Loss of the balance between CD4+Foxp3+ regulatory T cells and CD4+IL17A+ Th17 cells in patients with type 1 diabetes

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Loss of the balance between CD4+Foxp3+ regulatory T cells and CD4+IL17A+ Th17 cells in patients with type 1 diabetes

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Monika Ryba-Stanisławowska a,⇑, Maria Skrzypkowska a, Małgorzata Mys´liwiec b, Jolanta Mys´liwska a a b

´ sk, De˛binki 1, 80-210 Gdan ´ sk, Poland Department of Immunology, Medical University of Gdan ´ sk, 80-210 Gdan ´ sk, Poland Diabetological Department, Clinic of Pediatrics, Hematology, Oncology and Endocrinology, Medical University of Gdan

a r t i c l e

i n f o

Article history: Received 13 September 2012 Accepted 24 January 2013 Available online xxxx

a b s t r a c t The presence of low-grade chronic inflammation is a known feature of long standing diabetes type 1. Recently, two T cell subsets, that may contribute to the disease progression are under investigation. These are Treg cells, which are specialized T cell subset, that controls the activity of autoreactive and inflammatory cells and Th17 cells which are involved in the pathogenesis of inflammatory and autoimmune diseases. The balance between Treg and Th17 controls inflammation and is responsible for the proper function of the immune system. An decrease of Tregs and/or increase of Th17 may induce local inflammation, which in turn may hasten the development of diabetic complications. In the present study, we have demonstrated that the Treg/Th17 balance was broken in patients with diabetes type 1 and might contribute to the progression of microvascular angiopathy. Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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[14,15] the majority of papers provide data on the Tregs decrease as well as their impaired function, which could be the result of inflammatory environment present in DM1 patients [5,16–20]. As the balance between Th17 and Tregs may play important role in development of autoimmune/inflammatory diseases, the aim of this study was to find out if this was broken in patients with type 1 diabetes and how it contributes to the progression of diabetic microvascular complications.

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2. Subjects and methods

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2.1. Subjects

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Peripheral blood samples from 32 patients aged 16.1 (±3.5) years with long standing diabetes type 1 and 20 age matched healthy controls (17.6 ± 1.3 years) were collected. The mean duration of diabetes was 6.43 (±4.02) years. The patients were recruited from The Diabetic Department, Clinic of Pediatrics, Hematology, Oncology and Endocrinology Medical University of Gdan´sk. Type 1 diabetes was defined according to the criteria of the American Diabetes Association [21]. Patients with coexisting autoimmune, chronic and acute inflammatory diseases were excluded from the study. In all examined patients the C-peptide levels were below 0.5 ng/ml. All patients were treated with humanized insulin. A blood glucose level was taken at the time of sampling along with biochemical measurement of renal function, lipid status, C-reactive protein (CRP) and glycosylated hemoglobin (HbA1c).

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1. Introduction

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Diabetes type 1 (DM1) is an autoimmune disease caused by T cell-mediated destruction of insulin producing cells. So far, it was thought that the disease is associated only with an imbalance between the Th1 and Th2 components of the immune system. Nowadays, however, two T cell subsets, that may contribute to the disease progression are under investigation. These are Treg cells, which are specialized T cell subset, that controls the activity of autoreactive and inflammatory cells, protecting the inflamed tissue against deleterious immune response [1–5]. The other side of the coin is represented by Th17 cells which are involved in the pathogenesis of inflammatory and autoimmune diseases [6–9]. The elevated frequencies of Th17 cells were observed in patients with rheumatoid arthritis (RA) [10], inflammatory bowel disease [11], multiple sclerosis [12] and more. The role of Th17 cells in autoimmune diseases is thought to be associated with their ability to promote inflammatory cytokines and chemokines production by macrophages and other phagocytes [13]. The activity of inflammatory cells, including Th17 subpopulation is regulated by Tregs which include naturally arising CD4+Foxp3+ cells. Recent data clearly indicate the importance of this cell subset in preventing autoimmune diseases. Even though there are studies, which show that Tregs do not appear to be deficient in type 1 diabetic subjects

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⇑ Corresponding author. Fax: +48 58 3491430. E-mail address: [email protected] (M. Ryba-Stanisławowska).

