Evaluation of Experimental Autoimmune Uveitis in Mice Treated with FTY720

Immunology and Microbiology Evaluation of Experimental Autoimmune Uveitis in Mice Treated with FTY720

Alessandra Gonc¸alves Commodaro,1 Jean Pierre S. Peron,2 Camila Takao Lopes,3 Christina Arslanian,2 Rubens Belfort, Jr,1 Luiz Vicente Rizzo,2,4 and Valquiria Bueno3

PURPOSE. FTY720 (ﬁngolimod) is an immunomodulatory drug the study of human idiopathic uveitis. This disease can be capable of preventing T-cell migration to inﬂammatory sites by induced in susceptible primates and rodents after immuni- binding to and subsequently downregulating the expression of zation with retinal self-antigens, such as interphotoreceptor

sphingosine-1 phosphate receptor 1 (S1P1) leading in turn to retinoid-binding protein (IRBP) or S-antigen (arrestin), or by T-cell retention in lymphoid organs. Additional effects of the adoptive transfer of uveitis antigen-specific T cells.1–3 FTY720 by increasing functional activity of regulatory T cells It has been shown that autoreactive Th1 cells mediate have recently been demonstrated, raising the conversion of EAU4,5; thus, its induction is correlated with the production of conventional T cells into regulatory T cells and affecting the IFN-␥ by T cells. Moreover, inhibition of Th1 responses by sequestration of regulatory T cells in normal mice. In this anti–IL-12 treatment suppresses the disease.6 Recently, Th17 study, the action of FTY720 in the ocular autoimmune model in cells have also been suggested to be involved in EAU.7,8 mice was investigated. The EAU mouse model has contributed significantly to the METHODS. Mice were immunized with 161-180 peptide and establishment of parameters for the evaluation of possible 3 pertussis toxin and were treated with 1 mg/kg/d FTY720 by therapies for posterior uveitis in humans. Studies of genetic 9 gavage (7–21 days postimmunization [dpi]) or left untreated. susceptibility and resistance to EAU, characterization of 10 Spleen cells, harvested 21 dpi, were cultured and assayed for uveitogenic epitopes, and studies of tolerance in EAU by 11,12 cytokine production. Draining lymph node, spleen, and eye anterior chamber-associated immune deviation or by sys- 13,14 cells 21 dpi were assayed for quantification of T-cell popula- tems of oral tolerance have obtained success according to tions. Disease severity was evaluated by histologic examination this model. of the enucleated eyes at 21 and 49 dpi. In addition, anti–IRBP In 2005, our group demonstrated that treatment with an ␣4 antibodies were analyzed by ELISA. active peptide inhibitor, which targets the ␣4 integrin of the VLA-4 adhesion molecule, had a significant ameliorating effect RESULTS. FTY720 was effective in suppressing the experimental on EAU.15 Clinical ocular pathology can also be prevented by autoimmune uveitis score. Although there was a reduction in the administration of recombinant Galectin-1 (rGal-1) either the number of eye-infiltrating cells, FTY did not prevent Treg early or late during the course of EAU.16 Associated mecha- accumulation at this site. FTY720 leads to a significant increase ϩ ϩ ϩ ϩ nisms were recently described because Gal-1 induces dendritic of CD4 IFN-␥ and CD4 Foxp3 cell percentages in lymph cells to secrete IL-27, which induce T cells to become Tr1 nodes, suggesting that this site could be the source of Treg IL-10–secreting cells. In addition, IL-27 is able to directly sup- cells found in the eye. press Th17 cells.17 Altogether, these results have shown that CONCLUSIONS. The data showed that treatment in vivo with manipulating the immune system to upregulate Th2- and FTY720 was able to suppress EAU in mice. These results are T-regulatory cytokines can prevent inflammation and EAU de- indicative of the possible therapeutic use of FTY720 in ocular velopment.18–20 autoimmune processes. (Invest Ophthalmol Vis Sci. 2010;51: Recently, various reports clearly showed that some mol- 2568–2574) DOI:10.1167/iovs.09-4769 ecules, such as LX211 (voclosporin),21 anti–IL-17,22 and ␣-melanocyte–stimulating hormone,23 were effective in sup- xperimental autoimmune uveitis (EAU) is an organ-specific, pressing EAU. In this context, fingolimod (FTY720; Novartis, ET cell–mediated disease that targets the posterior pole of Basel, Switzerland) is an immunomodulatory drug capable of the eye. It is also a valuable, well-characterized model for preventing T-cell infiltration by binding to and subsequently downregulating the expression of sphingosine-1 phosphate 24 receptor 1 (S1P1). S1P1 signaling is required for T-cell 25 1 3 egress from secondary lymphoid tissue. Therefore, block- From the Vision Institute and the Immunology Division, Univer- ing this pathway leads to T-cell retention in lymphoid organs sidade Federal de Saõ Paulo, Saõ Paulo, Brazil; the 2Department of Immunology, University of Saõ Paulo and the 4Albert Einstein Jewish and prevents their migration to inflammatory sites. Institute for Education and Research, Saõ Paulo, Brazil. In transplantation, FTY720 prolonged mouse (C3H to Supported by Fundaçaõ de Amparo a Pesquisa do Estado de Saõ BALB/c) corneal allograft survival by reducing the infiltration of ϩ ϩ Paulo Grant 04/14727-0; Conselho Nacional de Desenvolvimento CD4 and F4/80 cells and decreasing allogeneic T-cell prolif- Científico e Tecnolo´gico; Pan-American Ophthalmological Foundation eration.26 In rats, penetrating keratoplasty was performed Grant 2007; and FADA-Universidade Federal de Saõ Paulo. (Fisher to Lewis), and FTY720 treatment caused significant Submitted for publication October 14, 2009; revised November rejection-free graft survival and reduction of CD4ϩ, CD8ϩ, and 18, 2009; accepted December 5, 2009. NK infiltrating cells.27 In addition, FTY720 treatment sup- Disclosure: , None; , A. Gonçalves Commodaro J.P.S. Peron pressed the incidence and intensity of EAU in Lewis rats im- None; C.T. Lopes, None; C. Arslanian, None; R. Belfort, Jr, None; munized with S-antigen. Suppression of antibody serum levels L.V. Rizzo, None; V. Bueno, None 28 Corresponding author: Valquiria Bueno, Laboratory of Experimental and antigen-specific lymphocyte proliferation was observed. Immunology, Immunology Division, Universidade Federal de Saõ Paulo, In B10.RIII mice induced to autoimmune uveoretinitis, the Rua Botucatu 862 4o andar, Saõ Paulo, Brazil; [email protected]. retinal infiltrate was prevented by FTY720 treatment 2 days

