CD4+ CD25+ regulatory T cells ameliorate Behcet's disease-like symptoms in a mouse model

Cytotherapy, 2011; 13: 835–847

CD4 CD25 regulatory T cells ameliorate Behcet’s disease-like symptoms in a mouse model

JUA SHIM1, EUN-SO LEE2, SUN PARK3, DONGSIK BANG4 & SEONGHYANG SOHN1,5 1Laboratory of Cell Biology, Ajou University Institute for Medical Sciences, Suwon, Republic of Korea, 2Department of Dermatology and 3Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea, 4Department of Dermatology,Yonsei University, Seoul, Republic of Korea, and 5Brain Korea 21 Project for Medical Science, Republic of Korea

Abstract Background aims. Behcet’s disease (BD) is a chronic, multisystemic inflammatory disorder with arthritic, gastrointestinal, mucocutaneous, ocular, vascular and central nervous system involvement. It is well known that CD4 CD25 T-regulatory (Treg) cells prevent harmful immune responses to self- and non-self-antigens. In the present study, the role of Treg cells in herpes simplex virus (HSV)-induced BD-like symptoms was investigated. Methods. HSV type 1 (F strain) inoculation of the earlobe of ICR mice has been shown to induce the development of BD-like symptoms. To determine whether the effect of Treg was associated with change in BD-like symptoms, CD4 CD25 T cells from the splenocytes of normal mice were adoptively transferred intravenously. Treg cells of splenocytes were significantly elevated following the transfer of 3  105 CD4 CD25 T cells to BD-like mice compared with the control group. Results. The transfer of CD4 CD25 T cells to BD mice improved the symptoms, and the serum protein levels of interleukin (IL)-10, IL-6 and IL-17 were significantly altered compared with the control groups. Intravenous injection of anti-CD25 antibody to BD mice reduced the frequency of CD4 CD25 T cells and increased the BD severity score. We confirmed the influence of CD4 CD25 T cells on BD-like mice. Conclusions. These results show that up-regulation of the CD4 CD25 T cells in BD-like mice improves the inflammatory symptoms, while down-regulation of CD25 T cells is associated with deteriorated symptoms. Furthermore, these findings are correlated with changes in pro-inflammatory and antiinflammatory cytokine levels. Key Words: Behcet’s disease, herpes simplex virus, mouse model, regulatory T cells, systemic inflammation

Introduction Behcet’s disease (BD) is a chronic, multisystemic disorder with arthritic, gastrointestinal, mucocutaneous, ocular, vascular and central nervous system involvement. This disease follows a chronic course, with periodic exacerbations and progressive deterioration (1). Although the etiology of BD is unclear, viral infection has long been postulated as one of the main factors. Since Hulusi Behcet first proposed a viral etiology (2), the viral hypothesis has been verified by detection of the virus in the saliva (3), intestinal ulcers (4) and genital ulcers (5,6) of patients with BD. Subsequent to these findings, inoculation of the earlobes of Institute for Cancer Research (ICR) mice with herpes simplex virus (HSV) resulted in the development of BD-like symptoms (7). Manifestations in mice after HSV inoculation include multiple symptoms, such as oral ulcers, genital ulcers, skin

ulcers, eye symptoms, gastrointestinal ulcers, arthritis and neural involvement, as well as skin crusting. The frequencies of these symptoms are similar to those of patients with BD (8). In 1995, Sakaguchi identified a subpopulation of CD4 T cells that constitutively expressed the interleukin (IL)-2 receptor α-chain (CD25) (9). CD4 CD25 cells are heterogeneous T-cell populations that prevent harmful immune responses to self- and non-self-antigens. These cells emerge from the thymus as part of normal T-lymphocyte development. CD4 CD25 T cells reside in peripheral tissues, where they maintain self-tolerance and prevent autoimmunity by inhibiting pathogenic lymphocytes. CD4 CD25 T cells can be classified into two major categories, natural CD4 CD25 T cells (nTreg) and inducible CD4 CD25 T cells (iTreg) (10–12). nTreg cells, which are characterized by the

Correspondence: Seonghyang Sohn, Laboratory of Cell Biology, Ajou University Institute for Medical Sciences, Suwon, 443–721, Republic of Korea. E-mail: [email protected] (Received 1 July 2010; accepted 6 March 2011) ISSN 1465-3249 print/ISSN 1477-2566 online © 2011 Informa Healthcare DOI: 10.3109/14653249.2011.571245

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expression of CD4, CD25 and the transcriptional factor Forkhead box P3 (forkhead/winged helix transcription factor; Foxp3), develop in the thymus and recognize specific self-antigens (13). The subset of Treg cells known as iTreg are also generated in the periphery during active immune response. The newly induced CD4 suppressor cells inhibit proliferation of CD4 T cells via either IL-10 (14) or transforming growth factor (TGF)-β (15) production. Foxp3 protein is currently considered to be the most specific marker of Treg cells, and a mutation of this transcription factor is strongly linked to immune dysregulation. Treg cells play an important role in the pathogenesis of autoimmune disorders, such as diabetes mellitus (16), arthritis (17,18) and lupus (19). These autoimmune disorders can be prevented by the infusion of Treg cells (20). More recently, Nanke et al. (21) reported that the percentage of Treg cells was reduced in the peripheral blood of patients with BD prior to ocular attack. In the present study, we investigated the role of Treg cells by identifying and analyzing the function of CD4 CD25 T cells in spleen tissues of HSV-induced BD mouse models. In addition, we analyzed the frequencies of CD4 CD25 and CD4 CD25 Foxp3 Treg cells in splenocytes and cytokines in sera and spleen tissues from CD4 CD25 cell-treated mice. Overall, this study was conducted to determine whether the progress of this disease could be inhibited by elevating CD4 CD25 T cells.

