases, specifically T1DM.
Introduction
Type 1 diabetes mellitus (T1DM), a chronic autoimmune disease, results in a significant loss of pancreatic β-cells. Autoreactive T cells are important players in the destruction of β-cells [1]. The function and role of B cells in the humoral immune system have long been studied. B cells produce not only antibodies and an antibody-mediated memory response against pathogens, but also cell-mediated immunity. Several studies have demonstrated that B cells can activate antigenspecific CD4 and CD8 T cells, which can have cytotoxic and regulatory effects and contribute to the onset of T1DM [2]. The breakdown of the immune system is primarily mediated by T-helper 1 (Th1) cells. On the other hand, islet infiltration, immune cell activation, and other mediators contribute to the destruction of pancreatic cells and to the apparent hyperglycemia observed in this disease [3].
Exogenous insulin replacement is the current mainstay of T1DM treatment, highlighting the need for specific immunotherapy to slow disease progression and improve clinical outcomes. Genetic and immunopathogenic studies have directly linked cytokines to the pathogenesis of T1DM. Cytokines are the main cause of inflammation and are essential for controlling progressive cell degeneration [4]. The modulation of cytokine function can be a therapeutic strategy, as evidenced by studies conducted in mouse models, especially in nonobese diabetic mice, an established animal model of T1DM. Several novel cytokines have been identified as potential therapeutic targets to counteract immune-mediated cell damage [5].
Three classes of cytokines exist: those with roles that are typically anti-inflammatory (e.g., IL-10 and TGF-β1 cytokines), those with roles that are typically proinflammatory (e.g., IL-1, IL-6, and tumor necrosis factor [TNF]-α), and those that belong to the IL-12 family (e.g., IL-21 and IL-33) [6].
However, the roles of cytokines in the pathophysiology of T1DM are currently unclear and complex, especially with regard to inflammation and the course of the disease, because many dysregulated cytokines are involved in the dynamics of cytokine regulation. In the context of T1DM, very few cytokines have shown pro- or anti-inflammatory effects. For instance, in children with newly diagnosed T1DM, blocking the action of the TNF preserves β-cell function [7], and, in patients with T1DM, IL-2 treatment can increase the percentage of regulatory T-cells (Tregs) without adverse effects [8]. These findings highlight the crucial role of cytokines in the development of T1DM. The cytokine TGF-β1 has been associated with the control of innate and adaptive immunity and plays a significant role in many pathological and physiological responses [9]. Moreover, TGF-β1 secreted by Tregs plays a role in autoreactive T-cell suppression, the induction of immune tolerance, and the inhibition of proinflammatory cytokine production [10]. The signaling sequence of the TGF-β1 protein is encoded by the TGF-β1 gene polymorphisms rs1800470 and rs1800471, which impact cytokine production [11].
Several studies have suggested that Th2 (IL-4) and Th3 (IL-10 and TGF-β1) cytokines play protective roles by preventing the production of proinflammatory and Th1 cytokines [12]. As IL-10 suppresses the immune system, it is essential for immune tolerance [13]. IL-10 participates in immunological and inflammatory responses, in addition to regulating cell growth and division. This cytokine is currently regarded as an immunosuppressive agent [14] and has been connected to many autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis [15]. Moreover, IL-10 is encoded by the 3 polymorphisms rs1800896, rs1800871, and rs1800872, which impact IL-10 production.
The role of the proinflammatory cytokine IL-6 is less understood, and existing evidence is inadequate to support claims that it damages or cytotoxically affects pancreatic cells [16]. A multifunctional cytokine called IL-6 is secreted by T cells and macrophages to stimulate the immune system in cases of inflammation and infection, including the inflammatory response associated with insulin resistance. A polymorphism in the IL-6 gene (rs1800795) has been documented to exert an effect on its secretion and function [17].
The genetic contributors of T1DM among Saudis have been unexplored. The aim of the present study was to examine cytokines, their gene polymorphisms, and their correlations with predisposition for T1DM. As such, this study was conducted to elucidate and illustrate the potential roles of circulating cytokine levels of TGF-β1, IL-10, and IL-6 and 6 single-nucleotide polymorphisms (SNPs) in 3 cytokine genes (TGF-β1 [rs1800470 and rs1800471], IL-10 [rs1800896, rs1800871, and rs1800872], and IL-6 [rs1800795]) in the incidence of T1DM in Saudi children.
