Experimental Eye Research 138 (2015) 145e152

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FTY720 ameliorates Dry Eye Disease in NOD mice: Involvement of leukocytes inhibition and goblet cells regeneration in ocular surface tissue Weibao Xiao a, Guo-Tong Xu b, Jingfa Zhang b, Jiaying Zhang a, Yu Zhang a, Wen Ye a, * a

Department of Ophthalmology, Huashan Hospital Affiliated to Fudan University, 12 Middle Wulumuqi Road, Buiding 3, Room 802, Shanghai 200040, China Tongji Eye Institute and Department of Regenerative Medicine, Tongji University School of Medicine, 1239 Siping Road, Medical School Building, Room 521, Shanghai 200092, China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 January 2015 Received in revised form 6 June 2015 Accepted in revised form 24 June 2015 Available online 14 July 2015

FTY720 is a promising drug in attenuating multiple sclerosis, prolonging survival of organ allograft, and many other protective effects. Its mechanism of action is considered to be mediated by the internalization of sphingosine 1-phosphate receptors (S1PRs). In the current study, we investigated the efficacy of FTY720 in Non-Obese Diabetic (NOD) mice, serving as a model of Dry Eye Disease (DED). NOD mice were divided into six study groups, i.e., FTY720-treated groups with 3 concentrations of FTY720 (0.05%, 0.005%, and 0.001%), 0.05% Cyclosporin A (CsA) treated group, normal saline treated group, and no treatment control group. FTY720 was reconstituted with normal saline and prepared as eye drop. The stability and production of tear film was measured by Tear Break up Time test (TBUT) and phenol red cotton thread test (PRCTT), respectively. Tear fluid washings were collected and assessed by ELISA. Cytokines were detected in lacrimal glands by RT-PCR. Inflammation in conjunctiva was assessed by immunohistochemistry, goblet cells and conjunctival epithelia were examined and evaluated by impression cytology. Our results indicated that FTY720 had a significantly therapeutic effect in NOD mice. After FTY720 intervention, TBUT and PRCTT data were greatly improved (p < 0.01), the interleukin 1b (IL-1b) level was markedly decreased in tear fluid washings compared to control and normal saline groups after 2 weeks (Control: 1.06 ± 0.12, Normal saline:0.97 ± 0.09 pg/ml, CsA:0.22 ± 0.02 pg/ml, 0.001% FTY720:0.23 ± 0.02 pg/ml, 0.005% FTY720:0.14 ± 0.03 pg/ml, 0.05% FTY720: 0.18 ± 0.03 pg/ml. CsA group and 3 FTY720 groups VS. control group and normal saline groups: p < 0.01). Proinflammatory factors were greatly decreased in lacrimal glands (p < 0.01). Leukocytes were identified and markedly decreased in conujnctiva (p < 0.01), inflammatory reaction of DED was greatly relieved. More importantly, the goblet cells were largely restored and ocular surface lesions were significantly ameliorated (p < 0.01). Thus, we observed FTY720 alleviated DED in NOD mice by inhibiting leukocytes, the function of ocular surface tissue in NOD mice was partially restored via inhibiting ocular surface inflammation and increasing the density of goblet cells and conjunctival epithelia. FTY720 may offer a novel strategy for the treatment of inflammatory disorders in the ocular surface. © 2015 Elsevier Ltd. All rights reserved.

Keywords: FTY720 Dry Eye Disease (DED) NOD mice Goblet cell Leukocyte

1. Introduction DED is a prevalent and multifactorial disorder involving multiple interacting mechanisms and with a great range of signs and

* Corresponding author. Department of Ophthalmology, Huashan Hospital Affiliated to Fudan University, 12 Middle Wulumuqi Road, buiding 3, Room 802, Shanghai 200040, China. E-mail address: [email protected] (W. Ye). http://dx.doi.org/10.1016/j.exer.2015.06.032 0014-4835/© 2015 Elsevier Ltd. All rights reserved.

symptoms. Dysfunction of any component of the ocular surface and/or tear film can lead to dry eye, alterations in tear composition and distribution, and eventually causing epithelial damage and inflammation (Lemp, 2007). An increasing number of studies imply that this disease is not simply a tear film deficiency. Inflammation of exocrine glands and cells in ocular tissues such as lacrimal glands and goblet cells are indicated. The pathogenesis is shown rooted in T cell-mediated autoimmune response (Schaumburg et al., 2011; Stern et al., 2012). A significant involvement of ICAM-1 plays a pivotal role in the immune-based ocular inflammation. Its