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before disease onset. This finding was associated with the blinded fashion by examining six sections cut at different levels for suppression of histologic disease. A single dose of fingolimod each eye. Severity of EAU was scored on a scale from 0 (no disease) to administered after disease onset (14 days after induction) not 4 (maximum disease) in half-point increments, according to a semi- only abolished retinal infiltrates but prevented disease relapse quantitative system described previously3 and according to lesion type, for at least 3 weeks.29 size, and number. In brief, the minimal criterion to score an eye as Additional effects of FTY720 have recently been demon- positive by histopathology was inflammatory cell infiltration of the strated by increasing the functional activity of regulatory T ciliary body, choroids, or retina (EAU grade 0.5). Progressively higher cells, enabling the conversion of conventional T cells into grades were assigned for the presence of discrete lesions in the tissue regulatory T cells30–32 and affecting the sequestration of regu- such as vasculitis, granuloma formation, retinal folding or detachment, latory T cells in normal mice.31 and photoreceptor damage. Daniel et al.32 used a model of experimental colitis to evaluate the role of FTY720 in disease development. It was Determination of Cytokine Production observed that FTY720 substantially reduced all clinical, his- Spleen cells harvested 21 dpi were cultured in 24-well plates (106 topathologic, macroscopic, and microscopic parameters of co- cells/well) and stimulated with 30 ␮g/mL IRBP. Supernatants were litis analyzed. FTY720 treatment caused a downregulation of collected for cytokine analysis after 48 or 72 hours and were stored at IL-12p70 and TNF-alpha expression in colon protein extracts. ϩ Ϫ80°C until assayed. The level of IFN-␥ was assessed by ELISA using a LP CD4 cells from Peyer’s patches were evaluated by ELISA kit from BD PharMingen (La Jolla, CA), and the level of IL-17 was and presented a dose-dependent increase of IL-10 and TGF-beta assessed by ELISA using a kit from eBioscience (San Diego, CA). All kits expression in FTY720-treated mice. In addition, induction of were used according to the manufacturers’ instructions. FoxP3 and CTLA4 protein expression was observed in colon. This findings provide evidence that, in addition to its well- established effects on migration, FTY720 leads to specific Flow Cytometry Analysis downregulation of proinflammatory signals while it simulta- Eyes. Eyes were nucleated and gently crushed with the flat surface ϩ ϩ neously induces the functional activity of CD4 CD25 Treg of a sterile syringe plunger in a plastic Petri dish containing PBS. The cells.32 cell suspension was then filtered through a 400-␮m stainless iron mesh Zhang et al.33 evaluated experimental autoimmune neuritis and a 70-␮m nylon cell strainer (BD Biosciences-Labware, Bedford, MA) (EAN) in rats and observed by flow cytometry that treatment and adjusted for 5 ϫ 105 cells/tube for staining with rat anti–mouse with FTY720 at disease onset (day 10 until day 18) caused an CD25Pe and rat anti–mouse CD4PerCP-Cy5.5 (eBioscience). After in- increase in Foxp3 cells in blood but a decrease in lymph node. cubation for 30 minutes, cells were washed with PBS, fixed for 15 In sciatic nerves of EAN rats, FTY720 increased the percent- minutes with 1% paraformaldehyde, permeabilized for 6 minutes with ϩ ages of Foxp3 cells as observed by immunohistochemistry.33 0.2% Triton X-100, and stained with rat anti–mouse Foxp3 FITC for 30 Considering that different T-cell subsets may present uveito- minutes (eBioscience). Stained cells were acquired (FACSCalibur Flow genic capability and