Sharon J. Hyduk

BACKGROUND: Tissue-resident macrophages (T-RMøs) accumulate at sites predisposed to atherosclerosis in the healthy aortic intima. T-RMøs require local cues for their maintenance, but the niche signals governing their persistence in arterial tissue remain poorly defined. Here, we identify distinct, spatially compartmentalized sources of CSF1 (colony-stimulating factor 1) that regulate T-RMø populations in the healthy aorta and during atherosclerosis, a chronic inflammatory disease. METHODS: Conditional Csf1 gene deletion (global, endothelial, arterial endothelial, and mesenchymal cell) or systemic antibody blockade was performed in normal or hypercholesterolemic Ldlr ^–/– mice. Cx3cr1 lineage tracing of T-RMøs initiated before hypercholesterolemia distinguished these cells from monocyte-derived macrophages in atherosclerotic lesions. Bone marrow transplantation enabled lineage tracing of T-RMøs and Csf1 deletion in endothelial cells. Ccr2 lineage tracing directly assessed monocyte-derived macrophages. A novel en face immunoconfocal microscopy approach accurately enumerated macrophages in aortic lesions. RESULTS: Conditional Csf1 deletion revealed that arterial endothelial cell-derived CSF1 is essential for T-RMø steady-state maintenance. In contrast, adventitial macrophages rely on CSF1 produced by PDGFRβ (platelet-derived growth factor-beta)-expressing mesenchymal, but not endothelial, cells. CSF1 primarily supports intimal macrophage proliferation and retention rather than survival. Postnatal parabiosis revealed only occasional clusters of parabiont blood monocyte-derived macrophages in the normal intima. During hypercholesterolemia-initiated atherogenesis, the abundance of T-RMøs increases then gradually declines, while an inflammatory response consisting of monocyte recruitment progressively expands the total number of lesion macrophages. Endothelial, but not mesenchymal, CSF1 is required for optimal macrophage accumulation in 4-week foam cell lesions. Lineage tracing showed that CSF1, specifically endothelial-derived CSF1, is required for the maintenance of T-RMøs, but not monocyte-derived macrophages, in atherosclerotic lesions. CONCLUSIONS: Our study uncovers a unique role of local endothelial cell CSF1 in maintaining the intimal, but not adventitial, T-RMø niche during homeostasis and early stages of atherosclerosis and reveals that maintenance of monocyte-derived macrophages in lesions is distinct.

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.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow–derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl−/− Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2–related factor 2 (Nrf2)–mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α– to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our in vitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.