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expression is not only essential in promoting infiltrating inflammatory cell homing to the ocular tissues, but also directly and/or indirectly modulating the physiological/functional stage of ocular resident epithelial cells, therefore, predisposing them to the immune-mediated ocular inflammation (Gao et al., 2004). The multifactorial and extremely complicated pathogenesis and mechanism of DED make its treatment quite difficult. Although a variety of treatments have been applied, such as tear film substitute, autologous serum, anti-inflammation, etc., there is no cure for this disease (Bron et al., 2009; Lemp, 2007; Pflugfelder, 2007; Schaumberg et al., 2009). FTY720 (fingolimod), 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3propanediol hydro-chloride, is a chemical derivative of the myriocin (ISP-1). It was first developed by Fujita Tetsuya in collaboration with Yoshitomi Pharmaceuticals Ltd (Fujita et al., 1994). FTY720 is phosphorylated by sphingosine kinases (SphK) to FTY720-phosphate, which acts as a sphingosine-1-phosphate (S1P) agonist to four out of the five known S1P receptors (S1PRs) except S1P2. FTY720 was reported to produce lymphopenia by suppressing the recirculation of lymphocyte and sequestering them into lymph nodes and Peyer's patches without affecting their normal induction, proliferation, and differentiation in secondary lymphoid organs (Brinkmann et al., 2002; Brinkmann, 2009; Mandala et al., 2002). FTY720 has been reported to be highly effective in attenuating multiple sclerosis, prolonging allograft survival, relieving ischemia-reperfusion injury, protecting nerve cells, inhibiting proliferation and metastasis of sarcoma cells, preventing diabetes as well as treating autoimmune diseases (Commodaro et al., 2010; € rns et al., 2010; Kappos et al., 2006; Li et al., Egom et al., 2010; Jo 2012; Noda et al., 2013). Recently, FTY720 was also reported efficacious in treating autoimmune uveitis and prolonging corneal graft survival (Commodaro et al., 2010; Liu et al., 2012). In view of the pathological characteristics of DED and the mechanism of action of FTY720, we hypothesize that FTY720 may produce lymphopenia and relieve inflammation in DED. Several mouse models are currently used for studying the mechanism of DED and its potential treatments (Haneji et al., 1994; Li et al., 2004a, 2004b; Saegusa and Kubota, 1997). Among them, male NOD mouse is one of the most commonly used models, for its autoimmune lesions and autoreactive Th1 cells secrete up-regulated cytokine genes, which is similar to that occurring in human DED. (Takahashi et al., 1997). Hence, NOD mouse was used as a DED model in our study. Our experiments clearly indicated that FTY720 had a significantly therapeutic effect in NOD mice, leukocytes in conjunctiva were identified to play a vital role in triggering and developing DED in NOD mice. After treated by FTY720, the number of leukocytes markedly decreased, inflammatory reaction of DED was greatly relieved. More importantly, the goblet cells in conjunctiva were largely restored and ocular surface lesions were significantly ameliorated. FTY720 may offer a novel strategy for the treatment of inflammatory disorders in the ocular surface. 2. Materials and methods 2.1. Animal and reagent One hundred and forty-four inbred 10-week-old male NOD mice (Shanghai SLAC Laboratory Animal Co., Ltd., Chinese Academy of Sciences, Shanghai, China) were kept in pathogen-free conditions under constant ambient temperature (20e23
C) and humidity. The experiments were carried out in accordance with the institutional animal care and use committee guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. FTY720 was a generous gift from Hangzhou Tongming Scientific and Technologic Co., Ltd. (Hangzhou, China). CsA was purchased from

the Eye Ear Nose and Throat Hospital, Fudan University, (Shanghai, China). 2.2. Study design NOD mice were randomly divided into six groups (24 mice per group), i.e., FTY720-treated groups with FTY720 at three different concentrations (0.05%, 0.005%, and 0.001%), 0.05% CsA-treated group, vehicle (normal saline) control group, and control group without any treatment. FTY720 and CsA were reconstituted in normal saline and prepared as eye drops (pH ¼ 7.3e7.5). Each NOD mouse received eye drops three times daily at 8:00, 12:00, and 17:00. Mice were examined at 2, 4, and 8 weeks after the commencement of treatment. 2.3. Quantitation of aqueous tear production Mice were anesthetized with 1% sodium pentobarbital (10 mL/g body weight, i.p.). Tear production was measured by the phenol red cotton thread test (PRCTT), using phenol red impregnated cotton threads (Zone-Quick, FCI Ophthalmics Inc.). The threads were held with a pair of jeweler forceps and put in the lateral canthus for 1 min without contacting the ocular surface tissue. The wetted lengths of the thread, which turned red when soaked with tears, were measured using the scale on the cotton thread in millimeters under a microscope. Both eyes were measured at the same time. Tests were conducted three times at each time point and the mean values reported. 2.4. Measurement of tear-film stability The stability of tear-film was measured by TBUT as previously described (Lin et al., 2011). Briefly, 1.5 mL of 0.1% sodium fluorescein solution was dropped with pipette tips into the conjunctival sac. Eyelids of anesthetized mice were blinked three times and then kept open. Tear film was observed under slit-lamp microscope with a cobalt blue filter (SLM-6, Chongqing Kang Hua Rui Ming technology co., LTD). The time between the last blink and the appearance of the first break in the tear film (formation of a dry spot in the central area of cornea) was recorded with a stopwatch. Tests were conducted three times at each time point and the mean values were reported. 2.5. Tear IL-1b assay Tear fluid washings were collected according to the method of Song et al. (Song et al., 2003). Briefly, 1.5 mL of 0.1% bovine serum albumin in phosphate-buffered saline was instilled into the conjunctival sac. A mixture of tear and buffer was collected with a 1 mL glass capillary tube via capillary action from the tear meniscus in the lateral canthus. Tear fluid washings of both eyes were collected three times on alternate days. Tear fluid washings in the same group at each time point were pooled and stored at 80
C until assayed. IL-1b levels in tear washing fluids were determined by ELISA kits for mouse IL-1b (Quantikine M Murine; R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. Absorbance at 450 nm was read by a Tecan Safire2 Multifuction Reader (Tecan Group, Ltd., Switzerland) with the reference wavelength of 570 nm. The mean data of the three measurements were reported. 2.6. Impression cytology Mice were anesthetized with 1% sodium pentobarbital (10 mL/g body weight, i.p.). A piece of 3  4 mm2 nitrocellulose filter (Millipore) was applied tightly to the upper palpebral conjunctiva of a NOD mouse for approximately 5 s and removed from the

