gip receptor In this study we have explored a
In this study we have explored a functional role of the oxysterol/EBI2 system in this process. In 2011 we and others reported the discovery of oxysterols as ligands for EBI2 , . Oxysterols are metabolites generated by hydroxylation of cholesterol and have been linked to a variety of fundamental physiological processes including sterol transportation, bile gip receptor biosynthesis as well as immune cell function , . For example oxysterols are ligands for several nuclear hormone receptors (e.g. LXR, RORα, RORγ) which play a role in the immune process. While LXR and their ligands are negative regulators of macrophage inflammatory gene expression  and constitute a metabolic checkpoint for immune cell proliferation , RORγt is the key transcription factor to orchestrate differentiation of pro-inflammatory Th17 cells .
For mouse macrophages it was reported that they up-regulate CH25H expression in a time-dependent manner upon immune challenge with LPS. The increase in mRNA and protein levels leads to a subsequent production of 25-OHC which is released into the cellular environment , . In contrast, EBI2 mRNA transcripts are down-regulated after LPS treatment , . While the oxysterol/EBI2 complex in murine B cells is well studied its role in primary human cells of the innate immune system, in particular macrophages, has not yet been investigated. In order to develop an integrated understanding of the transcriptional regulation and function of the oxysterol/EBI2 pathway here we describe the expression of the oxysterol-metabolizing enzymes CH25H, CYP27A1, CYP7B1, HSD3B7, and the receptor itself following inflammatory stimuli and show a functional role of this ligand/receptor pair in monocyte-derived macrophages.
Materials and methods Details on materials and methods can be found in the Supplemental material section.
Discussion In our experiments we mimicked inflammation by LPS challenge of M0 macrophages. We discovered a strong increase in expression of CH25H and CYP7B1 while CYP27A1 and HSD3B7 are down-regulated over time. CH25H up-regulation is transient with a peak around 2h. This is similar to the dramatic raise in expression of EBI2, even beyond expression levels of β-actin, which occurs after LPS treatment. Both findings suggest a feedback mechanism by which increasing amounts of oxysterols diminish further expression of enzyme and receptor mRNA. Interestingly, this result is in contrast to the murine system where EBI2 transcript levels are strongly down-regulated upon LPS challenge. PBMC-derived macrophages not only express EBI2 but also have the ability to respond to their natural agonist 7α,25-OHC by activating downstream signal transduction pathways. In line with previous findings, our data confirm that receptor stimulation induces calcium mobilization which can be blocked by the EBI2 antagonist NIBR189. As described in several studies for EBI2-expressing immune cells, the main function of the ligand/receptor complex is the promotion of chemotaxis , . Here, we demonstrate EBI2-dependent movement of macrophages. The observed bell-shaped curve, a classical hall mark of directed cell migration in transwell assays, suggests receptor desensitization. Increased expression of oxysterol-producing enzymes lead to an elevation of oxysterol levels. While detection of oxysterols in cell culture systems has been successful , ,  it remains challenging. Thus, we decided to use a bioassay in which supernatants from LPS-stimulated macrophages were transferred on other cells and the release of intracellular calcium was monitored. Supernatants induced calcium mobilization which is in part mediated by EBI2 as demonstrated through blockade by the receptor antagonist. These results confirm our hypothesis that an inflammatory challenge leads to enhanced generation of oxysterols which act as EBI2 agonists. To determine which specific oxysterol species are produced in this setting analysis by mass spectrometry will be needed.