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  • PBMC derived macrophages not only

    2020-03-21

    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 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. 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
    Acknowledgments
    Oxysterols are oxygenated derivatives of cholesterol produced by the oxidation by the CYP family of enzymes in the cells and falls in the bile 1295 synthesis or steroid synthesis pathway. Few oxysterols such as 7-hydroxy and 7-keto are also known to be produced in vivo by non-enzymatic radical oxidation mechanism. Although found in very low concentrations in most mammalian tissues, oxysterols play a crucial role in cholesterol and fatty acid metabolism, regulation of immune response and are also believed to be mediators in neurodegenerative disorders. While oxysterols were most extensively studied for its potent ability to mediate feedback regulation of cholesterol biosynthesis, some early investigations showed that the enzymes involved in its syntheses were highly upregulated in macrophages and dendritic cells—an indication of possible roles in immune related functions., Subsequent research has shown that oxysterols have a broad range of roles in innate and adaptive immune responses. For example, 25-hydroxycholesterol (25-HC) is induced in macrophages by type 1 interferon (IFN) signaling and has broad ability to prevent viral entry, replication and budding., In addition to the pro-inflammatory effects, many studies show that, 25-HC also mediate the anti-inflammatory effects downstream of the IFN pathway. Immunomodulation effects of certain oxysterols were previously shown to depend on activation of oxysterol-binding liver X receptors (LXRs). Recent work from our labs as well as from others has shown additional receptors linking oxysterols and immunity. For example, we found that 7α,25-dihydroxycholesterol as a key molecule involved in directing the migration of naive B cells, T cells and dendritic cells by engaging an orphan GPCR, GPR183, a gene which was initially found to be highly induced by Epstein–Barr virus infection of B cells. In another study, an oxysterol was found to be the most potent endogenous ligand of RORγt, an orphan nuclear receptor whose activation is the key step in the downstream production of IL-17—a key inflammatory pathway in several autoimmune disorders. Thus, many new biological functions of oxysterols, especially their broad range of roles as signaling molecules in the innate and adaptive immune system continues to be discovered and hold the promise of finding new targets for the therapeutic intervention for autoimmune disorders such as psoriasis, RA, IBD, and MS.,