0198-8859/$36.00 - see front matter Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2013.01.024

Please cite this article in press as: Ryba-Stanisławowska M et al. Loss of the balance between CD4+Foxp3+ regulatory T cells and CD4+IL17A+ Th17 cells in patients with type 1 diabetes. Hum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.01.024

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Table 1 General clinical characteristics of each analyzed group. Group

Age (years)

Disease duration (years)

HbA1c

Albumin excretion rate (mg/24 h)

CRP (mg/l)

DM1 (n = 32) Healthy (n = 20) p⁄

13.1 ± 3.5 17.6 ± 1.3

6.43 ± 4.02 –

8.62 ± 2.07 –

16.1 ± 7.03 –

2.49 ± 1.95 0.85 ± 0.28 0.0005

Data are shown as mean ± SD. The significance between DM1 patients and healthy individuals.

⁄

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The control group consisted of age and sex matched healthy individuals recruited during control visits in outpatient clinic. No signs of autoimmune, chronic, inflammatory, neoplastic disease at the time of sampling and no evidence of DM1 in their families was disclosed as confirmed by medical records, laboratory examination and laboratory tests. The study followed the principles of the Declaration of Helsinki and was approved by The Ethics Committee of The Medical University of Gdan´sk.

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2.2. Cell isolation and culture

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Heparinised venous blood samples (4–6 ml) were collected aseptically into the tubes and used to isolate peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMC) were separated by density gradient preparation over Ficoll-Uropoline. Mononuclear cells at the interface were carefully transferred into a Pasteur Pipette, then treated with Red Blood Cell (RBC) Lysis Buffer (BioLegend, USA) and washed twice in PBS. For Th17 analysis cells were suspended at a density of 2  106 cells/ml and cultured in RPMI 1640 supplemented with 5% heatinactivated fetal calf serum (FCS). Cultures were stimulated with 50 ng/ml of phorbol myristate acetate – PMA (Sigma, USA) plus 1 ll/ml of ionomycin (Sigma, USA) for 4 h in the presence of 1 ll/ ml of monensin (BioLegend, USA). After 4 h of culture in 37 °C with 5% CO2 the contents of the wells were transferred to 5 ml polystyrene round bottom test tubes (BD Bioscience, USA) and centrifuged at 200g for 5 min. For Treg analysis, fresh, resting PBMCs were suspended in 5 ml polystyrene round bottom test tubes (BD Bioscience, USA) at a density of 1  106 cells per 1 ml of RPMI 1640 and centrifuged at 200g for 5 min. Cell pellets were then destined for flow cytometric staining.

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2.3. Flow cytometric staining and analysis

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Before staining cells were washed with Cell Staining Buffer (BioLegend, USA). Cells were stained with anti-CD4 antibody (IgG1, j mouse Pe/Cy5, Clone RPA-T4, BioLegend, USA). After 20 min incubation at room temperature, cells were washed and stained for intracellular expression of Foxp3 in case of Treg and IL-17A in case of Th17 cells. The following monoclonal antibodies were used for Treg and Th17 intracellular staining respectively: anti-Foxp3 (IgG1, j mouse Alexa-Fluor 488 or PE, Clone 206D, BioLegend, USA) and anti-IL17A (IgG1, j mouse FITC, Clone BL168, BioLegend, USA). Intracellular staining for Foxp3 and IL-17A was performed with ready-to-use kits according to the manufacturers suggestions. (BioLegend, USA). Expression of cell surface and intracellular markers was assessed using flow cytometry (LSRII, Becton Dickinson, USA) after gating on live cells determined by scatter characteristics. Positive signal for each staining was established using appropriate isotype controls. The expression of Foxp3 in the CD4+Foxp3+ as well as expression of IL17-A in the CD4+IL-17A+ gates were quantified by determining mean fluorescence intensity (MFI). It was quantified as a ratio of mean fluorescence intensity for Foxp3

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Fig. 1. Representative staining of circulating CD4+IL17A+ and CD4+Foxp3+ T cells in patients with type 1 diabetes. (A) PBMCs from diabetic type 1 patients were cultured and stimulated as described in Methods and then stained with antibodies against CD4 and IL-17A. The percentage of CD4+IL17A+ T cells was determined by flow cytometry. Analyzing CD4+IL17A+ cells, dot plots representing anti-CD4 versus SS were carried out to establish CD4+ and CD4 lymphocyte gates. Then, the antiCD4 versus IL-17A from CD4+ gate dot plot was generated and the frequency of Th17 cells was determined. (B) Fresh, resting PBMCs from diabetic type 1 patients were stained with antibodies against CD4 and Foxp3 molecules and analyzed using flow cytometry. The gate was set on CD4+ lymphocytes according to forward scatter and staining with CD4 Pe/Cy5 and then the percentage of CD4+Foxp3+ T cells in CD4+ gate was determined.

or IL-17A to MFI for appropriate isotype control. Data were analyzed by FACSDiva 6.0 Software (Becton Dickinson, USA).

