Elevated Levels of Serum IL-12 and IL-18 are Associated with Lower Frequencies of CD4+CD25highFOXP3+ Regulatory T cells in Young Patients with Type 1 Diabetes

Type 1 diabetes is thought to involve chronic inflammation, which is manifested by the activation and expression of different inflammatory mediators. IL-12 and IL-18 are two cytokines that have been shown to exert strong proinflammatory activity and have been implicated in the pathogenesis of type 1 diabetes in mice and humans. The overproduction of proinflammatory mediators is controlled by specialized T cell subset, namely regulatory T cells that express FOXP3 transcription factor. Since IL-12 and IL-18 mediate inflammatory response and Tregs exhibit anti-inflammatory potential, we aimed to examine their reciprocal relationship in patients with type 1 diabetes. The study group consisted of 47 children diagnosed with type 1 diabetes and 28 healthy individuals. Serum levels of IL-12 and IL-18 were measured by ELISA, and the peripheral blood CD4+CD25high FOXP3+ regulatory T cell frequencies were analyzed by flow cytometry. Patients with type 1 diabetes had a decreased percentage of circulating CD4+CD25highFOXP3+ Tregs in comparison to their healthy counterparts. In addition, they produced more IL-12 and IL-18 than children from the control group. Concentrations of these cytokines positively correlated with one another, as well as with CRP and HbA1c. Moreover, the negative association between IL-12, IL-18, CRP serum levels, and the frequency of regulatory CD4+CD25highFOXP3+ Tregs was observed. IL-12 and IL-18 may have direct or indirect impact on regulatory T cell subset, which may contribute to their reduced frequency in peripheral blood of patients with type 1 diabetes mellitus.


INTRODUCTION
Type 1 diabetes mellitus (DM1) is thought to involve chronic inflammation, which is manifested by the activation and expression of different inflammatory mediators [1]. As a result, various diabetic complications develop leading to increased mortality and morbidity.
IL-12 is a proinflammatory cytokine produced by antigen presenting cells in response to PAMPs (pathogen-associated molecular patterns) and DAMPs (danger-associated molecular patterns). It induces the polarization of the immune response towards Th1 profile, which protects against intracellular pathogens [2,3]. IL-12 has been implicated in the pathogenesis of type 1 diabetes in the NOD (non-obese diabetic) mouse [4]. Alleva et al. showed that macrophages from NOD mice produced more IL-12 than NOR (non-obese resistant) mice macrophages [5]. A link between IL-12 and type 1 diabetes was also suggested in humans. Glucose-stimulated PBMCs (peripheral blood mononuclear cells) from healthy subjects produced more IL-12 than resting, unstimulated cells [6]. What's more, the production of IL-12 did not change even after insulin treatment [6]. Similar effect was seen in patients with type 2 diabetes. The LPS-stimulated PBMCs under glucose treatment produced elevated level of IL-12 [7]. Furthermore, it was shown that patients with longstanding DM1 show increased levels of IL-12 in both serum and aqueous humor [8]. The latter may provide an evidence for the involvement of IL-12 in pathogenesis of late diabetic microvascular complications.
Regulatory T cells (Tregs) play a crucial role in the maintenance of immune homeostasis in controlling autoimmunity and inflammation. They are responsible for suppressing the excessive ability of different cells to proliferate and/or produce proinflammatory cytokines [26,27]. They are characterized by the coexpression of CD4, CD25, and a t r a n s c r i p t i o n f a c t o r F O X P 3 , t h u s t h e CD4 + CD25 high FOXP3 + is the most widely accepted phenotype of Tregs [28,29]. Defects in Tregs have been reported by us and others in several autoimmune/ inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, or type 1 diabetes [27,[30][31][32][33]. Moreover, it was shown that depletion of Tregs deteriorated nephropathy in non-insulindependent diabetic mice, while adoptive transfer of Tregs exerted protective effect on kidneys [34]. Immune response studies in animal models of kidney injury also suggested the protective role for the CD4 + FOXP3 + regulatory T cell subset [35][36][37].
Since IL-12 and IL-18 mediate inflammatory response and Tregs exhibit anti-inflammatory potential, we aimed to examine the relation between them in patients with type 1 diabetes. It is particularly important because the disease can progress into clinically manifested vascular complications such as retinopathy or nephropathy.

