Extended Data Fig. 6: Dermal macrophages in the skin are derived from bone marrow.
(a) Time course (1–10 weeks after BMT) of myeloid cell chimerism in peripheral blood of WT mice received BMT from GFP mice. The proportions of GFP+ cells (positive ranges were indicated by bars) in total myeloid cells are expressed as percentages (n = 4). (b–d) Confocal microscopic images of the hind paw skin of WT mice that received BMT from GFP mice. Sections are double labeled for GFP and cell markers (CD206 (b), F4/80 (c), and Lyve1(d)). HF denotes hair follicles showing non-specific fluorescence. (Scale bar, 100 μm. (e) Percentage of MHCII+ (64.6%), CD206+ (27.7%), F4/80+ (22.3%), and Lyve1+ (4.3%) cells among GFP+ cells (5 weeks after transplantation) (MHCII: n = 8; CD206: n = 6; F4/80: n = 6; Lyve1: n = 8 hind paw skin sections from at least 3 different mice). (f) Percentage of GFP+ cells among MHCII+, CD206+, F4/80+, and Lyve1+ cells (5 weeks after transplantation) (MHCII: n = 8; CD206: n = 6; F4/80: n = 6; Lyve1: n = 8 hind paw skin sections from at least 3 different mice). Ratios are 70.0%, 15.1%, 31.5%, and 14.5%, respectively. (g–k) Confocal microscopic images of the sections stained for Gr1 (g), CD19 (h), CD8a (i), CD4 (j), and NK1.1 (k) of the hind paw skin of WT mice that received BMT from GFP mice. Each antibody was used as a marker for neutrophils, B cells, killer T cells, helper T cells, and NK cells, respectively. HF; hair follicles with non-specific fluorescence. Scale bars, 100 μm. (l) Flow cytometry strategy using Gr1, CD19, CD8a, CD4, and NK1.1 marker expression in the hind paw skin. (m) Flow cytometry strategy using MHCII, F4/80, Gr1, CD19, CD8a, CD4, and NK1.1 marker expression in the back skin. (n) Confocal microscopic images of the dorsal horn of the spinal cord (L4) of WT mice that received BMT from GFP mice. The gray matter is demarcated by the dotted line. Insets show magnified images of the boxed areas. Note the absence of GFP+ cells in the gray matter. Scale bar, 100 μm. (o) VF thresholds in WT mice before and after busulfan treatment (n = 6, p = 0.328). g, gram. (p) VF thresholds before and after BMT (WT, before BMT: n = 8; after BMT: n = 5, p = 0.748; Snx25+/− mice, before BMT: n = 6; after BMT: n = 4, p = 0.108) in the WT and Snx25+/− mice that received BMT from the mice of the same genotype. g, gram. Results are represented as mean ± SEM. Statistical analyses were performed using the two-tailed Student’s t-test. n.s., not significant. Representative of three independent experiments (b-d, g-k, and n).
