Limited proliferation capacity of aortic intima resident macrophages requires monocyte recruitment for atherosclerotic plaque progression

Abstract

Early atherosclerosis depends upon responses by immune cells resident in the intimal aortic wall. Specifically, the healthy intima is thought to be populated by vascular dendritic cells (DCs) that, during hypercholesterolemia, initiate atherosclerosis by being the first to accumulate cholesterol. Whether these cells remain key players in later stages of disease is unknown. Using murine lineage-tracing models and gene expression profiling, we reveal that myeloid cells present in the intima of the aortic arch are not DCs but instead specialized aortic intima resident macrophages (Mac^AIR) that depend upon colony-stimulating factor 1 and are sustained by local proliferation. Although Mac^AIR comprise the earliest foam cells in plaques, their proliferation during plaque progression is limited. After months of hypercholesterolemia, their presence in plaques is overtaken by recruited monocytes, which induce Mac^AIR-defining genes. These data redefine the lineage of intimal phagocytes and suggest that proliferation is insufficient to sustain generations of macrophages during plaque progression.

Extended Data Fig. 1

Profiling aorta macrophage populations by bulk RNA-seq Bulk sorted Mac^AIR from C57BL/6 mice and adventitia, intima non-foamy, and intima foamy macrophages from 26-week HFD fed ApoE^−/− mice were profiled for gene expression by RNA-seq. Gene groupings for (A) macrophage and DC genes, (B) foamy and adventitia macrophage genes, (C) Mac^AIR enriched genes, and (D) genes associated with chemokine signaling in myeloid cells. Data are mean expression values derived from pooled macrophages from two biological replicates for Mac^AIR derived from 10-pooled aorta each, and three biological replicates each for ApoE^−/− samples from 6-pooled aorta each.

Extended Data Fig. 2

Mac^AIR are detected across multiple scRNA-seq approaches (A) scRNA-seq data from C57BL/6 total CD45⁺ cells was integrated with two published studies where CD45⁺ aorta cells from chow-fed Ldlr^−/− mice and atherosclerosis regression samples, identifying 10 unique clusters. (B) Top 12 enriched Mac^AIR genes were used to define Mac^AIR cluster as cluster 4, presented as relative expression. (C) Mac^AIR cells were detectable in all three datasets with a relative abundance between 2.00-9.28%. (D) cDC1 genes (Zbtb46, Flt3, Itgae, Xcr1, Snx22, Mycl, and Rab43) were interrogated against the integrated dataset, defining a unique DC population. (E) Itgae and Xcr1 gene expression plotted on integrated t-SNE cluster map.

Extended Data Fig. 3

Differential gene expression between clusters of integrated dataset of C57BL/6, Ldlr^−/−, and regression scRNA-seq experiments Top differentially expressed genes are listed with expression level across the 10 unique groups identified by unsupervised clustering of the integrated data of C57BL/6, chow fed Ldlr^−/−, and regression scRNA-seq datasets.

Extended Data Fig. 4

Profiling myeloid cells in the aorta Spleen and aorta samples from (A) SNX22^gfp and (B) L-Myc^gfp reporter mice were assessed for presence of cDC1 in respective tissues, GFP (green) and Dapi (blue). In panel B aorta, sample was co-stained with CD45 (red) and MHC II (white) antibody. Aorta from XCR1-venus reporter mice were isolated and stained for MHC II (red) or CD45 (white) expression by antibody labeling, then imaged in whole-mount for presence of cDC1 in the (C) aortic arch intima, (D) adventitia, and (E) aortic valve. C57Bl/6 (WT) aorta was isolated and stained for CD103 (green), MHC II (red), and CD45 (white) by antibody labeling and imaged by confocal microscopy for cDC1 presence in the (F) intima of the aortic arch or (G) the adventitia of the aorta. Embryonic labeling was performed using CD115creER Rosa26-mTmG mice. Aorta were collected from adult animals and assessed for GFP (green) and Tomato (red) expression in mice labeled at (H) E11.5, and co-stained with CD45 (white) in mice labeled at (I) E14.5. Data are representative of (A) n=3, (B) n=3, (C-E) n=6 in two experiments, (F-G) n=6 in two experiments, (H) n=3, and (I) n=5 in two experiments.

