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 diseases1,2,3. 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)4. 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.
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Acknowledgements
We would like to thank the following scientists for their support: F. Su and J.-L. Vincent (Bruxelles Erasme Hospital) for access to tissue samples from the peritonitis sheep sepsis model; D. W. Russell (University of Texas Southwestern) for access to the Ch25h−/− mouse strain; J. Carballido and T. Rubic (all Novartis) for providing an EBV-infected B-cell line and help in preparation of mouse bone-marrow-derived dendritic cells; J. C. Reed (Burnham Institute for Medical Research) for the RS11846 cells; A.-G. Letombe (Euroscreen), A. Feige and N. Beluch for technical assistance; and J. Hamon, C. Bauer, A. Glaenzel, P. Bross, V. Techer-Etienne (all Novartis) and RC Tritec Ltd (Teufen, Switzerland) for support in the development of a radioligand binding assay.
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S.H., S.N., M.-O.R., M.D. and K.S. initiated the project and developed strategy. S.H., S.N. and M.O.R. purified activity from sheep tissue extracts. W.S., D.-I.K., C.Y.C. and P.G.S. carried out corresponding work on the pig tissue. J.Z., Ri.K., I.C., Ra.K., R.F. and E.C.P. conducted mass-spectrometry and NMR experiments. S.L., M.V., I.P., D.G., D.N., W.L., A.K., Y.A.C., B.U.B., K.S. and A.W.S. designed and conducted pharmacological characterization of the receptor and bioactivity assays. S.R., C.M., F.G., Ca.S., W.S., C.Y.C., S.H. and A.W.S. proposed and/or synthesized various oxysterol ligands. T.S., Ch.S. and B.W. supported in vivo experiments with mouse models. T.Y., J.P.P., L.M.K. and J.G.C. developed the bioassay, conducted the experiments in CH25H-deficient and transduced mice and performed the B-cell chemotaxis and immunization studies. A.W.S. wrote the first draft of the manuscript, which was completed by S.H., J.Z., D.N., B.W., S.R., J.G.C., C.Y.C., K.S. and commented on by all authors.
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All authors, except for T.Y., J.P.P., L.M.K., D.I.K. and J.G.C. are current or previous employees of either Euroscreen or Novartis and some of them do hold stock or stock options in their respective companies.
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Hannedouche, S., Zhang, J., Yi, T. et al. Oxysterols direct immune cell migration via EBI2. Nature 475, 524–527 (2011). https://doi.org/10.1038/nature10280
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DOI: https://doi.org/10.1038/nature10280
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