BD-like symptoms Manifestations in mice after HSV inoculation involved multiple symptoms. Of the total number of HSVinjected mice, 15% developed BD-like symptoms. Among the symptoms in human patients, mouth ulceration, genital ulceration, erythema, skin pustules, skin ulceration, joint arthritis, diarrhea, red eye (right, left), reduced vision (right, left), loss of balance, discoloration and swelling of the face were selected and analyzed as BD-like symptoms. The score of each symptom was 1 and the sum of the symptoms was used to determine the severity of BD. The disappearance of symptoms or a decrease in the lesion size of more than 20% was classified as improvement. The severity of BD was followed by determination of the Behcet’s disease activity index, as outlined in the BD Activity Form (www.behcet.ws/pdf/BehcetsDisease ActivityForm.pdf (4th April, 2011)). Splenocyte culture and generation of Treg cells The mice used in the experiments were 4–5-weekold male ICR mice. Splenocytes were isolated from the mice and erythrocytes were removed from splenocyte cell suspensions in lysis buffer for red blood cells (ACK) solution. The splenocytes were stimulated with anti-CD3 (0.1 μg/mL), anti-CD28 (0.2 μg/mL), rIL-2 (20 U/mL) and TGF-β1 (2 ng/mL) in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics for 1–4 days.

Methods Antibodies and reagents Anti-mouse antibodies (Ab) CD4 (fluorescein isothiocyanate; FITC), CD25 (phycoerythrin; PE), CD3 and CD28, and recombinant mouse IL-2 (rIL-2), were purchased from BD Pharmingen (San Diego, CA, USA). A mouse anti-Foxp3 (PE–cy5) staining kit and granulocyte–macrophage colony-stimulating factor (GM-CSF) were purchased from eBioscience (San Diego, CA, USA). Recombinant human TGFβ1 was purchased from R&D Systems (Minneapolis, MN, USA). Animal experiments Male ICR mice (4–5 weeks old) were infected with HSV type 1 (1  106 plaque forming unit (PFU) mL, F strain) grown in Vero cells, as described previously (7). Virus inoculation was performed twice with a 10-day interval, followed by 16 weeks of observation. Animals were handled in accordance with a protocol approved by the animal care committee of Ajou University School of Medicine (Suwon, Republic of Korea).

Isolation of CD4 CD25 T cells and adoptive transfer to BD-like mice The splenocytes from normal mice were cultured for 2 days with stimulants (anti-CD3, anti-CD28, rIL-2 and TGF-β1). CD4 CD25 or CD4 CD25– T cells were isolated from cultured splenocytes using a mouse CD4 CD25 regulatory T-cell isolation kit™ (Milteny Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. Briefly, CD4 T cells were isolated through negative selection by removing all other cell types. Pre-isolated CD4 T cells were incubated with magnetic beads conjugated with anti-CD25 Ab to separate the CD4 CD25 and CD4 CD25– T-cell populations. Isolated T cells were more than 80% pure upon re-analysis by flow cytometry. The isolated CD4 CD25 T cells were adoptively transferred to BD-like mice via the tail vein. Specifically, 3  103, 3  104 or 3  105 CD4 CD25 T cells/mouse were administered. The change in symptoms was photographed for 2 weeks after adoptive transfer. Two weeks later, the transferred mice were killed and the serum and spleen tissues then collected.

Treg in Behcet’s disease model Depletion of CD4 CD25 cells in BD-like mice with anti-CD25 Ab Anti-IL-2 receptor alpha chain-specific B-cell hybridomas (PC 61.5.3; American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS, 0.05 nM 2-mecaptoethanol and 1% antibiotics. Supernatants were routinely harvested. Briefly, supernatants were centrifuged at 400  g to remove cells and debris, after which they were filtered through a 0.2-μm filter and stored for less than 2 months at 4°C. To concentrate the samples, the supernatants were added to Vivaspin 20 (Sartorius, Aubagne, France) and then centrifuged at 1500  g for 60 min at 10°C. Protein pellets were sterilized with a 0.2-μm filter, resuspended in phosphate-buffered saline (PBS), and then stored at –20°C. The mice were injected intraperitoneally (i.p.) with 1 or 10 mg anti-CD25 Ab for selective inhibition of CD25 T cells. Flow cytometry The cells were stained with anti-mouse CD4 and anti-mouse CD25 Ab for 30 min at 4°C in the dark. For intracellular detection of Foxp3, an anti-mouse Foxp3 staining kit was used according to the manufacturer’s instructions. Briefly, cells were fixed using Fix/perm buffer after washing with 1  permeabilization buffer, then incubated with anti-mouse Foxp3 Ab for 30 min at 4°C in the dark. Stained cells were analyzed by flow cytometry (FACS Vantage; Becton Dickinson, Franklin Lakes, NJ, USA) with 10 000 gated lymphocytes. Isotype control Ab were used to estimate the non-specific binding of target primary Ab.

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manufacturer’s instructions. One microgram of total RNA was then used as a template for cDNA synthesis, which was conducted using a SuperScript III first-strand synthesis system for a reverse transcription–polymerase chain reaction (RT-PCR) kit (Invitrogen, Carlsbad, CA, USA). The cDNA was amplified by PCR with the primers shown in Table I. Amplified PCR products were visualized on 1.8% agarose gels. Real-time PCR For real-time SYBR green RT-PCR, the 20-μL reaction contained 10 μL 2  Quantitect SYBR green master mix (Qiagen, Valencia, CA, USA) composed of a hot-start Taq polymerase, 0.4 μL of two reverse transcriptases, 0.5 μL (10 ng/μL) template and 0.8 μL of primers. An ABI 7900 HT thermal cycler (Lab Centraal BV, Haarlem, the Netherlands) was used for real-time PCR assays. RT was conducted at 50°C for 30 min, followed by denaturation at 95°C for 15 min. DNA was amplified by 40 PCR cycles, which were as follows: 95°C (30 s), 55°C (30 s) and 72°C (30 s). Data were collected for 15 s at 75°C to avoid non-specific fluorescence as a result of primer dimers occurring at low template concentrations. For the generation of standard quantitation curves, the cycle threshold values were plotted proportionally to the logarithm of the input copy numbers. Negative controls were included in each run. Statistical analysis All data shown represent the mean  SE. Statistical differences between the experimental groups were Table I. The sequence of primers used for RT-PCR.