Extended Data Fig. 5

Atherosclerotic plaque development in the aorta of hypercholesterolemic mice following acute depletion of Mac^AIR (A) Following the schematic, CX3CR1^creER CD115-stop-DTR mice were used to conditionally express DTR on Mac^AIR cells by gavage with tamoxifen, waiting three weeks to allow circulating cells to repopulation from DTR-negative progenitors. Mac^AIR cells were depleted by i.p. injection of diphtheria toxin and hypercholesterolemia induced by i.v. injection of AAV-PCSK9. Mice were started on HFD the following day. Mice were sacrificed and assessed for plaque area in the aortic arch by oil red o (ORO) staining. (B) Representative images of ORO staining and en face imaging. (C) Quantification of plaque area on the aortic arch. Data are from n=7, Cntl and n=4 for DTR-group and are the result of a single experiment, error bars represent SEM.

Extended Data Fig. 6

Fate-mapping Mac^AIR during atherosclerosis progression in bone marrow transplant model Recipient Ldlr^−/− mice were lethally irradiated and reconstituted with CX3CR1^creER Rosa26-lsl-Tomato bone marrow. Mice were rested for 8-weeks for full hematopoietic reconstitution, then treated with tamoxifen by gavage to induce Tomato expression in CX3CR1-expressing cells, including Mac^AIR. (A) Three weeks after tamoxifen labeling, Mac^AIR remained Tomato⁺ (red) positive. Mice were then fed HFD and assessed for plaque development at (B) 10 days and (C) 28 days. Mac^AIR can be observed by Tomato-expression, whereas recruited cells were stained with CD68 (green) antibody. Samples are all en face whole mount confocal images of the aortic arch. Images are representative of n=3 for each time point.

Extended Data Fig. 7

Csf2rb^−/− Ldlr^−/− mice fed HFD for 12 days Csf2rb^+/− Ldlr^−/− or Csf2rb^−/− Ldlr^−/− mice were fed HFD for 12 days to induce atherosclerotic lesions in the aortic arch. Aorta were stained for MHC II (red) and imaged by tile scanning whole mount confocal microscopy for macrophage burden and morphologic changes associated with foamy cell development. Three representative images (n=7 for Csf2rb^+/− Ldlr^−/−) or (n=4 Csf2rb^−/− Ldlr^−/−) were selected from a single experiment.

Extended Data Fig. 8

Differential gene expression analysis of scRNA-seq of CD45⁺ cells from Ldlr^−/− aorta after 21-days HFD feeding The top 10 enriched genes for each cluster are reported for scRNA-seq analysis of total CD45⁺ cells from Ldlr^−/− mice fed HFD for 21 days.

Extended Data Fig. 9

Integrated scRNA-seq analysis across atherosclerosis progression (A) scRNA-seq data from sorted CD45⁺ aorta cells isolated from C57Bl/6, 21-day HFD Ldlr^−/−, and 12-week HFD Ldlr^−/− mice was integrated and clustered into 16 populations. (B) All clusters were represented across unique time points. (C) Mac^AIR geneset (Mmp12, Il1b, Lgals3, Nes, Rgs1, Acp5, Asb2, Itgax, Cadm1, Gngt2, Cd9, Bcl2a1a) expression analysis of integrated cluster map and across time points. (D) Foamy macrophage geneset (Fabp4, Ctsl, Atp6v0d2, Gpnmb, Fabp5, Htra1, Epb41l3, Pld3) expression analysis of integrated cluster map and across time points. (E) Human foamy macrophage geneset (Gpx1, Pfdn5, Tpt1, Eef1a1, Ctb, B2m, Tmsb4x, Fth1, Ftl, Tmsb10, Cd63, Lgals1, Fcer1g, Npc2, Serf2, Ybx1, Psap, Apoc1, Apoe, Cstb, Ctsb, Vim, RnaseI, Fabp5, Plin2, Ccl2) expression analysis in the murine integrated scRNA-seq cluster map, suggesting shared gene expression programs between human and murine foamy macrophage subsets.

Extended Data Fig. 10

Differential gene expression from integrated scRNA-seq analysis across atherosclerosis time course scRNA-seq data from sorted CD45⁺ aorta cells isolated from C57Bl/6, 21-day HFD Ldlr^−/−, and 12-week HFD Ldlr^−/− mice were integrated and clustered into 16 populations. Top 10 differentially expressed genes are shown for each cluster.

Figure 1.

