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 (MacAIR) that depend upon colony-stimulating factor 1 and are sustained by local proliferation. Although MacAIR 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 MacAIR-defining genes. These data redefine the lineage of intimal phagocytes and suggest that proliferation is insufficient to sustain generations of macrophages during plaque progression.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Gene expression data (bulk RNA-seq or scRNA-seq) have been uploaded to the Gene Expression Omnibus (GEO) repository for public availability under accession codes GSE116271, GSE116239, GSE154817 and GSE154921. All other data that support the findings of this study are available from the corresponding author upon reasonable request.
References
Geovanini, G. R. & Libby, P. Atherosclerosis and inflammation: overview and updates. Clin. Sci. 132, 1243–1252 (2018).
Benjamin, E. J. et al. Heart disease and stroke statistics—2019 update: a report from the American Heart Association. Circulation 139, e56–e528 (2019).
Libby, P., Lichtman, A. H. & Hansson, G. K. Immune effector mechanisms implicated in atherosclerosis: from mice to humans. Immunity 38, 1092–1104 (2013).
Williams, J. W., Huang, L. H. & Randolph, G. J. Cytokine circuits in cardiovascular disease. Immunity 50, 941–954 (2019).
Ridker, P. M. et al. Anti-inflammatory therapy with canakinumab for atherosclerotic disease. N. Engl. J. Med. 377, 1119–1131 (2017).
Randolph, G. J. Mechanisms that regulate macrophage burden in atherosclerosis. Circ. Res. 114, 1757–1771 (2014).
Dick, S. A., Zaman, R. & Epelman, S. Using high-dimensional approaches to probe monocytes and macrophages in cardiovascular disease. Front. Immunol. 10, 2146 (2019).
Jongstra-Bilen, J. et al. Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis. J. Exp. Med. 203, 2073–2083 (2006).
Choi, J. H. et al. Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves. J. Exp. Med. 206, 497–505 (2009).
Meredith, M. M. et al. Expression of the zinc finger transcription factor zDC (Zbtb46, Btbd4) defines the classical dendritic cell lineage. J. Exp. Med. 209, 1153–1165 (2012).
Satpathy, A. T. et al. Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages. J. Exp. Med. 209, 1135–1152 (2012).
Zhu, S. N., Chen, M., Jongstra-Bilen, J. & Cybulsky, M. I. GM-CSF regulates intimal cell proliferation in nascent atherosclerotic lesions. J. Exp. Med. 206, 2141–2149 (2009).
Kim, K.-W. et al. MHC II+ resident peritoneal and pleural macrophages rely on IRF4 for development from circulating monocytes. J. Exp. Med. 213, 1951–1959 (2016).
Choi, J. H. et al. Flt3 signaling-dependent dendritic cells protect against atherosclerosis. Immunity 35, 819–831 (2011).
Roufaiel, M. et al. CCL19-CCR7–dependent reverse transendothelial migration of myeloid cells clears Chlamydia muridarum from the arterial intima. Nat. Immunol. 17, 1263–1272 (2016).
Paulson, K. E. et al. Resident intimal dendritic cells accumulate lipid and contribute to the initiation of atherosclerosis. Circ. Res. 106, 383–390 (2010).
Robbins, C. S. et al. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nat. Med. 19, 1166–1172 (2013).
Gautier, E. L. et al. Gene expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat. Immunol. 13, 1118–1128 (2012).
Kim, K. et al. Transcriptome analysis reveals nonfoamy rather than foamy plaque macrophages are proinflammatory in atherosclerotic murine models. Circ. Res. 123, 1127–1142 (2018).
Ensan, S. et al. Self-renewing resident arterial macrophages arise from embryonic CX3CR1+ precursors and circulating monocytes immediately after birth. Nat. Immunol. 17, 159–168 (2016).
Lim, H. Y. et al. Hyaluronan receptor LYVE-1-expressing macrophages maintain arterial tone through hyaluronan-mediated regulation of smooth muscle cell collagen. Immunity 49, 326–341 (2018).
Cochain, C. et al. Single-cell RNA-seq reveals the transcriptional landscape and heterogeneity of aortic macrophages in murine atherosclerosis. Circ. Res. 122, 1661–1674 (2018).
Lin, J. D. et al. Single-cell analysis of fate-mapped macrophages reveals heterogeneity, including stem-like properties, during atherosclerosis progression and regression. JCI Insight 4, e124574 (2019).