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2.4. Statistical analysis

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All statistical analyses were performed using Statistica 8.0 (StatSoft, Inc USA). For comparison of the skew-distributed variables non-parametric U Mann Whitney tests was applied. Spearman’s correlations were used to compare the associations between analyzed parameters. Multiparametric regression analyses were also carried out. The level of significance was set at p < 0.05.

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a

percentage of CD4 Foxp3 cells (%) absolute number of CD4+Foxp3+ cells (cells/mm3) percentage of CD4+IL17A+ cells (%)a absolute number of CD4+IL17A+ cells (cells/mm3)

DM1 (n = 32)

Control group (n = 20)

p

1.15 (0.7/2.0) 0.12 (0.07/0.18) 2.25 (1.3/4.2) 1.0 (0.64/1.96)

4.75 (2.8/8.25) 0.57 (0.38/0.1) 1.1 (0.4/2.1) 0.48 (0.2/1.08)

0.0001 0.00002 0.00001 0.00001

a The percentage of cells among peripheral blood lymphocytes. The results are shown as median and 10./90. percentile. All the differences were calculated by the U-Mann Whitney test.

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3. Results

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3.1. Basic information about patients

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DM1 patients had higher values of CRP in comparison to the age and sex-matched healthy young individuals from the control group. The patients did not have microalbuminuria (Table 1).

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3.2. The Treg/Th17 status in children with type 1 diabetes

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3.2.1. Treg frequencies are decreased and Th17 are increased in patients with DM1 As shown in Fig. 1 and Table 2 the frequencies as well as absolute number of CD4+Foxp3+ regulatory T cells were significantly lower in patients with type 1 diabetes than in healthy individuals. In case of CD4+IL17A+ T cells we found inverse dependence. Children with type 1 diabetes mellitus were characterized by higher number and percentage of the CD4+IL17A+ T cells than their healthy counterparts (Table 2 and Fig. 1). Interestingly, in case of DM1 patients, the intensity of Foxp3 expression correlated with higher frequency of CD4+Foxp3+ Tregs (Fig. 2A; r = 0.7) as well as with lower intensity of IL17A expression (Fig. 2B; r = [ 0.38]). We performed the Sperman’s rank correlation between peripheral Treg and Th17 frequencies of DM1 patients and found these two cell lineages were significantly inversely correlated (Fig. 3A and B; r = [ 0.32]), suggesting the existence of disrupted balance between Treg and Th17 cells in diabetic type 1 patients. Such correlation was not seen in healthy individuals from the control group (r = 0.01). It has been recently shown that not all Th17 cells are pathogenic, which means that some of them may have regulatory

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phenotype [22–24]. Therefore, we have decided to check if Th17 cells from children with type 1 diabetes express IL17A and Foxp3 simultaneously. We did not however find this kind of population in the peripheral blood of analyzed DM1 patients (Fig. 4).

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3.3. The relationship between Treg/Th17 cells and factors that influence the development of late microvascular complications in type 1 diabetic patients

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Th17 cells are thought to be associated with promoting the inflammation, while Tregs control/suppress the production of inflammatory cytokines. As inflammation proceeds, the risk for vascular complications increases. This risk depends on both the duration and the severity of hyperglycemia. In view of these facts we have decided to find out if there is association between Treg and Th17 cell subsets and factors that play role in development of diabetic complications, which are duration of the disease and serum levels of HbA1c and CRP (Table 3).We found negative, statistically significant correlations between expression of Foxp3 in CD4+Foxp3+ Tregs defined as MFI as well as frequency of these cells and duration of type 1 diabetes. The negative statistically significant correlation was also found between MFI of Foxp3 in CD4+Foxp3+ cells and serum level of HbA1c. When analyzing Th17 cells, the only statistically significant correlation was found between the expression of IL17A defined as MFI and CRP value. The higher CRP level, the greater MFI of IL17A in CD4+IL17A+ T cells, was observed (Table 3). To find out if the frequency of Tregs depends on age in analyzed patients we performed the multiple regression analysis which showed no association between the level of CD4+Foxp3+ regulatory T cells and age in type 1 diabetic group (b = [ 0.06]; p = 0.2).

