Fig. 3: Aortic intimal macrophages seed the aorta immediately after birth and self-renew in areas of oscillatory and disturbed flow.

a, Experimental design. EdU injection at P7 2 h before euthanasia. Intimal CD45⁺ cells (green) proliferate (EdU⁺, white) in situ (yellow arrowheads). Endothelial cells are shown in red (ERG). Scale bar, 20 μm; n = 3 mice. b, For lineage tracing, Cx3cr1^CreERT2;R26^tdTomato neonates were injected with tamoxifen (tam) at P1, P3 and P5. At P7, aortae were collected from half of the litter as a baseline control. At 4 weeks, aortae were collected from the remaining littermates for retention of the reporter in Mac^AIR cells. c, Illustration depicting possible outcomes. tdTomato levels are negligible in blood (as shown in g). PB, peripheral blood. d, tdTomato⁺ immune cells in aortae at P7 (top) and 4 weeks (bottom) of Cx3cr1^CreERT2;R26^tdTomato mice after tamoxifen administration. Mac^AIR cells in aortae of Cx3cr1^CreERT2;R26^tdTomato mice at 4 weeks retained reporter (tdTomato⁺) expression when labeled postnatally, as shown by the comparison between P7 (baseline) and 4 weeks (lineage traced). CD45, green; VE-Cad, white. Scale bars, 80 μm and 10 μm, n = 5 (P7); 300 μm and 20 μm, n = 4 (4-week-old) mice. e, Percentage of intimal tdTomato⁺CD45⁺ cells in aortae of Cx3cr1^CreERT2;R26^tdTomato mice at P7 and 4 weeks when labeled with tamoxifen postnatally (n = 4 (4-week-old), n = 5 (P7) mice; Mann–Whitney t-test, P = 0.0159, two tailed, mean ± s.d., *P ≤ 0.05). f, Flow cytometry plots of tdTomato labeling of circulating cells at P7 and 4 weeks (n = 3 (4-week-old), n = 4 (P7) mice). g, Experimental design for the clonal tracing model and possible outcomes. Tamoxifen injection of Cx3cr1^CreERT2;R26^Rainbow mice at P1, P3 and P5. Aortae were collected at 8 weeks. Cer., Cerulean; mOr., mOrange; mCh., mCherry. h, Clones of Mac^AIR cells labeled at 8 weeks (initial tamoxifen injections at P1, P3 and P5). CD45⁺ cells are shown in white. Scale bar, 100 μm; n = 5 mice. i, Intimal CD11c⁺CD45⁺ cells from control and Ccr2^−/− mice at P5 (top, former ductus arteriosus region) and 8 weeks (bottom, lesser curvature). CD11c, red; CD45, green; ERG, white. Scale bar, 100 μm and 50 μm; n = 7 mice per time point. j, Comparison of CD11c⁺CD45⁺ cells in aortae from control and Ccr2^−/− mice at P5 (n = 6 (control), n = 7 (Ccr2^−/−) mice; Mann–Whitney t-test, mean ± s.d., P > 0.9999 (exact), two tailed) or in aortae from mice at 8 weeks (n = 7 (control) or n = 6 (Ccr2^−/−) mice; Mann–Whitney t-test, mean ± s.d., P = 0.0872, two tailed). NS, not significant. k, Summary of Figs. 1–3. Mac^AIR cells enter the aorta via the ductus arteriosus, expand through self-renewal and populate the lesser curvature. Macrophages also accumulate in descending aortae from aged mice. Illustrations (a,b,g) were created using https://biorender.com/.

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