Extended Data Fig. 5: Collagen-producing neutrophils localize near subepidermal fibres.
From: Matrix-producing neutrophils populate and shield the skin
a, Multiphoton sectional imaging of ear skin explants of LysMGFP mice with anti-Ly6G (for neutrophils) and wheat germ agglutinin (WGA, for vasculature), combined with SHG to show the position of the subepidermal collagen layer and, at right, SHG imaging of this layer above looser collagen in the rest of dermis, with several neutrophils shown from a side view. b, Reconstruction of a 1.5 × 1.5 mm region of the ear skin imaged from the top by two-photon imaging showing the network of CD31+ vessels as well as intravascular (yellow) and extravasated (pink) neutrophils as also shown in the inset at right. Images are representative of 4 mice. c, Distribution of distances for neutrophils to sup-epidermal and dermal collagen compared with random dots, illustrating the preferential localization of neutrophils near the subepidermal layer of collagen. Data are from 193 cells and 3 mice. P values were calculated using the two sample Kolmogorov–Smirnov tests comparing the sample distributions of the empirical distances against the simulated randomly generated dots. d, Single-cell RNA-seq of the ear skin, with UMAP showing the main identified clusters corresponding to the indicated cell types and distribution of the cells in control and neutrophil-depleted (anti-Ly6G, 2 days) skins. e,f, Violin plots showing expression of the Col3a1 gene across the different cell types in the skin (e) and expression of the matrix signature (f; genes from Fig. 1a) in the different clusters, showing the strongest expression in fibroblasts. g, Representative images of one neutrophil and one fibroblast stained for COL3 and specific markers (Mrp14 and Podoplanin, respectively). Right, quantification of COL3+ frequencies and COL3 signal intensity per cell area or in total cells from the immunofluorescence images. Data are shown as mean ± s.e.m. from 3 mice, with the number of cells (n) displayed in the figure. P-value determined by unpaired two-sided Student’s t-test.
