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IL-33 improves the suppressive potential of regulatory T cells in patients with type 1 diabetes
diabetes research and clinical practice
1 2 8 (2 0 17 ) 6 7–73
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locat e/dia bre s
IL-33 improves the suppressive potential of regulatory T cells in patients with type 1 diabetes Monika Ryba-Stanisławowska a,*, Laura Buksa a, Agnieszka Brandt b, Ulana Juhas a, Małgorzata Mys´liwiec b a b
Department of Immunology, Medical University of Gdan´sk, Poland Clinic of Pediatrics, Department of Diabetology and Endocrinology, Medical University of Gdan´sk, Poland
A R T I C L E I N F O
A B S T R A C T
Article history:
Aims: The presented study was aimed to analyze the influence of IL-33 on regulatory T cells
Received 30 January 2017
(Tregs) suppressive potential in patients with type 1 diabetes.
Received in revised form
Methods: We analyzed the ability of IL-33 treated Tregs to inhibit the production of IFN-c by
19 March 2017
effector T lymphocytes in an in vitro co-culture. The study group consisted of 22 patients
Accepted 7 April 2017
with type 1 diabetes and 12 age and sex-matched healthy individuals.
Available online 13 April 2017
Results: Our findings revealed that in vitro IL-33 treatment of Tregs derived from patients with type 1 diabetes resulted in quantitative as well as qualitative changes in this cell pop-
Keywords: Diabetes type 1 IL-33
ulation, confirming immunoregulatory features of IL-33. Conclusion: IL-33 could be considered as a potential therapeutic tool in adoptive therapies of type 1 diabetes.
Regulatory T cells
1.
Introduction
Deregulation of the immune system is an important factor for the occurrence of type 1 diabetes (DM1) as well as progression of its complications. CD4+CD25highFoxp3+ human regulatory T cells (Tregs) are essential for the active suppression of autoimmunity. They also exert control over excessive inflammatory response [1–3]. The capacity of Tregs to suppress the immune response may strongly depend on the level of Foxp3 expression, which is a transcription factor particularly responsible for their function and development [1]. In some autoimmune diseases, including rheumatoid arthritis, multiple sclerosis or type 1 diabetes, a defective suppressive function of Tregs was observed [4–6]. Some studies demonstrated that DM1 subjects have a defective peripheral Treg suppressive ability [7–10]. In contrast, others did not report any impairments in Treg suppressive function [11,12]. It is possi-
Ó 2017 Elsevier B.V. All rights reserved.
ble that Tregs in DM1 patients may have genetic defect, which leads to their compromised function [1,13,14] or that some reduced suppression may in part have resulted from resistance of effector lymphocytes to suppression mediated by Tregs [15,16]. The suppressive Treg potential may also depend on the IL-2 availability [17,18] or Foxp3 expression at a single cell level [11,19]. More recently, there has been increasing interest in the immunoregulatory properties of IL-33, which belongs to the IL-1 cytokine family, and has the ability to induce Th2 immune response, required to dampen overactive Th1 activity [20,21]. As to its direct effect on population of regulatory T cells, this cytokine was tested in animal models, in which their suppressive activity was compromised. In these studies, IL-33 has proven to be efficient in promoting Tregs regulatory phenotype [22–26].
* Corresponding author at: Department of Immunology, Medical University of Gdan´sk, Dez binki 1, 80-211 Gdan´sk, Poland. E-mail address: [email protected] (M. Ryba-Stanisławowska). http://dx.doi.org/10.1016/j.diabres.2017.04.011 0168-8227/Ó 2017 Elsevier B.V. All rights reserved.
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diabetes research and clinical practice
Mechanisms of action of IL-33 in patients with type 1 diabetes are poorly understood and still there are little data on the influence of this cytokine on diabetic regulatory T cells. Previously, we have shown that IL-33 has in vitro ability to increase frequencies of Treg cells [27] Here, continuing our research we would like to show IL-33 in vitro effect on the function of regulatory T cells derived from a group of patients with type 1 diabetes.
2.
Methods
2.1.
Human subjects
The study group consisted of 22 patients with type 1 diabetes and 12 healthy individuals. Mean age of the patients and disease duration was 14.7 ± 2.5 and 5.3 ± 3.6 years respectively. The patients were recruited from Clinic of Pediatrics, Department of Diabetology and Endocrinology, Medical University of Gdan´sk. Type 1 diabetes was defined according to the criteria of the American Diabetes Association [28]. A blood glucose level was taken at the time of sampling along with biochemical measurement of renal function, lipid status, and glycosylated hemoglobin (HbA1c). In all examined patients the Cpeptide levels were below 0.5 ng/ml. All patients were treated with humanized insulin. The study followed the principles of the Declaration of Helsinki and was approved by The Ethics Committee of The Medical University of Gdan´sk. The control group consisted of age and sex-matched healthy individuals with no signs of autoimmune, chronic, inflammatory, neoplastic disease at the time of sampling and no evidence of DM1 in their families. A summary of the clinical characterististics of analyzed subject groups is presented in Table 1.
