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  • To analyze EBI expression and its

    2019-07-11

    To analyze EBI2 expression and its function in T cells in vivo, we created an EGFP reporter/knockout mouse strain, termed EBI2EGFP. This mouse strain in heterozygous configuration allows for a systematic analysis of the expression of EBI2 in distinct cell types in steady-state and under inflammatory conditions. Among murine and human T cell subsets, EBI2 was highly and uniformly expressed by either naive CD4+ T cells or within the Th17 subset under inflammatory conditions. Its expression was strongly repressed under transforming growth factor β (TGF-β) + interleukin-6 (IL-6)-induced Th17 differentiation conditions but sustained by interleukin-1 beta (IL-1β) and interleukin-23 (IL-23) during T helper cell differentiation. Strikingly, transfer of myelin oligodendrocyte glycoprotein (MOG)-specific EBI2-deficient Th17 AICAR phosphate isolated from homozygous EBI2EGFP/EGFP mice induced EAE with a significantly delayed onset. Moreover, we found that expression of the enzymes CH25H and CYP7B1 changes dramatically during the course of EAE with a strong upregulation detected in the spinal cord (SC) leading to enhanced tissue levels of the EBI2 ligand in the SC in EAE. Additionally, we found that microglia start expressing CH25H early in EAE and that CYP7B1 is expressed in EAE by infiltrating monocytes and lymphocytes. In accordance with our mouse data, EBI2 was highly expressed by human Th17 cells derived from peripheral blood mononuclear cells (PBMCs). Furthermore, macrophages and a subset of T cells in lesions of multiple sclerosis (MS) patients stained positive for EBI2. Therefore, we suggest that EBI2 and its ligand(s) play important roles for efficient and early migration of encephalitogenic CD4+ T cells into the CNS. This mechanism may also be more generally implicated in the infiltration of human lymphocytes into inflamed tissues.
    Results
    Discussion Analysis of EBI2 expression showed that EBI2 is highly expressed in central memory CD8+ T cells that is in line with previous data from human T cells in which these cells express higher levels of EBI2 than naive or effector memory CD8+ T cells (Hannedouche et al., 2011). Such cells were shown to have a specific role in fast viral defense in secondary infections (Wherry et al., 2003). Recently, it was suggested that EBI2 has a function in egress of T cells from LNs, because immunized CH25H-deficient animals contained enriched numbers of CD44+ effector T cells in draining LNs (Chalmin et al., 2015). This, together with our findings of differential expression of EBI2 in distinct stages of T cell differentiation, may suggest that EBI2 has an important role in T cell re-localization and possibly in LN egress reminiscent of the S1P-S1P receptor system, similar to its role in germinal center B cells. In two different types of co-transfer experiments, we analyzed where encephalitogenic T cells reside either in the active EAE model or in the transfer model, either before EAE or at onset of EAE. In active EAE, we found similar numbers of proliferating effector T cells irrespective of the EBI2 genotype in draining LNs but an accumulation of EBI2-deficient T cells in the blood. In the transfer model, we found a clear ratio change of EBI2-expressing T cells over EBI2-deficient T cells from periphery to the CNS in early EAE. Both experiments point to a role of EBI2 and its ligand in CNS extravasation. Therefore, our data do not support the hypothesis that EBI2 plays a major role in the regulation of T cell LN egress but also does not exclude that certain T cell subpopulations might be differentially released of draining lymph nodes (dLNs) after priming in absence of EBI2. In accordance with its original discovery, EBI2 may play its major role in response to viral infections or in a pathological context such as in autoimmunity. Although we found a significant delay of onset in the Th17 transfer EAE model, active EAE was unchanged in absence of EBI2. At the moment, we can only speculate about this difference. It might be that active EAE induction using CFA/MOG induces a stronger inflammatory array of immune cells, pro-inflammatory factors, cytokines, and chemokines, which might override the need for EBI2-mediated migration. Alternatively, there might be compensatory mechanisms for the lack of this GPCR, so that mice are susceptible to EAE also independently of EBI2 in knockout animals. We showed in transfer experiments with EBI2 mutant T cells that EAE was significantly delayed but after onset, disease proceeded normally. This might be due to the previously described plasticity of Th17 cells, which tend to change their expression profile toward the Th1 lineage together with high levels of GM-CSF (Hirota et al., 2011, Kurschus et al., 2010) (and herein). In absence of EBI2, EAE initiation may more depend on Th1 cells co-expressing GM-CSF than on Th17 cells. This hypothesis is supported by our data showing a major population of Th1 cells co-expressing GM-CSF in the CNS during disease.