Profiling aorta intima resident macrophages (Mac^AIR) in the steady state. (A) scRNA-seq analysis of CD45⁺ cells isolated from 8-week old C57BL/6 mouse aorta. tSNE dimensionality reduction and unsupervised clustering were performed using Seurat package identifying 10 unique clusters. (B) Expression of principal myeloid markers (Fcgr1, Itgax, Mrc1, Csf1r, Cx3cr1 and Lyve1) shown on tSNE plot of C57BL/6 scRNA-seq data. (C) Heatmap displaying averaged gene expression profiles per cluster of unique gene signatures of C57BL/6 scRNA-seq data. (D) Violin plot of Nlrp3 and Il1b gene expression in C57BL/6 scRNA-seq data. (E) CD11c-YFP reporter animals were stained for MHC II antigen and Dapi (nuclei) then imaged for presence of myeloid cells in the intima of the aortic arch; CD11c (green), Dapi (blue) and MHC II (white). (F) CX3CR1^gfp/+ (green) reporter mouse aorta was immunostained for CD64 (red) and imaged by confocal microscopy. (G) LysM^cre Rosa26-lsl-Tomato mice were assessed for Tomato protein (magenta) expression by en face whole mount confocal microscopy, autofluorescence (cyan). (H) CD115-creER Rosa26-lsl-Tomato CX3CR1^gfp/+ mice were treated with tamoxifen diet for 3 weeks, then aorta were imaged for presence of Tomato (red) and GFP (green) in the intima of the aortic arch. (I) CX3CR1^gfp/+ (green) reporter mouse aorta was stained for MHC II (red) and imaged by confocal microscopy. (J-K) Zbtb46^gfp bone marrow was transplanted into irradiated C57BL/6 recipients, reconstituted mice were assayed for GFP⁺ (green) cells in spleen (J) and Aorta (K). (L) Flt3^−/− or (M) Flt3L^−/− aorta stained for CD45 (Magenta) and MHC II (Cyan) and imaged by confocal microscopy. (N) Violin plot of Flt3 expression in C57BL/6 scRNA-seq data. scRNA-seq data (A-D, N) were derived by pooling ten C57BL/6 mouse aorta for digestion and FACS sorting, then sequencing. Heat map (C) was derived by pooling mean expression data from defined clusters. Imaging data (E-I) are representative images of n=5, (J-M) are representative of n=3.

Figure 2.

Aorta intima resident macrophages (Mac^AIR) are Csf1-dependent. Aorta from (A) op/op (Csf1-mutant) and (B) IL-34^−/− mice were stained for CD68 (red) and CD45 (green) and imaged by confocal microscopy with Dapi (blue). Aorta from (C) control or (D) tamoxifen treated CD115creER Csf1r^f/f mice were stained for CD45 (white) and MHC II (red) in and imaged by confocal microscopy. (E) Quantification of Mac^AIR numbers in the aortic arch of CX3CR1^gfp/+ (Cntl), CCR2^−/−, CCR7^−/−, Csf2rb^−/−, and op/op mice. (F) CD11c-YFP reporter mice were irradiated, then reconstituted with Tomato-expressing bone marrow. Aorta were isolated from mice at 0, 1, and 4 weeks, then imaged for presence of YFP⁺ (green) and Tomato⁺ (red) cells by confocal microscope, Dapi (blue) identifies nuclei. (G-H) Competitive bone marrow chimera approach was performed with 50/50 ratio of CCR2^gfp/gfp:LysM^cre Rosa26-lsl-Tomato donor. After 8 weeks, (G) blood monocytes were >95% derived from GFP-negative bone marrow, confirming dependence on CCR2. (H) Aorta from bone marrow chimera mice reconstituted with 50/50 CCR2^gfp/gfp:LysM^cre Rosa26-stop-tomato donor bone marrow were stained for CD45 (blue) and imaged for GFP (green) and Tomato (red). Imaging data (A-D) are representative of n=3, (E) is n=2-4 mice per group as depicted in graph, (F) was performed with n=5 mice from two independent experiments, (G-H) was performed with n=3 mice from two independent experiments. Statistical analysis was performed by one-way ANOVA with multiple comparisons (CX3R1^gfp/+ (Cntl) vs Csf2rb^−/−, *P=0.0181), error bars are presented SEM.

Figure 3.