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Fig. 2. Correlation of Foxp3 expression with the frequency of CD4+Foxp3+ Treg cells and the expression of IL17A in CD4+IL17A+ Th17 cells in analyzed DM1 subjects. During the cytometric analysis of CD4+Foxp3+ and CD4+IL17A+ T cells the relative expression of Foxp3 in CD4+Foxp3+ and IL17A in CD4+IL17A+ T cells was quantified as a ratio of mean fluorescence intensity (MFI) for Foxp3 or IL-17A to MFI for appropriate isotype control. The Spearman test was used to calculate the strength of correlation. (A) The correlation between the Foxp3 expression defined as MFI and the frequency of CD4+Foxp3+ T cells in DM1 subjects. Spearman correlation; r = 0.7; p < 0.05. (B) The correlation between the Foxp3 expression defined as MFI and intensity of IL17A expression in DM1 subjects. Spearman correlation; r = [ 0.38]; p < 0.05.

Please cite this article in press as: Ryba-Stanisławowska M et al. Loss of the balance between CD4+Foxp3+ regulatory T cells and CD4+IL17A+ Th17 cells in patients with type 1 diabetes. Hum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.01.024

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Fig. 3. Disrupted balance between CD4+Foxp3+ Tregs and CD4+IL17A+ Th17 cells in DM1 patients. The CD4+Foxp3+ and CD4+IL17A+ T cells were analyzed by flow cytometry as described in Methods. The Spearman test was used to calculate the strength of quantitative correlation between these two subsets. (A) Graph showing the frequency of CD4+Foxp3+ and CD4+IL17A+ T cells in DM1 subjects. Data are presented as medians and 25./75. percentile. The median frequency of CD4+Foxp3+ and CD4+IL17A+ T cells among peripheral blood lymphocytes was 1.15 and 2.25% respectively. (B) A significant negative correlation between the frequency of CD4+Foxp3+ and CD4+IL17A+ T cells in DM1 subjects; r = [ 0.32]; p < 0.05.

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As it was mentioned, regulatory T cells play important role in controlling the activity of inflammatory cells such as monocytes and neutrophils. Monocytes and tissue macrophages are the source of proinflammatory cytokines engaged in pathogenesis of diabetic retinopathy. The onset and progression of diabetic retinopathy directly depends on increasing glycemia as well as long-term exposure of retinal endothelial cells to elevated glucose. When multiplying two measurements (the value of the HbA1c level by the years of disease duration) we obtain the RD (retinopathy development) value, which relates to the risk of retinopathy development [25]. The multiple regression analysis, with a change of RD as the dependent variable, showed that HbA1c (b = 0.33; p = 0.0001) and duration of the disease (b = 0.90; p = 0.0003) are the factors that significantly increased the RD value in patients with diabetes type 1. RD value was inversely correlated with the expression of Foxp3 in CD4+Foxp3+ Tregs as well as their frequency (Fig. 5; r = [ 0.49] and r = [ 0.3] respectively).

Fig. 4. Flow-cytometic analysis of Th17 cells with regulatory phenotype. PBMCs from diabetic type 1 patients were cultured and stimulated as described in Methods and then stained with antibodies against CD4, IL17A and Foxp3. Analyzing Th17 cells with simultaneous expression of Foxp3, dot plots representing anti-CD4 versus FSC were carried out to establish CD4+ and CD4 lymphocyte gates. Then, the following dot plots were generated: anti-CD4 versus IL17A from CD4+ gate (A). After gating on CD4+IL17A+ cells, the frequency of CD4+IL17A+ Foxp3+cells was determined (B).