Wumesh, K. C. et al. L-Myc expression by dendritic cells is required for optimal T-cell priming. Nature 507, 243–247 (2014).
Perdiguero, E. G. & Geissmann, F. The development and maintenance of resident macrophages. Nat. Immunol. 17, 2–8 (2016).
Wang, Y. et al. IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia. Nat. Immunol. 13, 753–760 (2012).
Williams, J. W., Giannarelli, C., Rahman, A., Randolph, G. J. & Kovacic, J. C. Macrophage biology, classification and phenotype in cardiovascular disease: JACC macrophage in CVD series (part 1). J. Am. Coll. Cardiol. 72, 2166–2180 (2018).
Serbina, N. V. & Pamer, E. G. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2. Nat. Immunol. 7, 311–317 (2006).
Lavin, Y. et al. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159, 1312–1326 (2014).
Zhu, Y. et al. Tissue-resident macrophages in pancreatic ductal adenocarcinoma originate from embryonic hematopoiesis and promote tumor progression. Immunity 47, 323–338 (2017).
Epelman, S. et al. Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation. Immunity 40, 91–104 (2014).
Hashimoto, D. et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity 38, 792–804 (2013).
Davies, L. C. et al. Distinct bone marrow-derived and tissue-resident macrophage lineages proliferate at key stages during inflammation. Nat. Commun. 4, 1886 (2013).
Scholzen, T. & Gerdes, J. The Ki-67 protein: from the known and the unknown. J. Cell. Physiol. 182, 311–322 (2000).
Williams, J. W. et al. Limited macrophage positional dynamics in progressing or regressing murine atherosclerotic plaques—brief report. Arterioscler. Thromb. Vasc. Biol. 38, 1702–1710 (2018).
Yona, S. et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 79–91 (2013).
Wang, M. et al. Interleukin-3/granulocyte macrophage colony-stimulating factor receptor promotes stem cell expansion, monocytosis and atheroma macrophage burden in mice with hematopoietic ApoE deficiency. Arterioscler. Thromb. Vasc. Biol. 34, 976–984 (2014).
Subramanian, M., Thorp, E. & Tabas, I. Identification of a non-growth factor role for GM-CSF in advanced atherosclerosis: promotion of macrophage apoptosis and plaque necrosis through IL-23 signaling. Circ. Res. 116, e13–e24 (2015).
Ingersoll, M. A. et al. Comparison of gene expression profiles between human and mouse monocyte subsets. Blood 115, e10–e19 (2010).
Fernandez, D. M. et al. Single-cell immune landscape of human atherosclerotic plaques. Nat. Med. 25, 1576–1588 (2019).
Jaitin, D. A. et al. Lipid-associated macrophages control metabolic homeostasis in a Trem2-dependent manner. Cell 178, 686–698 (2019).
Xiong, X. et al. Landscape of intercellular crosstalk in healthy and NASH liver revealed by single-cell secretome gene analysis. Mol. Cell 75, 644–660 (2019).
Ridker, P. M. et al. Low-dose methotrexate for the prevention of atherosclerotic events. N. Engl. J. Med. 380, 752–762 (2019).
Zernecke, A. et al. Meta-analysis of leukocyte diversity in atherosclerotic mouse aortas. Circ. Res. 127, 402–426 (2020).
Wiesmann, F. et al. Developmental changes of cardiac function and mass assessed with MRI in neonatal, juvenile and adult mice. Am. J. Physiol. Heart Circ. Physiol. 278, H652–H657 (2000).
Wagenseil, J. E. & Mecham, R. P. Vascular extracellular matrix and arterial mechanics. Physiol. Rev. 89, 957–989 (2009).
Mackarehtschian, K. et al. Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity 3, 147–161 (1995).
Benz, C., Martins, V. C., Radtke, F. & Bleul, C. C. The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development. J. Exp. Med. 205, 1187–1199 (2008).
Ohta, T. et al. Crucial roles of XCR1-expressing dendritic cells and the XCR1-XCL1 chemokine axis in intestinal immune homeostasis. Sci. Rep. 6, 23505 (2016).
Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902 (2019).
Finak, G. et al. MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol. 16, 278 (2015).
Hafemeister, C. & Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 20, 296 (2019).
Linderman, G. C., Rachh, M., Hoskins, J. G., Steinerberger, S. & Kluger, Y. Fast interpolation-based t-SNE for improved visualization of single-cell RNA-seq data. Nat. Methods 16, 243–245 (2019).
