Methods: THP-1 macrophages were incubated ± 1:500 dilution of human anti-IL-33 receptor-blocking antibody (3h), followed by 18h incubation ± 1, 10, 20, 50 ng/ml of human IL-33 (each condition in triplicate). Following incubation, total RNA was isolated. Expression level was determined by QRT-PCR for cholesterol efflux proteins ABCA1 and 27-hydroxylase, and for cholesterol uptake proteins CD36, LOX1, SRA1 and CXCL16. Expression was normalized to the housekeeper gene, GAPDH. (Dil)-oxLDL (Intracel, Frederick, MD) uptake was analyzed by fluorescence microscopy. Data were analyzed by one-way ANOVA with Bonferroni correction.
Results: IL-33 at 10 ng/ml augmented ABCA1 and 27-hydroxylase message to 191.1±14.9% and 227.4±28.8%, respectively (P<0.05, n=3). High concentrations of IL-33 (20 and 50ng/ml) reduced efflux protein expression to that of untreated cells while doubling expression of scavenger receptors CD36, LOX-1 and CXCL16 versus control cells (P<0.05, n=3). These changes contributed to increased oxLDL accumulation and at 50ng/ml IL-33, ox LDL reached 155.7±8.8% above control (P<0.05, n=5). Blocking of the IL-33 receptor abrogated IL-33-induced alterations in gene expression, confirming an IL-33 receptor-dependent mechanism for observed changes in cholesterol metabolism.
Conclusions: We have demonstrated a dose-dependent effect of IL-33 on the expression of ABCA1, CXCL16, CD36 and LOX1. The effect is mediated through the IL-33 receptor. High concentrations of IL-33 relevant to those observed in AP plasma resulted in a switch from an anti-atherogenic to a pro-atherogenic state. Such changes could support the development of a pro-atherogenic profile in macrophages that may be improved with treatment.