4. Discussion

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The balance between Treg and Th17 controls inflammation and is responsible for the proper function of the immune system. An decrease of Tregs and/or increase of Th17 may induce local inflammation, which in turn may hasten the development of diabetic complications. In this study, we demonstrate that the Treg/Th17 balance is broken in patients with diabetes type 1. We found that the frequency of Treg cells was significantly lower in patients with DM1 than in healthy individuals, which is consistent with previous studies done by us and others [5,14–17]. In contrast, the frequency of Th17 cells in type 1 diabetic patients was significantly higher in comparison to healthy subjects from the control group. In the current study the observed Treg/Th17 balance was specific to Th17 cells which levels were highly increased as compared to regulatory T cell numbers. Similarly, increased Th17 cells in the peripheral blood of children with diabetes has been recently reported by Honkanen et al., Marwaha et al., and Bradshaw et al. [26,27,8].

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Table 3 The results of the correlation analysis between Treg/Th17 subsets and factors that influence the development of late microvascular complications in type 1 diabetic patients.

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MFI of Foxp3 in CD4 Foxp3 T cells The percentage of CD4+Foxp3+ cells (%)a MFI of IL17A in CD4+IL17A+ T cells The percentage of CD4+IL17A+ T cells (%)a

HbA1c

Duration of the disease

CRP

r = [ 0.42] p < 0.05 r = [ 0.28] p > 0.05 r = 0.29 p > 0.05 r = 0.28 p > 0.05

r = [ 0.43] p < 0.05 r = [ 0.34] p < 0.05 r = 0.21 p > 0.05 r = 0.05 p > 0.05

r = [ 0.15] p > 0.05 r = [ 0.22] p > 0.05 r = 0.34 p < 0.05 r = 0.12 p > 0.05

The Spearman test was used to calculate the strength of correlation a the percentage of cells among peripheral blood lymphocytes; MFI – Mean Fluorescent Intensity; CRP – Creactive protein.

Fig. 5. Correlation of RD value with Foxp3 expression and frequency of Tregs in DM1 patients. The RD value describing risk of retinopathy development was calculated by multiplying the value of the HbA1c level by the years of type 1 diabetes duration. It was then correlated with the expression (defined as MFI) of Foxp3 in CD4+Foxp3+ Tregs as well as the frequency of these cells, determined by flow cytometry, in peripheral blood of DM1 patients. The Spearman test was used to calculate the strength of correlation. (A) The correlation between the RD and MFI of Foxp3 in CD4+Foxp3+ Tregs (r = [ 0.49]; p < 0.05). (B) The correlation between the RD and the frequency of CD4+Foxp3+ Tregs (r = [ 0.3]; p < 0.05).

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The authors analyzed patients with new onset (6–12 months) and long term (7–12 years) diabetes, which implies that our group is comparable (2–10 years). However, our results do not support the findings of Ferraro et al., who did not find signs of upregulated Th17 immunity in peripheral blood of diabetic patients [28]. The analyzed group of patients had the disease for an average of 26 years. The authors suggest that the level of peripheral Th17 cells together with diabetes duration decreases and Th17 cells accumulate in the target organ.

We found that there was an inverse correlation between the frequency of CD4+Foxp3+ and Th17 CD4+IL17A+ T cells in diabetic group. Additionally, the expression of Foxp3 in CD4+Foxp3+ Tregs was inversely correlated with the expression of IL17A in CD4+IL17A+ Th17 cells. Foxp3 is a key transcription factor of Tregs that may act as repressor or activator of certain genes [29]. It was shown that Th17 immune response in mice is restrained by binding of Foxp3 to Stat3 – a key factor in the initiation of Th17 differentiation [30]. In another study, the authors showed that overexpression of Foxp3 resulted in a strong reduction of IL17A gene expression [31]. Foxp3 does not directly bind to IL17A gene promoter but it acts through binding to RORct transcription factor which is sufficient for activation of IL17A transcription [31]. So, if overexpression of Foxp3 suppresses RORct-mediated IL17A mRNA transcription, then too low expression of Foxp3 probably is not able to maintain the Treg/Th17 balance. In view of these data, one can speculate that the impaired expression of Foxp3 may lead to defective control of Th17 cells. It is unclear why the expression of Foxp3 in Tregs of diabetic patients is downregulated. It can be modulated by inflammatory conditions seen in these patients, such that they are not able to control the activity of inflammatory cells [5,16,32]. Foxp3 expression is suppressed and thus RORct induces the CD4+IL17A+ T cell subset. The Treg/Th17 balance is shifted towards Th17 cells promoting inflammatory response, often harmful to the host. This in vivo connection between Tregs and Th17 has been supported by several authors. It was shown that under inflammatory conditions the suppressive function of Tregs may be lost and these cells start to produce IL-17 [24,33–35].On the other hand, Th17 cells can differentiate into ‘regulatory’ Th17 cells, which protect from inflammation [22,23,35]. It was shown that IL17 is produced early during antigenic stimulation and at later time points when anti-inflammatory mechanisms are induced, it’s production is suppressed [23]. In case of type 1 diabetes, which is characterized by a chronic inflammatory response, one can suspect that the suppression of Th17 activity may not occur. Indeed, in the current study when analyzing Th17 cells for their regulatory phenotype, we did not find cells with dual capacity to express Foxp3 and IL17A simultaneously. Such cells may not be present in analyzed individuals or a second explanation could be that the Foxp3+IL-17+ cells represent a relatively minor subset in the human peripheral blood and these cells reside within the memory CD4+CD25highDR population [33]. Moreover, it was shown that these Foxp3+IL-17+ cells also express CCR6 and RORct transcription factor, which define Th17 subpopulation [36]. This minor cell population probably takes part in immune defence against microbes while controling autoimmunity and inflammatory response. If non-pathogenic Th17 exist in DM1 patients, additional studies with more specific markers are required to characterize them. Chronic inflammation can lead to development of microvascular diabetic complications, one of which is retinopathy [37]. We demonstrated that there is an association between Treg/Th17 cell subsets and factors that play role in development of diabetic complications. The frequency of Th17 cells positively correlated with CRP level in patients with DM1. Patients with higher percentage of these cells had higher serum CRP, which elevated level may +

