Effect of Interferon Alfa-2 a on Peripheral Blood CD 4 + CD 25 + T Regulatory Cells in Patients with Behçet Uveitis : Preliminary Study

C l i n M e d International Library Citation: Koc A, Onal S, Tulunay A, Kazokoglu H, Demiralp EE, et al. (2015) Effect of Interferon Alfa-2a on Peripheral Blood CD4+CD25+ T Regulatory Cells in Patients with Behçet Uveitis: Preliminary Study. Int J Ophthalmol Clin Res 2:012 Received: January 08, 2015: Accepted: February 11, 2015: Published: February 13, 2015 Copyright: © 2015 Koc A. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Koc et al. Int J Ophthalmol Clin Res 2015, 2:1 ISSN: 2378-346X


Introduction
Uveitis associated with Behcet Disease (BD) is characterized by bilateral intraocular inflammation mostly affecting the posterior segment of the eye as retinal vasculitis and has a relapsing remitting course [1].Interferon alfa-2a is a cytokine proven to be effective in controlling refractory and sight-threatening uveitis in patients with BD [2][3][4][5][6][7][8][9][10][11][12].However, the exact mechanism by which interferon alfa-2a controls the intraocular inflammation in patients with BD remains to be elucidated.CD4+CD25+ regulatory T (Treg) cells are characterized by their immunoregulatory ability to inhibit the development of certain autoimmune diseases in animal models [13,14].It has been reported that approximately 5% to 10% of the human CD4+ T-cell subpopulation from peripheral blood expresses CD25 and that of these cells only 1% to 2% express high levels of CD25.These CD4+CD25 high cells have regulatory properties and are designated CD4+CD25 high regulatory T (Treg) cells [15].Forkhead box p3 (Foxp3) has been a reliable marker for CD4+CD25+Treg cells and is critical for maintaining immune tolerance and preventing autoimmune diseases [16,17].Either reduced frequency or impaired function of CD4+CD25+Treg cells has been reported in patients with a number of autoimmune diseases, including multiple sclerosis, psoriasis, systemic lupus erythematosus, and rheumatoid arthritis [18][19][20][21].The possible role of CD4+CD25+Treg cells in regulating active inflammation in BD has been emphasized in previous studies [22,23].One study also pointed on the predictive value of decreased percentage of CD4+CD25+Treg cells as a marker of uveitis flare up in BD patients [24].The effect of interferon alfa-2a on lymphocyte subpopulations and monocytes has also been studied in patients with BD [25,26].Our study aimed to specifically focus on the effect of interferon alfa-2a therapy on CD4+CD25+Treg cells; with known immunoregulatory function(s), in patients with Behçet uveitis [27].
staining of the surface receptors, cells were fixed and permeabilized by using Foxp3 Buffer Set (BD Biosciences), then incubated with Phycoerythrin (PE) conjugated anti-Foxp3 monoclonal antibody for 30 minutes.Samples were acquired with FACSCanto flow cytometry using Diva software (BD Biosciences).Both percentage and Mean Fluorescence Intensity (MFI) of Foxp3 were quantified in the CD4+CD25+ gate.

Cell purification
CD4+CD25+ Treg and CD4+CD25-T cells were purified by magnetic separation with CD4+CD25+ Regulatory T Cell Isolation Kit (MACS, MiltenyiBiotec, Germany).Peripheral blood mononuclear cells were isolated from heparinized blood by Ficoll-Hypaque gradient centrifugation for cell purification.First, non-CD4 cells were labeled with a cocktail of biotin-conjugated antibodies against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ/δ, and CD235a.Then labeled cells were incubated with microbeads conjugated to anti-biotin monoclonal antibodies.Indirectly labeled non-CD4+ T cells were magnetically depleted and CD4+ T cells were collected using a LS MACS Column with MidiMACS Separator (both MiltenyiBiotec).Further purified CD4+ T cells were labeled with CD25 microbeads for the isolation of CD4+CD25+ and CD4+CD25-T cells.Directly labeled CD25+ T cells were isolated with a MS MACS Column with MiniMACS Separator (both MiltenyiBiotec).CD25+ Treg cells were enriched with positive selection and CD25 depleted CD4+ T cells were purified with negative selection.Purification of enriched cell populations and purified cells were analyzed by FACS.The purity of the cell populations was >50% for CD4+CD25+T cells and >95% for CD4+CD25-T cells.