2.2.
Reagents and antibodies
The following monoclonal antibodies (mAbs) were used in cytometric studies (fluorochromes and clones in the brackets): anti-CD3 (PE-Cy7, SK7), anti-CD4 (APC or PE-Cy5 RPAT4), anti-CD25 (PE, 2A3), anti-CD127 (PE-Cy7, AO19D5). Items were purchased from BD Biosciences, USA or BioLegend, USA. Intracellular staining for Foxp3 (FITC, 206D) and IFN-c (PerCP-Cy.5.5, B27) was performed with ready-to-use kits according to the manufacturers suggestions. (BioLegend, USA and BD Biosciences, USA, respectively). Additionally, the following reagents were used for stimulation of cell cultures: anti-CD3 (clone UCHT1, eBioscience, USA), anti-CD28 (clone CD28.2, eBioscience, USA) antibodies, rIL-33 (BioLegend, USA), rIL-2 (BioLegend, USA), PMA (Sigma, Poland), ionomycin (Sigma, Poland) and GolgiStop (BD Biosciences, USA).
2.3.
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procedure with a negative selection of CD4+CD127low cells followed by positive selection of CD25+ cells. CD4+CD127low T cells were isolated from whole blood using RosseteSep Human CD4+CD127low Regulatory T Cell Pre-Enrichment Cocktail (Stem Cell, USA). Then cells expressing CD25 molecule were isolated from enriched CD4+CD127low fraction by positive selection with an EasySep Human CD25 Positive Selection Kit (Stem Cell, USA). The purity of isolated cells was assessed by flow cytometry. Starting with fresh peripheral blood, the CD4+ cell content of the enriched fraction was typically 94 ± 5% and the purity of selected CD4+CD127lowCD25+ Tregs, ranged from 86% to 96%. Magnetically selected CD4+CD127lowCD25+ T cells were then cultured on the round bottom 96-well plates. For each study individual the isolated Tregs were placed in two wells. First, control well (starting point) was designated ‘nonstimulated’ and second ‘stimulated’. Cells in both wells were treated with anti-CD3 and anti-CD28 antibodies, both at a concentration 5 lg/ml. Wells designated as ‘stimulated’ were additionally treated with recombinant human IL-33 at a concentration 25 ng/ml. Cells in both wells were also stimulated with recombinant human IL-2 (100 U/ml). Cultures were incubated at 37 °C with 5% CO2 for 24 h. At the same time, from each study individual, the autologous PBMCs were isolated by density gradient preparation over Histopaque-1083 (Sigma) and cultured for 24 h in RPMI medium supplemented with 5% heat-inactivated FSC.
2.4.
Quality check of stimulated Tregs
The quality of stimulated Tregs was checked after 24 h of culture. It consisted of intracellular Foxp3 staining and IFN-c suppression assay. The assay started from making cocultures of Tregs with autologous PBMCs in 1:1 ratio. For each co-culture 50,000 isolated Tregs as well as PBMCs were used. The stimulation consisted of 50 ng/ml of phorbol 12myristate 13-acetate, 500 ng/ml of ionomycin and 2 ml/ml of GolgiStop (BDBiosciences, USA). After 5 h the cells were stained to CD3+CD4+IFN-c+ phenotype and analyzed on flow cytometer.
2.5.
Flow cytometric analysis
Cells were harvested and then stained with fluorochrome labeled monoclonal antibodies. 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 an appropriate isotype control. Data were analyzed by FACSDiva 6.0 Software (Becton Dickinson, USA).
Isolation and culture of CD4+CD127lowCD25+ Tregs 2.6.
Venous blood samples were collected aseptically into the test tubes containing heparin and used to isolate CD4+CD127lowCD25+ Tregs by a two-step magnetic separation
Statistics
All statistical analyses were performed by Statistica 12.0 (StatSoft, Inc USA) using either the nonparametric Wilcoxon test
0.7 ± 0.11 – 12.5 ± 3.34 – 10.0 ± 1.96; [84.9 ± 22.6] – 21.1 ± 4.0 18 ± 2.0
2.06 ± 0.5 0.65 ± 0.1
Serum Creatinine level (mg/dl) Serum Albumin level (g/l) HbA1c (%); (mmol/mol) BMI (kg/m2)
69
or the U Mann-Whitney test with p values less than 0.05 defined as significant.
3.