Enzyme-linked immunosorbent assay

Gene

Two weeks after the CD4 CD25 T cells were trans-

β-actin

ferred to BD-like mice, the serum was obtained and analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits for the detection of mouse IL-6, tumor necrosis factor (TNF)-α, TGF-β, IL-17, interferon (IFN)-γ and IL-10. All ELISA kits were purchased from R&D Systems. ELISA was conducted according to the manufacturer’s instructions. The means and standard deviations were calculated using ELISA values determined for each well. The ELISA reader was a Bio-Rad model 170–6850 microplate reader and the wavelength was 450 nm.

IL-6 ROR-γt TNF-α TGF-β IL-17A IL-17F IFN-γ

Reverse transcription–polymerase chain reaction Total RNA was isolated with TRIzol (Life Technologies, Helgerman, CT, USA) according to the

Sequence F R F R F R F R F R F R F R F R

5′-TGGAATCCTGTGGCATCCATGAAAC-3′ 5′-TAAAACGCAGCTCAGTAACA GTCCG-3′ 5′-TGGAGTCACAGAAGGAGTGGCTAAG-3′ 5′-TCTGACCACAGTGAGGAATGTCCAC-3′ 5′-GCGGAGCAGACACACTTACA-3′ 3′-TTGGCAAACTCCACCACATA-5′ 5′-GGCAGGTCTACTTTGGAGTCATTGC-3′ 5′-ACATTCGAGGCTCCAGTGAATTCGG-3′ 5′-AGGAGACGGAATACAGGGCTTTCG-3′ 5′-ATCCACTTCCAACCCAGGTCCTTC-3′ 5′-AGGCCCTCAGACTACCTCAACC-3′ 5′-GCCTCTGAATCCACATTCCTTG-3′ 5′-CAACGCTGCATACAAAAATCA-3′ 5′-TTAAGTGAGGCATTGGGAACA-3′ 5′-AGCGGCTGACTGAACTCAGATTG TAGCTTGTACCTTTACTTCACTG-3′ 5′-GTCACAGTTTTCAGCTGTATAGGG-3′

F, forward; R, reverse. ROR-γt (22), TNF-α (23), TGF-β (24), IL-17A (25), IL-17F (26).

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determined using a chi-square test, Student’s t-test and Bonferroni’s correction. Statistical analysis was performed using MedCalc® version 9.3.0.0.

A

CD4 CD25 T cells were amplified in primary cultures of spleen tissues Splenocytes were cultured for 1–4 days with stimulants, after which CD4 CD25 T cells and Treg cells were analyzed by FACS analysis. As shown in Figure 2, the frequency of CD4 CD25 T cells in BD-like mice on day 0 was 0.59  0.42%, while it was 2.52  1.31% (P  0.008) in normal mice and 1.14  0.4% (P  0.012) in BDN mice. In addition, the frequency of Treg cells in BD-like mice was 0.41  0.35%, while it was 1.15  0.83% (P  0.028) in normal mice and 0.79  0.56% (P  0.098) in BDN mice. On day 1, the frequency of CD4 CD25 T cells in BD-like mice was 4.05  0.29%, while it was 16.99  3.04% (P  0.01) in normal mice and 12.02  2.34% (P  0.01) in BDN mice. Moreover, the level of Treg cells in BD-like mice was 0.64  0.59% on day 1, while it was 3.34  0.85% (P  0.01) in normal mice and 1.75  1.57% in BDN mice. The frequency of Treg cells in BDN was 2.4 times higher than that in BD-like mice, but this difference was not significant. On day 2, the frequency of CD4 CD25 T cells in BD-like mice was 4.67  1.39%, while it was 22.68  5.96% (P  0.01) in normal

p=0.012

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The frequencies of CD4 CD25 T and Treg cells in the splenocytes of normal healthy and BDN (BD asymptomatic; HSV-inoculated but no symptoms were observed) mice were compared with those of BD-like mice by fluorescence-activated cell sorter (FACS) analysis. The levels of CD4 CD25 T cells in BDlike mice (n  9) were significantly lower than those in BDN mice (n  9; 0.59  0.42% versus 1.14  0.4%; P  0.012) and normal mice (n  8; 0.59  0.42% versus 2.52  1.31%; P  0.008). In addition, the frequencies of CD4 CD25 T cells in BDN mice were lower than in normal mice (1.14  0.4% versus 2.52  1.31%, P  0.009; Figure 1A). The frequency of CD4 CD25 Foxp3 Treg cells in BD-like mice was lower than in BDN mice (0.39  0.37% versus 0.78  0.56%, P  0.098) and normal mice (0.39  0.37% versus 1.15  0.83%, P  0.028; Figure 1B). Furthermore, the frequencies of CD4 CD25 T and Treg cells in BDN mice were twice as great as those in BD-like mice. The frequency of Treg cells in BD-like mice was also reduced.

p=0.008 p=0.009

Results CD4 CD25 Treg cells in BD-like mice compared with BDN mice

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Figure 1. The frequencies of CD4 CD25 T and Treg cells in splenocytes of normal, BDN and BD-like mice were compared by FACS analysis. CD4 CD25 T- and Treg-cell levels were lower in BD-like mice than in BDN and normal mice (A, B). The short bars indicate the means. BD, Behcet’s disease-like mice; BDN: BD normal (asymptomatic) mice; Nor, normal healthy mice; CD4 CD25, CD4 CD25 T cells; Treg, regulatory T (CD4 CD25 Foxp3) cells.