Aorta intima resident macrophages (Mac^AIR) develop from bone marrow progenitors and seed the aorta at birth. (A) Embryonic labeling at E8.5 was performed using CD115creER Rosa26-mTmG mice. Aorta were collected from adult animals and assessed for GFP (green) and Tomato (red) expression in Mac^AIR (left) and adventitia (right) by confocal microscopy (Dapi, blue). (B) Embryonic labeling of CX3CR1^creER/+ Rosa26-lsl-Tomato mice was performed at E18.5. Immune cells were identified in the aortic arch by CD45 (white) staining, along with Tomato (red) expression in the aortic arch (left) and adventitia (right) by confocal microscopy. (C) Aorta from Flt3cre Rosa26-mTmG mice were stained with CD45 antibody (white) and imaged for GFP (green) and Tomato (red) expression in the aortic arch. (D) CCR2^gfp/+ postnatal day 1 (P1) pups were stained for CD45 (white) and MHC II (red), and were imaged by en face confocal microscopy showing intima (left) and adventitia (right). (E) CCR2^gfp/+ postnatal day 4 (P4) pups were stained for CD45 (white) and MHC II (red), then imaged by confocal microscopy, showing intima (left) and adventitia (right) macrophages expressing GFP (green), CD45, and MHC II. Images are representative of (A) n=8 from three independent experiments (B) n=3 from two independent experiments, (C) n=5 from two independent experiments, (D) n=4 from two independent experiments (E) n=5 animals from two independent experiments.

Figure 4.

Aorta intima resident macrophages (Mac^AIR) maintain independent of circulating progenitors and proliferate within the tissue. (A) Parabiosis was performed to surgically link the microvasculature of CX3CR1^gfp/+ reporter mice with C57BL/6 for 2 weeks, then sacrificed and assessed for transfer of cells between animals. Whole-mount immunostaining of MHC II (red) shows no cross-over of GFP⁺ cells between parabionts. (B) Blood analysis of 2-week parabiotic pairs shows donor/host ratios for monocytes. (C) Parabiosis was performed surgically linking the microvasculature of (cre-negative) Rosa26-mTmG reporter mice (all cells Tomato⁺) with C57BL/6 for 5 months, then sacrificed and assessed for cellular turn over. Whole mount confocal imaging of MHCII (green) identified resident macrophages and rare Tomato⁺ (red) cells were observed. (D) Aorta from C57BL/6 mice were stained for Ki67 (white) and MHC II (red) expression, imaged by confocal microscopy, and counted for the frequency of Ki67⁺ cells within the Mac^AIR population in the intima. (E) CX3CR1^creER Rosa26-lsl-Confetti mice were pulsed with tamoxifen to induce recombination of stochastic fluorescent reporter alleles. Mice were left on chow diet for 4 weeks, then assessed by confocal imaging for clustering of cells of similar color to represent cellular proliferation within the tissue. Data are representative of (A) n=9 from three independent experiments, (B) representative of n=6 pairs from three independent experiments, (C) n=5 mice from two independent experiments, (D) n=4 mice across two independent experiments, and (E) n=3 from independent experiments. Error bars on graphs are presented as SEM.

Figure 5.

Aorta intima resident macrophages (Mac^AIR) promote monocyte recruitment in early atherosclerotic lesions. (A) C57BL/6 (Cntl) or CD11c-DTR (DTR+) mice were assessed for Mac^AIR depletion in the aortic arch following diphtheria toxin (Dtx) injection, staining for CD45 (white). (B) C57BL/6 (Cntl) or CD11c-DTR bone marrow was adoptively transferred into irradiated Ldlr^−/− mice, followed by 8-week reconstitution. Mice were then injected with diphtheria toxin on day 0 and fed HFD for 5 days, then aorta were collected and stained for oil red o (neutral lipid) in the aortic arch and quantified. (C) CX3CR1^creER/+ x CD115-stop-DTR mice were treated with tamoxifen to conditionally express DTR on Mac^AIR. Mice were rested for three weeks, then given Dtx to determine depletion and recovery kinetics of Mac^AIR; Control (day 0, no Dtx), 36 hr post Dtx treatment, 5 day post Dtx treatment, and 7 days post treatment. Samples are co-stained for CD45 (white) and MHCII (red). (D-E) CX3CR1^creER/+ x CD115-stop-DTR or control mice were treated with tamoxifen, rested for three weeks, then given Dtx to determine depletion of circulating myeloid cells at 24 hours. (F) CX3CR1^creER/+ x CD115-stop-DTR or control mice were treated with tamoxifen, then rested on chow diet. Mice then received i.v. injection of PCSK9-AAV virus to promote hypercholesterolemia and the following day mice were given Dtx and placed on HFD. After 5 days, aorta were isolated and assessed for CD45⁺ cells (red) and co-stained with bodipy (neutral lipid, green) and VE-cadherin (blue). Data are representative of (A-B) represent 2 independent experiments with 5 animals per group. (C-E) representative images from three independent experiments. (F) Representative images from 2 independent experiments with 5 animals per group. Statistical analysis was performed using nonparametric T-test (*p=0.0079), and error bars represent SEM.