Subjects
The study group consisted of 47 young patients (mean age; 14.25±3.5 years) diagnosed with type 1 diabetes that were recruited from Clinic of Pediatrics, Department of Diabetology and Endocrinology Medical University of Gdańsk. Type 1 diabetes was defined according to the criteria of the American Diabetes Association. Patients with microvascular complications, as well as those with coexisting autoimmune, chronic, and acute inflammatory diseases were excluded from the study. The mean duration of the disease was 7.39±3.8 years. In all examined patients, the C-peptide levels were below 0.5 ng/ml. All patients were treated with humanized insulin at doses of 0.87± 0.2 mg/kg. At the time of sampling, a blood glucose level along with biochemical measurement of renal function, lipid status, C-reactive protein (CRP), and glycosylated hemoglobin (HbA1c) were monitored.
The control group consisted of 28 age and sexmatched individuals recruited during control visits in outpatient clinic. No signs of autoimmune, chronic, inflammatory, and neoplastic disease at the time of sampling and no evidence of DM1 in their families were disclosed as confirmed by medical records, laboratory examination, and laboratory tests.
All subjects gave informed consent and the study followed the principles of the Declaration of Helsinki and was approved by The Ethics Committee of The Medical University of Gdańsk.

Isolation and Flow Cytometric Analysis of Peripheral
Blood CD4 + CD25 high FOXP3 + Regulatory T cells Heparinised venous blood samples were collected and used to isolate PBMCs (peripheral blood mononuclear cells) by density gradient preparation over Ficoll-Uropoline. 1 × 10 6 freshly isolated PBMCs were destined for flow cytometric staining. Cells were stained with anti-CD4 (IgG1,κ mouse Pe/Cy5, Clone RPA-T4, BioLegend, USA) and anti-CD25 (IgG1, ĸ mouse PE, Clone BC96, BioLegend, USA) antibodies and incubated for 30 min in the dark, fixed, and stained for intracellular expression of FOXP3 (IgG1,κ mouse Alexa-Fluor 488, Clone 206D, BioLegend, USA). Measurements were performed on the LSRII flow cytometer (BD Biosciences). Dead cells were excluded by forward and side scatter. Positive signal for each staining was established using appropriate isotype control. Data were analyzed by FACSDiva 6.0 Software (Becton Dickinson, USA).

Determination of Serum IL-12 and IL-18 Levels
Serum levels of IL-12 and IL-18 were measured by ELISA method (Quantikine R&D Systems, Minneapolis, Minn., USA and Quantitative test, MBL International, USA, respectively). According to the manufacturer protocol, minimum detectable concentrations were determined by the manufacturer as 0.1 pg/ml for IL-12 and 12.5 pg/ml for IL-18.

Statistical Analysis
The results were analyzed using the Statistica, ver. 10.0 (StatSoft Inc, USA). For comparison of the skewdistributed variables, non-parametric Mann-Whitney U test was applied. Spearman's correlations were used to compare the associations between analyzed parameters. The level of significance was set at p≤0.05.

Clinical Characteristics of the Study Groups
The basic characteristic of children enrolled in the study is presented in Table 1. No significant difference was detected between diabetic and control group by means of age, gender, and BMI. Patients with type 1 diabetes had significantly higher levels of HbA1c, as well as CRP in comparison to the age and sex-matched healthy individuals from the control group.

