Hemoglobin Directs Macrophage Differentiation and Prevents Foam Cell Formation in Human Atherosclerotic Plaques

Abstract

Objectives To examine selective macrophage differentiation occurring in areas of intraplaque hemorrhage in human atherosclerosis. Background Macrophage subsets are recognized in atherosclerosis but the stimulus for and importance of differentiation programs remains unknown. Methods We used freshly isolated human monocytes, a rabbit model, and human atherosclerotic plaques to analyze macrophage differentiation in response to hemorrhage. Results Macrophages characterized by high expression of both mannose and CD163 receptors preferentially exist in atherosclerotic lesions at sites of intraplaque hemorrhage. These hemoglobin (Hb)-stimulated macrophages, M(Hb), are devoid of neutral lipids typical of foam cells. In vivo modeling of hemorrhage in the rabbit model demonstrated that sponges exposed to red cells showed an increase in mannose receptor positive macrophages only when these cells contained hemoglobin (Hb). Cultured human monocytes exposed to hemoglobin:haptoglobin complexes (Hb:Hp), but not IL-4, expressed the M(Hb) phenotype and were characterized by their resistance to cholesterol loading and upregulation of ABC transporters. M(Hb) demonstrated increased ferroportin (FPN) expression, reduced intracellular iron, and reactive oxygen species (ROS). Degradation of FPN using hepcidin increased ROS, inhibited ABCA1 expression, and cholesterol efflux to ApoAI, suggesting reduced ROS triggers these effects. Knockdown of liver x receptor alpha (LXRα) inhibited ABC transporter expression in M(Hb) and macrophages differentiated in the anti-oxidant superoxide dismutase. Lastly, liver X receptor α (LXR) luciferase reporter activity was increased in M(Hb) and significantly reduced by overnight treatment with hepcidin. Collectively, these data suggest reduced ROS triggers LXRα activation and macrophage reverse cholesterol transport (RCT). Conclusions Hb is a stimulus for macrophage differentiation in human atherosclerotic plaques. A reduction of macrophage intracellular iron plays an important role in this non- foam cell phenotype by reducing ROS, which drives transcription of ABC transporters through activation of LXRα. Reduction of macrophage intracellular iron may be a promising avenue to increase macrophage RCT.