Toll-like Receptor 4 Mediates Oxidized LDL-Induced Macrophage Differentiation to Foam Cells

Abstract

Macrophage foam cells are central in the development of atherosclerosis, but the mechanism of foam cell formation is unclear. Toll-like receptor 4 (TLR4) signaling is known to participate in the pathogenesis of atherosclerosis. Further, oxidized low density lipoprotein (oxLDL) enhances TLR4 expression in macrophages. We hypothesized that TLR4 mediates macrophage differentiation to foam cells. Peritoneal macrophages were isolated by lavage from TLR4 competent C3H/HeN mice and TLR4 defective C3H/HeJ mice. Cells were treated with oxLDL, lipopolysaccharide (LPS), or oxLDL plus LPS. Cells were also treated with a TLR4 blocking antibody before oxLDL treatment. Foam cells were identified by Oil red O staining for intracellular lipids. Percent macrophage differentiation into foam cells were compared between C3H/HeN and C3H/HeJ macrophages. Following oxLDL treatment 29% of TLR4 competent macrophages differentiated into foam cells compared to 5.8% of TLR4 defective macrophages (P < 0.01). Pretreatment with a TLR4 blocking antibody decreased the differentiation of TLR4 competent cells to foam cells from 29% to 13% (P < 0.01). Stimulation of TLR4 with LPS in the presence of oxLDL increased differentiation of TLR4 competent cells to foam cells from 29% to 60% (P < 0.01). In addition, there was a pattern in the spatial relationship between foam cells that were consistently observed in clusters rather than as isolated cells. TLR4 is necessary for oxLDL-induced macrophage differentiation to foam cells. Macrophage foam cell clustering may indicate an underlying intercellular signaling mechanism that facilitates foam cell formation. We conclude that TLR4 contributes to the pathogenesis of atherosclerosis by promoting foam cell formation.