Statistical analysis
SPSS statistical software, version 16.0 (SPSS Inc, Chicago, Illinois), was used for the statistical analysis.The Friedman and Wilcoxon tests and the Mann-Whitney test used to test for differences in the paired and unpaired blood samples, respectively.No correction was made for multiple testing.before initiation of any systemic therapy while the patients had active Behcet uveitis with posterior segment involvement (sample 1), ( 2) at the termination of conventional immunosuppressive agent due to unresponsiveness of uveitis/before the initiation of interferon alfa-2a (sample 2), and (3) while Behcet uveitis was inactive on interferon alfa-2atherapy (sample 3).All five patients had panuveitis that was refractory to azathioprine.Interferon alfa-2a (Roferon-A; Roche Pharmaceuticals, Whitehouse Station, New Jersey) was administered based on a low-dose and dose escalating treatment protocol described previously by us [10][11][12].Based on this protocol interferon alfa-2a is given 3 million international-units (IU) subcutaneously (sc) daily for 14 days for induction.Maintenance is achieved with interferon alfa-2a given as 3 million IU 3x/week sc.The dose is increased sequentially to 4,5,6 and 9 million IU 3x/week, if uveitis relapses occur.Inactive anterior uveitis was defined as 0.5 cells or less [29].Vitritis, evidenced by the presence of cells, not haze, was graded from 0 to 4 and considered inactive when there were 0.5 cells or less [30].Inflammation of the posterior segment was documented by the presence of retinal vasculitis, retinitis, cystoid macular edema, and papillitis.Inactivity of uveitis while on maintenance therapy of IFN α-2a was defined as inactivity of anterior chamber and vitreous inflammation along with absence of posterior segment intraocular inflammatory signs [29,30].In case the uveitis specialist decided that uveitis was inactive, a fluorescein angiography was also performed.Absence of any pathologic leakage on fluorescein angiography was also set as a criteria in order to collect the third blood sample from the patients.High-dose systemic corticosteroids were used to control uveitis flare up at presentation and during follow-up.Blood samples were always collected before the initiation of highdose systemic corticosteroid therapy.Systemic corticosteroids were ideally discontinued or tapered to a maximum dose of 8mg/day methyl-prednisolone during maintenance therapy with conventional immunosuppressive and interferon alfa-2a therapy.Blood was also collected from 5 age and sex-matched healthy control subjects (sample 4).
The study was approved by the ethics committee of the School of Medicine, Marmara University (approval number: MR-YC-08-0105), and conducted according to the tenets of the Declaration of Helsinki.Informed consent was obtained from all patients and control subjects.

Flow cytometry
EDTA-preserved whole blood samples obtained from Behçet uveitis patients and control subjects were used to determine the frequencies of Foxp3+ expressions on CD4+CD25+Treg cells.Foxp3 Staining Kit (BD Biosciences, Franklin Lakes, NJ, USA) was used for intracellular staining according to the manufacturer's instructions.Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by erythrocyte lysing solution (155mM NH 4 Cl, 10mM KHCO 3 , 0.1mM EDTA) followed by washing the cells with phosphate buffered saline (PBS).Cells were then stained with Fluorescein Isothiocyanate (FITC) conjugated Anti-CD4 and Allophycocyanin (APC) conjugated anti-CD25 monoclonal antibodies and their isotypic controls.After

Results
Demographical and clinical features and treatment properties at the time of blood sampling of patients with Behçet uveitis are shown in Table 1.Sex-matched controls composed of 3 male and 2 female subjects.Median age of Behçet uveitis patients and control subjects was 29 years (range: 21-54 years) and 28 years (range: 26-32 years), respectively and there was no significant difference (p=0.54).Median duration of Behçet uveitis was 6 years (range: 2-25 years).All patients included in the study had panuveitis with vitritis and retinal vasculitis.Patients were started on azathioprine 2mg/kg/day upon diagnosis.At the time of the second blood sampling median duration of azathioprine therapy was 3 months (range: 3-7 months).Maintenance dose of interferon alfa-2a was and 4.5 million IU 3x/ week sc in 3 patients and 3 million IU 3x/week in 1 patient.The median duration of interferon alfa-2a therapy was 6 months (range 4-6 months) at the time of the third blood sampling.

