Fig 1.
Monocyte/macrophage deficiency in ACKR2–/–embryonic skin.
A) Flow cytometry plot showing CD11b/F4/80 staining of CD45+ myelomonocytic cells in E15.5 WT and ACKR2–/–skin. The red arrow marks the intermediate population. B) i) Flow cytometric analysis of CD45/F4/80 staining in the total CD11bhi gate from skin of E14.5 WT (left-hand plot) and ACKR2–/–embryos (right-hand plot), showing a selective reduction in the F4/80Mid population (arrowed). ii) Quantitation of the percentage of F4/80lo and F4/80mid cells in WT and ACKR2–/–(KO) E15.5 embryonic skin. Results are representative of 2 separate experiments. Mann–Whitney U test was used for statistical analysis. C) Flow cytometric assessment of CSF1R and Ter119 staining of the i) monocyte (M), ii) intermediate (I), and iii) yolk-sac (YS)–derived cellular populations from E14.5 skin of MacGreen embryos. D) Quantitative assessment of multiple flow cytometric analyses showing numbers of i) monocyte- and yolk-sac–derived and ii) intermediate cells in E14.5 WT and ACKR2–/–(KO) skin. Results are from 1 of 4 independent experiments. Statistical analysis used the Mann–Whitney U test. E) Flow cytometric analysis of CD11b/F4/80 expression by CD45+ myelomonocytic cells from WT and ACKR2–/–(KO) skin at E14.5 and E17.5. Data are representative of at least 2 repeat experiments. Data were pooled from 3 independent experiments. Mann–Whitney test (monocytes) and unpaired two-tailed Student t test (yolk-sac–derived macrophages) were used for statistical analysis. Data associated with this figure can be found in the supplemental data file (S2 Data). ACKR, atypical chemokine receptor; CSF1R, Colony-stimulating factor 1 receptor; E, embryonic day; KO, knockout; WT, wild type.
Fig 2.
The intermediate cellular population is transcriptionally distinct from other embryonic monocyte/macrophage populations.
A) The E14.5 monocyte and intermediate populations were purified by BD index sorting with gating on the extremes of the CD11bhi population (as marked in blue). B) Principal component analysis showing the lack of transcriptional relatedness of the monocyte and intermediate populations. C) Funnel plots showing the extent of i) transcriptional differences between the monocyte and intermediate populations in WT embryos and ii) transcriptional relatedness between the residual monocyte population in ACKR2–/–skin and the equivalent population in WT skin. D) Venn diagram showing limited overlap in the genes identified as being differentially expressed between monocyte and intermediate cells in the current study (blue circle) and those identified as being differentially expressed between YS and bulk monocytic cells in a previous study (red circle) [14]. Data associated with this figure can be found in the supplemental data files S1 Data and S2 Data. ACKR, atypical chemokine receptor; BD, Becton Dickinson; E, embryonic day; padj, adjusted p value; WT, wild type; YS, yolk sac.
Fig 3.
The intermediate cellular population expresses high levels of chemokines and their receptors.
A) Heat map showing elevated expression of inflammatory chemokines and their receptors in the intermediate population compared to the monocyte population. B) CXCL4 is much more strongly expressed in intermediate population than in the monocyte population. C) i) Flow cytometric analysis demonstrating separate populations of CXCL4– and CXCL4hi cells within the overall CD11bhi gate (left-hand panel) and showing the CXCL4hi cells to be predominantly F4/80mid (right-hand panel; blue line, CXCL4– and red line, CXCL4hi). ii) Flow cytometric analysis demonstrating GP49 expression by CXCL4– and CXCL4hi myelomonocytic cells. Data associated with this figure can be found in the supplemental data file (S2 Data). ACKR, atypical chemokine receptor; CCL, CC ligand; CCR, CC chemokine receptor; CXCL, CXC ligand; CXCR, CXC chemokine receptor; GP49, glycoprotein 49; Pf4, platelet factor 4.
Fig 4.
CCR2–/–embryos phenocopy ACKR2–/–embryos.
