gms | German Medical Science

Objectives: Our previous studies revealed elevated levels of phospholipids (PLs) in human knee synovial fluid (SF) during osteoarthritis (OA), which may derive from blood and from local cells such as fibroblast-like synoviocytes (FLS). However, the mechanisms controlling the PL level in SF remains largely unknown. Natural agonists of the nuclear liver X receptors (LXRs) such as oxysterols are involved in the release and biosynthesis of cholesterol, PLs respectively fatty acids. In FLS, cholesterol can be oxidised to oxysterols by cholesterol 25-hydroxylase (CH25H) and 25-hydroxycholesterol 7-α-hydroxylase (CYP7B1). Here, we investigated the effects of IL-1ß and two LXRα agonists on the release of PLs from FLS, and the mechanisms controlling PL efflux.

Methods: Cultured FLS isolated from synovial tissue of OA patients were treated for 24 h, 48 h and 72 h with 1 ng/mL IL-1ß alone or with 0.1 µM of the synthetic LXRα-agonist TO901317 or GW3965. Furthermore, FLS were treated with 10 µM Nf-κB-, JNK- or p38 MAPK- pathway inhibitors, either singly or in combination with IL-1ß. RT-qPCR analysis and/or western blot analysis were performed on ABC-transporters ABCA1, ABCA3, ABCC1, ABCG1, and on apolipoprotein (Apo) A1, ApoE, LXRα and LXRß. Additionally, the release of [³H]-choline-labelled PLs from FLS into the nutrient media was analysed during a 48h period. A one factorial ANOVA or a linear mixed effect model was performed to determine statistical significance between treated samples and untreated controls. A Benjamini and Yekutieli posthoc method was applied for multiple testing to control the false discovery rate at 10%. Statistical analysis was performed using program R V3.6.1. The local ethics committee of our University approved the study.

Results and Conclusion: IL-1ß induced a 1.4-fold elevated release of [³H]-labelled-PLs from FLS, which was not antagonized by the addition of pathway inhibitors. TO901317 and GW3965 induced a 1.5- and 1.8-fold enhanced release of [³H]-labelled-PLs respectively, which was further enhanced in the presence of IL-1ß (2.7- and 2.9-folds, respectively). Simultaneously, IL-1ß markedly elevated the expression of CH25H, CYP7B1, and ABCA1. Treatment with TO901317 significantly elevated the expression of LXRα, ABCA1, ABCG1, and ApoE.

Our results indicate for the first time that IL-1ß, being elevated in SF during OA, is part of the mechanisms regulating PL release from FLS into SF during OA by inducing the biosynthesis of oxysterols. Both synthetic LXRα-agonists can markedly stimulate the efflux of PLs from FLS confirming the regulatory role of the natural LXRα-agonist oxysterol on PL level in SF.