Materials and methods
1. Study population
A total of 182 unrelated Saudi subjects was selected and included in this study. The patient group, which consisted of 91 patients with T1DM (median age, 9 years; age range, 6–12 years), of whom 37 were male (median age, 9 years; age range, 7–12 years) and 54 were female (median age, 9 years; age range, 6–12 years), were enrolled from Al-Baha Region, Saudi Arabia. The control group included 91 unrelated healthy subjects (39 boys and 52 girls; median age, 9 years; age range, 6–12 years) free from autoimmune diseases, symptoms, and family history of T1DM. The diagnosis of T1DM was established by referring clinicians using a criterion from the American Diabetes Association— specifically, by the presence of a hemoglobin A1c level ≥6.5% (≥48 mmol/mol). Adherence to the diagnostic criterion was based on plasma glucose measured 2 hours after an oral glucose load (≥200 mg/dL [≥11.10 mmol/L]) and while fasting (≥126 mg/dL [7 mmol/L]) [18]. After recruitment, informed consent for genetic analysis was obtained from all study participants' parents before the start of the study.
2. Sample collection
Each child underwent aseptic venous blood sample collection, during which 10 mL of blood was extracted. Subsequently, collected blood samples were allowed to clot for 15 minutes. The serum was extracted through centrifugation at 1,500 rpm for 10 minutes at room temperature and was then divided into 2 sterile vacutainer tubes for immediate assessment of biochemical parameters.
3. Measurement of circulating TGF-β1, IL-10, and IL-6 levels
A second sterile vacutainer tube of serum was used to measure the levels of the selected cytokines (TGF-β1, IL-10, and IL-6). TGF-β1 was quantitatively detected in the serum by enzyme-linked immunosorbent assay (ELISA) using a human TGF-β1 ELISA kit (Invitrogen cat. no. BMS249-4; Thermo Fisher Scientific, Waltham, MA, USA). Meanwhile, an IL-10 human uncoated ELISA kit (Invitrogen cat. no. 88-7106-88; Thermo Fisher Scientific) was used to measure serum levels of IL-10, and a sandwich ELISA IL-6 human kit (Invitrogen cat no. EH2IL6; Thermo Fisher Scientific) was used to measure serum levels of IL-6. As previously mentioned, the samples were prepared and tested in duplicate in accordance with the manufacturer's instructions. The assays we used are specific for human TGF-β1, IL-10, or IL-6 and do not cross-react with other known cytokines, according to the information provided by their respective manufacturers.
4. DNA extraction
Ethylenediaminetetraacetic acid (EDTA) was added to a sterile vacutainer tube containing the venous blood sample (5 mL) from each participant. Genomic DNA was extracted from the whole-blood EDTA samples using the Gentra Puregene blood kit (Qiagen N.V., Hilden, Germany), in accordance with the manufacturer's instructions. The extracted DNA was loaded onto a 1% agarose gel to confirm its integrity. The DNA concentration was measured in each sample using the NanoDrop 2000c system (Thermo Fisher Scientific) [19].
5. Genotyping
Genomic DNA was extracted from whole blood in accordance with the manufacturer's recommendations, and the subjects' genotypes were assessed for TGF-β1 (rs1800470 and rs1800471), IL-10 (rs1800896, rs1800871, and rs1800872), and IL-6 (rs1800795) polymorphisms. Cytokine genotyping was performed with polymerase chain reaction (PCR) with sequence-specific primers using a commercial kit (One Lambda, Canoga Park, CA, USA). Based on their previously determined genotypes, study subjects were classified as low, high, or intermediate producers of the phenotypes predicted for these cytokines [20], as shown in Fig. 1A and B. PCR amplifications were performed using the PTC-200 thermal cycler (MJ Research, Inc., Waltham, MA, USA), in accordance with the manufacturer's instructions.
6. Statistical analysis
Data are summarized as follows: quantitative variables are expressed as mean±standard deviation and median with range, while frequencies are reported for categorical variables to describe the distribution of the categories within the dataset. To compare the data between the groups, an independent t-test was performed, while the chi-square and Fisher exact tests were used to compare categorical variables. A probability value (P-value) <0.05 was considered statistically significant.
Results
1. Clinical and demographic characteristics of healthy controls and patients with T1DM
Patients with T1DM who were screened and deemed eligible were included in this study. Separately, subjects who were found to be healthy (no diabetes) during screening were recruited for the control group. Each study group consisted of 91 participants (39 boys and 52 girls in the control group and 37 boys and 54 girls in the T1DM group). Table 1 displays the study participants' baseline clinical and demographic characteristics. Compared to the healthy controls, the patients with T1DM had significantly higher mean body mass index (P<0.001) and mean creatinine, fasting glucose, postprandial blood glucose, and HbA1c levels (P < 0.001) than controls. The serum lipid profile of the patients with T1DM was significantly different from that of the controls, characterized by significantly higher total cholesterol levels and significantly lower high-density lipoprotein levels (P<0.001 for all comparisons). The patients with T1DM showed no significant difference in triglyceride level between the 2 sexes or compared to the subjects in the control group.
