Scavenger Receptor CD36 Directs Nonclassical Monocyte Patrolling Along the Endothelium During Early Atherogenesis

Abstract

Objective: Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature.

Approach and results: To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE^-/- (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet-fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36^-/- mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein.

Conclusions: Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.

Keywords: atherosclerosis; humans; mice; monocytes; phosphorylation.

Figure 1

Nonclassical monocyte patrolling activity increases in large peripheral blood vessels of mice fed a Western Diet. Age and sex-matched ApoE^−/− and B6 mice were fed a WD for the times indicated and assessed for patrolling activity and monocyte frequency A, The number of CX₃CR1^high patrolling monocytes per surface area of blood vessel was determined for each recording (n=3 videos per mouse). Each point represents the total number of patrolling monocytes per total surface area of blood vessel for each mouse. n=6-11 mice per group. A Kruskal-Wallis test with Dunn’s multiple comparison correction was used to analyze WD and Regression groups using Chow as the control group. B, Representative images of patrolling monocytes (highlighted by white arrows) in large blood vessels of ApoE^−/− mice: CX₃CR1 (Ly6C⁻ NCM), CCR2 (Ly6C⁺ CM) and injected with anti-CD31 (endothelium). scale bars: 30 μM C, Patrolling speeds for NCM from ApoE^−/− mice fed a Western diet (28 days) or chow. D, NCM numbers in blood were obtained by flow cytometry from chow and WD-fed mice. n=4-5 mice per group. Error bars represent mean ± SEM for all graphs. *P < 0.05, **P < 0.01, ****P < 0.0001, ns indicates not significant.

Figure 2

Nonclassical monocytes from WD-fed mice are atheroprotective. A, Differential gene expression of RNA transcripts between monocyte subsets of WD or chow-fed mice. Ly6C⁺ and Ly6C⁻ monocyte subsets were sorted from chow or WD-fed mice. Genes of interest related to lipid metabolism, cell migration, and inflammation resolution that were uniquely upregulated in nonclassical monocytes from WD-fed mice are listed. n=30 mice (3 groups of 10 mice pooled). Statistical significance of upregulated genes was determined by False Discovery Rate (FDR) correction (Benjamini-Hochburg) with filtering of variance outliers by tagwise dispersion (>99.9%), P>0.001. B, Comparison of atherosclerotic plaque formation in B6 or E2 mice by en face Oil Red O (ORO) staining of aortas from mice fed a 15-week Western diet. Quantification of plaque area as a percentage of total aortic area. n=8-9 mice per group. C, Representative images of aortic root ORO staining. D, Bodipy staining of neutral lipids in blood monocyte subsets from chow and WD-fed mice by flow cytometry. n=4 mice per group. E, Filipin III staining of free cholesterol in blood monocyte subsets from chow-fed mice. n=5 mice. F, Bodipy staining of neutral lipids of blood monocyte subsets from healthy human donors by FACS. n=3 individuals. Error bars represent means ± SEM. **P < 0.01, ***P < 0.001, ns indicates not significant.

Figure 3

Increased F-actin formation in nonclassical monocytes from mice fed a Western diet. A, Representative images of Ly6C⁺ (classical) and Ly6C⁻ (nonclassical) monocytes stained for F-actin formation using phalloidin-AF488 by confocal microscopy. scale bar = 2μM B, Mean intensity of F-actin staining (phalloidin-AF488) of monocyte subsets sorted from chow or WD-fed mice by confocal microscopy. n=10 cells per group. Error bars represent mean ± SEM for all graphs. *P < 0.05, ****P < 0.0001

