Age-related changes in the phenotype and function of mouse skin macrophages

Skin ageing is accompanied by chronic low-grade inflammation (“inflammaging”) and progressive remodelling of the dermal extracellular matrix, yet the cellular changes that initiate these processes early in life remain poorly defined. Here we used single-cell RNA sequencing (scRNA-seq) of dorsal skin from young (8-week), middle-aged (26-week) and aged (52-week) male C57BL/6 mice to map age-related shifts in cellular composition, with a focus on macrophages. Whereas the relative proportions of most cell types remained largely 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 only 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 signalling in aged skin, including the pro-fibrotic mediators Thbs1 and Spp1, accompanied by up-regulation of collagen and fibrosis-associated genes in a subset of fibroblasts (FB3/FB4). Notably, collagen transcripts increased while COL5A3 protein decreased and dermal collagen architecture appeared disorganised by second-harmonic-generation imaging, pointing to post-transcriptional control of matrix remodelling. Ex vivo treatment of aged skin with the secretome of γ-irradiated peripheral blood mononuclear cells (PBMC-sec) reduced the macrophage expansion towards a more youthful composition and down-regulated fibrosis-associated fibroblast genes. Together, 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.