Actions of a picomolar short-acting S1P₁ agonist in S1P₁-eGFP knock-in mice

Abstract

Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.

Figure 1. RP-001 is an orthosteric, short-duration S1P₁ selective agonist

(a) Chemical structures of CYM-5442 and its derivative, RP-001. (b) Left graph represents dose- response curves of RP-001 activation of S1P₁, expressed as a percent of S1P-induced activation. Right graph is a Schild Plot demonstrating competition between RP-001 and the S1P₁ antagonist W146. (c) RP-001 induces sustained S1P₁ signaling in internalized vesicles. S1P₁ CRE-β-lactamase expressing CHO-K1 cells were pretreated with 1μM of the indicated compounds for 1 hour, washed, and rested for 5 hours. Cells were then treated with 2μM forskolin with or without 10nM FTY720 as indicated, and cyclic AMP induced beta lactamase expression was detected by cleavage of CC4-AM. (d) RP-001 elicits dose-dependent lymphopenia of CD4⁺ T cells. (e) RP-001 induces acute lymphopenia with rapid recovery to untreated levels. Graph for d and e display number of CD4⁺ T cells per mL of blood, while e additionally displays concentration of RP-001 found in the blood as quantified by mass spectrometry with a dashed line. The bottom horizontal line represents the lower limit of detection of RP-001 (1nM). All intravital experiments are representative of at least 3 experiments, 3–4 mice per group per experiment and are presented as mean ±SEM.

Figure 2. Expression and function of S1P₁-eGFP in Edg1^eGFP/eGFP mice

(a) Schematic for the final locus for mice expressing Edg1-eGFP from the Edg1 locus. E1 and E2 represent exons 1 and 2 of Edg1, triangle represent loxP recombination site (b) S1P₁ is broadly expressed across many tissues by Western blot for eGFP from tissues listed under non- denaturing conditions. (c) S1P₁-eGFP is expressed similarly to wild-type S1P₁. Brain lysates were immunoprecipitated with either an antibody specific to the carboxyl-terminus of S1P₁ or an antibody specific to GFP. These lysates were separated by SDS-PAGE, transferred onto membrane, and incubated with antibodies specific to either S1P₁ or GFP. (d) S1P₁-eGFP is N- Glycosylated in vivo. Homogenized brain or lung tissues from Edg1^eGFP/eGFP mice were immunoprecipitated with an antibody specific to GFP then incubated with or without PNGase F. The smaller size of S1P₁-eGFP following incubation with PNGase F indicates loss of N-linked sugars from S1P₁-eGFP. (e) T and B lymphocytes from Edg1^eGFP/eGFP and Edg1^+/+ mice are sequestered following S1P₁ agonist RP-001 treatment. Lymphocyte counts were taken two hours after intraperitoneal injection of either 0.3mg/kg RP-001 or vehicle. (f) Enhancement of vascular leakage following S1P₁ antagonism. Graphs represent leakage of Evans’ Blue Dye into the lungs 1 hour after treatment with 3mg/kg W146 or vehicle intraperitoneally, standardized to wild-type mice treated with W146. Graphs in e and f represent mean +/− SEM for 2 pooled experiments, 3–4 mice per group per experiment. *p<0.05, **p<.01, ***p<0.001 for e and f using unpaired t-test.

Figure 3. RP-001 causes changes in lymphcytic and endothelial S1P₁-eGFP expression and localization

(a) Flow cytometry histograms for eGFP fluorescence on specified lymphocyte populations isolated from lymph nodes of Edg1^eGFP/eGFP (colored) and wild-type (gray) mice at indicated timepoints following IP injection of RP-001. (b) Lymphocytes were homogenized and mixed 1:1 with 90% sucrose. These lysates were placed in centrifuge tubes and overlaid sequentially with equal volumes of 35% sucrose followed by 5% sucrose. Following centrifugation at 100,000×g for 16 hours, twelve equal volume fractions were collected and Western blotted for GFP, Caveolin-1 (Cav1), Early Endosomal Antigen 1 (EEA-1). c) Equal numbers of lymphocytes were isolated and centrifuged as above. S1P₁-eGFP in Cav1-rich fractions is highly preserved following treatment with RP-001, but endosomal-associated S1P₁-eGFP is lost, particularly at 1mg/kg. (d) Left panel represents scatter plot of GP38 and CD31 expression on CD45.2⁻ cells isolated from Collagenase/DNaseI digested lymph nodes. Right panels are histograms for eGFP fluorescence on blood endothelial cells (BECs), lymphatic endothelial cells (LECs), and fibroblast reticular cells (FRCs). (e) Flow cytometry histograms for eGFP from lung BECs and LECs from the same mice as in a. (f) Snapshots from intravital two-photon microscopy of lymph nodes treated as indicated. Internalization of S1P₁-eGFP is not detected at 0.1mg/kg at timepoints up to 80 minutes. Flow cytometry plots in a and e are representative of 3 experiments, 3 mice per group per experiment.