mice and 16.80  5.66% (P  0.05) in BDN mice. The frequency of Treg cells in BD-like mice was 0.74  0.46%, while it was 3.37  1.86% (P  0.01) in normal mice and 1.75  0.94% in BDN mice. The level of Treg cells in BDN mice was 2.3 times greater than in BD-like mice, although this difference was not significant. On days 3 and 4, the levels of CD4 CD25 T and Treg cells in BD-like mice were lower than in BDN or normal mice, revealing a decreasing tendency. On day 3, the frequency of CD4 CD25 T cells in BD-like mice was 3.41  1.11%, while it was 8.92  2.56% in normal mice (P  0.05) and 12.78  2.78% in BDN mice (P  0.01). Furthermore, the level of Treg cells in BD-like mice on day 3 was 0.85  0.74%, while it was 1.35  0.84% in normal mice and 1.18  1.02% in BDN mice. On day 4, the frequency of CD4 CD25 T cells in BD mice was 1.29  0.72%, while it was 6.20  1.71% in normal mice (P  0.01) and 4.55  0.39% in BDN mice (P  0.01). Additionally, the level of Treg cells in BD-like mice was 0.04  0.08%

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Figure 2. CD4 CD25 T cells were amplified in primary cultures of spleen tissues, and the amplified range was different among normal, BDN and BD-like mice. The frequencies of CD4 CD25 T and Treg cells of splenocytes cultured with stimulators (antiCD3 Ab, anti-CD28 Ab, rIL-2 and TGF-β) from normal, BDN and BD-like mice were serially measured at days 0 (no culture), 1, 2, 3 and 4. The frequency of CD4 CD25 T cells in normal mice was significantly higher than in BDN and BD-like mice. The frequency of CD4 CD25 T cells in BDN was significantly higher than in BD-like mice (A). Treg cells were also significantly higher in normal mice than in BD-like mice at days 0, 1, 2 and 4. (B). Data shown are the means  SD of three independent experiments. ∗P  0.1, ∗∗P  0.05, ∗∗∗P  0.01; NC, no culture (day 0).

on day 3, while it was 0.65  0.14% in normal mice (P  0.01) and 0.78  0.43% in BDN mice (P  0.1). Taken together, these findings indicated that the frequencies of CD4 CD25 T and Treg cells in splenocytes induced to proliferate by stimulators in BD-like mice were lower than those in BDN or normal mice. The frequencies of CD4 CD25 T and Treg cells that were cultured for 2 days were higher than those that were cultured for different lengths of time. Therefore, we used 2-day cultured splenocytes for the following experiments. Adoptive transfer of CD4 CD25 T cells up-regulated the frequencies of Treg cells and then improved the symptoms in BD-like mice The CD4 CD25 T cells from the splenocytes of normal healthy mice were cultured with stimulators

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and then isolated by Magnetic Cell Sorter (MACS; Mitenyi Biotec Company, Bergisch Gladbach, Germany). FACS analysis revealed that  80% of the CD4 CD25 T cells were present in sorted fractions. The proportions of Foxp3-positive cells were  25% in the CD4 CD25 T cells and  3% in the CD4 CD25– T cells. Next, CD4 CD25 (3  103, 3  104 and 3  105) T cells and CD4 CD25– (3  105) T cells were adoptively administered to BD-like mice via intravenous injection. Two weeks after treatment, the mice were killed and changes in the frequency of Treg cells from spleen tissues were evaluated (Figure 3). The frequencies of CD4 CD25 T cells following treatment with CD4 CD25 T cells were as follows: 0.59  0.42% for the non-treated group (n  9), 0.50  0.04% for the 3  103 group (n  3), 0.55  0.16% for the 3  104 group (n  5) and 0.95  0.39% for the 3  105 group (n  16) (non-treated versus 3  105, P  0.04; 3  103 versus 3  105, P  0.06; 3  104 versus 3  105, P  0.04). The percentages of Treg cells transferred with the CD4 CD25 T cells were as follows: 0.41  0.35% for the non-treated group, 0.31  0.01% for the 3  103 group, 0.26  0.14% for the 3  104 group and 0.69  0.30% for the 3  105 group (non-treated versus 3  105, P  0.04; 3  103 versus 3  105, P  0.04; 3  104 versus 3  105, P  0.004; Figure 3A). Taken together, our results showed that administration of 3  105 CD4 CD25 T cells resulted in a significantly higher percentage of Treg cells than administration of 3  103 and 3  104 of CD4 CD25 T cells. Before and 2 weeks after treatment with CD4 CD25 T cells, the symptoms in BD-like mice were photographed. As shown in Figure 3C, BD-like symptoms such as skin ulcer, scrotum enlargement, genital inflammation and arthritis were improved. As shown in Table II, treatment with 3  105 CD4 CD25 T cells effectively decreased the BD-like symptoms in 15 of 18 cases (83%); however, treatment with CD4 CD25– T cells decreased BD-like symptoms in four of nine cases (44%) and treatment with 3  104 CD4 CD25 T cells decreased BDlike symptoms in four of seven cases (57%), indicating that treatment with CD4 CD25 T cells led to the improvement of BD-like symptoms. The percentage of improved mice was correlated with the number of CD4 CD25 T cells. As shown in Figure 3B, the severity score following the adoptive transfer of 3  104 and 3  105 CD4 CD25 T cells in BD-like mice was significantly lower than that of BD-like mice treated with CD4 CD25– T cells or 3  103 CD4 CD25 T cells. Specifically, the severity score of mice treated with 3  103 CD4 CD25 T cells was 2.7  1.2 before injection and

