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.