Fingolimod Suppresses a Cascade of Core Vicious Cycle in Dry Eye NOD Mouse Model: Involvement of Sphingosine-1-Phosphate Receptors in Infiltrating Leukocytes.

Purpose: The purpose of this study was to evaluate the inhibitory mechanism of fingolimod and the involvement of sphingosine-1-phosphate receptors (S1PRs) and cytokines-matrix metalloproteinases (MMPs)/MAP kinases (MAPKs) signaling in a dry eye disease (DED) mouse model. Methods: Sixty-four male NOD mice (DED model) and 16 age-matched BALB/c mice were used. In a preliminary experiment, 16 NOD mice were randomly divided into a positive control group and fingolimod-treated groups, with 8 BALB/c mice serving as wild-type control. In a subsequent, separate study, 48 NOD mice were randomly divided into 6 groups: fingolimod-treated groups at three different concentrations (0.05%, 0.005%, and 0.001%), normal saline group, untreated group, and fingolimod+W146 group. Animals received normal saline or fingolimod eyedrops three times daily until euthanasia 2 months later. Mice in the fingolimod+W146 group received daily intraperitoneal injections of W146 (0.1 mg/kg/day). Proinflammatory mediators were assessed by a protein array. Activities of MMP-2 and MMP-9 were evaluated by zymography. MAPKs and S1PRs were examined by Western blots and immunohistochemistry. Infiltrating cells and inhibitory mechanisms were assessed. Results: In the positive control group, levels of inflammatory mediators and S1PRs were upregulated. By comparison, fingolimod treatment significantly suppressed such markers which were significantly reversed by W146 (P < 0.01). Importantly, by double immunofluorescence staining, leukocytes were confirmed involved in DED in the NOD mouse model. Conclusions: Leukocytes are involved in DED in the NOD mouse model. The therapeutic mechanisms of fingolimod may be associated with inhibitory roles of "cytokines-MMPs/MAPKs" cycle in NOD mouse ocular surface tissues by mediating S1PRs in infiltrating leukocytes.

Adverse Effect Profile of Topical Ocular Administration of Fingolimod for Treatment of Dry Eye Disease.

Fingolimod is a promising prodrug in attenuating multiple sclerosis and prolonging survival of organ allograft, with many other protective effects. Its mechanism of action is related to the internalization of sphingosine 1-phosphate receptors (S1PRs). Our previous study indicated that fingolimod eyedrops was efficacious in inhibiting ocular inflammation in a dry eye disease (DED) model of non-obese diabetic (NOD) mice. In the current study, we evaluated potential adverse effects of fingolimod eyedrops. Inbred 10-week-old BALB/c mice were randomly divided into four groups, fingolimod-treated groups at three different concentrations (0.01%, 0.1% and 0.5%) and a negative control group without intervention. Our results showed that in the 0.5% fingolimod group, adverse effects such as photophobia, catacleisis and corneal oedema were observed after 1 week of treatment. 1 month later, corneal opacity, oedema and neovascularization persisted till the mice were killed 2 months later. In contrast, there was no significant abnormality in the negative control group, and 0.01% and 0.1% fingolimod-treated groups. During a 2-month treatment period, we did not detect fingolimod, nor significant change in blood cells in peripheral blood, nor pathological changes in retina and systemic organs. Combined with our previous study and the current results, we recommend that an optimal range of safe and effective concentration of fingolimod as eyedrops is between 0.005% and 0.1%.

FTY720 ameliorates Dry Eye Disease in NOD mice: Involvement of leukocytes inhibition and goblet cells regeneration in ocular surface tissue.

UNLABELLED: 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 1ß (IL-1ß) 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.

Selecting highly sensitive non-obese diabetic mice for improving the study of Sjögren's syndrome.

BACKGROUND: Non-obese diabetic (NOD) mice are a commonly used murine model for the study of Sjögren's syndrome. However, variations in susceptibility to the disease among the mice has often yielded less stable results. Based on the correlation between the pathological changes and the tear tests, we attempt to establish a simple screening procedure to assure the validity of experimental results by excluding those mice with poor susceptibility to dry eyes. METHODS: Seventy male NOD mice were recruited. The tear film break-up test (BUT) and the phenol red cotton thread test (CTT) were implemented while the mice were under anesthesia. The mice were divided into four groups (grades 1 to 4) based on their BUT readings, and four similar groups based on CTT measurements. Tear samples in each grade were collected for IL-1beta detection with ELISA. The lacrimal glands and conjunctiva of the mice were used to detect the levels of leucocyte common antigen (LCA). LCA-Positive staining was considered as the "gold standard" in the receiver operating characteristic curve (ROC curve) analysis. C57BL/6 mice were used as wild-type controls. RESULTS: There were 13 (18.57%), 43 (61.43%), 10 (14.29%) and 4 (5.71%) mice in grades 1, 2, 3 and 4 by BUT test, and 34 (48.57%), 15 (21.43%), 14 (20.00%) and 7 (10.00%) in grades 1, 2, 3 and 4 by CTT test respectively. Fifty-one out of the 70 mice (72.86%) were detected LCA-positive, and they were mainly in grades 1 and 2 of both the BUT and CCT grading systems. ELISA showed significant variances of IL-beta levels among the four groups (p < 0.01), with much lower IL-beta levels in group 3 and 4 when both BUT and CTT were used for grouping. The tear IL-beta level in the wild-type mice was similar to those of the grade 4 mice, using either BUT or CTT for grouping. The ROC curve analysis provided optimal cutting lines, which were 2 seconds in BUT readings and 4 mm/min in CTT measurements respectively. CONCLUSION: BUT and CTT tests are useful methods in screening high susceptible NOD mice. Cutting lines at BUT < or = 2 seconds and CTT < or = 4 mm/min provide a good balance between the assurance of susceptibility and the maximization of use of NOD mice for the study of Sjögren's syndrome.