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conjunctiva in a peeling motion to ensure maximal collection of surface cells. The filter was fixed by 95% ethanol for 1 h and stained by the modified Nelson procedure. The steps are conducted as below: Specimens were rinsed the fixed in distilled water for 5 min, submerged in 0.5% periodic acid for 5 min, rinsed in distilled water for 2 min, immersed in Schiff's reagent for 5 min, rinsed in tap water for 5 min, stained with hematoxylin for 1 min, rinsed in tap water for 30 min, dried overnight, They were then dehydrated by 95% and 100% ethanol for 5 min consecutively and cleared by dimethylbenzene for 3 min. The filters were then sealed by neutralbalsam and analyzed under a microscope. Grading of cytology was done according to Nelson's grading system (Nelson et al., 1983). Grade 0: Epithelial cells are small and round with large nuclei; goblet cells are abundant, plump and oval. Grade 1: Epithelial cells are slightly larger and more polygonal; nuclei are smaller; goblet cells are decreased in number, but still maintain their plump and oval shape. Grade 2: Epithelial cells are larger, more polygonal and occasionally multinucleated; nuclei are small; goblet cells are markedly decreased in number and are smaller with well-defined cellular borders. Grade 3: Epithelial cells are larger and more polygonal with small and pyknotic nuclei; goblet cells are completely absent. Grade 0 and 1 were regarded as normal, whereas grade 2 and 3 abnormal cytology. 2.7. Isolation and detection of mRNA level of cytokines in lacrimal glands by RT-PCR Total RNA was extracted from lacrimal glands using Trizol reagent (Invitrogen Life Technologies, USA) according to the manufacturer's instructions. The RT product (1 mL) was then amplified by PCR. The specific primers were designed by software primer premier (Version 5) and purchased from Shanghai DNA Biotechnology Corporation, Ltd. The information for primers and the product size are summarized as below: IL-1b: Sense: AATCTCACAGCAGCACAT, anti-sense: AGGTTATCATCATCATCCC. Product size: 189 bps. TNF-a: Sense: ATTTGAAGGTGGTCGTCT, anti-sense: CATCGCTCACTCCTGTTG. Product size: 192 bps. Amplification conditions included an initial denaturation at 95
C for 5 min (min), followed by 35 cycles of denaturation at 94
C for 1 min, annealing at 50.5e54.5
C (IL-1b: 50.5
C, TNF-ɑ: 51.7
C, ICAM-1:54.5
C and GAPDH: 53.5
C) for 30 s, and 30 s extending at 72
C, and a final extension at 72
C for 10 min. PCR products were electrophoretically separated on 2% agarose gel in 1  TBE buffer. The optical densities of the detections were determined by the Quantity One software (Bio-Rad). The densitometric values were normalized by GAPDH. 2.8. Inflammation in ocular surface was assessed by immunohistochemistry At the end of each time points, 8 mice were euthanized. The palpebral conjunctiva were collected and fixed with 4% PBSbuffered paraformaldehyde for 24 h, and then incubated sequentially with 10%, 20%, and 30% sucrose solutions. After OCTembedding, conjunctival specimens were cryo-sectioned at 5 mm thickness. The sections were air-dried at room temperature for at least 2 h, then rinsed with PBS and treated with 3% hydrogen peroxide for 5 min sections were then blocked by 1% normal goat serum for 15 min at room temperature. After the goat serum was removed, the sections were incubated with primary antibodies, i.e., rat monoclonal anti-mouse ICAM-1 (anti-ICAM-1, 1:100; BioLegend), rat leucocyte common antigen monoclonal antibody (antiLCA, 1:100; eBioscience), in a humidified chamber at 4
C overnight. After being washed three times with PBS, the sections were treated with a peroxidase-labeled goat anti-rat IgG antibody (1:500,