Results
Magnetically selected CD4+CD127lowCD25+ T cells were cultured with or without IL-33 for 24 h. After this time cells were stained to CD4+CD25highFoxp3+ phenotype and analyzed on flow cytometer. The analysis revealed that in diabetic group, after IL-33 treatment, there was a significant increase in the percentage of CD4+CD25high T cells that were Foxp3+ (Figs. 1 and 2). In the control, healthy group there was no significant difference in the frequency of isolated Tregs after treatment with IL-33 in comparison to resting, nonstimulated cells (Fig. 2). When comparing patients with type 1 diabetes and healthy individuals we found lower frequency of CD4+CD25highFoxp3+ T cells in diabetic group in comparison to healthy subjects at a starting point (cells without IL-33 stimulation – ‘nonstimulated’ cells) as well as after IL-33 treatment (stimulated cells) (Fig. 2).
3.2. Qualitative analysis of isolated regulatory T cells after in vitro IL-33 treatment Magnetically selected CD4+CD127lowCD25+ regulatory T cells after 24 h in vitro culture with or without IL-33 were added to isolated autologous PBMCs and IFN-c suppression assay was performed in order to determine the ability of Tregs to suppress the production of IFN-c by effector T lymphocytes. The analysis revealed that in patients with type 1 diabetes IL-33 treated Tregs more efficiently suppressed the production of IFN-c by effector T lymphocytes compared to Tregs not stimulated with IL-33 (Fig. 3). The percentage of CD3+CD4+IFN-c+ effector T lymphocytes among PBMCs was lower when Tregs were treated with IL-33 in comparison to non IL-33-stimulated Tregs. In the healthy, control group the frequency of effector T lymphocytes lowered only when Tregs were added to the culture of PBMCs and practically did not changed upon IL-33 stimulation (Fig. 3).
Data are shown as mean ± standard deviation.
14.7 ± 2.5 15.2 ± .2.3 DM1 (n = 22) Healthy (n = 12)
5.3 ± 3.6 –
4.
Age (years)
Duration of diabetes (years)
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3.1. Quantitative analysis of isolated regulatory T cells after in vitro IL-33 treatment
Group
Table 1 – Clinical characteristics of analyzed subject groups.
CRP (mg/l)
diabetes research and clinical practice
Discussion
The role of IL-33 in type 1 diabetes is not well defined. Little is known about its potential involvement in pathogenesis of this autoimmune and inflammatory disease. Moreover, there are no data on IL-33 influence on regulatory T cells isolated from DM1 patients’ blood. Comparing magnetically sorted Treg cells in unstimulated and IL-33 stimulated cultures one can assume that this cytokine exerts positive effect on patients’ derived Foxp3+ Tregs by increasing their numbers. Our previous study has revealed that in vitro IL-33 treatment of PBMCs from patients with type 1 diabetes acts on quantitative features of regulatory T cells by increasing their numbers as well as the expression of Foxp3 transcription factor and membrane bound form of ST2 [27]. Foxp3 is required for the proper immunoregulatory function of Tregs [1], while
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Fig. 1 – Representative flow cytometric analysis of CD4+CD25highFoxp3+ cells after in vitro IL-33 treatment in analyzed subjects. CD4+CD127lowCD25+ T cells were isolated by immunomagnetic method and cultured as described in Section 2. The percentage of CD4+CD25highFoxp3+ T cells was determined by flow cytometry. 10,000 events were acquired. Gate on lymphocytes was established based on scatter characteristic. Cells in the lymphocyte gate were magnetically selected CD4+CD25+ T lymphocytes, so the anti-CD4 versus CD25 dot plot was generated. The gate was set on CD4+CD25+ lymphocytes and only cells with high expression of CD25 were taken into account. After gating on CD4+CD25high cells, the frequency of nonstimulated (A, C) and IL-33 stimulated (B, D) CD4+CD25highFoxp3+ cells was determined.
