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 Sa˜o Paulo, Sa˜o Paulo, Brazil; the 2Department of Immunology, University of Sa˜o Paulo and the 4Albert Einstein Jewish and prevents their migration to inflammatory sites. Institute for Education and Research, Sa˜o Paulo, Brazil. In transplantation, FTY720 prolonged mouse (C3H to Supported by Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o 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 Sa˜o 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. Gonc¸alves 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 Sa˜o Paulo, In B10.RIII mice induced to autoimmune uveoretinitis, the Rua Botucatu 862 4o andar, Sa˜o Paulo, Brazil; [email protected]. retinal infiltrate was prevented by FTY720 treatment 2 days Investigative Ophthalmology & Visual Science, May 2010, Vol. 51, No. 5 2568 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 10/01/2021 IOVS, May 2010, Vol. 51, No. 5 EAU in Mice 2569 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).