Serum Concentrations of IL-12 and IL-18 in Patients with Type 1 Diabetes
Patients with type 1 diabetes showed statistically higher serum levels of IL-12 and IL-18 than children from the control group ( Table 2). Concentrations of these cytokines positively correlated with CRP level (Fig. 1a, b) as well as demonstrated a positive correlation with one another (Fig. 1c). As to the association of analyzed cytokines with HbA1c, we found correlation only in case of IL-12 (Fig. 1d). We could not found significant correlation between serum IL-18 and HbA1c (Fig. 1e).
Peripheral Blood CD4 + CD25 high FOXP3 + Regulatory T Cell Counts in Patients with Type 1 Diabetes Peripheral blood from the two groups of children was analyzed with regard to the frequency of CD4 + CD25 high T cells expressing FOXP3 transcription factor. As shown in Figs. 2 and 3, the frequencies of circulating CD4 + CD25 high FOXP3 + Tregs were significantly lower in DM1 patients in comparison to their healthy counterparts from the control group.
Our study was extended by performing the correlation analysis between frequency of peripheral blood Tregs and serum level of CRP, IL-12, and IL-18 in DM1 group. The results of this analysis are presented in Table 3. A significant inverse association between the percentage of regulatory CD4 + CD25 high FOXP3 + Tregs and serum level of CRP, IL-12, and IL-18 (R = −0.69; R = −0.42; and R = −0.66, respectively) was observed.

DISCUSSION
Chronic inflammation in type 1 diabetes has been confirmed by the number of studies [38][39][40][41]. Several proinflammatory cytokines were shown to be elevated in serum of diabetic patients with a new onset of diabetes or a longstanding disease [38,42,43]. IL-12 and IL-18 are additional two cytokines that have been shown to exert strong proinflammatory activity and that synergize in action with each other, as well as with TNF-α or IL-1 [44]. In the present study, patients with type 1 diabetes had higher serum level of IL-12 and IL-18 in comparison to the healthy subjects from the control group. What's more, Data are shown as mean ± SD *The significance between DM1 patients and healthy subjects serum levels of IL-12 and IL-18 were positively associated with CRP, which is one of the most important biomarkers of chronic inflammation [40,42]. Recently, Devaraj et al.
showed that CRP has the ability to polarize human monocytes towards proinflammatory M1 phenotype [45]. The overproduction of CRP may disrupt the balance between M1 and M2 macrophages and thus induce the production of IL-12 and/or IL-18 in an indirect way [10]. Our results demonstrating increased circulating IL-12 and IL-18 concentrations in DM1 patients are consistent with the studies done by Blazhev et al. [46], which showed that the levels of IL-12 and IL-18 have been increasing along with DM1 progression [46]. IL-12 as well as IL-18 may have a role in pathophysiology of the late microvascular complications; however, studies have yielded contradictory findings so far [24,47,48]. Besides CRP, a positive relationship between IL-12 serum level and glycosylated hemoglobin was found. However, we found no significant correlation between plasma IL-18 and HbA1c. HbA1c is an indicator of metabolic control and is measured to provide an index of average blood glucose for the previous 3 to 4 months. Esposito et al. have shown that acute hyperglycemia, contrary to chronic state, raised IL-18 level to a peak at 2 h, which returned to basal value after next hour [49]. This could explain the observed lack of correlation between concentration of IL-18 and HbA1c in our patient group. Similar findings regarding no association between serum