that FTY720 not only increases Treg cells Cytometer; BD Biosciences), and data were analyzed (Cell Quest Pro generation but also affects their homing properties,27 it was software; BD Biosciences). At least 30,000 events were acquired for our aim to investigate the effects of FTY720 administration in each analysis. the EAU model and possible associated mechanisms. Spleen and Lymph Nodes. The frequency of CD4ϩ and CD8ϩ in spleen cells and CD4ϩCD25ϩFoxp3ϩ T cells in lymph nodes was evaluated in cell suspensions first incubated with anti–CD32/CD16 (Fc ATERIALS AND ETHODS M M block) antibody for 20 minutes at 4°C. Cell suspensions were further Animals incubated with combinations of fluorescent antibody for 30 minutes at 4°C (all purchased from eBioscience). All samples were acquired (FACS- Six- to 8-week-old B10.RIII mice were obtained from the animal facil- Calibur; BD Biosciences), and data were analyzed with appropriate soft- ities of the University of Sao Paulo in Brazil. All animals were housed ware (FlowJo; TreeStar, Ashland, OR). under specific pathogen-free conditions and treated in accordance with institutional guidelines and with the ARVO Statement for the Use Intracellular Cytokine Staining and FACS Analysis of Animals in Ophthalmic and Vision Research. The animal care and For detection of intracellular expression of IL-17 and IFN-␥ by FACS use committee at the Institute of Biomedical Sciences of the University analysis, cells were collected and were left unstimulated or were of Sao Paulo approved all the procedures used in this study. stimulated for 5 hours with 50 ng/mL phorbol 12-myristate 13-acetate Induction of EAU and In Vivo Treatment (PMA; Sigma, St. Louis, MO) and 750 ng/mL ionomycin (Calbiochem, La Jolla, CA) in the presence of either protein transport inhibitor with FTY720 containing Brefeldin A (Golgiplug; BD PharMingen) or protein trans- Mice were immunized subcutaneously at the base of the tail with 30 ␮g port inhibitor containing monensin (Golgistop; BD PharMingen) at the 161-180 peptide emulsified in 0.2 mL CFA (vol/vol). At the same time, recommended concentration. Standard intracellular cytokine staining mice were injected intraperitoneally with 0.5 ␮g PTX in 0.1 mL as an was performed. Briefly, cells were stained extracellularly with fluores- additional adjuvant. Animals were treated with 1 mg/kg/d FTY720 cein-conjugated anti–CD4ϩ and were fixed. Then they were perme- administered in 200 ␮L PBS by gavage (7–21 days postimmunization abilized with solution (Cytofix/Cytoperm; BD PharMingen) and stained [dpi]) or were left untreated. intracellularly with phycoerythrin-conjugated anti–IL-17 and anti– IFN-␥. All samples were acquired (FACSCalibur; BD Biosciences), and Histopathology EAU data were analyzed (FlowJo; TreeStar). Eyes were collected and prepared for histopathologic evaluation at the end of each experiment (21 and 49 dpi). The eyes were immersed Analysis of Anti–IRBP Antibodies for 1 hour in phosphate-buffered glutaraldehyde 4%, transferred to Levels of anti–IRBP IgG1 and IgG2a isotypes were determined by ELISA phosphate-buffered formaldehyde 10% for 24 hours, and replaced with in untreated and in FTY-treated mice sera. Briefly, 96-well microtiter ethanol 70% until processing. Fixed and dehydrated tissue was embed- plates (MaxiSorp; Nunc, Roskilde, Denmark) were coated with 1 ␮g/ ␮ Ϫ ded in paraffin wax, and 4- to 6- m sections were cut through the well IRBP diluted in 0.1 M NaHCO3 , pH 9.6, overnight at 4°C. Plates papillary-optic nerve plane. Sections were stained by hematoxylin and were washed three times with PBS containing 0.05% Tween 20. Serum eosin. Presence or absence of disease was evaluated in a double- was serially diluted starting at 1:16. Peroxidase-labeled anti–mouse