Figure 6.

Fate-mapping aorta intima resident macrophages in the progression of atherosclerosis. (A) CX3CR1^creER Rosa26-lsl-Tomato Ldlr^−/− mice were treated with tamoxifen by oral gavage three times over a week, then sacrificed and imaged by en face whole mount confocal microscopy. (B) Blood monocyte labeling efficiency following tamoxifen treatment of CX3CR1^creER Rosa26-lsl-Tomato Ldlr^−/− mice, showing Tomato expression as assessed by flow cytometry for classical monocytes (CD115⁺ CD11b⁺ Ly6G⁻ Ly6C⁺) at day 0 (left panel). Labeling kinetics for classical and non-classical monocytes in the blood tracked for 14 days after tamoxifen treatment (right panel). (C) Following tamoxifen treatment, CX3CR1^creER Rosa26-lsl-tomato Ldlr^−/− mice were rested on chow diet for three weeks, then assayed by whole mount en face confocal microscopy for Tomato (red) labeling of Mac^AIR co-stained with CD45 (white). (D) Serum cholesterol levels of CX3CR1^creER Rosa26-lsl-tomato Ldlr^−/− mice following labeling regiment and started on HFD feeding.(E-I) Following tamoxifen treatment, CX3CR1^creER Rosa26-lsl-tomato Ldlr^−/− mice were rested on chow diet for three weeks, then fed HFD for 10-days (E-F), 14 days (G), 28 days (H), and 84 days (I). Mac^AIR were tracked based on expression of Tomato and recruited cells were stained by antibody labeling, as indicated on the micrographs. (J) Following Mac^AIR labeling, the frequency of Tomato+ cells was calculated across the course of disease, from 0-84 days of HFD feeding. Data are derived from (A) n=9 from three independent experiments, (B) n=6 from two independent experiments, (C) n=6 from two independent experiments, (D) n=13 from a single experiment, (E-J) n=26 across all time points from three independent experiments. Error bars are presented as SEM.

Figure 7.

Aorta intima microenvironment promotes shared gene programs between resident macrophages in the steady state and disease. (A) scRNA-seq analysis of CD45⁺ cells isolated from atherosclerotic mice fed HFD for 21-days, tSNE dimensionality reduction and unsupervised clustering were performed to identify 11 unique clusters. (B) Expression of Mmp12, Fabp5, Lyve1 and Mafb is shown by tSNE plot with color corresponding to expression levels. (C) Mac^AIR, foamy macrophages, and monocytes are identified by previously defined gene sets, with all populations being restricted to the cluster 4 population. With a single-foamy macrophage population present, suggesting convergence from origins corresponding to MMP12⁺ Mac^AIR or Ly6C⁺ monocytes (marked with red arrow). (D) tSNE plot of scRNA-seq data derived from total CD45⁺ cells isolated from 12-week HFD fed Ldlr^−/− mice (Kim et. al. 2018), identifying 12-unique clusters. (E) The top 100 foamy macrophage genes were identified by differential expression of cluster 4 vs all other macrophage clusters (0, 1, 2, 3, 5, 7, 8) in scRNA-seq dataset of 12-week HFD fed Ldlr^−/− mice. Averaged expression of these 100 genes is shown in original dataset (left panel) and in C57Bl/6 scRNA-seq dataset (right panel) localizing to the Mac^AIR population (cluster 8). (F) Differential expression comparison between cluster 8 enrichment in C57BL/6 compared to all other macrophages on Y-axis and cluster 4 (foamy macrophage) compared to all other macrophages in Ldlr^−/− 12-week HFD atherosclerotic aorta on X-axis. Grey dots represent non-significant genes, yellow represent genes enriched only in foamy macrophages, blue represents genes enriched in B6 cluster 8, and red represents genes that are significantly modulated in both foamy cells and also in cluster 8. scRNA-seq data (A-F) were derived by pooling aorta from 10 mice for each group, followed by digestion and FACS sorting, then sequencing.