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Chemicon) for 1 h at room temperature. A sample without the primary antibody was used as a negative control. Visualization was performed with treatment of diaminobenzidine (DAB) for 5 min. 2.9. Statistical analyses Data are expressed as mean ± SD. Student's t-test was used to compare values between two groups. One-way ANOVA followed by F test was used to compare values among three or more groups. A p value of 0.05 or less was considered statistically significant. 3. Results 3.1. Aqueous tear production and tear-film stability TBUT showed a time-dependent decrease in control group and normal saline-treated group, while it was significantly prolonged after 2 weeks in the FTY720 and CsA treated groups (Table 1, p < 0.01). PRCTT showed a similar tendency in FTY720 and CsA treated groups compared to the control group and the saline groups (Table 2, p < 0.01). 3.2. Inflammatory factors were detected in tear film, lacrimal glands and conjunctiva In order to explore the change of inflammatory factors in tear film, ELISA was used to detect the level of IL-1b in tear film samples. We found the level of IL-1b was up-regulated steadily from 2 weeks to 8 weeks in the control and saline-treated groups, while it was sharply decreased in drug-treated groups early in 2 weeks observation. After that, it kept in a low level until the mice were sacrificed (Fig. 1, p < 0.01). Also, we observed a similar trend by RT-PCR in lacrimal glands. The mRNA levels of IL-1b and TNF-a were significantly down-regulated in CsA-treated and FTY720-treated groups when compared to untreated and saline-treated groups (Fig. 2, p < 0.01). ICAM-1 showed a similar change as IL-1b and TNFa till 8 weeks (Fig. 3, p < 0.01). Additionally, immunohistochemistry showed that ICAM-1 was substantially expressed in conjunctiva of NOD mice in both untreated and normal saline groups. In contrast, it was greatly down regulated after FTY720 treatment in the 0.05% and 0.005% groups after 4 weeks (Fig. 4, p < 0.01). 3.3. FTY720 (0.005%e0.05%) seemed to be the most efficacious concentration as an eye drop In order to explore the optimal concentration as an eye drop for ocular surface application, FTY720 were diluted into 3 concentrations, i.e., 0.05%, 0.005% and 0.001%. Our experiments indicated that both 0.005% and 0.05% produced significantly therapeutic effects. Comparatively, FTY720 at 0.001% was less efficacious in most tests except IL-1b ELISA level in tear film. We also tried a higher concentration (0.5% FTY720), which caused adverse effects such as photophobia, catacleisis, hyperemia, and edema in the conjunctiva, 4 weeks later, opacity of cornea was also found in a few NOD mice (data not shown). 3.4. Leucocytes were identified and inhibited by FTY720 According to the previous reports, FTY720 produced lymphopenia by suppressing the recirculation of lymphocyte and sequestering them into lymph nodes and Peyer's patches. In our study, we observed definitely LCA-positive staining cells infiltrated between tarsus and palpebral conjunctiva in untreated and saline-treated groups. In contrast, the staining leucocytes were much less or hardly detected in the FTY720-treated groups (Fig. 5, p < 0.01).

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Table 1 Comparison of TBUT in different groups. Groups

Control

Baseline 2wk 4wk 8wk

2.16 1.86 1.70 1.58

± ± ± ±

0.21 0.16 0.28 0.24

NS 2.19 2.10 1.92 1.86

0.05% CsA ± ± ± ±

0.18 0.23 0.15 0.27

2.06 2.62 2.98 3.38

± ± ± ±

0.24 0.15* 0.19* 0.32*

0.001% F 2.22 2.49 2.43 2.68

± ± ± ±

0.32 0.19* 0.22* 0.31*

0.005% F 2.18 3.56 3.89 4.14

± ± ± ±

0.17 0.18*# 0.21*# 0.26*#

0.05% F 2.31 2.91 3.25 3.50

± ± ± ±

0.18 0.16* 0.18* 0.19*

TBUT and PRT data were recorded in each group in 2 weeks, 4 weeks and 8 weeks after intervention. The results were expressed as mean ± SEM from three separate TBUT and PRCTT experiments (8 mice per group). *: FTY720 and 0.05% CsA treated groups vs. normal saline and positive control groups, p < 0.01; *#: 0.005% FTY720 vs. other FTY720 treated groups and 0.05% CsA treated group, p < 0.05. (F: FTY720). Table 2 Comparison of PRCTT in different groups. Groups