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IgG1 (1:1000) or IgG2a (1:1000) was obtained from BD PharMingen.

Antibody titers were expressed as log2 of the reciprocal serum dilution giving an absorbance value of 20% of the saturation level.

Statistical Analysis Data are expressed as mean Ϯ SD. Statistical analyses were performed (Prism version 5.00 for Windows; GraphPad Software, San Diego CA). Parametric Student’s t-test was used, and P Ͻ0.05 was considered signiﬁcant.

RESULTS FTY720 Was Associated with a Decreased EAU Score Mice were immunized and treated with oral FTY during the migration of cells to the eye. We observed that treatment with FTY was able to inhibit the development of the disease (Fig. 1A). Histopathologic examination showed retinal disorganiza- tion and the presence of inflammatory cells in the vitreous in mice with EAU on day 21 (Fig. 1B). In treated animals (EAU ϫ FTY) it is possible to observe a normal organization of the retina. FTY720 Prevents Cell Migration to the Eye It has been shown that FTY720 sequesters T lymphocytes in secondary lymphoid organs and inhibits their migration to the inflammatory site. In agreement with this finding, our results show that 21 dpi nontreated mice had a significantly higher number of infiltrating cells in the eye than did FTY-treated mice (Fig. 2). FTY720 Reduces the Percentage of CD4؉CD25؉ T-Cell Population in the Eye It is known that in animals with EAU, CD4 T cells migrate to the eye and are responsible for the induction of the inflammatory response, leading to the lesions observed in disease pro- gression. In addition, it is known that FTY prevents T-cell FIGURE 1. Treatment with FTY prevents ocular pathology. B10.RIII migration to the inflammatory site with a more important mice were immunized with 25 ␮g 161-180 IRBP peptide on day 0 and effect in CD4ϩ T cells.34 Therefore, we analyzed the percent- were treated (gavage) with FTY or saline solution (vehicle control). ages of cells in the eyes of mice treated with FTY and of mice Eyes were collected for histopathology on day 21 after immunization. that did not receive treatment. Lower percentages and absolute (A) EAU score was assigned by histopathologic examination of the eyes cell numbers of CD4ϩCD25ϩ T cells were observed in the eyes on a scale from 0 to 4 according to the extent of inflammation and tissue damage. EAU, n ϭ 9; EAU ϩ FTY, n ϭ 8(P ϭ 0.0001). (B) of treated mice than of nontreated mice (Fig. 3A). Also noted ϩ ϩ ϩ Histopathologic features representative of the EAU scores: mice immu- was a decrease in the frequency of CD4 CD25 Foxp3 regu- nized with IRBP and treated with vehicle control (EAU) show ocular latory T cells inside the eye of treated mice, though the abso- lesions characterized by cells infiltrating the vitreous and retinal folds; lute cell numbers were similar to those of nontreated mice mice treated with FTY (EAU ϩ FTY) exhibit a normal retinal architec- (Fig. 3B). On the other hand, although there was no difference ture corresponding to nonimmunized naïve mice. Representative pho- in the percentage of the CD4ϩIL-17ϩ T-cell population when tographs (hematoxylin-eosin staining). Original magnifications: 100ϫ EAU and EAU ϩ FTY mice were compared, we observed a (left); 400ϫ (right). decrease in absolute cell numbers of this cell population in FTY-treated mice (Fig. 3C). were similar when EAU mice were compared to EAU ϩ FTY mice. On the other hand, percentages and absolute numbers of Treatment with FTY720 Causes a Change in the ϩ ϩ ؉ ؉ ؉ ؉ CD4 IL-17 T cells were similar in control and treated animals Percentage of CD4 Foxp3 and CD4 IFN-␥ T (Fig. 4D). Cells in Lymph Nodes FTY720 Is Capable of Decreasing the Absolute We analyzed the percentages and absolute cell numbers of ؉ CD4ϩCD25ϩ and CD4ϩFoxp3ϩ T cells in the lymph nodes of Cell Numbers of CD4 T Cells in the Spleen animals treated with FTY and those left untreated. Our study It has been shown that FTY720 has a more prominent effect in showed a significant increase in the percentage of CD4ϩ than in CD8ϩ T-cell populations.34 In agreement with CD4ϩFoxp3ϩ cells in treated animals when compared with this, our data from spleen cells showed a significant decrease in control animals (Fig. 4A), but no difference was found in absolute cell numbers of CD4ϩ compared with CD8ϩ T cells absolute cell numbers. (Fig. 5A). Considering that autoreactive Th1 cells mediate Our study also showed a significant increase in the fre- EAU,4,5 this decrease corroborates with the lower level of quency of CD4ϩIFN-␥ϩ T cells in the lymph nodes of treated disease observed in FTY720-treated mice. We also found in this animals (Fig. 4B), whereas CD4ϩIFN-␥ϩ absolute cell numbers group a reduced absolute cell (lymphocyte) number in spleen

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FIGURE 2. FTY720 prevents cell migration to the eye. Mice were immunized with IRBP peptide and either treated or not treated with FTY720 (7–21 days). Eyes were collected in order to obtain inﬁltrating cells. Cell count is shown in absolute numbers (ϫ106) for EAU and EAU ϩ FTY (P ϭ 0.0001).

and lymph node that did not reach statistical signiﬁcance (Fig. 5B). Analysis of IFN-␥ and IL-17 Levels in Spleen Spleen cells of treated animals produced the same amount of IFN-␥ that the cells of control animals produced (Fig. 6A).