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Figure 3. Adoptive transfer of CD4 CD25 T cells up-regulated the frequencies of Treg cells and then improved the symptoms of BDlike mice. The splenocytes of normal healthy mice were cultured for 2 days with stimulators. Next, CD4 CD25 T or CD4 CD25–T cells were isolated. Isolated CD4 CD25 T cells were transferred to BD-like mice via tail vein injection. Two weeks after the transfer, the mice were killed and the frequencies of CD4 CD25 T and Treg cells in spleen tissues were analyzed by FACS. (A) The frequencies of CD4 CD25 T and Treg cells were dependent on the amount of transferred cells. The transfer of higher levels of CD4 CD25 T cells was associated with higher levels of CD4 CD25 T and Treg cells detected in BD-like mice. (B). The disease severity score was calculated before and after transfer of CD4 CD25 T or CD4 CD25– T cells. The severity score was decreased in BD-like mice treated with 3  104 and 3  105 CD4 CD25 T cells, and this decrease was statistically significant. (C). Photographs of mice were taken before and after the transfer of CD4 CD25 T and CD4 CD25– T cells to BD-like mice. Treatment with 3  105 CD4 CD25 T cells showed the greatest improvement. CD4 CD25, CD4 CD25 T cells; Treg, regulatory T (CD4 CD25 Foxp3) cells; no transfer, n  9; 3  103, n  3; 3  104, n  5; 3  105, n  18.

Treg in Behcet’s disease model 1.7  0.6 after 2 weeks of treatment (P  0.225, n  3). Similarly, these values were 2.8  0.8 before and 1.8  1.3 after (P  0.034, n  5) treatment with 3  104 CD4 CD25 T cells, while they were 2.8  0.9 before and 1.3  0.9 after (P  0.0001, n  16) treatment with 3  105 CD4 CD25 T cells, and 2.7  0.5 before and 2.4  1.0 after (P  0.4, n  10) treatment with CD4 CD25– T cells. These findings indicated that, although the symptoms of BD-like mice that received CD4 CD25 T cells were improved, treatment with CD4 CD25– T cells had no effect on the BD severity score. Treatment with CD4 CD25 T cells up-regulated IL-10 and TGF-β levels and down-regulated IFN-γ, TNF-α, IL-6 and IL-17 levels Many studies have investigated the secretion of TGF-β and IL-10 by CD4 CD25 T cells (2729). It is well known that the roles of IL-10 and TGF-β in the suppressive effects of inflammation are mediated by CD4 CD25 T cells. As shown in Figure 4A, ELISA revealed that the IL-10 protein level was 84.49  45.94 pg/mL in the serum of the BD-like mice (n  6) that did not receive the cells, while it was 167.08  72.86 pg/mL in the BD-like mice that were treated with 3  105 CD4 CD25 T cells (n  12; P  0.02), 73.04  69.96 pg/mL in those that were treated with 3  104 cells (n  3; 3  104 versus 3  105, P  0.06) and 101.96  83.91 pg/mL in the BDN mice (n  6). The IL-10 levels were significantly up-regulated after treatment with 3  105 CD4 CD25 T cells in the BD-like mice. The importance of TGF-β in the immune system was highlighted by the discovery that TGFβ-deficient mice developed multiple inflammatory diseases (30,31). As shown in Figure 4B, TGF-β expression was higher in BD-like mice that received 3  105 cells than in those that did not. ELISA revealed that the TGF-β protein level was 19.8  20.0 pg/mL in BD-like mice that received 3  105 CD4 CD25 T cells (n  15), 4.7  1.9 pg/mL in BD-like micethat did not receive the cells (n  8), 10.4  7.3 pg/mL in BDN mice (n  6) and 5.0  2.4 pg/mL in BD-like mice that received 3  104 CD4 CD25 T cells (n  5; non-treated versus 3  105, P  0.08; BD-like versus BDN, P  0.07). The intensity of TGF-β messenger RNA (mRNA) expression in the spleen tissues of BD-like mice treated with 3  105 CD4 CD25 T cells was stronger than that of those that did not receive the cells (Figure 4G). After treatment with 3  105 CD4 CD25 T cells, the IL-10 and TGF-β levels were higher in BD-like mice than in the BDN mice. IFN-γ is the key cytokine produced by T helper type 1 (Th1) cells, and IFN-γ protein levels have

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been shown to be higher in BD-like mice than in BDN mice (32). Therefore the IFN-γ levels of BDlike mice treated with 3  105 CD4 CD25 T cells and those that were not were compared. The results indicated that the IFN-γ levels were significantly lower after treatment with 3  105 CD4 CD25 T cells compared with mice that did not receive the cells. Specifically, ELISA revealed that the IFN-γ levels were 10.0  6.2 pg/mL in BDlike mice treated with 3  105 CD4 CD25 T cells (n  12), 37.4  6.6 pg/mL in BD-like mice that were not treated with T cells (n  5), 5.3  7.4 pg/mL in BDN mice (n  4) and 14.1  10.7 pg/mL in BD-like mice that were treated with 3  104 CD4 CD25 T cells (n  5; non-treated versus 3  105, P  0.000001; BD-like mice versus BDN, P  0.002; non-treated versus 3  104, P  0.03; Figure 4C). mRNA expression of IFN-γ in the spleen tissues of BD-like mice that were treated with 3  105 CD4 CD25 T cells was lower than that of untreated BD-like mice (Figure 4G). TNF-α is a potent paracrine and endocrine mediator of inflammatory and immune functions. Over-expression of TNF-α has been implicated in acute and chronic inflammatory diseases, such as rheumatoid arthritis, atopic dermatitis, psoriasis and Behcet’s disease (33). In the present study, the TNFα levels were found to be lower in BD-like mice treated with 3  105 CD4 CD25 T cells than in untreated BD-like mice. Specifically, ELISA revealed that the TNF-α level was 42.2  10.1 pg/mL in BD-like mice treated with 3  105 CD4 CD25 T cells (n  16), 67.8  30.3 pg/mL in BD-like mice that were not treated (n  9), 5.9  5.6 pg/mL in BDN mice (n  8) and 53.8  9.2 pg/mL in BDlike mice treated with 3  104 CD4 CD25 T cells (n  5; non-treated versus 3  105, P  0.002; BDlike mice versus BDN, P  0.00005; 3  104 versus 3  105, P  0.002; Figure 4D). mRNA expression of TNF-α in the spleen tissues showed similar patterns to the results of ELISA (Figure 4G). It is well known that IL-6 in combination with TGF-β induces Th17 cell generation from naive T cells and inhibits TGF-β-induced Foxp3 expression Table II. Changes in symptoms after CD4 CD25 T-cell transfer in BD-like mice. Improved/total number (%) CD4 CD25

CD4 CD25–

3 3 3 3

   

103 104 105 105

2/5 4/7 15/18 4/9

(40) (57) (83) (44)

Improvement and deterioration were determined based on the severity score.

