Abstract
Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1+ precursors and postnatally from bone marrow–derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.
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Acknowledgements
We acknowledge the administrative assistance of B. Bali. Supported by a Canadian Institutes of Health Research (CIHR) New Investigator Award (MSH136670), a CIHR operating grant (MOP133390), an Ontario Lung Association/Pfizer Award and the Peter Munk Chair in Aortic Disease Research (C.S.R.) and an Ontario Graduate Scholarship (S.E.).
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S.E., A.L. and R.B. conceived the project, designed and performed experiments and analyzed and interpreted data. N.D., J.C., M.R., M.E.M., J.W., E.S., C.L., B.L., L.R., T.J.Y., J.S.L., P.W., I.H., S.E., R.K. and K.F. performed experiments and helped interpret the data. M.O., J.B., C.C.J.Z., G.A.L., C.M.T.B., P.L., M.H., F.K.S., C.C., M.P., I.H., G.J.R., S.E., A.O.G., M.C. and B.B.R. provided materials and intellectual input and edited the manuscript. C.S.R. conceived the project, designed and performed experiments, supervised the study and wrote the manuscript.
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Supplementary Figure 1 Phenotype and gene-expression profiling of arterial macrophages.
(a) Representative dot plots identifying macrophages in the liver, lung, and brain by flow cytometry. (b) Heat map of mRNA transcripts increased in each single macrophage population by fivefold or more relative to their expression in the remaining 3 populations. (c) Heat map of mRNA transcripts decreased in each single macrophage population by fivefold or more relative to their expression in the remaining 3 populations. (d) Flow cytometry analysis of blood monocytes and arterial macrophages following treatment with clodronate liposomes (n = 3). (e) Number of CD45+ leukocytes isolated from adventitia and intima/media of aorta (mean ± SEM; adventitia n = 27, intima/media n = 22).* P = 0.0018 (unpaired Student’s t-test). (f) Visualization of arterial dendritic cells (DC) in CD11ceYFP/+ mice. (g) Representative dot plots showing presence of macrophages in carotid and femoral arteries.
Supplementary Figure 2 Arterial macrophages have embryonic and postnatal origins.
E8.5-induced CX3CR1-TdTomato expression in F4/80highCD11blow liver macrophages and F4/80intCD11bhigh liver monocytes at E16.5 (mean ± SD; n = 6). Data show a representative histogram from one of 6 mice analyzed. (b) E-selectin, ICAM-1, and CCL5 expression detected by RT-PCR and conducted on aortic tissue taken from mice at indicated time points (mean ± SD; n = 4 mice per time point except t = 1 where n = 3).* P < 0.05 (unpaired t-test).
Supplementary Figure 3 CX3CL1-CX3CR1 interactions determine survival of arterial macrophages.
(a) Enumeration of aortic macrophages in Csf2r−/− mice (mean ± SEM, n = 5). P = 0.1776 (unpaired Student’s t-test). (b) CD68 staining of aortic adventitia in wild type and Csf1−/− mice (mean ± SEM; wild type n = 9, M-CSF−/− n = 10).* P = 0.0002 (unpaired Student’s t-test). (c) Representative dot plots represent negative controls (C57BL6/J mice) for identifying GFP+ staining in CX3CR1gfp/+ mice. (d) IF showing association of adventitial aortic macrophages (CD68+) with CX3CL1+ cells in CX3CL1cherry mice. Shown is a representative image from one of several mice examined. (e) Representative dot plots showing absence of CX3CL1 expression in CD45+ cells isolated from aortae of CX3CL1cherry mice. 3 animals were analyzed (mean ± SD). (f) Representative IF section showing isotype staining for mcherry (rabbit IgG) and PDGFRα and CD31 (ratIgG2a).
Supplementary Figure 4 Arterial macrophages are maintained independently of monocytes in adulthood.
(a) Representative histogram showing Tdtomato labeling of blood monocytes in CX3CR1creER R26Tomato mice 9 and 11 months after tamoxifen exposure. (b) Adult TetOP-H2B-GFP mice were administered doxycycline for 4 weeks to induce H2B-GFP expression and then loss of fluorescence in aortic macrophages (indicative of cell division) was monitored during a 2 month chase period. (c) Representative histogram showing 5-bromodeoxyuridine (BrdU) incorporation by aortic MΦ.
Supplementary Figure 5 Arterial macrophages self-renew after exposure to bacteria.
(a) Enumeration of aortic macrophages in wild type mice following induction of sepsis by cecal puncture (mean ± SEM; day 0 n = 4, day 1 n = 4, day 7 n = 5). (b) Enumeration of neutrophils following exposure to LPS. Data were collected at multiple time points (mean ± SD; day 0 n = 8, 4h n = 3, 12h n = 3, 24h n = 3, 72h n = 4, 120h n = 4, 336h n = 3, 648h n = 3). (c) Flow cytometry characterization of CD11b (left panel) and Ly6C (right panel) expression on CD115+Lyve-1+ and CD115−Lyve-1− macrophages. (d) Representative dot plots showing purification of aortic macrophages following cell sorting. (e) Phagocytosis of pHrodo™ E. coli BioParticles® 7 days after induction of sepsis by cecal puncture (mean ± SD, n = 4). P = 0.26 (unpaired t-test).
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Ensan, S., Li, A., Besla, R. et al. Self-renewing resident arterial macrophages arise from embryonic CX3CR1+ precursors and circulating monocytes immediately after birth. Nat Immunol 17, 159–168 (2016). https://doi.org/10.1038/ni.3343
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DOI: https://doi.org/10.1038/ni.3343
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