Control

Baseline 2wk 4wk 8wk

3.36 2.93 2.41 1.98

± ± ± ±

0.57 0.64 0.77 0.37

NS 3.67 3.17 2.81 1.86

0.05% CsA ± ± ± ±

0.36 0.48 0.40 0.46

3.52 6.65 7.46 8.61

± ± ± ±

0.34 0.88* 0.70* 0.98*

0.001% F 3.42 4.14 4.76 4.98

± ± ± ±

0.52 0.33* 0.62* 0.47*

0.005% F 3.31 8.61 9.50 11.15

± ± ± ±

0.36 1.31*# 1.05*# 1.54*#

0.05% F 3.40 6.75 7.67 9.20

± ± ± ±

0.33 1.19* 0.70* 1.75*

TBUT and PRT data were recorded in each group in 2 weeks, 4 weeks and 8 weeks after intervention. The results were expressed as mean ± SEM from three separate TBUT and PRCTT experiments (8 mice per group). *: FTY720 and 0.05% CsA treated groups vs. normal saline and positive control groups, p < 0.01; *#: 0.005% FTY720 vs. other FTY720 treated groups and 0.05% CsA treated group, p < 0.05. (F: FTY720).

Fig. 1. IL-1b level in tear fluid washings were collected in each group before (baseline) and at 2 weeks, 4 weeks and 8 weeks after intervention (24 mice per group in all). Tear fluid washings were separately collected in baseline, 2 weeks, 4 weeks and 8 weeks after treatment in each group. After 1.5 mL of phosphate-buffered saline containing 0.1% bovine serum albumin were instilled into the conjunctival sac, 1.0 mL of mixture of tear fluid and phosphate-buffered saline were collected with a 1 mL glass capillary tube via capillary action from the tear meniscus in the lateral canthus. Tear fluid washings were collected three times, the averaged values were reported. The data were the mean ± SEM (n ¼ 8). *: p < 0.01, FTY720-treated groups and 0.05% CsA group vs. normal sodium group and positive control group.

Moreover, our experiments showed, in most cases, the conjunctival epithelial layer in the treated groups was smooth and orderly, the edema of conjunctiva was greatly relieved compared to the untreated and saline-treated groups (Figs. 4 and 5). 3.5. The goblet cells and conjunctival epithelia were partially restored and regenerated Goblet cells and conjunctival epithelial cells play a vital role in maintaining the normal physiological function of tear film. DED stimulates death of the epithelial surface cells and a cascade of inflammatory events, which lead to the loss of mucin-producing goblet cells. This exacerbates the tear film instability and contributes to the vicious circle of events that perpetuate DED (Baudouin, 2007). In control and saline-treated groups, we found the conjunctival epithelial cells were much less attached in filter papers, the cells were larger while the nuclei were polygonal. The density of goblet cells was also scare or even hardly found in conjunctival samples. Comparatively, the densities of conjunctival epithelial cells

Fig. 2. The mRNA expression of TNF-a and IL-1b in Lacrimal glands among groups by RT-PCR (8 mice per group). A: Both TNF-a and IL-1b were up-regulated in positive control and normal saline groups, while they were down regulated in FTY720 and CsA treated groups. B: Densitometries were compared in each groups, the results were the ratio of TNF-a or IL-1b vs. GAPDH, final data were the mean ± SEM. *: The mRNA levels of TNF-a in FTY720-treated group vs. positive control group and normal saline group, p < 0.005. #: The mRNA levels of IL-1b in FTY720-treated group vs. positive control group and normal saline group, p < 0.001. (F: FTY720).

and goblet cells were greatly alleviated in FTY720 and CsA treated groups especially in 0.005% and 0.05% FTY720 groups (p < 0.01), the goblet cells were plump and oval, densely PAS-stained (Fig. 6). 4. Discussion FTY720 was approved by the U.S. Food and Drug Administration as a first-line treatment for relapsing forms of multiple sclerosis. Previous studies showed that FTY720 at 0.1 mg/kg or higher oral doses almost completely prevent paralysis in experimental

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Fig. 3. The mRNA expression of ICAM-1 in Lacrimal glands among groups in 2, 4 and 8 weeks (24 mice per group in all). A: ICAM-1 was up-regulated in positive control and normal saline groups, while they were down regulated in FTY720 and CsA treated groups in 2 weeks. After 4 weeks, the mRNA expression of ICAM-1 was greatly down-regulated. B: Oneway ANOVA was used to compare values among groups. Densitometries were compared in each groups, the results were the ratio of mRNA expression of ICAM-1 vs. GAPDH, final data were the mean ± SEM. *: The mRNA levels of ICAM-1 in FTY720 group vs. positive control group and normal saline group, p < 0.001. #: The mRNA levels of ICAM-1 in 0.005% and 0.05% FTY720 group vs. 0.05% CsA group and 0.001% FTY720 groups, p < 0.005. (F: FTY720).