ST2 is a IL-33 receptor [20]. IL-33 interaction with membrane bound ST2 stimulates its action, in contrast to ST2 soluble form, which acts as a decoy receptor blocking IL-33 signaling [29]. According to the results obtained by us, it can be assumed that an increase in Treg frequency, as well as expression of Foxp3 protein may be due to an increased expression of mST2 after IL-33 treatment. This is consistent with studies done by Schiering et al. [25]. A significant difference in the frequency of CD4+CD25highFoxp3+ among CD4+CD25high cells between DM1 and healthy individuals was also seen at the baseline (starting point) and it was about 55% and 82% respectively. This is not a surprising result, as in several autoimmune diseases the balance between Tregs and effector T lymphocytes is shifted towards the latter and Treg deficiency frequently appear. However, our result does not clearly confirm the difference in the total amount of Foxp3+ T lymphocytes between analyzed groups. In the present study, only cells with high expression of CD25 (CD25high) were taken into account. CD4+CD25+ cells expressing Foxp3 were not considered in the analysis,
because these cells may exhibit only transient expression of Foxp3 transcription factor upon activation [30]. The effects of IL-33 on the immune response are contradictory and depend on the disease model, cell type, and cytokine microenvironment. IL-33 was shown to reduce inflammation in the intestine by increasing the stability and function of colonic ST2+ Treg cells in a colitis mouse model [25], while its administration exacerbated the disease course in a transgenic mouse model of EAE through an increased production of IFN-c and IL-17 [31]. In the current study we aimed to define the in vitro effect of IL-33 on the suppressive potential of magnetically sorted Tregs from patients with type 1 diabetes. To check this, we tested the ability of Tregs to inhibit the production of IFN-c by effector CD3+CD4+ T lymphocytes in mixed co-cultures. The cells analyzed by flow cytometry were stained to CD3+CD4+IFN-c+ phenotype. In our study we used two different clones of anti-CD3 antibodies. One for Treg stimulation, and the second for flow cytometric analysis of IFN-c producing effector T lymphocytes in co-cultures with Tregs. The used antibodies recognize over-
diabetes research and clinical practice
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Fig. 2 – Effect of IL-33 on the quantity of magnetically selected Tregs in diabetic and control groups. Magnetically selected CD4+CD127lowCD25+ T cells were cultured with or without IL-33 for 24 h as described in Section 2. After this time cells were stained with antibodies against CD4, CD25 and Foxp3 molecules and then the frequency of CD4+CD25highFoxp3+ cells was determined using flow cytometry. Data were calculated with the nonparametric Wilcoxon test and are shown as median and 25/75 percentile. **Percentage of cells among CD4+CD25+ T lymphocytes.
Fig. 3 – Percentage of effector T lymphocytes producing IFN-c in mixed cultures of Tregs and PBMCs. The diagram represents inhibition of IFN-c synthesis by effector T lymphocytes in a presence of autologous Tregs (1:1). Magnetically selected Tregs were cultured with or without IL-33 for 24 h as described in Section 2. Cells were stained with fluorochrome conjugated antibodies against CD3, CD4 and IFN-c. During flow cytometric analysis 5000 events were acquired. After gating on CD3+CD4+ cells, the frequency of cells producing IFN-c was determined. Data were calculated with the nonparametric Wilcoxon test and are shown as median and 25/75 percentile. **Percentage of CD3+CD4+IFN-c+ cells among PBMCs.
lapping epitopes, so during the cytometric analysis only CD3+CD4+IFN-c+ effector T lymphocytes among PBMCs, not Treg cells were taken into account. In case of pure PBMC cultures, there are effector as well as regulatory T lymphocytes,
but the frequency of the latter is insignificant and although there are data reporting the production of IFN-c by Tregs upon stimulation, such cells represent a small percentage, and may be omitted from analysis [32,33]. Our study has revealed that
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Tregs from both DM1 patients and healthy individuals exhibit suppressive activity. The frequency of CD3+CD4+IFN-c+ effector T lymphocytes among PBMCs was lower in co-cultures of PBMCs with non IL-33-stimulated Tregs in comparison to pure PBMCs. However, the frequency of CD3+CD4+IFN-c+ effector T lymphocytes among PBMCs was still higher in patients with type 1 diabetes than in healthy subjects. This was seen when comparing the co-cultures of PBMCs with non IL-33-stimulated Tregs as well as co-cultures of PBMCs with IL-33 stimulated Tregs. These results suggest that Tregs from patients with type 1 diabetes are somewhat functional, which is in agreement with other studies [11,12,32], but they probably exert lower in vitro suppressive activity in comparison to their counterparts derived from healthy subjects. This is consistent with previous reports, which confirmed that regulatory T cells in patients with type 1 diabetes exhibit reduced suppressive properties [7,8]. In vitro IL-33-treated Tregs, however, seem to increase their suppressive potential, because we observed a significant lower percentage of CD3+CD4+IFNc+ effector T lymphocytes among PBMCs in comparison to co-cultures of PBMCs with non IL-33-stimulated Tregs. This effect was only seen in DM1 patients but not in the healthy individuals. Our studies indicated that IL-33 has beneficial effect on regulatory T cells, which is manifested by an increase in their numbers as well as enhancement of their ability to suppress the production of IFN-c by effector T lymphocytes in mixed co-cultures. As the number of newly diagnosed individuals with type 1 diabetes has been increasing in recent years, it is of great importance to develop new strategies that would help alleviate the course of the disease and prevent subsequent complications. Given that it can be assumed that treatment of sorted Tregs with IL-33 could improve their properties in existing cellular therapies and this cytokine could be considered as a potential therapeutic tool. However, more studies on a larger group of patients are required to confirm the usefulness of IL-33 as a therapeutic agent.
Disclosures The authors declare that they have no competing financial interests. All authors have approved the final article.