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Figure 4. Expressions of cytokines were compared between BD-like mice treated with CD4 CD25 T cells and untreated BD-like mice. Serum was collected from the hearts of mice at 2 weeks after treatment with CD4 CD25 T cells. BD-like mice that received 3  104 and 3  105 CD4 CD25 T cells were compared with untreated BD-like and BDN mice. (A, B) IL-10 and TGF-β protein levels were increased in BD-like mice treated with 3  105 CD4 CD25 T cells. (C–F) IFN-γ, TNF-α, IL-6 and IL-17 protein levels were decreased in BD-like mice treated with 3  105 CD4 CD25 T cells. The short bars indicate the means. (G). mRNA expressions of cytokines in spleen tissues of BD-like mice. BDN and BD-like mice treated with 3  104 and 3  105 CD4 CD25 T cells were revealed by RT-PCR. In addition, real-time PCR was used to evaluate the effects of treatment with 3  105 CD4 CD25 T cells in BD-like mice. RT-PCR and real-time PCR results showed similar patterns. The mRNA expression of IFN-γ, TNF-α, IL-6, IL-17A, IL-17 and ROR-γt in BD-like mice treated with 3  105 CD4 CD25 T cells was lower than in untreated BD-like mice. However, mRNA expression of TGF-β in BD-like mice treated with 3  105 CD4 CD25 T cells was higher than in those that did not receive cells. RT-PCR: lane 1, BD-like mice; lane 2, BDN; lane 3, BD-like mice treated with 3  104 CD4 CD25 T cells; lane 4, BD-like mice treated with 3  105 CD4 CD25 T cells. ∗P  0.1, ∗∗P  0.05, ∗∗∗P  0.01.

Treg in Behcet’s disease model (22,34). Th17 cells produce IL-17 family specially IL-17A and IL-17F, and TNF (35) and are present in human patients with various autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus and asthma (36–39). As shown in Figure 4E, the serum IL-6 levels were higher in BD-like mice than BDN mice, and the levels in BD-like mice were down-regulated by the transfer of CD4 CD25 T cells. The IL-6 protein level was 215.6  133.3 pg/mL for untreated BD-like mice (n  6), 39.2  50.8 pg/mL for BDlike mice treated with 3  105 CD4 CD25 T cells (n  16) (P  0.0002) and 75.8  60.9 pg/mL for BD-like mice treated with 3  104 CD4 CD25 T cells (n  5; non-treated versus 3  104, P  0.05). In BDN mice (n  6), the IL-6 levels were 19.9  20.3 pg/mL (BD-like versus BDN, P  0.005). The levels in all mice that received cells were significantly lower than in BD-like mice that did not receive cells. To determine whether the adoptive transfer of CD4 CD25 T cells influenced the expression of Th17, IL-17 protein production was measured by ELISA. The IL-17 protein level was 19.1  8.1 pg/mL in BD-like mice that received 3  105 CD4 CD25 T cells (n  16), 33.4  7.2 pg/mL in those that received 3  104 CD4 CD25 T cells (n  5) and 37.9  20.5 pg/mL in untreated BD-like mice (n  13; non-treated versus 3  105, P  0.002). In BDN (n  10), the IL-17 level was 22.9  3.9 pg/mL, which was significantly lower than in BD-like mice (P  0.03; Figure 4F). mRNA expression of IL-17A and IL-17F in the spleen tissues of mice treated with 3  105 CD4 CD25 T cells and untreated BD-like mice showed similar patterns to the results of ELISA (Figure 4G). The mRNA expression of retinoic acid receptor (RAR)related orphan receptor-gamma t (ROR-γt), which is a transcription factor of Th17, was lower in BDlike mice that were treated with 3  105 CD4 CD25 T cells than in untreated BD-like mice. These results indicated that adoptive transfer of CD4 CD25 T cells altered the cytokine expression in BD-like symptoms. Influence of CD25 inhibition in BD-like mice by i.p. injection of anti-CD25 Ab Ten milligrams of anti-CD25 Ab produced by the PC 61 cell line were injected i.p. once into BD-like mice. The injected BD-like mice were then killed at days 1, 3 and 7, after which the frequencies of CD4 CD25 T and Treg cells in the splenocytes were analyzed by FACS and compared with those in the IgG1-injected groups (Figure 5A). One day after injection, the frequency of CD4 CD25T cells in the