For the diagnosis of T1DM, a thorough examination of a cohort of 91 recently diagnosed children with diabetes was conducted, focusing on antibodies to glutamic acid decarboxylase (GAD) auto-antibodies and C-peptide levels. Among them, 69 individuals (75.8%) exhibited GAD auto-antibodies at levels above the normal reference range (38.56±4.23 IU/mL), while the remaining 22 (24.2%) showed levels within the normal range (6.72±0.84 IU/mL). Notably, 100% of individuals in this group demonstrated decreased serum C-peptide levels below the normal reference range (0.43±0.08 ng/mL). In contrast, in the control group of 91 healthy individuals, all participants exhibited levels of both GAD auto-antibodies (5.51±0.59 IU/mL) and C-peptide (2.86±0.52 ng/mL) within the normal reference range.
2. Circulating cytokine serum levels of the healthy controls and patients with T1DM
Using an ELISA kit, TGF-β1, IL-10, and IL-6 levels were quantitatively detected in serum. The patient group had a significantly lower TGF-β1 serum level than the control group (P<0.001). However, no statistically significant difference in IL-10 or IL-6 serum level was found between the control and patient groups (Table 2).
3. Cytokine genotype and production
The genotype distributions of polymorphic cytokine (TGF-β1, IL-10, and IL-6) genes were observed in T1DM patients and the T1DM-free control group. Table 3 lists the genotypes for the TGF-β1 (rs1800470 and rs1800471), IL-10 (rs1800896, rs1800871, and rs1800872), and IL-6 (rs1800795) gene polymorphisms. Serum levels of TGF-β1 in T1DM patients were significantly low compared to those in the T1DM-free group (P<0.001). On the other hand, both the serum levels of Il-10 and IL-6 showed a non-significant difference between T1DM patients and control participants. The genotypic frequencies of the cytokine genes in the T1DM and control groups are clearly illustrated in Table 3. Genotypes of cytokines TGF-β1, IL-10, and IL-6 are illustrated and categorized into 3 categories according to protein production: (A) homozygotes, corresponding to high protein production; (B) heterozygotes, corresponding to intermediate protein production; and (C) homozygotes, corresponding to low protein production [21].
The patient group exhibited significantly lower TGF-β1 production (P<0.001) than the control group. No significant changes in TGF-β1 production were observed, even though significant differences in IL-10 genotypes were found between the control and patient groups. No significant difference in the IL-6 genotype was evident between the T1DM and control subjects. Nonetheless, a significant increase in IL-6 level with a low cytokine-production phenotype was found in patients with T1DM compared to the control subjects (P<0.001).
Discussion
Diabetes is a disease with the fastest-growing global incidence rates and a linearly increasing prevalence worldwide. According to estimates from the International Diabetes Federation, more than 382 million people worldwide currently have diabetes, and this number is expected to increase to 592 million by 2035. The incidence of T1DM, which primarily affects the youth, is alarmingly increasing at a rate of 3% annually. An estimated 78,000 children younger than 14 years of age are estimated to develop T1DM each year [21].
TGF-β1 serum levels regulate important cellular functions such as the proliferation rate and production of extracellular matrix proteins in different cell types. Circulating levels of TGF-β1 might be altered in various disease states. Given the established pathophysiological role of plasma TGF-β1 level, it may be used as a target for therapeutic interventions and a prognostic indicator of the future risk of disease and/or its complications. Our findings show elevated TGF-β1 levels in the control group, consistent with the findings of Ichinose et al. [22], who suggested that high TGF-β1 levels may prevent or delay the immunosuppressive and regulatory cytokine production of many cells, including those of the Th3 and Treg subsets, possibly reducing insulin production, which is responsible for autoimmune-mediated destruction of the pancreatic islets of Langerhans [22]. This contrasts with the finding of Jakus et al. [23] who reported that TGF-β1 levels were markedly elevated in subjects with T1DM. In addition, these authors determined that serum TGF-β1 levels were significantly higher in patients with diabetes than in controls, as TGF-β1 may participate in the development and progression of diabetic microvascular and macrovascular complications.
The polymorphism (rs1800470) C/C at position +869 in the promoter region of the TGF-β1 gene is associated with higher TGF-β1 concentrations in plasma, which confirms our observation of the circulating TGF-β1 level in the control subjects. Our results also show that the polymorphism with high TGF-β1 secretion was significantly more frequent in the control subjects than in the patients with T1DM.
Moreover, our data demonstrate no appreciable differences in circulating serum level or the genotype and phenotype of IL-10 between the patients and the controls. This is in agreement with the finding of Ide et al. in a study of Japanese patients that the mean polymorphisms in the promoter region of the IL-10 gene do not correlate with a genetic predisposition to T1DM [24].
