Oxysterols direct immune cell migration via EBI2

Abstract

Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.

Figure 1. Identification, structure, and pharmacological characterization of 7α, 25-OHC

a: Bioactivity profile measured by mobilization of intracellular calcium of fractions from septic sheep liver extracts tested on EBI2 expressing cells (blue) or tested on a control cell line (green) expressing an unrelated GPCR called CCRL2. Activity is plotted relative to an ATP response of an endogenous ATP receptor. b: Chemical structure of 5-cholesten-3β, 7α, 25-triol (7α, 25-OHC). c: Calcium mobilization: Dose response curve induced by several related oxysterols in a CHO cell line stably expressing EBI2. Data for 7α, 25-OHC on the parental cell line lacking expression of EBI2 is given as (7α, 25-OHC, no EBI2). Data shown are from a duplicate experiment. d: Radioligand binding assay: Displacement of 10 nM ³H-7α, 25-OHC bound to membranes from EBI2 expressing CHO cells by increasing concentrations of unlabeled oxysterols. Data from quadruplicate experiments are shown.

Figure 2. Oxysterol-mediated immune cell migration

a: Migration of EBV-infected B cells towards 7α, 25-OHC and closely related oxysterols. b: Migration of RS11846 cells towards 7α, 25-OHC and closely related oxysterols. c: Oxysterol-mediated RS11846 cell migration in the presence (red) or absence (blue) of pertussis toxin (PTX). d: Migration of bone marrow-derived dendritic cells (BMDC) from wild type (blue) or Ebi2(−/−) mice (red). Data for all four experiments were conducted in a transwell assay system with duplicate (a,b,c) or quadruplicate (d) samples.

Figure 3. CH25H expression regulates EBI2 bioactivity in vivo and is required for mounting a T-dependent antibody response

a: Quantitative PCR analysis of Ch25h and Cyp7b1 transcript abundance in the indicated tissues or cell preparations, relative to HPRT. b: Bioassay of spleen extracts from Ch25h(−/−) or wild type animals using a reporter cell line (M12) with or without EBI2. c: Bioassay of spleen extracts from mice reconstituted with bone marrow transduced with a CH25H expressing retroviral vector (CH25H-Thy1.1) or a control vector (Thy1.1). d: Spleen sections from the indicated mice after immune challenge (day 2). Visualization of antigen-sepcific transferred (IgM^a, blue) B cells in Ch25h(+/+) mice indicates that many of the activated B cells have moved to the back of the follicle and interfollicular regions, near Moma1+ marginal metallophilic macrophages (brown). In the Ch25h(−/−), the activated B cells largely fail to move to these regions and instead remain near the follicle/T cell zone interface or move into the follicle. Dashed lines indicate the follicle (F) T zone (T) boundary, identified in serial sections using IgD staining. These images are representative of multiple sections from three mice of each genotype. e: Fraction of day 2 activated B cells located in the outer follicle. Sections were stained as in d and IgMa cells within ~70um of the Moma1+ cells were enumerated and divided by the total number of IgMa cells in the follicle. Each point corresponds to an individual follicle. Enumeration was performed on sections from three mice. f: Migration of antigen-specific (MD4) B cells and non-cognate B cells from spleens of day 2 immunized wild type mice in response to 7α, 25-OHC. g: Plasma cell response in Ch25h(+/+) and Ch25h(−/−) mice at day 5 following immunization with sheep red blood cells. Left profiles show spleen cells stained for the indicated markers with plasma cells identified as B220^loSyndecan^hi and further stained to detect intracellular IgG1. Numbers indicate percent of total cells in the indicated gate. Right bar graph shows a summary of the data for 9 mice of each type (mean±SE). *p<0.05, **p<0.01 (unpaired student's T-test)