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anti-CD25-injected group (n  5) was 1.84  1.24%, while it was 2.15  0.58% in the IgG1-injected group (n  4; P  0.66). Three days after injection, the level of CD4 CD25 T cells in the anti-CD25injected group (n  5) was 1.06  0.21%, while it was 3.14  0.64% in the IgG1-injected group (n  4; P  0.0002). Seven days after injection, the level of CD4 CD25 T cells in the antiCD25-injected group was 1.39  0.18%, while it was3.92  0.03% in the IgG1-injected group (P  0.003). The level of Treg cells in the antiCD25-injected group was 0.53  0.33% at 1 day after injection, while it was 0.97  0.26% in the IgG1-injected group (P  0.07). After 3 days, the level of Treg cells in the anti-CD25-injected group was 0.20  0.12%, while it was 1.78  0.03% in the IgG1-injected group (P  0.00003). Seven days after injection, the level of Treg cells in the anti-CD25-injected group was 1.39  0.18%, while it was 3.92  0.03% in the IgG1 injection group (P  0.005). To evaluate the change in BD symptoms induced by injection of anti-CD25 Ab, 1 mg or 10 mg of anti-CD25 Ab was injected i.p. six times with 1-week intervals, and photographs taken before the first injection and 3 days after the final injection were compared. BD-like symptoms such as oral ulcer, skin ulcer, scrotum enlargement, genital inflammation and arthritis had deteriorated (Figure 5B). Following photography, the mice were killed and spleen tissues were analyzed for CD4 CD25 T and Treg cells by FACS analysis (Figure 5C). The frequency of CD4 CD25 T cells in the 10 mg antiCD25-injected group was 1.45  0.08%, while it was 2.05  0.38% in the IgG1-injected group (P  0.05) and 1.98  0.43% in the 1 mg anti-CD25injected group (n  4). The level of Treg cells in the 10 mg anti-CD25-injected group was 0.90  0.13%, while it was 1.23  0.22% in the IgG1-injected group (P  0.09) and 1.21  0.37% in the 1 mg anti-CD25-injected group (n  4). The decreased level of CD25 expression was related to deterioration of the BD symptoms and the increase in severity score of BD-like mice. In the 10 mg anti-CD25 Ab-treated group, the severity score was increased by 40% compared with before the injection. Conversely, the severity score in IgG1-treated BD-like mice was increased by 17% (P  0.05; Figure 5D). Discussion The results of this study have revealed that the number of CD4 CD25 T cells in BD-like mice was significantly lower than in both BDN and normal mice, and that the frequency of Treg cells in BD-like mice was also lower than in BDN and normal mice. It has

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been reported that the frequency of CD4 CD25 Treg cells is lower in autoimmune and inflammatory diseases, such as Crohn’s disease (40), multiple sclerosis (MS) (41) and systemic lupus erythematosus (42), than in healthy control and inactive patients.

A

Mouse models of autoimmune and autoinflammatory disease, such as collagen-induced arthritis (43) and experimental autoimmune encephalomyelitis (EAE) (44), have also been shown to have lower levels of CD4 CD25 Treg cells than healthy mice.

Treg

CD4+CD25 5 IgG1 anti-CD25 p=0.0002

p=0.005

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Anti-CD25

Figure 5. Influence of CD25 inhibition by injection of anti-CD25 Ab in BD-like mice. (A) Ten milligrams of anti-CD25 Ab produced by the PC 61 cell line were injected i.p. once into BD-like mice. The treated BD-like mice were killed at days 1, 3 and 7, and the frequencies of CD4 CD25 T and Treg cells in splenocytes were analyzed by FACS and then compared with control Ab (IgG1)-injected BD-like mice (n  4). (B) Anti-CD25 Ab injection into BD-like mice resulted in deteriorated BD symptoms. BD-like mice were injected i.p. with 1 or 10 mg of anti-CD25 Ab six times at 1-week intervals. IgG1 was injected as a control. Photographs were compared before the injection and 3 days after the final injection. After photography, the mice were killed and the levels of CD4 CD25 T and Treg cells in the spleens were analyzed by FACS analysis. (C). After six injections with 10 mg anti-CD25 Ab, the frequencies of CD4 CD25 T and Treg cells were lower than in the IgG1-injected or 1 mg anti-CD25-injected groups. There was a significant difference between the 10 mg-injected group and the IgG1-injected group in CD4 CD25 T cells (P  0.05). (D) The severity score of the 10 mg anti-CD25 Ab-injected group was significantly up-regulated compared with before the injection. The severity score of the 1-mg injected and IgG1-injected group did not differ significantly before and after treatment.

Treg in Behcet’s disease model However, in rheumatoid arthritis, the frequency of CD4 CD25 Treg cells is not different (42) or higher than that of healthy controls (45). Interestingly, the percentage of Treg cells among CD4 T cells in BD patients that have suffered an ocular attack was significantly lower before the ocular attack than after the attack (21). Treg cells of BD-like mice with symptoms are maintained at lower levels than in normal mice. It has also been reported that Treg cells suppress natural killer (NK) cells (46). NK cells play a role in the induction and regulation of various types of immune responses, including several autoimmune diseases, through cytotoxicity and cytokine production (47). Indeed, several studies have shown NK-mediated cytotoxicity and cytokine secretion are believed to play roles in the immunopathogenesis of Behcet’s disease (48,49). Moreover, the frequencies of NK cells in BD mice have been shown to be higher than in BDN mice (50). Therefore, transferred Treg cells may influence the frequencies of NK cells in BD mice. IL-2 and TGF-β are T-cell growth factors that can induce CD4 CD25 Treg cells (51). In addition, these cytokines play important roles in the development, survival and function of CD4 CD25 Treg cells (52). The results of the present study showed that these cytokines expand CD4 CD25 Treg cells effectively, and that the levels of CD4 CD25 T and Treg cells were higher in primary cultures of normal splenocytes than in BD-like and BDN mice. Increased regulatory T-cell numbers or functions have been shown to be associated with improved inflammatory symptoms, such as MS, EAE and diabetes, in mice and humans (10,16,53–55). CD4 CD25 Treg cells were found to play a role in maintaining immunologic self-tolerance, which led to a decrease in the spontaneous development of various autoimmune diseases (56). Therefore, it was expected that the transfer of CD4 CD25 T cells would improve BD symptoms in mice. Indeed, the frequencies of CD4 CD25 T and Treg cells were up-regulated in the splenocytes of BD-like mice following treatment with CD4 CD25 T cells. The frequencies of CD4 CD25 T and Treg cells in the splenocytes of BD-like mice were dependent on the amount of CD4 CD25 T cells administered. Furthermore, we observed a change in symptoms in BD-like mice after treatment with CD4 CD25 T cells. Two weeks after treatment, the symptoms of BD-like mice showed improvement and the disease severity score was decreased compared with the transfer of CD4 CD25– T cells. The disease severity score has been reported to decrease in response to the transfer of CD4 CD25 T cells in EAE (57) and collagen-induced arthritis (CIA) (43). In the present