Acknowledgments This study was supported by the State Committee for Scientific Research ST28 (Medical University of Gdan´sk, Poland).
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Contents available at ScienceDirect
Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locat e/dia bre s
IL-33 improves the suppressive potential of regulatory T cells in patients with type 1 diabetes Monika Ryba-Stanisławowska a,*, Laura Buksa a, Agnieszka Brandt b, Ulana Juhas a, Małgorzata Mys´liwiec b a b
Department of Immunology, Medical University of Gdan´sk, Poland Clinic of Pediatrics, Department of Diabetology and Endocrinology, Medical University of Gdan´sk, Poland
A R T I C L E I N F O
A B S T R A C T
Article history:
Aims: The presented study was aimed to analyze the influence of IL-33 on regulatory T cells
Received 30 January 2017
(Tregs) suppressive potential in patients with type 1 diabetes.
Received in revised form
Methods: We analyzed the ability of IL-33 treated Tregs to inhibit the production of IFN-c by
19 March 2017
effector T lymphocytes in an in vitro co-culture. The study group consisted of 22 patients
Accepted 7 April 2017
with type 1 diabetes and 12 age and sex-matched healthy individuals.
Available online 13 April 2017
Results: Our findings revealed that in vitro IL-33 treatment of Tregs derived from patients with type 1 diabetes resulted in quantitative as well as qualitative changes in this cell pop-
Keywords: Diabetes type 1 IL-33
ulation, confirming immunoregulatory features of IL-33. Conclusion: IL-33 could be considered as a potential therapeutic tool in adoptive therapies of type 1 diabetes.
Regulatory T cells
1.
Introduction
Deregulation of the immune system is an important factor for the occurrence of type 1 diabetes (DM1) as well as progression of its complications. CD4+CD25highFoxp3+ human regulatory T cells (Tregs) are essential for the active suppression of autoimmunity. They also exert control over excessive inflammatory response [1–3]. The capacity of Tregs to suppress the immune response may strongly depend on the level of Foxp3 expression, which is a transcription factor particularly responsible for their function and development [1]. In some autoimmune diseases, including rheumatoid arthritis, multiple sclerosis or type 1 diabetes, a defective suppressive function of Tregs was observed [4–6]. Some studies demonstrated that DM1 subjects have a defective peripheral Treg suppressive ability [7–10]. In contrast, others did not report any impairments in Treg suppressive function [11,12]. It is possi-
Ó 2017 Elsevier B.V. All rights reserved.
ble that Tregs in DM1 patients may have genetic defect, which leads to their compromised function [1,13,14] or that some reduced suppression may in part have resulted from resistance of effector lymphocytes to suppression mediated by Tregs [15,16]. The suppressive Treg potential may also depend on the IL-2 availability [17,18] or Foxp3 expression at a single cell level [11,19]. More recently, there has been increasing interest in the immunoregulatory properties of IL-33, which belongs to the IL-1 cytokine family, and has the ability to induce Th2 immune response, required to dampen overactive Th1 activity [20,21]. As to its direct effect on population of regulatory T cells, this cytokine was tested in animal models, in which their suppressive activity was compromised. In these studies, IL-33 has proven to be efficient in promoting Tregs regulatory phenotype [22–26].
* Corresponding author at: Department of Immunology, Medical University of Gdan´sk, Dez binki 1, 80-211 Gdan´sk, Poland. E-mail address: [email protected] (M. Ryba-Stanisławowska). http://dx.doi.org/10.1016/j.diabres.2017.04.011 0168-8227/Ó 2017 Elsevier B.V. All rights reserved.
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Mechanisms of action of IL-33 in patients with type 1 diabetes are poorly understood and still there are little data on the influence of this cytokine on diabetic regulatory T cells. Previously, we have shown that IL-33 has in vitro ability to increase frequencies of Treg cells [27] Here, continuing our research we would like to show IL-33 in vitro effect on the function of regulatory T cells derived from a group of patients with type 1 diabetes.
2.
Methods
2.1.
Human subjects
The study group consisted of 22 patients with type 1 diabetes and 12 healthy individuals. Mean age of the patients and disease duration was 14.7 ± 2.5 and 5.3 ± 3.6 years respectively. The patients were recruited from Clinic of Pediatrics, Department of Diabetology and Endocrinology, Medical University of Gdan´sk. Type 1 diabetes was defined according to the criteria of the American Diabetes Association [28]. A blood glucose level was taken at the time of sampling along with biochemical measurement of renal function, lipid status, and glycosylated hemoglobin (HbA1c). In all examined patients the Cpeptide levels were below 0.5 ng/ml. All patients were treated with humanized insulin. The study followed the principles of the Declaration of Helsinki and was approved by The Ethics Committee of The Medical University of Gdan´sk. The control group consisted of age and sex-matched healthy individuals with no signs of autoimmune, chronic, inflammatory, neoplastic disease at the time of sampling and no evidence of DM1 in their families. A summary of the clinical characterististics of analyzed subject groups is presented in Table 1.