FIGURE 4. FTY treatment sequesters T cells in lymph nodes and causes increases in the percentage of CD4ϩFoxp3ϩ, CD4ϩIFN-␥ϩ, and CD4ϩIL-17ϩ T cells. Cells from lymph nodes were collected at 21 dpi, labeled, ﬁxed and permeabilized, stained intracellularly, and analyzed by ﬂow cytometry evaluating the expression of CD4ϩFoxp3ϩ (A; P ϭ 0.0349), CD4ϩCD25ϩ (B), CD4ϩIFN-␥ϩ (C; P ϭ 0.0138), and CD4ϩIL- 17ϩ (D). Results are expressed as mean Ϯ SD.

Similarly, with regard to the production of IL-17, there was no difference in cytokine production between treated animals and control animals (Fig. 6B). Anti–IRBP IgG2a and IgG1 Levels Presented No Change after FTY Treatment As shown in Figure 7A, anti–IRBP IgG2a levels were not altered in the sera of treated animals compared with control. In addition, no significant difference was observed in the levels of anti–IRBP IgG1 antibodies in both groups (Fig. 7B). FTY720 Inhibits the Severity of EAU after Drug Withdrawal FTY-treated (7–21 days) animals were killed at 49 dpi of EAU FIGURE 3. Treatment with FTY causes decreases in the percentage induction. Histopathologic analysis showed that FTY was able and absolute cell number of CD4ϩCD25ϩ T cells, the percentage of ϩ ϩ ϩ ϩ ϩ to maintain the animals without disease recurrence (Fig. 8). CD4 CD25 Foxp3 T cells, and the absolute number of CD4 IL-17 Figure 8A shows that treated mice were compared with un- in the eye. Cells from the eye were collected at day 21 after immuni- treated mice but had no evidence of disease. Figure 8B shows zation, labeled with anti–CD4 and anti–CD25 mAb, fixed and permeabilized, stained intracellularly, and analyzed by flow cytometry evalu- that treated animals continued without intraocular lesions after ating the CD4ϩCD25ϩ (A) and CD4ϩCD25ϩFoxp3ϩ (B) expression. the drug was withdrawn. Data (mean Ϯ SD) represent the results of two experiments using ϩ ϩ pooled eyes. (C) Similar percentage of CD4 IL-17 T cells in EAU and DISCUSSION EAU ϩ FTY groups but significant decrease of total absolute number of these cells in EAU ϩ FTY. Data represent flow cytometry from three The model of EAU in mice contributes significantly to the pooled eyes. establishment of parameters for possible therapies for poste-

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ϩ ϩ FIGURE 5. CD4 and CD8 T cells decrease in FTY-treated mice (EAU ϩ FTY). Mouse spleen cells were harvested at day 21, labeled, and analyzed by ﬂow cytometry for (A) CD4ϩ and CD8ϩ expression. (B) Cell count in spleen and lymph node show discrete decreases in FTY720-treated mice. Data are shown as mean Ϯ SD (P ϭ 0.0499).