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study, the improvement of symptoms and increase of Treg in peripheral tissues occurred after treatment with CD4 CD25 T cells; therefore, adoptive transfer appears to lead to the improvement of BD-like symptoms in mice. CD4 CD25 T cells cultured with TGF-β and IL-2 showed increased levels of IL-10 and TGF-β, whereas the levels of IFN-γ were decreased (58,59). In the present study, we showed that treatment with CD4 CD25 T cells improved the symptoms through up-regulation of TGF-β and IL-10, and that the TNF-α and IFN-γ levels decreased after treatment compared with BD-like mice that did not receive the cells. IFN-γ is a Th1 cytokine (60) and TNF-α is a mediator of inflammatory and immune functions. Th17 cells are highly pro-inflammatory cells that orchestrate tissue inflammation and organ-specific autoimmune diseases (61). IL-17 is a cytokine secreted by Th17 cells and IL-6 inhibits CD4 CD25 T-cell regulatory functions (62). IL-6 was found to be highly elevated in the culture supernatants of peripheral blood mononuclear cells (PBMC) of patients with active Behcet’s disease (63). The results of the present study have revealed that IL-17 and IL-6 proteins decrease in response to the transfer of CD4 CD25 T cells to BD-like mice and show levels similar to those of BDN mice. Foxp3 inhibits ROR-γt-mediated IL-17A mRNA transcription (64). In the present study, the mRNA levels of ROR-γt in BD-like mice treated with 3  105 CD4 CD25 T cells were lower than in untreated BD-like mice, while the levels of IL-17A (IL-17) and IL-17F, which were produced by Th17 cells in BD-like mice that were treated with 3  105 CD4 CD25 T cells, were lower than in the untreated BD-like mice. Therefore, Th17 cell-mediated cytokines and Th17 cell-associated transcription factors were inhibited by treatment with CD4 CD25 Treg cells. We have confirmed that the frequency of CD4 CD25 T cells in BD-like mice is lower than in BDN mice. CD4 CD25 T- and Treg-cell levels in primary cultures of splenocytes were also lower in BD-like mice than in BDN mice. In vivo, the frequency of CD4 CD25 T cells was higher in BD-like mice treated with 3  105 CD4 CD25 T cells than in those treated with 3  103 and 3  104 CD4 CD25 T cells. Additionally, the transfer of CD4 CD25 T cells to BD-like mice improved the BD-like symptoms. The severity scores decreased accordingly compared with the transfer of CD4 CD25–T cells. The serum protein levels of TGF-β and IL-10 were higher in BD-like mice treated with 3  105 CD4 CD25 T cells, but those of IFNγ, TNF-α, IL-6 and IL-17 were lower compared with untreated BD-like mice. The mRNA level of TGF-β was higher in BD-like mice treated with

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CD4 CD25 T cells than in untreated BD-like mice, whereas the levels of IFN-γ, TNF-α, IL-17A, IL-17F and ROR-γt of BD-like mice treated with CD4 CD25 T cells were lower than in untreated BD-like mice. The expression levels of CD4 CD25 regulatory T cells were correlated with the symptoms of HSV-induced BD-like mice. Therefore, the low level of CD4 CD25 regulatory T cells can be a factor in the etiopathogenesis of BD, adding to the activation of macrophages (32), excessive expression of cytokines such as IL-6 (65), TNF-α (66) and IL-17 (65), and the low level of functional homologue of HLA-G (Qa-2) (50) in BD mice. Acknowledgments This work was supported by the Korea Research Foundation Grant (MOEHRD, Basic Research Promotion Fund) (KRF-2007-313-E00349) and the Korean Health Technology R&D Project, Ministry for Health, Welfare &Family Affairs, Republic of Korea (A100535). Declaration of interest: The authors have no competing interests to declare. References 1. Shimizu T. Behcet disease (Behcet syndrome). Semin Arthritis Rheum. 1979;8:223–60. 2. Behcet H. Ueber rezidivierende, aphthoese durch ein Virus verursachte Geschwuere am Mund, am Auge und an den Genitalien. Dermatol Wochenschr. 1937;105:1152–7. 3. Lee S, Bang D, Cho YH, Lee ES, Sohn S. Polymerase chain reaction reveals herpes simplex virus DNA in saliva of patients with Behcet’s disease. Arch Dermatol Res. 1996;288:179–83. 4. Lee ES, Lee S, Bang D, Sohn S, Park C, Lee K. Herpes simplex virus detection by polymerase chain reaction in intestinal ulcer of patients with Behcet’s disease. J Invest Dermatol. 1993;101:474. 5. Bang D, Yoon KH, Chung HG, Choi EH, Lee ES, Lee S. Epidemiological and clinical features of Behcet’s disease in Korea. Yonsei Med J. 1997;38:428–36. 6. Lee ES, Lee S, Bang D, Sohn S. Detection of herpes simplex virus DNA by polymerase chain reaction in genital ulcer of patients with Behcet’s disease. Revue du Rhumatisme. 1996;63:532. 7. Sohn S, Lee ES, Bang D, Lee S. Behçet’s disease-like symptoms induced by the Herpes simplex virus in ICR mice. Eur J Dermatol. 1998;8:21–3. 8. Kim HJ, Bang D, Lee SH, Yang DS, Kim DH, Lee KH, et al. Behcet’s syndrome in Korea: a look at the clinical picture. Yonsei Med J. 1988;29:72–8. 9. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155:1151–64. 10. Sakaguchi S. Naturally arising Foxp3-expressing CD25 CD4 regulatory T cells in immunological tolerance to self and non-self. Nat Immunol. 2005;6:345–52.

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