The sphingosine-1-phosphate receptor-1 (S1P1) agonist ozanimod ameliorates ulcerative colitis, yet its mechanism of action is unknown. Here we examine the cell subsets that express S1P1 in intestine using S1P1-eGFP mice, the regulation of S1P1 expression in lymphocytes after administration of DSS, after colitis induced by transfer of CD4+CD45RBhi cells and by crossing a mouse with TNF-driven ileitis with S1P1-eGFP mice. We then assayed the expression of enzymes that regulate intestinal S1P levels, and the effect of FTY720 on lymphocyte behavior and S1P1 expression. We found that not only T and B cells express S1P1, but also dendritic (DC) and endothelial cells. Furthermore, chronic but not acute inflammatory signals increased S1P1 expression, while the enzymes that control tissue S1P levels in mice and humans with IBD were uniformly dysregulated, favoring synthesis over degradation. Finally, we observed that FTY720 reduced T cell velocity and induced S1P1 degradation and retention of naïve but not effector T cells. Our data demonstrate that chronic inflammation modulates S1P1 expression and tissue S1P levels and suggests that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on DC migration and vascular barrier function.

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BACKGROUND: Pharmacological activation of the S1P1 receptor (S1P1R) results in, 1) disruption of lymphocyte egress from secondary lymphoid organs, and 2) therapeutic efficacy in preclinical models of inflammatory bowel disease (IBD). However, in addition to sequestration of lymphocytes within lymph nodes, other mechanisms of action may contribute to the anti-inflammatory effect of S1P1R agonists. Aim: The aim of this study was to begin to understand the regulation of the S1P metabolic pathway in patients with ulcerative colitis (UC) and mouse models of IBD and to assess the efficacy and mechanism of action of RPC1063, a potent S1P1R agonist, in the SAMP1YitFc mouse model of IBD. METHODS: Biopsies (n ¼ 11) were collected from involved and uninvolved sites during routine endoscopy of patients with ulcerative colitis. Tissue was collected from SAM-P1YitFc and non-inflamed AKR mice (n ¼ 6-9) and RAG2/2 mice adoptively transferred with CD4+CD45RBhi or CD4+CD45RBhi+lo cells (n ¼ 3-4). S1P metabolic pathway enzymes were analyzed by RT-PCR. After the onset of ileitis RPC1063 (1.2 mg/kg/po/ d), dexamethasone (4 mg/kg/po/d) or vehicle was administered for 14 days to SAM-P1YitFc mice (n ¼ 5-7). Ileitis severity was analyzed in a blinded manner by a pathologist, and tissue extracts analyzed for cytokine and chemokine protein expression. RESULTS: At inflamed sites, UC patient biopsies demonstrated decreased expression of S1P lyase (S1PL; 0.85 6 0.15-fold, P ¼ 0.001) and increased sphingosine-1 kinase (SPHK1; 5.6 6 5.7-fold, P ¼ 0.001). In agreement, ileum from SAMP1YitFc mice and colon from RAG2/2 CD4+CD45RBhi adoptive transfer mice also show a decrease in S1PL (5.6-fold and 2.6-fold, P < 0.05) and increase in SPHK1 (1.8-and 1.9-fold, P < 0.05). Changes in expression levels correlated with disease severity in SAMP1YitFc mice. In agreement with prior studies in colitis models, a dose of RPC1063 that achieves a 68% reduction in circulating lymphocytes significantly suppressed chronic inflammation (2.1-fold, P < 0.05) and mucosal thickening (1.9-fold, P < 0.05) in the small intestine of SAMP1YitFc mice. A reduction in 18/22 elevated cytokines and chemokines was observed, with TNFa, MCP-1, MIP-1b, MIP-2, LIF, IL-6, IL-12p40, and IL-13 reaching statistical significance. CONCLUSIONS: These results suggest that there is dysregulation of the S1P metabolic pathway that may result in elevated S1P at sites of inflammation within the intestine of patients with UC and in animal models of IBD. RPC1063 treatment restricted lymphocyte trafficking, blunted proinflammatory cytokine and chemokine expression and ameliorated histopathological features of IBD in SAMP1YitFc mice. RPC1063 is currently in clinical development for the treatment of patients with UC and multiple sclerosis.