Age‐related changes in the phenotype and function of mouse skin macrophages and their modulation by the secretome of peripheral blood mononuclear cells

Skin ageing is accompanied by chronic low-grade inflammation (“inflammaging”) and progressive remodeling of the dermal extracellular matrix, yet the cellular events that initiate these processes early in life remain incompletely understood. Here, we used single-cell RNA sequencing (scRNA-seq) of dorsal skin from young (8-week-old), middle-aged (26-week-old), and aged (52-week-old) male C57BL/6 mice to map age-related shifts in cellular composition, with particular emphasis on macrophages. Although the relative proportions of most cell types remained broadly stable, the macrophage compartment expanded markedly in the oldest skin. Sub-clustering resolved four macrophage populations, three of which expanded or emerged with age. The Mac4 population, marked by S100a8 and S100a9 and a Cd14 -high monocyte-derived signature, was detected mainly in aged skin and was confirmed in situ by F4/80, S100a8, and S100a9 co-staining. Cell–cell communication analysis predicted increased macrophage-to-fibroblast signaling in aged skin, including the pro-fibrotic mediators Thbs1 and Spp1 , together with upregulation of collagen and fibrosis-associated genes in a subset of fibroblasts (FB3/FB4). Notably, collagen transcripts increased, whereas COL5A3 protein decreased, and dermal collagen architecture appeared disorganized by second-harmonic-generation imaging, suggesting post-transcriptional control of matrix remodeling. Ex vivo treatment of aged skin with the secretome of γ-irradiated peripheral blood mononuclear cells (PBMC-sec) shifted the macrophage compartment toward a younger-like composition and downregulated fibrosis-associated fibroblast genes. Overall, these findings identify the early emergence of monocyte-derived, pro-inflammatory, and potentially pro-fibrotic macrophage subsets as a feature of mouse skin ageing and suggest that PBMC-sec can partially counteract these changes.