IL-18 concentrations and the level of glycemic control
were reported by other groups [23,50,51]. However, there are some studies with opposite results, suggesting that elevated IL-18 levels could, at least in part, contribute to the development of diabetic complications [47,48]. This is even more likely as the value of HbA1c is strongly associated with complications of diabetes [52]. DM1 patients with poor glycemic control are characterized by elevated serum level of TNF-α, so more intense inflammatory response [24]. Importantly, IL-12, as well IL-18 were shown to induce the synthesis of TNF-α which makes them complications-accelerating indirect factors [48,53]. In addition, IL-12 was found to be involved in the progression of retinopathy [8] and CRP may upregulate its synthesis [54], which is in agreement with our results. In view of the facts that control of inflammatory response is closely related to regulatory T cells, we've decided to analyze the CD4 + CD25 high FOXP3 + T cell subset in the context of IL-12 and IL-18 cytokine milleu in DM1 patients. We found a decreased percentage of these cells in peripheral blood of diabetic type 1 patients in comparison to their healthy counterparts, but what's more Fig. 2. Representative staining of circulating CD4 + CD25 high FOXP3 + T cells in patient with type 1 diabetes and healthy individual. Fresh, resting PBMCs from diabetic type 1 patients and healthy individuals were stained with antibodies against CD4, CD25, and FOXP3 molecules and analyzed using flow cytometry. The gate was set on CD4 + CD25 + lymphocytes a Based on the CD4 + CD25 + gate, cells were further gated based on CD25 and FOXP3 expression and the frequency of CD4 + CD25 high FOXP3 + cells was determined b. Fig. 3. Frequency of CD4 + CD25 high FOXP3 + T cells in peripheral blood of diabetic type 1 patients and healthy individuals. Fresh, resting PBMCs from diabetic type 1 patients (DM1) and healthy individuals were stained with anti-CD4, anti-CD25, and anti-FOXP3 mAbs and then the frequency of CD4 + CD25 high FOXP3 + cells among CD4 + lymphocytes was determined using flow cytometry. The mean percentage of cells (25/75 percentiles) in DM1 and healthy group was 1.6 (1.1/2.3) and 6 (4.5/7.2), respectively. Data were calculated with Mann-Whitney U test. Horizontal lines represent the mean frequency of cells. **The percentage of cells among peripheral blood CD4 + lymphocytes. *Indicates significant difference versus healthy group (p=0.0001).
interesting we observed the inverse association between serum level of IL-12, IL-18, and frequencies of Tregs. It is difficult to decide whether Tregs are unable to suppress production of inflammatory cytokines, or IL-12/IL-18 has detrimental effect on Tregs numbers. There are only few published mouse model studies on the association between IL-12/IL-18 cytokines and regulatory T cell subset.
A possible link between IL-12 and the induction of Tregs was suggested by Morrow et al. [54], who showed that IL-12 increased the number of splenic regulatory T cells in immunized mice [54]. Zhao et al. [55] also demonstrated that IL-12 signaling pathway may have a role in regulation of Tregs numbers. The authors showed that mice lacking β2 chain of IL-12 receptor had more CD4 + CD25 − effector T cells but fewer CD4 + CD25 + Tregs than wild-type mice upon activation [55]. Previous studies by King et al. [56] suggested that IL-12 acts directly on CD4 + CD25 − effector T cells rather than Tregs. IL-12 restores CD4 + CD25 − T cell activation, even in the presence of regulatory T cells [56]. In other studies, IL-12 was shown to induce IFN-γ production by Tregs in vitro and in vivo [57][58][59]. Induction of IFN-γ expression by Tregs upon IL-12 treatment reduced Treg numbers and expression of FOXP3 transcription factor [57,58]. Recent study by Zhao et al. [59] showed that IL-12 increased IL-2R expression on effector T cells, diminished its expression on Tregs, and decreased IL-2 production by effector T cells [59]. IL-2 is essential for the maintenance of regulatory T cells [30]. Low level of IL-2 may contribute to reduced Treg proliferation, which perhaps leads to their diminished numbers.
Similarly to IL-12, IL-18 was shown to increase the ratio of effector T cells to Tregs [60]. However, there are contradictory results showing that IL-18 is essential for inducing antigen-specific regulatory T cells and oral tolerance [61]. In contrast to these data, Zeiser et al. [62] demonstrated that IL-18 is not required for Treg expansion [62]. One of the know feature of IL-18 is its ability to stimulate the Th17 cells [10]. Th17 cells are involved in the pathogenesis of inflammatory and autoimmune diseases, and they also predominate in patients with type 1 diabetes, which was shown by us and others [63,64]. Upregulated Th17 immune response may have impact on Treg numbers.
In view of the limited data regarding the relation between proinflammatory IL-12/IL-18 and regulatory T cells, further studies in humans are needed to properly verify this. However, the results of our analysis lead us to conclude that patients with type 1 diabetes have enhanced inflammatory response, which is manifested by increased values of CRP, HbA1c, IL-12, and IL-18. These mediators of inflammation may have direct or indirect impact on regulatory T cell subset, which may contribute to their reduced frequency in peripheral blood.
In the larger context, the data presented by us are mainly observation, and future in vitro studies are needed to determine the impact of IL-12 and IL-18 on Tregs quantitative as well as qualitative changes.

ACKNOWLEDGMENTS
This work was supported by The State Committee for Scientific Research ST28 (Medical University of Gdańsk).
Disclosures. The authors declare that they have no competing financial interests.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. The Spearman test was used to calculate the strength of correlation *The percentage of cells among peripheral blood lymphocytes