In Spanish patients with T1DM, the IL-10 genotype was assumed to have a minor influence on the risk of autoimmune diabetes [25]. Our results conflict with those of a Polish study that found a correlation between T1DM and the IL-10 (rs1800896) A/G polymorphism, especially in AA genotypes [26]. However, such differences in results may be explained by the hypothesis that these genotypes are population-specific and may cosegregate with the disease genes in different ways among ethnic groups. Our findings also differ from those of the previously published study by Gouda et al. [27], who found a significant increase in serum IL-10 level in diabetic patients compared to controls. Such results were was also confirmed by He et al., who demonstrated that elevated IL-10 levels in patients with T1DM may be due to a compensatory mechanism to increase the levels of proinflammatory cytokines [28].
Furthermore, Chen et al. [29] reported that a subgroup analysis of patient age, disease duration, and ethnicity revealed elevated IL-6 serum levels in patients with T1DM. The inflammatory cytokines from peripheral blood T lymphocytes play a vital role in the induction and development of diabetes. Recently, accumulating research has demonstrated that cytokines are associated with the inhibition of cytotoxicity in pancreatic cells [30]. Among all cytokines, IL-6 is an efficient cytokine involved in various kinds of biological activities, such as inflammatory reactions and immune responses [31]. In addition, IL-6 is a promoting cytokine that regulates insulin secretion [32]. Our study reveals no significant difference in IL-6 serum level between the control subjects and patients with T1DM, although the genetic study of the IL-6 (rs1800795) C/G SNP revealed a significant increase among subjects in the control group, who showed a low production of the IL-6 phenotype, compared to the patients with T1DM. This finding is consistent with those of studies suggesting that low IL-6 levels can induce insulin secretion, whereas high IL-6 levels inhibit insulin production [33]. Other studies have reported that the IL-6 gene may contribute to genetic susceptibility to T1DM [34]. In recent years, increasing studies have suggested that IL-6 can prevent pancreatic islet cells from experiencing apoptosis and functional lesions in vitro and in vivo [35].
Our results are in line with those of a large U.K. case-control study [36], which also found a marginally positive association between T1DM and the IL-6-174 C allele. However, our results contradict those of one of the first case-control studies on the IL-6 (rs1800795) SNP, which found that individuals who were GG-homozygous, a phenotype with low cytokine production, were more likely to develop T1DM [34]. In this investigation, those with the IL-6-174 GG genotype had lower risk of T1DM [37]. However, whether the IL-6-174 GG genotype plays a role in the pathophysiology of T1DM remains unclear, and no hard evidence suggests that it damages or even kills pancreatic cells [35]. Studies on nonobese diabetic mice, which are genetically predisposed to autoimmune diabetes, have also demonstrated that IL-6 protects against cytokine-induced cell death and functional impairment [36]. Genetic studies have shown conflicting evidence regarding the relationship between IL-6 SNPs and T1DM [38,39]. The exact mechanism through which this polymorphism influences the genetic determination of T1DM is unknown, as cytokine gene SNPs are linked to phenotypes that are both high and low producers [40]. Nevertheless, some studies have not found an association between the genotypes and the levels of cytokines secreted [38]. According to Reuss et al. [39], genetic factors account for only 50% of the observed variability in cytokine secretion; environmental factors may also have an impact. This can help explain the lack of a full understanding of how IL-6 functions in the pathophysiology of T1DM and how the rs1800795 SNP affects function.
The limitations of this study include its relatively small sample size, which impacts the generalizability of its findings. The study also focused on a specific population of Saudi children, and caution is advised when extrapolating these results to other demographics. Additionally, the exclusive examination of TGF-β1, IL-10, and IL-6 cytokines may not capture the full complexity of the immune response in T1DM. Future studies with larger, diverse cohorts exploring additional variables and broader investigations into various cytokines and immune markers are necessary for validation and extension of these findings for a more comprehensive understanding of T1DM pathogenesis.
In conclusion, cytokine networks play crucial roles in orchestrating inflammation during both the initiation and destructive phases of T1DM. The notable significant difference in TGF-β1 level that we observed greatly supports its beneficial role in inhibiting the development of T1DM. This might encourage the development of new therapeutic strategies to target TGF-β1. Our findings support the protective roles of high TGF-β1 serum levels and TGF-β1 with a low-production phenotype against susceptibility to T1DM. In Saudi children, a SNP (rs1800795) with low IL-6 production might be a preventive factor against T1DM. Our results highlight the role of cytokine SNPs in controlling autoimmune diseases, particularly T1DM, within the population under investigation. Our findings may prompt larger cohort studies to further corroborate the impacts of these SNPs on the release of these cytokines and their ensuing effects on T1DM.
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