The G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2) is activated by 7α, 25-dihydroxycholesterol (7α25HC) and plays a role in T cell-dependant antibody response and B cell migration. Aberrant EBI2 signaling is implicated in a range of autoimmune disorders however its role in the CNS remains unknown. Here we characterize the functional role of EBI2 in GLIA cells using primary human astrocytes and EBI2 knockout animals. We find human and mouse astrocytes express EBI2 and the enzymes necessary for synthesis and degradation of 7α25HC. In astrocytes, EBI2 activation stimulates ERK phosphorylation, Ca(2+) signaling and induces cellular migration. These results, for the first time, demonstrate a role for EBI2 in astrocyte function and suggest that modulation of this receptor may be beneficial in neuroinflammatory disorders.

Background and Purpose The sphingosine 1‐phosphate receptor subtype 1 (S1P1R) is modulated by phosphorylated FTY720 (pFTY720), which causes S1P1R internalization preventing lymphocyte migration thus limiting autoimmune response. Studies indicate that internalized S1P1Rs continue to signal, maintaining an inhibition of cAMP, thus raising question whether the effects of pFTY720 are due to transient initial agonism, functional antagonism and/or continued signalling. To further investigate this, the current study first determined if continued S1P1R activation is pathway specific. Experimental Approach Using human and rat astrocyte cultures, the effects of S1P1R activation on cAMP, pERK and Ca2+ signalling was investigated. In addition, to examine the role of S1P1R redistribution on these events, a novel biologic (MNP301) that prevented pFTY720‐mediated S1P1R redistribution was engineered. Key Results The data showed that pFTY720 induced long‐lasting S1P1R redistribution and continued cAMP signalling in rat astrocytes. In contrast, pFTY720 induced a transient increase of Ca2+ in astrocytes and subsequent antagonism of Ca2+ signalling. Notably, while leaving pFTY720‐induced cAMP signalling intact, the novel MNP301 peptide attenuated S1P1R‐mediated Ca2+ and pERK signalling in cultured rat astrocytes. Conclusions and Implications These findings suggested that pFTY720 causes continued cAMP signalling that is not dependent on S1P1R redistribution and induces functional antagonism of Ca2+ signalling after transient stimulation. To our knowledge, this is the first report demonstrating that pFTY720 causes continued signalling in one pathway (cAMP) versus functional antagonism of another pathway (Ca2+) and which also suggests that redistributed S1P1Rs may have differing signalling properties from those expressed at the surface.

The family of interleukin 17 receptors (IL17Rs), subtypes IL17RA-IL17RE, is targeted by the group of pro-inflammatory IL17 cytokines (IL17A-F) and moreover the newly developed anti-IL17A antibody secukinumab (AIN457) has shown promise in Phase II trials in multiple sclerosis. Here, we show that human astrocytes, isolated from a fetal cerebral cortex, express IL17RA and IL17RC and in vitro treatment with IL17A increases protein levels of IL6 in human astrocytes, which is enhanced in the presence of TNFα, as determined by homogeneous time resolved fluorescence. Studies on acutely isolated mouse astrocytes are comparable to human astrocytes although the protein levels of IL6 are lower in mouse astrocytes, which also show a lower response to IL17F and IL1β in promoting IL6 levels. In human astrocytes, IL17A and TNFα also induce mRNA expression of IL6, IL8 and the Th17 cytokines CXCL1, CXCL2, and CCL20, with little effect on Th1 cytokines CXCL9, CXCL10, and CXCL11. The effects of IL17A are associated with nuclear translocation of the NF-κB transcription factor, as determined by immunocytochemistry, where treatment of human astrocytes with the inhibitors of the NF-κB pathway and with secukinumab inhibits the IL17A and IL17A/TNFα-induced increase in nuclear translocation of NF-κB and levels of IL6. Taken together the data shows that IL17A signaling plays a key role in regulating the levels of cytokines, such as IL6, in human astrocytes via a mechanism that involves NF-κB signaling and that selective inhibition of IL17A signaling attenuates levels of pro-inflammatory molecules in astrocytes.

Objective:Smoking cessation is crucial for patients with coronary heart disease (CHD), yet depression may impede cessation success. We systematically reviewed the longitudinal association between depression and subsequent smoking cessation in individuals with CHD in order to quantify this effect. Methods:Electronic databases (PsychInfo, PubMed, CINAHL) were searched for prospective studies of CHD patients which measured depression at baseline (scales, diagnostic interview or antidepressant prescription) and reported smoking continuation/cessation at follow-up. Inclusive dates were 1 st January 1990 to 22 nd May 2013. Standardized mean differences (SMD) and associated 95% confidence interval (CI) were estimated using random effects meta-analysis. Sensitivity analysis explored the impact of limiting metaanalysis to studies using different depression measures (validated scales, diagnostic interviews, antidepressant prescription), different durations of follow-up, or higher quality studies. Results:From 1451 citations retrieved, 28 relevant articles were identified. Meta-analysis of all available data from 20 unique datasets found that depressed CHD patients were significantly less likely to quit smoking at follow-up (SMD=-.39, 95% CI -.50 to -.29; I 2 =51.2%, p=.005). Estimates remained largely unchanged for each sensitivity analysis, except for two studies that used antidepressants, which showed a much larger effect (SMD=-.94, -1.38 to -.51; I 2 =57.7%, p=.124).--2 Conclusions:CHD patients with depressive symptoms are significantly less likely to quit smoking than their non-depressed counterparts. This may have implications for cardiovascular prognosis, and CHD smokers may require aggressive depression treatment to enhance their chances of quitting.Keywords: Depression; coronary heart disease; health behaviors; smoking; smoking

The endogenous oxysterol 7α, 25-dihydroxycholesterol (7α25HC) ligand activates the G protein-coupled receptor EBI2 to regulate T cell-dependant antibody response and B cell migration. We have demonstrated that EBI2 is expressed in human and mouse astrocytes, that 7α25HC induces intracellular signalling and astrocyte migration, and that EBI2 plays a role in the crosstalk between astrocytes and macrophages. Recently, we demonstrate that EBI2 regulates myelin development and inhibits LPC-induced demyelination. Here, we show that 7α25HC inhibits LPS- and IL17/TNF-induced pro-inflammatory cytokine release in astrocytes. We observe the following: 1. Human astrocytes treated with IL17/TNF increases the nuclear translocation of NFκB, which is attenuated by pre-treatment with 7α25HC; 2. IL17/TNF increases cell impedance in human astrocytes, which is also attenuated by pre-treatment with 7α25HC; 3. The EBI2 antagonist NIBR189 inhibits these effects of 7α25HC, supporting the role of EBI2; 4. in vivo data corroborate these in vitro findings, showing that EBI2 knock-out (KO) animals display enhanced pro-inflammatory cytokine in response to LPS challenge, in the brain. These results demonstrate a role for oxysterol/EBI2 signalling in attenuating the response of astrocytes to pro-inflammatory signals as well as limiting the levels of pro-inflammatory cytokines in the brain.