Figure 4

OxLDL is preferentially taken up by nonclassical monocytes and induces NCM patrolling. A, Chow-fed ApoE^−/− mice were injected with AF633-labeled OxLDL, LDL, mmLDL, or BHT-LDL. 15 minutes after injection, mice were analyzed for LDL uptake by flow cytometry (n=1 mouse per injection). Mice were anesthetized and bled prior to injection to obtain a baseline (Pre-injection). B, Comparison of AF633-labeled OxLDL uptake in Ly6C⁺ (CM) and Ly6C⁻ (NCM) blood monocyte subsets ex vivo by flow cytometry. n=3 mice. C, Representative images of F-actin staining in Ly6C⁻ monocytes incubated with or without OxLDL. Scale bar: 2 μM D, Quantification of F-actin mean fluorescent intensity of Ly6C⁻ monocytes with or without DiI-OxLDL. E, CX₃CR1^gfp/+ApoE^−/− mice were imaged to obtain a baseline of patrolling frequency in a large blood vessel of a mouse leg (left column). PBS (100 μL) or OxLDL (100 μg) solutions were injected during imaging after obtaining a baseline. Timelapse images were obtained post-injection from the same pre-injected areas (right column). White arrows highlight patrolling NCM. Quantification of patrolling frequency before and after injection of PBS (upper graph) or OxLDL (lower graph) were graphed for each condition. n=3 independent experiments, one representative experiment shown. scale bar: 30 μM F, Representative figure of CX₃CR1^gfp/+ApoE^−/− mice injected with DiI-OxLDL during imaging to observe OxLDL uptake by nonclassical monocytes while patrolling. n=2 independent experiments. scale bar: 30 μM. Error bars represent mean ± SEM for all graphs. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

CD36 is critical for nonclassical monocytes to patrol the vasculature in WD-fed mice. A, Mice were fed a WD for 28 days and imaged for patrolling activity. CD115 (shown in green) and FcɣRIV antibodies were used to label nonclassical monocytes in vivo, blood vessels were labeled with CD31 antibody. Ratios of patrolling monocytes per surface area of each blood vessel recording (n=3 per mouse) were summed and reported per mouse. n=5-11 mice per group. B, Representative images of patrolling NCM (highlighted with white arrows) in mouse femoral vasculature obtained by intravital confocal microscopy. scale bar: 30 μM. C, Mean speeds of NCM for each imaging experiment shown in graph A. D, Frequencies of Ly6C⁻ monocytes from retro-orbital bleeds of B6 or CD36^−/− mice measured by FACS. n=3-4 mice per group. E, Blood monocytes were incubated ex vivo with AF633-labeled OxLDL and analyzed for uptake by flow cytometry. n=3 mice per group. F, WT CX₃CR1^gfp/+ bone marrow was transplanted into either WT recipients or CD36^−/− recipients. Graph represents ratios of patrolling NCM per surface area of each blood vessel recording (n=3 per mouse) that were summed and reported per mouse. n=5-6 mice per group. G, Representative histogram of CD36 expression on CD16⁺ monocytes from healthy human donors determined by flow cytometry. n=4 donors. H, Healthy human blood samples were incubated for 20 minutes at 37°C with AF633-labeled OxLDL, LDL, mmLDL, or BHT-LDL (and lineage antibodies) and measured by FACS for LDL uptake. Representative histogram of n=3 donors. Error bars represent mean ± SEM for all graphs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates not significant

Figure 6

Src family kinases mediates signaling downstream of CD36 through Dap12. A, DAP12^−/− and B6 bone marrow chimeras were fed a WD and imaged by intravital microscopy for patrolling. Graph represents ratios of patrolling monocytes per surface area of each blood vessel recording (n=3 per mouse) that were summed and reported per mouse. n=9-10 mice per group. B, Mean speed of patrolling monocytes from mice fed a WD for 28 days. n=10 mice per group C, Representative western blot of phosphorylated Src family kinases (SFK) at Y416 with β-actin loading control. Blood monocytes were pooled and sorted from B6 mice fed chow (lane 1), WD (lane 2), CD36^−/− WD-fed mice (lane 3), or monocytes from WD-fed mice incubated with PP1 (lane 4). Blots were stripped and reprobed for total protein for Src protein. D, CX₃CR1^gfp/+CCR2^rfp/+ApoE^−/− mice on WD were imaged before (baseline) and after (post injection, p.i) injections of DMSO (vehicle, DMSO+PBS), Piceatannol (Syk inhibitor), or PP1 (SFK inhibitor). Each mouse was injected with 1 reagent and imaged intravitally to obtain patrolling speeds and behavior (arrest or detachment). Patrolling speed heat map represents each mouse injected with either DMSO or PP1 and plotted as each cell in the field of view in each mouse over time. Quantification of patrolling speeds were obtained with Imaris software and plotted in Excel. Bar graph shows what percent of NCM were arrested or detached after at 20 minutes post-injection listed on X-axis. n=2-3 mice per group. Error bars represent mean ± SEM for all graphs. *P < 0.05, **P < 0.01