Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

BACKGROUND: Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. METHODS: Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. RESULTS: In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. CONCLUSIONS: Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Oxidized Low-Density Lipoprotein Accumulation in Macrophages Impairs Lipopolysaccharide-Induced Activation of AKT2, ATP Citrate Lyase, Acetyl-Coenzyme A Production, and Inflammatory Gene H3K27 Acetylation.

The accumulation of lipid and the formation of macrophage foam cells is a hallmark of atherosclerosis, a chronic inflammatory disease. To better understand the role of macrophage lipid accumulation in inflammation during atherogenesis, we studied early molecular events that follow the accumulation of oxidized low-density lipoprotein (oxLDL) in cultured mouse macrophages. We previously showed that oxLDL accumulation downregulates the inflammatory response in conjunction with downregulation of late-phase glycolysis. In this study, we show that within hours after LPS stimulation, macrophages with accumulated oxLDL maintain early-phase glycolysis but selectively downregulate activation of AKT2, one of three AKT isoforms. The inhibition of AKT2 activation reduced LPS-induced ATP citrate lyase activation, acetyl-CoA production, and acetylation of histone 3 lysine 27 (H3K27ac) in certain inflammatory gene promoters. In contrast to oxLDL, multiple early LPS-induced signaling pathways were inhibited in macrophages with accumulated cholesterol, including TBK1, AKT1, AKT2, MAPK, and NF-κB, and early-phase glycolysis. The selective inhibition of LPS-induced AKT2 activation was dependent on the generation of mitochondrial oxygen radicals during the accumulation of oxLDL in macrophages prior to LPS stimulation. This is consistent with increased oxidative phosphorylation, fatty acid synthesis, and oxidation pathways found by comparative transcriptomic analyses of oxLDL-loaded versus control macrophages. Our study shows a functional connection between oxLDL accumulation, inactivation of AKT2, and the inhibition of certain inflammatory genes through epigenetic changes that occur soon after LPS stimulation, independent of early-phase glycolysis.