rior human uveitis.3 Studies of genetic susceptibility and resis- FIGURE 7. FTY showed no effect on serum levels of anti–IRBP IgG2a tance to EAU,9 characterization of uveitogenic epitopes,10 and and IgG1 antibodies. Anti– IRBP IgG2a (A) and IgG1 (B) antibody studies of tolerance in EAU by anterior chamber-associated production in treated (EAU ϩ FTY) or untreated (EAU) mice were ϩ immune deviation10–12 or systems of oral tolerance13,14 have determined by ELISA at 21 dpi. Results are expressed in titers SD. been successful according to this model. Our study showed that the treatment with FTY (fingolimod) was able to inhibit retinal folds, and inflammatory infiltrating cells in the vitreous. the development of disease in animals immunized with the We also observed, by harvesting and counting eye-infiltrating IRBP peptide. This can be seen in histopathologic analysis in lymphocytes, that FTY significantly impaired the migration of these cells to the eye. In agreement with our data, Raveney et which treated animals did not have retinal lesions, in contrast 29 to untreated animals with disorganized retinal layers, intense al. showed that treatment with fingolimod rapidly reduces ocular infiltrates in EAU in mice. EAU development in mice is provided by CD4 T cells with a Th1-like phenotype, high levels of IFN-␥, and low levels of IL-4 production.35,36 We evaluated by flow cytometry the phenotype of eye-infiltrating cells and observed that there was a decrease in the percentage of CD4ϩCD25ϩ and CD4ϩ CD25ϩFoxp3ϩ cells, whereas the percentage of CD4ϩIL-17ϩ cells was similar when compared with nontreated animals. Moreover, the absolute numbers of CD4ϩCD25ϩ and CD4ϩIL- 17ϩ infiltrating cells was lower in FTY-treated mice. CD4ϩ CD25ϩFoxp3ϩ absolute cell numbers infiltrating the eye were similar in EAU and FTYϩEAU groups. Our findings in the eye confirm the already known role played by FTY, which is to prevent cell migration to the inflammatory site. The results also suggest an additional role for FTY: the improvement of CD4ϩCD25ϩFoxp3ϩ function. Sim- ilar absolute numbers of eye-infiltrating cells with Treg phenotype were observed in both groups, but disease was prevented only in FTY-treated mice (Figs. 1, 3). Therefore, we can argue that FTY720 promoted the improvement of Treg function. This cell population was capable of inhibiting CD4ϩ-IL-17ϩ–produc- ing cells. In accordance with our data, Zhang et al.33 showed that FTY720 administered at the onset of EAN caused the accumulation of Foxp3ϩ cell in sciatic nerve during the period of recovery from neurologic disease, suggesting a contribution of Foxp3ϩ cells to the resolution of EAN. To evaluate a possible effect of FTY720 treatment in the periphery, we counted spleen and lymph nodes cells of mice induced with EAU and FIGURE 6. FTY has no effect on IFN-␥ production in spleen cells. Treated (EAU ϩ FTY) or untreated (EAU) mouse spleen cells were performed flow cytometry. 6 FTY-treated mice showed significantly higher expression of harvested at 21 dpi and stimulated in vitro (10 cells/mL) with 30 ϩ ϩ ϩ ϩ ␮g/mL IRBP (A, B). After 48 hours, IFN-␥ (A) and IL-17 (B) levels were CD4 Foxp3 and CD4 IFN-␥ cells in lymph nodes. We be- determined by ELISA. Results are expressed as mean Ϯ SD. lieve that in our model, the increase in IFN-␥ production in