Fig. 4. ICAM-1 expression assay in palpebral conjunctiva of NOD mice by immunohistochemistry 8 weeks after treatment (8 mice per group). The brown were positive staining, the blue were restained by haematoxylin. A and B: ICAM-1 was abundantly expressed in the conjunctiva of positive group and normal saline group (yellow arrow heads), CeF: ICAM-1 sharply down-regulated in FTY720 and 0.05% CsA treated groups especially 0.05% CsA-treated group, 0.05% FTY720-treated group and 0.005% FTY720-treated group which even hardly observed in 8-week samples. One-way ANOVA was used to compare values among groups. The values of ICAM-1 in FTY720 and CsA treated groups were significantly down regulated compared with that in positive group and normal saline group (P < 0.01) (200). G: Negative control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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autoimmune encephalomyelitis (EAE) induced by myelin basic protein in LEW rats (Fujino et al., 2003). Also in the same dose range, FTY720 showed a markedly prophylactic and immunomodulating effect on lupus nephritis in autoimmune MRL/lpr mice (Okazaki et al., 2002). Recently it was reported to be effective in treating experimental autoimmune uveitis in mice (Commodaro et al., 2010). FTY720 proved to be effective in inhibiting the inflammation of DED in NOD mice. We and others reported that most of the male NOD mice showed significant inflammatory lesions in the lacrimal glands from the age of 8 weeks, providing a good animal model for DED study (Van Blokland and Versnel, 2002; Xiao et al., 2009).In the present study, we provided clear evidences that FTY720 markedly ameliorated DED in NOD mice. We studied the efficacy of FTY720 eye drop in ocular surface of NOD mice by vivid observations including TBUT and PRCTT. We also detected the levels of inflammatory factors such as IL-1b, TNF-ɑ and ICAM-1 in tear film, lacrimal glands and conjunctiva respectively by ELISA, RT-PCR and immunohistochemistry. Our study showed that TBUT and PRCTT were markedly improved after FTY720 administration (Tables 1 and 2). The levels of proinflammatory factors such as TNF-ɑ and IL-1b were apparently down-regulated in tear film and lacrimal glands in FTY720-treated groups after 2 weeks (Figs. 1 and 2). ICAM-1, an important immne-based inflammation marker (Gao et al., 2004), was also observed to be significantly downregulated after FTY720 administration. The mRNA expression of ICAM-1 was kept in a low level in lacrimal glands of 0.05% CsA and three FTY720 groups till 8 weeks (Fig. 3). Leucocytes were found abundantly infiltrated in conjunctiva in both control and normal

saline-treated groups, whereas they were greatly reduced by FTY720 (Fig. 5). These results provided clear evidences that FTY720 was an effective potential drug in curing some ocular inflammatory diseases such as DED. FTY720 (0.005%e0.05%) seemed to be the most effective concentration as eye drop. In order to explore the optimal concentration as an eye drop for ocular surface application, FTY720 were diluted into 3 concentrations, i.e., 0.05%, 0.005% and 0.001%. Our experiments indicated that the optimal concentration was seemingly between 0.005% and 0.05%, FTY720 at 0.001% was less efficacious (Figs. 2e6). We did not find apparent side effects in three FTY720 groups. In addition, since this chemical derivative is reported to inhibit lymphocyte egress from secondary lymphoid tissues and thymus [Brinkmann, 2009], we observed the leukocytes levels in the same time by examining peripheral blood of NOD mice after treatment, we did not find a significant lymphocytopenia in FTY720 treated groups even after 2 months (data not shown). These evidences clearly imply that 0.005%~0.05% FTY720 may be a safe therapeutic range in its future clinical application. FTY720 seemed to have advantage over CsA, it had priority in lower concentration, prompt effect and better regeneration of epithelial cells and goblet cells. Previous clinical trials showed CsA was found to significantly decrease conjunctival rose bengal staining, superficial punctate keratitis, and ocular irritation symptoms (Stevenson et al., 2000). Treated eyes had a greatly increase in conjunctival goblet cell density (Kunert et al., 2002). Furthermore, there was a significant decrease in the number of cells expressing the lymphocyte activation markers CD11a and HLA-DR, indicating less activation of lymphocytes compared with vehicle-treated eyes