2.2.
Reagents and antibodies
The following monoclonal antibodies (mAbs) were used in cytometric studies (fluorochromes and clones in the brackets): anti-CD3 (PE-Cy7, SK7), anti-CD4 (APC or PE-Cy5 RPAT4), anti-CD25 (PE, 2A3), anti-CD127 (PE-Cy7, AO19D5). Items were purchased from BD Biosciences, USA or BioLegend, USA. Intracellular staining for Foxp3 (FITC, 206D) and IFN-c (PerCP-Cy.5.5, B27) was performed with ready-to-use kits according to the manufacturers suggestions. (BioLegend, USA and BD Biosciences, USA, respectively). Additionally, the following reagents were used for stimulation of cell cultures: anti-CD3 (clone UCHT1, eBioscience, USA), anti-CD28 (clone CD28.2, eBioscience, USA) antibodies, rIL-33 (BioLegend, USA), rIL-2 (BioLegend, USA), PMA (Sigma, Poland), ionomycin (Sigma, Poland) and GolgiStop (BD Biosciences, USA).
2.3.
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procedure with a negative selection of CD4+CD127low cells followed by positive selection of CD25+ cells. CD4+CD127low T cells were isolated from whole blood using RosseteSep Human CD4+CD127low Regulatory T Cell Pre-Enrichment Cocktail (Stem Cell, USA). Then cells expressing CD25 molecule were isolated from enriched CD4+CD127low fraction by positive selection with an EasySep Human CD25 Positive Selection Kit (Stem Cell, USA). The purity of isolated cells was assessed by flow cytometry. Starting with fresh peripheral blood, the CD4+ cell content of the enriched fraction was typically 94 ± 5% and the purity of selected CD4+CD127lowCD25+ Tregs, ranged from 86% to 96%. Magnetically selected CD4+CD127lowCD25+ T cells were then cultured on the round bottom 96-well plates. For each study individual the isolated Tregs were placed in two wells. First, control well (starting point) was designated ‘nonstimulated’ and second ‘stimulated’. Cells in both wells were treated with anti-CD3 and anti-CD28 antibodies, both at a concentration 5 lg/ml. Wells designated as ‘stimulated’ were additionally treated with recombinant human IL-33 at a concentration 25 ng/ml. Cells in both wells were also stimulated with recombinant human IL-2 (100 U/ml). Cultures were incubated at 37 °C with 5% CO2 for 24 h. At the same time, from each study individual, the autologous PBMCs were isolated by density gradient preparation over Histopaque-1083 (Sigma) and cultured for 24 h in RPMI medium supplemented with 5% heat-inactivated FSC.
2.4.
Quality check of stimulated Tregs
The quality of stimulated Tregs was checked after 24 h of culture. It consisted of intracellular Foxp3 staining and IFN-c suppression assay. The assay started from making cocultures of Tregs with autologous PBMCs in 1:1 ratio. For each co-culture 50,000 isolated Tregs as well as PBMCs were used. The stimulation consisted of 50 ng/ml of phorbol 12myristate 13-acetate, 500 ng/ml of ionomycin and 2 ml/ml of GolgiStop (BDBiosciences, USA). After 5 h the cells were stained to CD3+CD4+IFN-c+ phenotype and analyzed on flow cytometer.
2.5.
Flow cytometric analysis
Cells were harvested and then stained with fluorochrome labeled monoclonal antibodies. 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 an appropriate isotype control. Data were analyzed by FACSDiva 6.0 Software (Becton Dickinson, USA).
Isolation and culture of CD4+CD127lowCD25+ Tregs 2.6.
Venous blood samples were collected aseptically into the test tubes containing heparin and used to isolate CD4+CD127lowCD25+ Tregs by a two-step magnetic separation
Statistics
All statistical analyses were performed by Statistica 12.0 (StatSoft, Inc USA) using either the nonparametric Wilcoxon test
0.7 ± 0.11 – 12.5 ± 3.34 – 10.0 ± 1.96; [84.9 ± 22.6] – 21.1 ± 4.0 18 ± 2.0
2.06 ± 0.5 0.65 ± 0.1
Serum Creatinine level (mg/dl) Serum Albumin level (g/l) HbA1c (%); (mmol/mol) BMI (kg/m2)
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or the U Mann-Whitney test with p values less than 0.05 defined as significant.
3.
