title: Oxysterols exert proinflammatory effects in placental trophoblasts via TLR4-dependent, cholesterol-sensitive activation of NF-κB.
abstract: Oxidized cholesterol metabolites (oxysterols) promote inflammation in a variety of cell types and are thought to be involved in a number of disease pathologies. Oxysterol concentrations are increased in pregnancy, together with systemic oxidative stress and inflammation. We tested the hypothesis that oxysterols 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-ketoC) promote placental trophoblast inflammation, and determined the mechanisms involved. Treatment of primary trophoblasts in culture with 25-OHC and 7-ketoC increased the production of proinflammatory cytokines (interleukin-6, macrophage inflammatory protein-1β and tumour necrosis factor-α) in a concentration-dependent fashion. Inhibition of TLR4 activation using selective inhibitors of TLR4 complex formation (OxPAPC) or signalling transmission (CLI095) prevented lipopolysaccharide (LPS)- and oxysterol-induced inflammatory cytokine production. Pretreatment of trophoblasts with selective inhibitors of I-kB kinase activity (parthenolide and TPCA-1) reduced oxysterol- and LPS-stimulated inflammatory responses, consistent with the involvement of the nuclear factor kappa B (NF-κB) pathway downstream of TLR4 signalling. Both oxysterols also increased the phosphorylation and nuclear localization of NF-κB subunit p65/RelA. Oxysterols are also known to activate liver X receptors (LXRs) which can inhibit inflammatory signalling, either directly or indirectly via membrane cholesterol reduction. Treatment with the LXR agonist, T0901317, exerted significant anti-inflammatory effects, reducing LPS- and oxysterol-driven cytokine production. Treatment with methyl-β-cyclodextrin to deplete membrane microdomain cholesterol and thereby disrupt TLR4 signalling, similarly abrogated their effects. Together, these findings indicate that although oxysterols likely activate both pro- and anti-inflammatory pathways in the placenta, the predominant effect is the promotion of placental inflammation via TLR4-dependent activation of NF-κB.
authors.affiliation: School of Women's and Infants' Health, Faculty of Medicine, Dentistry and Health Sciences, King Edward Memorial Hospital, 374 Bagot Rd, Subiaco, The University of Western Australia, Perth, WA 6008, Australia. iaye@meddent.uwa.edu.au
authors.forename: Irving L M H
authors.initials: IL
authors.lastname: Aye
authors.forename: Brendan J
authors.initials: BJ
authors.lastname: Waddell
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Mark
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Keelan
mesh_terms: D002478:Cells, Cultured; D054407:Chemokine CCL4 / Q000378:metabolism; D005260:Female; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007150:Immunohistochemistry; D015850:Interleukin-6 / Q000378:metabolism; D007653:Ketocholesterols / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D011247:Pregnancy; D014327:Trophoblasts / Q000187:drug effects* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D054407:Chemokine CCL4; D006888:Hydroxycholesterols; D015850:Interleukin-6; D007653:Ketocholesterols; D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; C007997:25-hydroxycholesterol; C003001:7-ketocholesterol