Williams, J. W. et al. Thermoneutrality but not UCP1 deficiency suppresses monocyte mobilization into blood. Circ. Res. 121, 662–676 (2017).
Acknowledgements
Flt3Cre mice were provided by D. Mann (Washington University School of Medicine; WUSM); Flt3−/−, Flt3l−/−, SNZ22GFP, L-MycGFP and Zbtb46GFP mice were provided by K. Murphy (WUSM); IL-34−/− and CCR2GFP mice were provided by M. Colonna (WUSM); op/op mice were provided by E. Unanue (WUSM); and Csf2rb−/− mice were provided B. Edelson (WUSM). We also thank M. Vail (University of Minnesota), the WUSM Flow Cytometry Core Facility, WUSM McDonnell Genome Institute and WUSM Genome Technology Access Center for technical assistance on this study. Research was supported by the National Institutes of Health (NIH) R00 HL138163 (to J.W.W.), AHA 16SDGG30480008 (to B.H.Z.), T32 AI007313 (to C.G.T.), P01 AI35296 (to B.T.F.) and NIH R37 AI049653 and DP1DK109668 (to G.J.R.). J.W.W. was supported by NIH 2T32DK007120-41 and AHA 17POST33410473. J-H.C. was supported by the Korean Health Technology R&D project HI15C0399 and Ministry of Health, Welfare & Family Affairs (South Korea). K.Z. was supported by the Government of Russian Federation (grant no. 08-08).
Author information
Authors and Affiliations
Contributions
This project was conceived and designed by J.W.W., M.N.A., B.H.Z., J-H.C. and G.J.R. Samples and materials were provided by J.W.W., B.T.F., B.H.Z., J-H.C. and G.J.R. Experiments were performed and analyzed by J.W.W., K.Z., K-W.K., S.I., B.T.S., P.R.S., K.K., A.E., S.H.K., C.G.T., M.W., S.E., K.J.L., B.H.Z. and J-H.C. All authors contributed to the preparation of this manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Jamie D. K. Wilson was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Profiling aorta macrophage populations by bulk RNA-seq.
Bulk sorted MacAIR 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-seq19. Gene groupings for (a) macrophage and DC genes, (b) foamy and adventitia macrophage genes, (c) MacAIR 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 MacAIR derived from 10-pooled aorta each, and three biological replicates each for ApoE−/− samples from 6-pooled aorta each.
Extended Data Fig. 2 MacAIR 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−/− mice22 and atherosclerosis regression samples23, identifying 10 unique clusters. (b) Top 12 enriched MacAIR genes were used to define MacAIR cluster as cluster 4, presented as relative expression. (c) MacAIR 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. 4 Profiling myeloid cells in the aorta.
Spleen and aorta samples from (a) SNX22gfp and (b) L-Mycgfp 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 MacAIR.
(a) Following the schematic, CX3CR1creER CD115-stop-DTR mice were used to conditionally express DTR on MacAIR by gavage with tamoxifen, waiting three weeks to allow circulating cells to repopulation from DTR-negative progenitors. MacAIR 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 MacAIR during atherosclerosis progression in bone marrow transplant model.
Recipient Ldlr−/− mice were lethally irradiated and reconstituted with CX3CR1creER 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 MacAIR. (a) Three weeks after tamoxifen labeling, MacAIR remained Tomato+ (red) positive. Mice were then fed HFD and assessed for plaque development at (b) 10 days and (c) 28 days. MacAIR 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) MacAIR 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.
Supplementary information
Supplementary Information
Supplementary Fig. 1.
Rights and permissions
About this article
Cite this article
Williams, J.W., Zaitsev, K., Kim, KW. et al. Limited proliferation capacity of aortic intima resident macrophages requires monocyte recruitment for atherosclerotic plaque progression. Nat Immunol 21, 1194–1204 (2020). https://doi.org/10.1038/s41590-020-0768-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41590-020-0768-4
This article is cited by
-
Analysis of brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke
Nature Immunology (2024)
-
Macrophage-based therapeutic approaches for cardiovascular diseases
Basic Research in Cardiology (2024)
-
Macrophage polarization and metabolism in atherosclerosis
Cell Death & Disease (2023)
-
Lipid-associated macrophages transition to an inflammatory state in human atherosclerosis, increasing the risk of cerebrovascular complications
Nature Cardiovascular Research (2023)
-
Loss-of-function mutations in Dnmt3a and Tet2 lead to accelerated atherosclerosis and concordant macrophage phenotypes
Nature Cardiovascular Research (2023)