Figure 1. CCR2⁺ and CCR2⁻ macrophage subsets with distinct transcriptomic profiles in the dermis and hypodermis.

(A) Flow cytometry analysis of live CD45⁺ cells isolated from indicated skin layers of C57BL/6 mice. Graph indicates the percentage of CD45⁺ CD11b⁺ in both skin layers. Each dot represents an individual mouse. Data representative of two independent experiments (n=5–6 per group). (B) Flow cytometry analysis of live CD45⁺CD11b⁺EpCAM⁻Ly6C^lo cells from indicated skin layers. Data representative of more than 2 independent experiments (n=2–3 per experiment). (C) Giemsa stain on sorted dermal and hypodermal CCR2⁺ and CCR2⁻ macrophages from C57BL/6 mice. Scale=10μm (D) Principal component analysis of RNA-seq transcriptome analysis of indicated sorted cells from C57BL/6 mouse skin. (E and F) Volcano plots presenting the differentially expressed genes (p-value <0.05) between dermal (n=4467) and hypodermal (n=2493) CCR2⁺ and CCR2⁻ macrophages (blue). Genes with p-value <0.05 and an absolute value of Log₂ fold change >2 are presented in red, with genes of interest showed in yellow. (G-H) UMAP of unsupervised clustering analysis from scRNA-seq performed on dermal and hypodermal immune cells. Feature plots show the expression of characteristic genes for indicated cell lineage. (I) Schematic representation of the strategy used to obtain gene sets specific for macrophages in both dermis and hypodermis (top). Enrichment score for bulk RNA-seq macrophage gene set projected onto UMAP plots of dermal and hypodermal myeloid cells (bottom left). Cell identity annotation based on enrichment scores (bottom right). (J and L) Unsupervised heatmap of top 20 DEG between macrophage clusters of dermis and hypodermis. Selected genes are depicted. (K and M) Violin plot depicting enrichment score in scRNA-seq macrophages clusters of gene sets for CCR2⁺ and CCR2⁻ macrophages that were generated from macrophage bulk RNA-seq. (B-M) Macs: macrophages, DCs: dendritic cells.