Fig. 5. LCA expression assay in palpebral conjunctiva of NOD mice by immunohistochemistry 8 weeks after treatment (8 mice per group). The brown were positive staining, the blue were restained by haematoxylin. A and B: LCA-positive cells densely infiltrated between palpebral and glandulae tarsale of conjunctiva in positive group and normal saline group (yellow arrow heads). CeF: By contrast, the infiltrated cells markedly decreased in FTY720 and CsA treated groups especially in 0.05% FTY720 and 0.005% FTY720 treated group. Oneway ANOVA was used to compare LCA-positive cells among groups. The amount of infiltrated cells in FTY720 and CsA treated groups sharply decreased compared with the amount in positive group and normal saline group (P < 0.01) (200). G: Negative control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Impression cytology of conjunctiva after 8 weeks treatment (8 mice per group). The NOD mice were treated by FTY720 and 0.05% CsA for 2 months, the positive control and NS groups were compared. A and B: Grade 3. In positive control and NS groups, the density of epithelial cells was significantly decreased, the epithelial cells were large and the nuclei were polygonal. There were hardly goblet cells in impression samples. C: Grade 2. The density of epithelial cells was increased significantly after 0.05% CsA intervention. However, the goblet cells were still markedly decreased in number. D: grade 2 ~ grade 3. The density of epithelial cells was increased compared to the positive control and NS groups. However, it was still greatly decreased compared to the normal conjunctiva. Moreover, the goblet cells were scare. E and F: Grade 1. The epithelial cells were densely stained, the density of goblet cells were markedly increased compared to the positive control group and maintained plump and oval shape. (Yellow arrow heads: Goblet cells). One-way ANOVA was used to compare goblet cells among groups. The amount of goblet cells in FTY720-treated groups and CsA-treated group greatly increased compared with positive control and normal saline groups (P < 0.01). The amount of goblet cells in 0.05% and 0.005% FTY720-treated group significantly increased compared with the amount in CsA-treated group (P < 0.05) (200). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Kunert et al., 2000). However, previous clinical trial reported some patient complaint burning symptoms after instillation though it doesn't exhibit significantly systemic or ocular adverse events (Sall et al., 2000). In the current study, we found FTY720 significantly alleviated ocular inflammation of NOD mice. Compared to CsA, it had priority in promptly improving the health of tear film and inhibiting inflammatory factors and leukocytes (Tables 1 and 2, Figs. 3 and 5). The effective concentration is much lower than CsA. More importantly, we found the amount of goblet cells and conjunctival epithelia in 0.05% and 0.005% FTY720 treated groups was invariably more than that in 0.05% CsA group even after 2 months (Fig. 6). This may indicate FTY720 has priority over CsA not only in a lower effective concentration, prompt effect, but in reestablishment of physiological function of ocular surface tissues as well. Leucocytes were identified to be the target and were markedly decreased after FTY720 administration accompanied with a relived inflammatory infiltration and ocular surface lesions in NOD mice. In our study, we observed a clearly anti-inflammatory effect by FTY720 in the ocular surface of NOD mice. Meanwhile, a lot of infiltrating cells were found in palpebral conjunctiva. Comparatively, they were much less or hardly found in FTY-720 treated groups. We speculate these cells might be infiltrating leukocytes. In view of this, LCA, the specific marker of leucocytes was used to verify and compare these cells between FTY720-treated groups and positive control group by immunohistochemistry. We confirmed our speculation that these cells were surely leukocytes and were expressed in the same area, namely, between tarsal glands and palpebral conjunctiva, which were much less or hardly found in FTY-720 treated groups. Additionally, we also found the conjunctival edema was relieved and the structure of the conjunctiva was always more smooth and orderly in

FTY720 treated groups (Figs. 4 and 5). The function of ocular surface tissue was partially restored and reestablished. The ocular surface is highly exposed, and efficient tear production and turnover is essential for its continued health. The tear film, lacrimal glands (main and accessory lacrimal glands, meibomian glands, goblet cells, and all ocular surface secretory cells), lacrimal outflow pathways, and corneal and conjunctival epithelia work together as a lacrimal functional unit (LFU) to maintain the tear film and protect the transparency of the cornea and the integrity of the ocular surface (Stern et al, 1998. Stern et al, 2004). Disease or damage to any of these components can destabilize the tear film and lead to ocular surface disease that expresses itself as dry eye. In our study, we observed a prolonged PRCTT in FTY720-treated groups which indicated the function of lacrimal glands was partially restored. Meanwhile, after FTY720 treated, the TBUT time was also prolonged, which implied a sign of regeneration and functional restoration of goblet cells. Impression cytology provided further evidences to verify our conjecture. The impression filter papers showed the density and morphology of goblet cells and conjunctival epithelia were greatly ameliorated compared to the positive control and NS-treated groups (Fig. 6). According to the recent consensus by Dry Eye Workshop in 2007, the core mechanisms of all dry eye types are driven by tear hyperosmolarity and tear film instability (Lemp 2007). Classically, the tear film is reported to be composed of three layers: the mucous, aqueous, and lipid layers (Mantelli et al., 2007). Mucins, produced by goblet cells, prevent adhesion of foreign debris, inflammatory cells or pathogens to the ocular surface and stabilize the tear film (Sumiyoshi et al., 2008). Mucins are also essential to maintaining proper epithelial protection by forming an epithelial barrier through the

