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  • br Materials and methods Details


    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 [6], [17]. 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 [20], [21], [22] it remains challenging. Thus, we decided to use a bioassay in which supernatants from LPS-stimulated macrophages were transferred on other AG-126 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. It is interesting to note that Eibinger and colleagues [23] recently reported chemotactic movement to 25-OHC in THP1 cells as well as in primary human monocytes. RNA interference suggested that in part this migration was mediated by EBI2.
    Competing financial interest statement
    Introduction Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2, also known as GPR183) was discovered in 1993 in a screen of genes induced by in vitro Epstein-Barr virus (EBV) infection of a Burkitt’s lymphoma cell line (Birkenbach et al., 1993) and identified by sequence similarity as a G protein-coupled receptor (GPCR). EBI2 was shown to have an important role in B cell positioning in the germinal center reaction (Gatto et al., 2009, Pereira et al., 2009), resulting in fewer plasma cells and reduced antibody titers in EBI2-deficient animals. Also, CD4+ conventional dendritic cells (cDCs) are profoundly diminished in the spleen of EBI2 mutant mice (Gatto et al., 2013, Yi and Cyster, 2013). Recently, a role for EBI2 in differentiation of T follicular helper (Tfh) cells was demonstrated. Activated T cells were shown to migrate in an EBI2-dependent manner to the outer T zone to receive inducible T cell co-stimulator (ICOS) stimulation under IL-2 deprived conditions by IL-2 quenching DCs that fosters Tfh differentiation (Li et al., 2016). Furthermore, Suan et al. (2015) showed that Tfh cells in the germinal centers downregulate expression of EBI2. Thus, EBI2 regulates positioning of immune cells in secondary lymphoid organs. Oxysterols were recently identified as natural ligands for EBI2 (Hannedouche et al., 2011, Liu et al., 2011). The most active ligand, 7α,25-dihydroxycholesterol (7α,25-OHC), is generated via sequential hydroxylation of cholesterol by cholesterol-25-hydroxylase (CH25H) and 25-hydroxycholesterol by 7-alpha-hydroxylase (CYP7B1). In naive mice, CH25H is highly expressed in the spleen, whereas CYP7B1 is expressed quite ubiquitously with highest expression in the liver. In addition, both enzymes are expressed in lymphoid stromal cells (Hannedouche et al., 2011, Yi et al., 2012). Contradictory data were published about the role of CH25H in experimental autoimmune encephalomyelitis (EAE) (Chalmin et al., 2015, Reboldi et al., 2014), and no direct contribution of EBI2 in EAE pathogenesis has been described.