In mice our EBI EGFP reporter showed that
In mice, our EBI2EGFP reporter showed that EBI2 is expressed consistently in naive T helper cells. Already single positive CD4+ thymocytes express EBI2 (not shown). In effector and regulatory T cells in naive mice, the percentage of EBI2 expression was lower (∼40%). Like others (Liu et al., 2011), we found that for efficient in vitro migration, polyclonal pre-stimulation of the T cells was needed (not shown). Our findings that 2D2 ON123300 upregulate EBI2 in vivo during priming is in line with enhanced levels of EBI2 expression necessary for directed migration toward 7α,25-OHC. Concomitant with a function of EBI2 for effector cell migration, human CD45RO− memory/Teff cells expressed higher surface EBI2 protein than naive T helper cells. Our data indicate that EBI2 is one of several molecules mediating migration of T cells into the inflamed CNS. Because other important molecules for tissue infiltration, such as CD44, VLA4, and LFA1, are not expressed on naive T cells, those cells may be precluded from immigration to the inflamed CNS despite expression of EBI2. Here, we show that enzymes responsible for synthesis of 7α,25-OHC from cholesterol are highly expressed in the CNS of mice in response to EAE induction. This was paralleled by increased levels of the EBI2 ligands 7α,25-OHC and 7α,27-OHC in SCs of mice with EAE. Therefore, we suggest that during inflammation, expression of CH25H and CYP7B1 sustain lymphocyte accumulation at sites of inflammation/infection. It has been suggested that expression of CH25H supports transmigration of activated CD44+ T helper cells into activated tissues such as the inflamed CNS. Similarly to our data obtained from the EAE transfer model, the lack of CH25H delays onset of EAE with similar end scores and similar infiltration and demyelination at the end of the experiments (Chalmin et al., 2015). Bone marrow chimeric experiments by the same group suggested that CH25H was needed to be expressed in cells of hematopoietic origin (supposedly monocytes/macrophages or moDCs) rather than in cells of the CNS tissue itself to promote EAE development. Contrary to this, we found that Ch25h is expressed by CNS-derived microglia in EAE and not by infiltrating myeloid cell populations. Interestingly, the second enzyme for generating 7α,25-OHC CYP7B1 was expressed by infiltrating lymphocytes and monocytes. This suggests that activated microglia and infiltrating cells have to cooperate in production of the cell-permeable ligand as it was demonstrated previously for different cell lines expressing these enzymes separately (Yi et al., 2012). We did not define the lymphocyte population expressing CYP7B1. Interestingly, NK cells can express this enzyme (Liu et al., 2011) and were also shown to infiltrate the CNS in EAE (Huang et al., 2006). We also did not analyze astrocytes for expression of these enzymes in EAE. It was recently shown that microglia (Butovsky et al., 2014, Olah et al., 2012) as well as murine astrocytes can express the necessary enzymes for production of the EBI2 ligand (Rutkowska et al., 2015, Rutkowska et al., 2016). Alternative sources for EBI2 ligand production in EAE under investigation by us may be endothelial cells. How microglia cells become activated to express Ch25h in EAE is not clear. Because we observe major differences in transfer EAE of Th17 cells, we assume that expression of Ch25h is not a response to CFA used in active EAE induction but rather induced by inflammatory cytokines. Interestingly, our analyses of human PBMCs using a monoclonal antibody against EBI2 showed that the expression pattern of human EBI2 is largely overlapping with that in mice. Especially Th17 cells showed homogeneous high expression of EBI2 compared with Th1 cells, which contained a fraction of cells negative for EBI2. Furthermore, when we analyzed histological sections of lesions from MS patients, we found expression of EBI2 restricted to mononuclear cells present within inflamed white matter regions. Most cells expressing EBI2 morphologically appeared as infiltrating macrophages. We found T cells that were clearly negative and T cells with strong expression of EBI2 in MS lesions. This finding is in accordance with our flow cytometry data showing that only a part of all T cells express EBI2 and the fact that MS lesions are known to be dominated by CD8 cells (Babbe et al., 2000), which are largely negative for EBI2.