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reflect an inflammatory state. Inflammatory response may be associated with more intense Th17 response [13]. Similar results were also obtained in patients with rheumatoid arthritis [10,38]. We also found that the level of HbA1c, which is an indicator of metabolic control in diabetic patients, was in significant relation with the expression of Foxp3 in CD4+Foxp3+ Tregs. Higher HbA1c is associated with more intense inflammatory response and this may further cause quantitative and/or qualitative disruption in regulatory T cell population. In addition, patients with longer disease duration had lower frequency of Tregs in peripheral blood than patients with an early disease. Interestingly, their CD4+Foxp3+ T cells showed lower Foxp3 expression in comparison to Tregs from patients with juvenile diabetes mellitus of short duration. Longer disease is associated with longer chronic inflammation, and so we can suspect that it may have impact on the expression of Foxp3 in Tregs. The incidence of retinopathy is associated with the duration of diabetes as well as the degree of metabolic control [25,39]. When we multiply these two values we get the RD which describes the risk of retinopathy development. Our studies revealed that children with lower frequency of Tregs in their blood as well as lower expression level of Tregs’ Foxp3 are more susceptible to develop retinopathy as diabetic complication. It is well known that the pathogenesis of diabetic retinopathy includes glucose-mediated microvascular damage, but how this can be related to Tregs? Prolonged exposure to hyperglycemia leads to the formation of advanced glycation end products (AGEs). These are involved in the tissue damage associated with diabetic complications. AGEs and high glucose can induce the expression of proinflammatory cytokines produced mainly by monocytes and tissue macrophages [40–43]. As inflammation proceeds, the Treg subset may become more and more impaired/exhausted, the control over activity of inflammatory cells is lost and thus the risk for vascular complications increases. Our work support the idea that type 1 diabetic patients have an unbalanced Treg/Th17 cell ratio, which may contribute to the accelerated progression of diabetic micro-vascular complications. However, more studies are needed to determine the true nature of these two cell subsets. Elucidation of strategies for enhancing Treg and/or attenuation of Th17 subsets will have important implications for therapy of diabetes.

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Disclosures

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The authors declare that they have no competing financial interests.

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Acknowledgments

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This work was supported by the National Committee for Scientific Research grant no. N N402 0975 33 and The State Committee for Scientific Research ST28 (Medical University of Gdan´sk).

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Please cite this article in press as: Ryba-Stanisławowska M et al. Loss of the balance between CD4+Foxp3+ regulatory T cells and CD4+IL17A+ Th17 cells in patients with type 1 diabetes. Hum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.01.024

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Please cite this article in press as: Ryba-Stanisławowska M et al. Loss of the balance between CD4+Foxp3+ regulatory T cells and CD4+IL17A+ Th17 cells in patients with type 1 diabetes. Hum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.01.024