br Results br Discussion Oxysterols and EBI

Results
Discussion Oxysterols and EBI2 have received growing attention in the field of immunology, with works highlighting an important role for EBI2 in immune cell migration. However, these studies are mainly limited to animal models (Chalmin et al., 2015, Hannedouche et al., 2011, Kelly et al., 2011, Liu et al., 2011, Pereira et al., 2009, Suan et al., 2015), while in human and particularly in a disease setting, such as MS, the relevance of EBI2 expression and function remains a critical question to potentially translate research findings from the bench side to the patient. We here report that EBI2 is functional and differentially expressed at the surface of human lymphocytes, with a strong predominance for CD4+ T and B cell memory subsets. These observations obtained at the protein level are in line with previous mRNA data (Hannedouche et al., 2011) showing predominant EBI2 expression in human memory subsets of buy flumazenil and CD4+ and CD8+ T cells, and they corroborate murine data showing a higher EBI2 expression in mature B cells compared to naive correlates (Pereira et al., 2009). Curiously, despite similar EBI2 expression levels between memory CD4+ T cells and B cells, CD4+ T cells migrate twice as much as their B cell counterparts. Technically, this could be secondary to differences between B and CD4+ T cells about the optimal oxysterol concentrations for migration in our assay or to a higher sensitivity of B cells to the thawing process. However, previous murine data show a similar migration between B220+ B cells and CD4+ T cells (Liu et al., 2011). Therefore, possible differences between human B and CD4+ T cell migration patterns cannot be dismissed, and they could be secondary to differences in EBI2 signaling or to distinct interactions with other adhesion or activation molecules. No positive correlation between the migration rate toward 7α,25-OHC and EBI2 expression was observed in different subsets of lymphocytes obtained from HDs. On the contrary, a positive correlation between EBI2 expression and migration toward 7α,25-OHC was observed in RRMS patients (both untreated or under DMTs). This is particularly relevant for CD4+ and CD8+ memory T cells but not for CD19+ B cells. Those correlation differences between HDs and RRMS patients could indicate that EBI2 signaling or interactions with other adhesion molecules differ during autoimmunity. In addition, the similar levels of EBI2 expression in untreated RRMS patients might not exclude changes in EBI2 expression and/or function in more severe disease states or at precise moments during the disease evolution. Because it was described that upon certain stimuli (i.e., lipopolysaccharides [LPS] challenge), human macrophages upregulated EBI2 RNA expression in only 2 hr with return to basal level after 4 hr (Preuss et al., 2014), we can hypothesize that EBI2 expression and/or function may vary in a transient and possibly highly dynamic course in lymphocytes depending on the disease evolution. Accurate time points according to the relapses may be necessary to highlight such changes. EBI2 is important for the migration of myeloid and lymphoid cells in steady-state and inflammatory conditions (Chalmin et al., 2015, Chiang et al., 2013, Gatto et al., 2013, Hannedouche et al., 2011, Kelly et al., 2011, Liu et al., 2011, Pereira et al., 2009, Preuss et al., 2014, Rutkowska et al., 2015, Suan et al., 2015, Yi et al., 2012). We can therefore hypothesize that EBI2 may also play a role in organ-specific migration of memory cells during normal or pathologic conditions or participate in CD4+ T cell homing to secondary lymphoid organs. Regarding the latter hypothesis, given that EBI2 expression in peripheral blood naive CD4+ T cells is very low and that, in the memory subsets, we did not see any difference in expression between central and effector memory T cells, it is unlikely that EBI2 might specifically participate in this process. However, the observation by Preuss et al. (2014) that EBI2 RNA transcripts are maximal in human primary monocytes and upregulated in M0 macrophages upon LPS challenge brings arguments for a potential role of EBI2 for site-specific migration of immune cells during inflammatory settings. Future investigations are required to clarify this point.