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  • Oxysterols modulate the immune responses and


    Oxysterols modulate the immune responses and as such could be effectors of the tumor environment: 25-OHC impairs IgA production in B-lymphocytes [26] and induces the secretion of the proinflammatory and angiogenic cytokine IL-8 [27], [28]. Of note, oxysterols (in particular 7α,25-OHC) are potent chemoattractants for immune cells via Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2; also termed GPR183) [29], [30]. Besides regulating normal function of the immune system this pathway might be of importance in the tumor environment, contributing to chemotactic recruitment of monocytes across the tumor vasculature and subsequent deposition of tumor-associated macrophages. The present study aimed at investigating CH25H expression on mRNA and protein level in two GBM cell lines with different in silico CH25H mRNA expression ( We explored the effects of TNFα and IL-1β (cytokines secreted by GBM cells [31], [32], [33]) on CH25H transcription and translation, and quantitated its product 25-OHC by GC–MS analysis. Using THP-1 and primary human blood monocytes we studied the effects of exogenous 25-OHC and GBM-conditioned medium on cell migration, since monocyte-derived macrophages are known to contribute to increased aggressiveness and invasiveness of glioblastoma [34]. Finally, the involvement of the G protein-coupled receptor EBI2 in 25-OHC-mediated migration of THP-1 cells was investigated.
    Materials and methods
    Discussion Macrophages and dendritic cells respond to Toll-like receptor (TLR) ligands by upregulating CH25H expression [4], [5]. In the latter cell type TLR-dependent upregulation is mediated via a signalling pathway that involves NFκB and IFNβ secretion and converges on activation of the JAK/STAT1 pathway [4]. In vivo, exposure of mice to the selective TLR4 agonist KDO induced strong upregulation of CH25H in fluorescent probes [26]. Results of the present study revealed upregulated CH25H transcription and translation in GBM cells. Interestingly, within the NCI60 data set contained in the BioGPS gene portal GBM cells appear to take a unique role in terms of highest CH25H expression ( The response to TNFα and IL1β was different with respect to cytokine selectivity in the two cell lines studied here (Fig. 1). Whether this is due to different cytokine receptor expression levels is currently not clear. However, both cytokines used here may activate JAK/STAT pathways. TNFα activates JAK/STAT1 [42] and IL1β activates STAT1 [43] and a STAT-like factor, leading to activation of gene transcription [44]. Therefore our observations are compatible with a pathway inducing transcriptional activation of CH25H identified in dendritic cells and macrophages [4]. In line with findings reported for lipopolysaccharide activated macrophages [5] we have observed efficient secretion of 25-OHC into the cellular supernatant. In experiments analyzing free transfer of cholesterol or 25-OHC from erythrocytes and plasma, exchange of 25-OHC was found to occur about 2000 times faster than that of cholesterol [45]. The rate of transfer of oxysterols from a monolayer to acceptor particles followed a clear rank order with the highest rate of transfer observed for 25-OHC [46]. As reported for the quantitatively dominating oxysterols 24S- and 27-OHC, also 25-OHC is transported in association with circulating lipoproteins including high-density lipoproteins [47]. This might be of functional importance for gliomagenesis since high-density lipoproteins containing sphingosine-1-phosphate (a potent lipid GBM mitogen; [48]) induced increased DNA synthesis, ERK phosphorylation and Ca2+ mobilization in glioma cells [49]. Significant progress has been made in elucidating the functional significance of oxysterol-induced B- and T-cell migration in lymphoid organs [29], [30], [50], [51], [52]. It is now widely accepted that EBI2-mediated chemotaxis represents an important molecular mechanism directing follicular B cell migration and localization. Of note, oxysterols represent natural ligands for EBI2 activation [29], [30], [51]. In line with findings from the present study (Fig. 6), PTX suppressed 7α,25-OHC stimulated and EBI2-mediated binding of GTP [30]. However, PTX-insensitive ERK activation in response to EBI2 engagement was also reported [52]. The functional potency of different oxysterols towards EBI2 is 7α,25-OHC>7α,27-OHC>7α–OHC>25-OHC>27-OHC [50]. Of relevance for the present study 25-OHC also confers agonist activity towards EBI2 albeit with lower affinity [32], [33]. In line, silencing of EBI2 using 21-mer siRNA reduced 25-OHC-induced THP-1 migration by 46% (Fig. 7). Whether EBI2 is responsible for increased migration of primary human monocytes in response to 25-OHC remains to be elucidated. The reason why mock transfection (GeneMute) and transfection with scrambled siRNA reduced migration of THP-1 cells in response to 25-OHC is currently not clear. However, lipid-based transfection reagents impact on cellular phospholipid composition (enrichment in 16:0 or 22:6-containing phosphatidylcholine species; unpublished observation; S.F.), alterations that could account for different receptor responsiveness.