The model of EAU in mice contributes significantly to the establishment of parameters for possible therapies for posterior human uveitis.
3 Studies of genetic susceptibility and resistance to EAU,
9 characterization of uveitogenic epitopes,
10 and studies of tolerance in EAU by anterior chamber-associated immune deviation
10–12 or systems of oral tolerance
13,14 have been successful according to this model. Our study showed that the treatment with FTY (fingolimod) was able to inhibit the development of disease in animals immunized with the IRBP peptide. This can be seen in histopathologic analysis in which treated animals did not have retinal lesions, in contrast to untreated animals with disorganized retinal layers, intense retinal folds, and inflammatory infiltrating cells in the vitreous. We also observed, by harvesting and counting eye-infiltrating lymphocytes, that FTY significantly impaired the migration of these cells to the eye. In agreement with our data, Raveney et al.
29 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-γ, 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 CD4
+CD25
+ and CD4
+CD25
+Foxp3
+ cells, whereas the percentage of CD4
+IL-17
+ cells was similar when compared with nontreated animals. Moreover, the absolute numbers of CD4
+CD25
+ and CD4
+IL-17
+ infiltrating cells was lower in FTY-treated mice. CD4
+CD25
+Foxp3
+ absolute cell numbers infiltrating the eye were similar in EAU and FTY
+EAU 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 CD4
+CD25
+Foxp3
+ function. Similar 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 CD4
+-IL-17
+–producing cells. In accordance with our data, Zhang et al.
33 showed that FTY720 administered at the onset of EAN caused the accumulation of Foxp3
+ cell in sciatic nerve during the period of recovery from neurologic disease, suggesting a contribution of Foxp3
+ 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 performed flow cytometry.
FTY-treated mice showed significantly higher expression of CD4
+Foxp3
+ and CD4
+IFN-γ
+ cells in lymph nodes. We believe that in our model, the increase in IFN-γ production in lymph node was associated with the enhancement of CD4
+Foxp3
+ cells in this site (
Figs. 4A, B). Consistent with our hypothesis, Kelchtermanns et al.
37 showed that in the experimental model of collagen-induced arthritis, the absence of IFN-γ was associated with disease susceptibility and impaired function of CD4
+CD25
+ Treg cells. In transplantation, mice tolerized to alloantigen experience enhanced IFN-γ production by CD4
+CD25
+Foxp3
+ cells. The binding of IFN-γ to its receptor on Tregs upregulates STAT1 activation, causing in turn the maintenance of these cells in the graft and an enhancement of their regulatory role.
38 Altogether these findings suggest that, in our model, FTY treatment led to the accumulation of a regulatory T-cell population and to improved function in lymph nodes that was supported by the production of IFN-γ. Evaluating the profile of the migration of these cells is needed to clarify their role in EAU. Yopp et al.
39 showed that T cells migrate from peripheral lymph nodes in response to both chemokine (CXCL12) and S1PR activation. It is possible that in our model, FTY caused a preferential migration of Treg cells from lymph nodes to the eye and, thus, contributed to the inhibition of EAU development.
Absolute cell numbers in lymph nodes from FTY-treated mice decreased but did not reach statistical significance. Treatment caused a significant decrease in splenic CD4
+ T cells, but the decrease in CD8
+ T cells did not reach statistical significance. The major effect of FTY on CD4
+ T cells has been reported by others.
34 At early periods (as early as 3 hours) after FTY720 administration, an important increase of cells in lymph node has been shown.
40 However, in C57BL/6 mice treated for 21 days with FTY720, Morris et al.
41 found that after a transient increase in peripheral lymph nodes and Peyer's patches, lymphocyte 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 confirmed the previously described
16 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, CD4
+ 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 extended to 21 days also enabled the development of Treg cells capable of regulating EAU antigen-specific effector cells.
It is important to emphasize the need to study the effect of FTY during relapse, which, according to Chan et al.,
44 occurs 5 and 10 weeks after immunization. Studies of long-term treatment are important; the effect on the production of specific antibody has been previously reported.
44 Our group has observed that treatment with iVLA-4 in the efferent phase is effective at reducing ocular damage in animals killed in 49 days.
15
In conclusion, FTY was able to prevent EAU development when administered between 7 and 21 dpi. In addition, the drug withdrawn did not lead to disease, suggesting the development of a regulatory state. We believe that FTY caused a decrease of eye-infiltrating cells without inhibiting Treg accumulation in this site, that Treg cells in the eye were enhanced by FTY treatment and thus impaired the effects of CD4+IL-17+ cells, and that, in lymph nodes, the increased percentage of CD4+IFN-γ+ cells was associated with the higher percentage of CD4+Foxp3+ cells, which could have been the source of Treg cells found in the eye. Our results suggest a possible therapeutic use of this immunomodulatory drug for the treatment of ocular autoimmune disease of autoimmune etiology.
Supported by Fundação de Amparo a Pesquisa do Estado de São Paulo Grant 04/14727-0; Conselho Nacional de Desenvolvimento Científico e Tecnológico; Pan-American Ophthalmological Foundation Grant 2007; and FADA-Universidade Federal de São Paulo.
The authors thank Paulo Albe for assistance in preparation of the histopathology slide and Robson L. Melo for synthesis of the IRBP peptide 161-180.