In addition to playing important roles

In addition to playing important roles in the immune system, EBI2 is also expressed in the Tamoxifen of the central nervous system (CNS), namely astrocytes, and this receptor regulates astrocyte signalling, as well as astrocyte cell migration (Rutkowska et al., 2015, Rutkowska et al., 2016a). Moreover, it has been shown that LPS induces the release of oxysterols such as 25HC, 7α25HC and 7β25HC in astrocytes and that the oxysterol/EBI2 signalling pathway is involved in the crosstalk between the immune cells, namely macrophages, and the CNS cells, namely astrocytes (Rutkowska et al., 2016b). Most recently, we have demonstrated that EBI2 regulates myelin development and inhibits LPC-induced demyelination (Rutkowska et al., 2017). Therefore, in the immune system as well as the CNS, oxysterols and EBI2 signalling appears responsive to pro-inflammatory signals including, TLR4 stimulation or type I/II interferons, which may provide a mechanism against infection and neuroinflammation (Blanc et al., 2013, Diczfalusy et al., 2009, Rutkowska et al., 2016b). Here, to investigate further the glial cell functions of EBI2, we examined its role in regulating the response to and release of pro-inflammatory cytokines in human and rodent astrocytes, and in vivo using EBI2 knockout mice.
Materials and methods
Results
Discussion We have recently demonstrated that EBI2 receptor, as well as the endogenous agonist 7α25HC and enzymes necessary for synthesis and degradation of 7α25HC, are expressed in human and mouse astrocytes (Rutkowska et al., 2015, Rutkowska et al., 2016b). We have also shown that 7α25HC induces pERK and Ca2+intracellular signalling in astrocytes. In addition, we find that EBI2 induces astrocyte cell migration (Rutkowska et al., 2015) and that EBI2/oxysterol signalling is involved in the crosstalk between macrophages and astrocytes (Rutkowska et al., 2016b). Most recently we demonstrate that EBI2 regulates myelin development and inhibits LPC-induced demyelination. In the current study, we show that human, mouse and rat astrocytes release pro-inflammatory cytokines when stimulated with TNFα/IL17 or LPS, respectively and that pre-treatment with EBI2 agonist 7α25HC attenuates this response. We find that 7α25HC attenuates the TNFα/IL17-induced protein levels of IL6 in human astrocytes, via a mechanism that likely involves reduced levels of IL6 mRNA and via altered NFκB signalling. We also report that IL17/TNFα increases cell impedance in human astrocytes and that pre-treatment with 7α25HC significantly reduces this response. Moreover, we demonstrate that the EBI2 antagonist NIBR189 blocks 7α25HC-mediated attenuation of IL17/TNF-induced signalling, supporting a direct EBI2 involvement. Lastly, our in vivo LPS challenge study supports that EBI2 regulates pro-inflammatory cytokine signalling, whereby EBI2 KO mice exhibited enhanced pro-inflammatory cytokine release after LPS challenge as compared to WT mice. Taken together, these results demonstrate a role for EBI2/oxysterol signalling in limiting the levels of pro-inflammatory cytokines in the brain.