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interaction with carbohydratebinding proteins called galectins (galactose binding lectins) that are highly expressed by the conjunctival epithelium (Argüeso et al., 2009; Mantelli et al., 2009). That is to say, a normal morphology and function of goblet cells is critical for normal tear film stability, essential refraction and necessary ocular surface tissue nutrition. By our experiment, we showed clearly therapeutic results including prolonged TBUT, PRCTT, much less leukocytes infiltration as well as greatly ameliorated goblet cells and conjunctival epithelia. These results provided persuasive evidences of a partially restored LFU including reestablished tear film and lacrimal glands. Conclusively, we observed a produced efficacy of FTY720 as an eye drop to inhibit ocular inflammation of DED in NOD mice. TBUT and PRCTT were significantly prolonged, IL-1b level was markedly decreased in tear fluid washings. The mRNA expression levels of IL1b, TNF-ɑ and ICAM-1 were greatly down regulated in lacrimal glands and, the leukocytes were sharply inhibited in conjunctiva. More importantly, the density and morphology of goblet cells and conjunctival epithelial were significantly ameliorated, the function of tear film were partially reestablished. FTY720 may be a promising potential drug in curing some ocular diseases such as DED. Conflicts of interest None. References Argüeso, P., Guzman-Aranguez, A., Mantelli, F., Cao, Z., Ricciuto, J., Panjwani, N., 2009. Association of cell surface mucins with galectin-3 contributes to the ocular surface epithelial barrier. J. Biol. Chem. 284, 23037e23045. Baudouin, C., 2007. A new approach for better comprehension of diseases of the ocular surface. J. Fr. Ophtalmol. 30, 239e246. Brinkmann, V., Davis, M.D., Heise, C.E., Albert, R., Cottens, S., Hof, R., Bruns, C., Prieschl, E., Baumruker, T., Hiestand, P., Foster, C.A., Zollinger, M., Lynch, K.R., 2002. The immune modulator FTY720 targets sphingosine 1-phosphate receptors. J. Biol. Chem. 277, 21453e21457. Brinkmann, V., 2009. FTY720 (fingolimod) in multiple sclerosis: therapeutic effects in the immune and the central nervous system. Br. J. Pharmacol. 158, 1173e1182. Bron, A.J., Yokoi, N., Gaffney, E., Tiffany, J.M., 2009. Predicted phenotypes of dry eye: proposed consequences of its natural history. Ocular Surf. 7, 78e92. Commodaro, A.G., Peron, J.P.S., Lopes, C.T., Arslanian, C., Belfort, R., Rizzo, L.V., Bueno, V., 2010. Evaluation of experimental autoimmune uveitis in mice treated with FTY720. Invest. Ophthalmol. Vis. Sci. 51, 2568e2574. Egom, E., Ke, Y., Musa, H., Mohamed, T., Wang, T., Cartwright, E., Solaro, R.J., Lei, M., 2010. FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling. J. Mol. Cell. Cardiol. 48, 406e414. Fujino, M., Funeshima, N., Kitazawa, Y., Kimura, H., Amemiya, H., Suzuki, S., Li, X.K., 2003. Amelioration of experimental autoimmune encephalomyelitis in Lewis rats by FTY720 treatment. J. Pharmacol. Exp. Ther. 305, 70e77. Fujita, T., Inoue, K., Yamamoto, S., Ikumoto, T., Sasaki, S., Toyama, R., Chiba, K., Hoshino, Y., Okumoto, T., 1994. A potent immunosuppressive activity found in Isaria sinclairii metabolite. J. Antibiot. (Tokyo) 47, 208e215. Gao, J., Morgan, G., Tieu, D., Schwalb, T.A., Luo, J.Y., Wheeler, L.A., Stern, M.E., 2004. ICAM-1 expression predisposes ocular tissues to immune-based inflammation €grens syndrome-like MRL/lpr mice. Exp. Eye. Res. 78, in dry eye patients and Sjo 823e835. Haneji, N., Hamano, H., Yanagi, K., Hayashi, Y., 1994. A new animal model for primary Sjogren's syndrome in NFS/sld mutant mice. J. Immunol. 153, 2769e2777. €rns, A., Rath, K.J., Terbish, T., Arndt, T., Meyer, Zu, Vilsendorf, A., Wedekind, D., Jo Hedrich, H.J., Lenzen, S., 2010. Diabetes prevention by immunomodulatory FTY720 treatment in the LEW. 1AR1-iddm rat despite immune cell activation. Endocrinology 151, 3555e3565. Kappos, L., Antel, J., Comi, G., Montalban, X., O'Connor, P., Polman, C.H., Haas, T., Korn, A.A., Karlsson, G., Radue, E.W., 2006. Oral fingolimod (FTY720) for relapsing multiple sclerosis. N. Engl. J. Med. 355, 1124e1140. Kunert, K.S., Tisdale, A.S., Gipson, I.K., 2002. Goblet cell numbers and epithelial proliferation in the conjunctiva of patients with dry eye syndrome treated with cyclosporine. Arch. Ophthalmol. 120, 330e337. Kunert, K.S., Tisdale, A.S., Stern, M.E., Smith, J.A., 2000. Analysis of topical cyclosporine treatment of patients with dry eye syndrome: effect on conjunctival

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