Discussion
In this in-vitro study, we showed that interferon alfa-2a decreased Foxp3+CD4+CD25+ Treg cells and increased the level of IL-10 in the culture supernatant of refractory Behçet uveitis patients.The Foxp3+CD4+CD25+ Treg cells are a unique lineage of T cells, capable of suppressing effector cell responses.T regulatory cells have been extensively studied in many autoimmune and inflammatory diseases [31].Although there is a controversy among the studies regarding their frequency in peripheral blood of BD patients, Hamzaoui et al. [22] reported that CD4+ CD25+ Treg cells are increased in the peripheral blood of active BD patients [22].Similar to their results we found a slightly elevated percentage of CD4+CD25+Foxp3+ Treg cells in patients with active Behçet uveitis.
Behçet disease is a systemic inflammatory disorder in which the presence of prolonged inflammation causes clinical manifestations including ocular involvement.It has been shown that the frequency of Treg cells is higher in the synovial fluid compared to peripheral blood of patients with rheumatic diseases [33][34][35].According to these studies, Treg cells accumulate at sites of inflammation where they perform their suppressive functions on reactive T cells.However, for the efficient suppression of reactive cells that reside at inflammation sites, functional trafficking of Treg cells is required.In various autoimmune and inflammatory diseases, homing receptors on Treg cells have been shown to be defective and suggested to be involved in the pathogenesis of diseases [36,37].Our finding of elevated percentage of CD4+CD25+Foxp3+ Treg cells at the peripheral blood of patients with active Behçet uveitis may indicate the defective migration of these cells to the inflamed ocular tissue.Further studies Table 2: Comparison of CD4+CD25+Treg cells, Foxp3 mean fluorescence index, and cytokine levels from CD4+CD25+Treg cell supernatants offive Behçet uveitis patients at baseline-during acute panuveitis attack (sample 1) and five healthy control subjects (sample 4).
Two important classes of Treg cells within the CD4+ subset are CD4+CD25+Foxp3+Treg cells and T regulatory type 1 (Tr1) cells [36][37][38].These two regulatory subsets differ in a number of biological features, including their cytokine profile, cellular markers, transcription factors, and mechanism of immune suppression.The Tr1 subset are CD4+ T lymphocytes defined by their production of IL-10 and suppression of helper T cells.T regulatory 1 cells are inducible cells, arise from naive precursors, and can be differentiated both exvivoand in-vivo.T regulatory 1 cell differentiation is characterized by a massive secretion of IL-10 and bystander CD4+T cell suppression [38].The increased level of IL-10 after interferon alfa-2a therapy can be accepted as clear evidence that this treatment affects the altered Treg cells, either enabling them to regain their suppressive functions or by inducing the Trl cell subset.Furthermore, the protective role of IL-10 in patients with Behçetuveitis has been shown recently [39].Interleukin-10 significantly reduced the production of nitric oxide in peripheral mononuclear cell culture supernatants of patients with Behçet uveitis.
However, there are some limitations of our study.First, our patient had a severe form of Behçet uveitis that was refractory to conventional immunosuppressive therapy.Therefore we had to switch to interferon alfa-2a therapy immediately after azathioprine had failed to control uveitis flare-ups.Despite the fact that IL-10 level did not seem to be affected by azathioprine therapy, the effect(s) of azathioprine on the results can not to be ruled out.However, the current practice of Behçet uveitis management dictates to start interferon alfa-2a therapy in those who fail conventional treatment and interferon alfa-2a is used as second-line agent.Second, our study group was rather small to draw any strong conclusions.
In summary, our results suggest that IFN α-2a therapy might cause Treg cells to regain their suppressive function and thereof beneficial in suppressing the intraocular inflammation as well as in preventing uveitis flare-ups to occur in patients with refractory Behçet uveitis.Further studies are warranted to understand the exact mechanisms of interferon alfa-2a in controlling Behçet uveitis.

Table 1 :
Demographic and ocular clinical features and treatment properties at the time of blood sampling of patients with Behçet uveitis.
MFI: Mean Fluorescence Index, IFN: Interferon, TNF: Tumor Necrosis Factor, *: Friedman; †: Wilcoxon are needed to clarify the involvement of homing receptors in the pathogenesis of BD.