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phocyte recirculation reaches a new steady state. All lymphatic organs showed a decline in lymphocyte numbers as the treatment time was extended to 21 days. Antibodies were investigated in mice sera, and we con- firmed the previously described16 higher levels of IgG2a than IgG1, suggesting that the inhibition of EAU development by FTY was not the result of immune deviation. Moreover, FTY treatment did not cause any statistical difference in the levels of evaluated antibodies compared with those of nontreated mice. The inflammatory process in the eye occurs between 6 and 9 dpi.42 After 9 days it is possible to observe an infiltrate of small cells in the retina and choroid in the eyes of B10.RIII mice. Among these cells are macrophages, CD4ϩ T lymphocytes, and dendritic cells that increase in number between 12 and 15 dpi, causing the onset of structural damage in retina. In 3 weeks (21–26 dpi), there was a decrease in the infiltration of inflammatory cells because of lower numbers of cells migrating to the eye. However, the intense intraocular inflammatory response already set causes retinal degeneration.43 We started treatment with FTY early (7 days after EAU induction) in cell migration to the eye. Even when the drug was withdrawn (21 dpi), EAU development was inhibited. This result suggests that FTY, which prevents cell migration to the eye during the inflammatory period, is a drug with an early mechanism of action capable of inhibiting EAU development. Treatment ex- FIGURE 8. Treatment with FTY prevents ocular pathology until 49 dpi even after the drug is withdrawn. B10.RIII mice were immunized with tended to 21 days also enabled the development of Treg cells 25 ␮g 161-180 IRBP peptide on day 0 and were treated (gavage) with capable of regulating EAU antigen-specific effector cells. FTY or saline solution (vehicle control) until 21 dpi. Eyes were col- It is important to emphasize the need to study the effect of lected for histopathology on 49 dpi. (A) EAU score was assigned by FTY during relapse, which, according to Chan et al.,44 occurs histopathologic examination of the eyes. (B) Histopathologic features 5 and 10 weeks after immunization. Studies of long-term treat- representative of the EAU scores: mice immunized with IRBP and ment are important; the effect on the production of specific treated with vehicle control (EAU) show ocular lesions characterized antibody has been previously reported.44 Our group has ob- by cells infiltrating the vitreous and retinal folds; mice treated with FTY ϩ served that treatment with iVLA-4 in the efferent phase is (EAU FTY) exhibit a normal retinal architecture corresponding to effective at reducing ocular damage in animals killed in 49 nonimmunized naive mice. Representative photographs (hematoxylin- 15 eosin staining). Original magnifications: 100ϫ (left); 400ϫ (right). days. In conclusion, FTY was able to prevent EAU development when administered between 7 and 21 dpi. In addition, the drug lymph node was associated with the enhancement of withdrawn did not lead to disease, suggesting the development CD4ϩFoxp3ϩ cells in this site (Figs. 4A, B). Consistent with our of a regulatory state. We believe that FTY caused a decrease of hypothesis, Kelchtermanns et al.37 showed that in the experi- eye-infiltrating cells without inhibiting Treg accumulation in mental model of collagen-induced arthritis, the absence of this site, that Treg cells in the eye were enhanced by FTY ϩ ϩ IFN-␥ was associated with disease susceptibility and impaired treatment and thus impaired the effects of CD4 IL-17 cells, function of CD4ϩCD25ϩ Treg cells. In transplantation, mice and that, in lymph nodes, the increased percentage of ϩ ϩ tolerized to alloantigen experience enhanced IFN-␥ production CD4 IFN-␥ cells was associated with the higher percentage ϩ ϩ by CD4ϩCD25ϩFoxp3ϩ cells. The binding of IFN-␥ to its re- of CD4 Foxp3 cells, which could have been the source of ceptor on Tregs upregulates STAT1 activation, causing in turn Treg cells found in the eye. Our results suggest a possible the maintenance of these cells in the graft and an enhancement therapeutic use of this immunomodulatory drug for the treat- of their regulatory role.38 Altogether these findings suggest ment of ocular autoimmune disease of autoimmune etiology. that, in our model, FTY treatment led to the accumulation of a regulatory T-cell population and to improved function in Acknowledgments lymph nodes that was supported by the production of IFN-␥. Evaluating the profile of the migration of these cells is needed The authors thank Paulo Albe for assistance in preparation of the to clarify their role in EAU. Yopp et al.39 showed that T cells histopathology slide and Robson L. Melo for synthesis of the IRBP migrate from peripheral lymph nodes in response to both peptide 161-180. chemokine (CXCL12) and S1PR activation. It is possible that in our model, FTY caused a preferential migration of Treg cells References from lymph nodes to the eye and, thus, contributed to the inhibition of EAU development. 1. Rizzo LV, Silver P, Wiggert B, et al. Establishment and character- ϩ Absolute cell numbers in lymph nodes from FTY-treated ization of a murine CD4 T cell line and clone that induce exper- mice decreased but did not reach statistical significance. Treat- imental autoimmune uveoretinitis in B10.A mice. J Immunol. ment caused a significant decrease in splenic CD4ϩ T cells, but 1996;156:1654–1660. the decrease in CD8ϩ T cells did not reach statistical signifi- 2. Mochizuki M, Kuwabara T, McAllister C, et al. Adoptive transfer of ϩ experimental autoimmune uveoretinitis in rats: immunopatho- cance. 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