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

Abstract

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. 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. 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. 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.