Results
Magnetically selected CD4+CD127lowCD25+ T cells were cultured with or without IL-33 for 24 h. After this time cells were stained to CD4+CD25highFoxp3+ phenotype and analyzed on flow cytometer. The analysis revealed that in diabetic group, after IL-33 treatment, there was a significant increase in the percentage of CD4+CD25high T cells that were Foxp3+ (Figs. 1 and 2). In the control, healthy group there was no significant difference in the frequency of isolated Tregs after treatment with IL-33 in comparison to resting, nonstimulated cells (Fig. 2). When comparing patients with type 1 diabetes and healthy individuals we found lower frequency of CD4+CD25highFoxp3+ T cells in diabetic group in comparison to healthy subjects at a starting point (cells without IL-33 stimulation – ‘nonstimulated’ cells) as well as after IL-33 treatment (stimulated cells) (Fig. 2).
3.2. Qualitative analysis of isolated regulatory T cells after in vitro IL-33 treatment Magnetically selected CD4+CD127lowCD25+ regulatory T cells after 24 h in vitro culture with or without IL-33 were added to isolated autologous PBMCs and IFN-c suppression assay was performed in order to determine the ability of Tregs to suppress the production of IFN-c by effector T lymphocytes. The analysis revealed that in patients with type 1 diabetes IL-33 treated Tregs more efficiently suppressed the production of IFN-c by effector T lymphocytes compared to Tregs not stimulated with IL-33 (Fig. 3). The percentage of CD3+CD4+IFN-c+ effector T lymphocytes among PBMCs was lower when Tregs were treated with IL-33 in comparison to non IL-33-stimulated Tregs. In the healthy, control group the frequency of effector T lymphocytes lowered only when Tregs were added to the culture of PBMCs and practically did not changed upon IL-33 stimulation (Fig. 3).
Data are shown as mean ± standard deviation.
14.7 ± 2.5 15.2 ± .2.3 DM1 (n = 22) Healthy (n = 12)
5.3 ± 3.6 –
4.
Age (years)
Duration of diabetes (years)
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3.1. Quantitative analysis of isolated regulatory T cells after in vitro IL-33 treatment
Group
Table 1 – Clinical characteristics of analyzed subject groups.
CRP (mg/l)
diabetes research and clinical practice
Discussion
The role of IL-33 in type 1 diabetes is not well defined. Little is known about its potential involvement in pathogenesis of this autoimmune and inflammatory disease. Moreover, there are no data on IL-33 influence on regulatory T cells isolated from DM1 patients’ blood. Comparing magnetically sorted Treg cells in unstimulated and IL-33 stimulated cultures one can assume that this cytokine exerts positive effect on patients’ derived Foxp3+ Tregs by increasing their numbers. Our previous study has revealed that in vitro IL-33 treatment of PBMCs from patients with type 1 diabetes acts on quantitative features of regulatory T cells by increasing their numbers as well as the expression of Foxp3 transcription factor and membrane bound form of ST2 [27]. Foxp3 is required for the proper immunoregulatory function of Tregs [1], while
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Fig. 1 – Representative flow cytometric analysis of CD4+CD25highFoxp3+ cells after in vitro IL-33 treatment in analyzed subjects. CD4+CD127lowCD25+ T cells were isolated by immunomagnetic method and cultured as described in Section 2. The percentage of CD4+CD25highFoxp3+ T cells was determined by flow cytometry. 10,000 events were acquired. Gate on lymphocytes was established based on scatter characteristic. Cells in the lymphocyte gate were magnetically selected CD4+CD25+ T lymphocytes, so the anti-CD4 versus CD25 dot plot was generated. The gate was set on CD4+CD25+ lymphocytes and only cells with high expression of CD25 were taken into account. After gating on CD4+CD25high cells, the frequency of nonstimulated (A, C) and IL-33 stimulated (B, D) CD4+CD25highFoxp3+ cells was determined.
