Fig 2: Gate strategy for flow cytometry analysis.A, B and C = representative experiment after ficoll gradient followed by two steps of cold-aggregation and by cold-aggregation + percoll gradient for isolation of monocytes. D, E and F = representative experiment after ficoll gradient followed by Immunomagnetic beads for isolation of monocytes. G, H and I = representative experiment for monocyte differentiation to moDC. A = Dotplot of size FSC-A vs FSC-H for doublets exclusion, gated on singlets. B = Dotplot of FSC-A vs SSC-A for PBMCs, inside singlets gate. C = Dotplot of CD14 vs CD3, inside PBMC gate, where Q1 represents monocytes (CD14+) and Q3 represents T lymphocytes (CD3+). D = Dotplot of FSC-A vs FSC-H for doublets exclusion, gated on singlets. E = Dotplot of FSC-A vs SSC-A for PBMCs, inside singlets gate. F = Dotplot of FSC-A vs SSC-A inside the PBMC gate, where lymphocytes and monocytes were gated according to their size and granulosity characteristics. G = Dotplot FSC-A vs FSC-H for doublets exclusion of moDC after the differentiation protocol, gated on singlets. H = Dotplot of FSC-A vs SSC-A for moDCs, inside singlets gate. I = Dotplot of CD14 vs CD209, inside moDCs gate, where Q1 represents moDCs (CD209+) and Q3 represents non-differentiated monocytes (CD14+). All percentage and statistical analysis were performed inside the PBMC gate, thus excluding cell debris.

Fig 3: Flow cytometry overlay histograms for side scatter (A-D) and dot plots for CD14 and CD209 staining (E-L) demonstrating typical cell profiles from a representative experiment out of three. Side scatter (SSC, granularity) in non-differentiated monocytes (red shaded histogram) and differentiated moDCs (blue shaded histogram) cultured in either RPMI (A and B) or DMEM medium (C and D), supplemented with either FBS (A and C) or HS (B and D). CD14 and CD209 surface marker expression on non-differentiated monocytes (E-H) and moDCs (I-L) cultured in either RPMI (E, F, I and J) or DMEM medium (G, H, K and L), supplemented with either FBS (E, G, I and K) or HS (F, H, J and L). Cells were stained with CD14-FITC (y-axis) and CD209-PE (x-axis). The percentages of cells are indicated in each quadrant.

Fig 4: Expression levels of interleukin 1 family members in the skin.a Dynamic range plot illustrating the abundance of interleukin (IL) 1 family members in the skin. b, c Estimated intensity of the IL-1 family members IL-36? and IL-37 throughout the skin. IL-36? is primarily found to be expressed in the outer epidermal layer whereas IL-37 is both found to be expressed in the outer- and inner epidermal layer. d Immunohistochemical staining of healthy skin obtained from the Human Protein Atlas (Methods, Supplementary Data 17) showing IL-18 protein expression primarily in the inner epidermal layer (dark brown), scale bar: 50 µm. e, f Estimated intensity of the IL-1 family members IL-18 and IL-36a throughout the skin. IL-18 is mainly found in the inner- and outer epidermis whereas IL-36a is found in subcutis. g Immunohistochemical staining of IL-33 expression on healthy skin obtained from the Human Protein Atlas (Methods, Supplementary Data 17), scale bar: 50 µm. h Estimated intensity of recently discovered IL-33, here depicted in FACS-sorted skin-derived endothelial cells (EC), and to lesser extent in macrophages (MF) and dendritic cells (DC). i Estimated intensity of IL-1ß expression in FACS-sorted cellular subsets from healthy skin. All barplots show estimated intensity (IBAQ, log2) based on one library of pooled samples (N = number of pooled samples) of skin layers (N = 5; b, c, e, f), CD1A+dendritic cells (N = 3; h, i), CD14+ dendritic cells (N = 3; h, i), MF; macrophages (N = 4; h, i), mast cells (N = 6; h, i), EC; endothelial cells (N = 4; h, i), Mel; melanocytes (N = 5; h, i). Source data are provided as a Source Data file.

Fig 5: In-depth MS-based proteomic analysis of skin layers and its cellular subsets.a Workflow for measuring the skin proteome including skin layers, cultivated keratinocytes, and fibroblasts and FACS-sorted melanocytes, endothelial cells, and immune cells. b High-resolution MS analyses of fractionated, pooled samples in data-dependent acquisition mode (DDA) and subsequent (c) single-run analyses of three to six biological replicates in data-independent (DIA) acquisition mode. d Number of protein groups for each major cell subset and skin layers based on pooled samples (one independent experiment) for cell subsets and skin layers, respectively examined over one independent experiment (CD1A+dendritic cells (N = 3), CD14+ dendritic cells (N = 3), macrophages (N = 4), mast cells (N = 6), fibroblast (N = 5), keratinocytes (N = 5), endothelial cells (N = 4), melanocytes (N = 5), skin layers (N = 5). A total of 10,701 protein were identified. e Principal component analysis (PCA) of all proteomes from skin layers, primary cells and immune cell types. Color code from panel (d). The fractionated proteome of PBMC (green dot) was included as library for skin-associated T cells. Component 1 and 2 account for 24 and 11% of the total data variation, respectively.