A) CCR2 expression is higher in the intermediate compared to the monocyte population of cells. These data represent raw counts of CCR2 expression from the bulk RNA sequencing data. B) Flow cytometric analysis of CD11b and F4/80 expression in CD45+ cells from skins of E15.5 WT and CCR2–/–embryos. C) Quantification of multiple flow cytometric analyses of the monocyte, intermediate, and YS populations in skin from E15.5 WT, CCR2–/–, and ACKR2–/–embryos. Data shown here are from 1 of 2 repeated experiments, and Mann–Whitney U test was used to test for significance. D) Flow cytometric analysis of CD11b and F4/80 expression in CD45+ cells from skins of E15.5 WT and iCCR–/–embryos. E) Relative expression (normalised counts of DESeq2 outputs) of genes preferentially expressed in the monocyte population from WT (n = 3 embryos), ACKR2–/–and CCR2–/–E14.5 embryonic skin (n = 4 embryos). Data associated with this figure can be found in the supplemental data file (S2 Data). ACKR, atypical chemokine receptor; CCR, CC chemokine receptor; Cdk4, cyclin-dependent kinase 4; DESeq2, differential expression sequencing 2; E, embryonic day; iCCR, inflammatory CC chemokine receptor; KO, knockout; lrf1, liver regeneration factor 1; n.s., not significant; Rsad2, Radical S-Adenosyl-Methionine-Domain–Containing 2; smad3, smad family member 3; Tgfb2, transforming growth factor beta 2; WT, wild type; YS, yolk sac.
Fig 5.
Analysis of CCL2 expression in embryonic tissues.
A) Flow cytometric analysis of E12.5 total YS cells from CCL2-reporter mice showing i) a discrete CCL2-expressing population and ii) CD11b and F4/80 gating, demonstrating that this population is largely CD11bhiF4/80lo. B) Flow cytometric analysis of E12.5 total skin cells from CCL2-reporter mice showing i) a discrete CCL2-expressing population and ii) CD11b and F4/80 gating, demonstrating that this population is CD11bhiF4/80lo. C) Flow cytometric analysis of E12.5 total foetal liver cells from CCL2-reporter mice indicating the absence of CCL2 expression. Data are representative of 3 repeat experiments. D) Flow cytometric analysis of CCL2-expressing cells from i) E14.5 and ii) E17.5 skin from CCL2-reporter mice. The left-hand panels show total cell CCL2 expression, and the right-hand panels assign expression to CD45+ or–cells. iii) Quantification of CCL2 expression amongst CD45+ and–cells from E14.5 and E17.5 embryonic skin. iv) Dual Lyve-1 (Green) staining showing CCL2 (red) expression by skin resident macrophage-like cells in E15.5 skin whole mounts. E) i) Flow cytometric analysis of CCL2 expression in the distinct E14.5 monocyte, intermediate, and YS populations. Quantification as ii) percentage of live CCL2+ cells and iii) MFI of CCL2 expression amongst the monocyte (M), intermediate (I), and YS populations. One-way ANOVA with Bonferroni post-test for comparisons between two groups. F) Flow cytometric analysis of CCL2 expression in i) lungs and ii) livers from E14.5 and E17.5 embryos. iii) Comparative quantification of CCL2 expression in E14.5 skin and liver (dotted line = background level from nonreporter foetal tissues). Data associated with this figure can be found in the supplemental data file (S2 Data). CCL, CC ligand; E, embryonic day; Lyve-1, Lymphatic vessel endothelial hyaluronan receptor 1; MFI, mean fluorescent intensity; SSC, side scatter; YS, yolk sac.
Fig 6.
Placental ACKR2 limits chemokine movement from the maternal decidua to the embryo.
A) MSD-based analysis of CCL2 concentrations (normalised to total protein concentration) in plasma from i) E14.5 and ii) E15.5 WT and ACKR2–/–embryos. iii) CCL2 levels in adult pregnant mouse plasma expressed as pg/ml. Data shown here were derived from 1 of 2 repeated experiments. Unpaired Student t test without (i) and with Welch’s correction (ii) was used for statistical comparison between groups. B) In situ hybridisation showing (in red) i) ACKR2 expression (red coloration) in trophoblastic cells (grey coloration) in the E14.5 WT murine placenta and ii) lack of expression in ACKR2–/–placenta. F = foetal side of the placenta, J = foetal/maternal junction, and M = maternal decidua. Scale bar = 100 μm. C) MSD analysis of CCL2 levels (normalised to total protein concentration) in the maternal decidua and placenta of WT and ACKR2–/–E14.5 embryos. Data shown here are from 1 experiment. Similar results were independently obtained with E13.5 placenta lysates. D) Quantification of i) human CCL2 and ii) human CCL7 levels (normalised to total protein concentration) in plasma of embryos from mothers, systemically, injected i.v. with 500 ng of each chemokine. Data shown here are from 1 experiment. Unpaired Student t test without Welch’s correction was used for statistical comparison of injected human CCL2 (i) between genotypes. Data associated with this figure can be found in the supplemental data file (S2 Data). ACKR, atypical chemokine receptor; CCL, CC ligand; E, embryonic day; hCCL, human CCL; i.v., intravenously; KO, knockout; MSD, mesoscale discovery; n.s., not significant; WT, wild type.