ST2 is a IL-33 receptor [20]. IL-33 interaction with membrane bound ST2 stimulates its action, in contrast to ST2 soluble form, which acts as a decoy receptor blocking IL-33 signaling [29]. According to the results obtained by us, it can be assumed that an increase in Treg frequency, as well as expression of Foxp3 protein may be due to an increased expression of mST2 after IL-33 treatment. This is consistent with studies done by Schiering et al. [25]. A significant difference in the frequency of CD4+CD25highFoxp3+ among CD4+CD25high cells between DM1 and healthy individuals was also seen at the baseline (starting point) and it was about 55% and 82% respectively. This is not a surprising result, as in several autoimmune diseases the balance between Tregs and effector T lymphocytes is shifted towards the latter and Treg deficiency frequently appear. However, our result does not clearly confirm the difference in the total amount of Foxp3+ T lymphocytes between analyzed groups. In the present study, only cells with high expression of CD25 (CD25high) were taken into account. CD4+CD25+ cells expressing Foxp3 were not considered in the analysis,
because these cells may exhibit only transient expression of Foxp3 transcription factor upon activation [30]. The effects of IL-33 on the immune response are contradictory and depend on the disease model, cell type, and cytokine microenvironment. IL-33 was shown to reduce inflammation in the intestine by increasing the stability and function of colonic ST2+ Treg cells in a colitis mouse model [25], while its administration exacerbated the disease course in a transgenic mouse model of EAE through an increased production of IFN-c and IL-17 [31]. In the current study we aimed to define the in vitro effect of IL-33 on the suppressive potential of magnetically sorted Tregs from patients with type 1 diabetes. To check this, we tested the ability of Tregs to inhibit the production of IFN-c by effector CD3+CD4+ T lymphocytes in mixed co-cultures. The cells analyzed by flow cytometry were stained to CD3+CD4+IFN-c+ phenotype. In our study we used two different clones of anti-CD3 antibodies. One for Treg stimulation, and the second for flow cytometric analysis of IFN-c producing effector T lymphocytes in co-cultures with Tregs. The used antibodies recognize over-
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Fig. 2 – Effect of IL-33 on the quantity of magnetically selected Tregs in diabetic and control groups. Magnetically selected CD4+CD127lowCD25+ T cells were cultured with or without IL-33 for 24 h as described in Section 2. After this time cells were stained with antibodies against CD4, CD25 and Foxp3 molecules and then the frequency of CD4+CD25highFoxp3+ cells was determined using flow cytometry. Data were calculated with the nonparametric Wilcoxon test and are shown as median and 25/75 percentile. **Percentage of cells among CD4+CD25+ T lymphocytes.
Fig. 3 – Percentage of effector T lymphocytes producing IFN-c in mixed cultures of Tregs and PBMCs. The diagram represents inhibition of IFN-c synthesis by effector T lymphocytes in a presence of autologous Tregs (1:1). Magnetically selected Tregs were cultured with or without IL-33 for 24 h as described in Section 2. Cells were stained with fluorochrome conjugated antibodies against CD3, CD4 and IFN-c. During flow cytometric analysis 5000 events were acquired. After gating on CD3+CD4+ cells, the frequency of cells producing IFN-c was determined. Data were calculated with the nonparametric Wilcoxon test and are shown as median and 25/75 percentile. **Percentage of CD3+CD4+IFN-c+ cells among PBMCs.
lapping epitopes, so during the cytometric analysis only CD3+CD4+IFN-c+ effector T lymphocytes among PBMCs, not Treg cells were taken into account. In case of pure PBMC cultures, there are effector as well as regulatory T lymphocytes,
but the frequency of the latter is insignificant and although there are data reporting the production of IFN-c by Tregs upon stimulation, such cells represent a small percentage, and may be omitted from analysis [32,33]. Our study has revealed that
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Tregs from both DM1 patients and healthy individuals exhibit suppressive activity. The frequency of CD3+CD4+IFN-c+ effector T lymphocytes among PBMCs was lower in co-cultures of PBMCs with non IL-33-stimulated Tregs in comparison to pure PBMCs. However, the frequency of CD3+CD4+IFN-c+ effector T lymphocytes among PBMCs was still higher in patients with type 1 diabetes than in healthy subjects. This was seen when comparing the co-cultures of PBMCs with non IL-33-stimulated Tregs as well as co-cultures of PBMCs with IL-33 stimulated Tregs. These results suggest that Tregs from patients with type 1 diabetes are somewhat functional, which is in agreement with other studies [11,12,32], but they probably exert lower in vitro suppressive activity in comparison to their counterparts derived from healthy subjects. This is consistent with previous reports, which confirmed that regulatory T cells in patients with type 1 diabetes exhibit reduced suppressive properties [7,8]. In vitro IL-33-treated Tregs, however, seem to increase their suppressive potential, because we observed a significant lower percentage of CD3+CD4+IFNc+ effector T lymphocytes among PBMCs in comparison to co-cultures of PBMCs with non IL-33-stimulated Tregs. This effect was only seen in DM1 patients but not in the healthy individuals. Our studies indicated that IL-33 has beneficial effect on regulatory T cells, which is manifested by an increase in their numbers as well as enhancement of their ability to suppress the production of IFN-c by effector T lymphocytes in mixed co-cultures. As the number of newly diagnosed individuals with type 1 diabetes has been increasing in recent years, it is of great importance to develop new strategies that would help alleviate the course of the disease and prevent subsequent complications. Given that it can be assumed that treatment of sorted Tregs with IL-33 could improve their properties in existing cellular therapies and this cytokine could be considered as a potential therapeutic tool. However, more studies on a larger group of patients are required to confirm the usefulness of IL-33 as a therapeutic agent.
Disclosures The authors declare that they have no competing financial interests. All authors have approved the final article.
Acknowledgments This study was supported by the State Committee for Scientific Research ST28 (Medical University of Gdan´sk, Poland).
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