The age and sex of studied animals profoundly impact experimental outcomes in biomedical research. However, most preclinical studies in mice use a wide-spanning age range from 4 to 20 weeks and do not assess male and female mice in parallel. This raises concerns regarding reproducibility and neglects potentially relevant age and sex differences, which are largely unknown at the molecular level in naïve mice. Here, we employed an optimized quantitative proteomics workflow in order to deeply profile mouse paw skin and sciatic nerves (SCN) – two tissues implicated in nociception and pain as well as diseases linked to inflammation, injury, and demyelination. Remarkably, we uncovered significant differences when comparing male and female mice at adolescent (4 weeks) and adult (14 weeks) age. Our analysis deciphered protein subsets and networks that were correlated with the age and/or sex of mice. Notably, among these were proteins/biological pathways with known (patho)physiological relevance, e.g., homeostasis and epidermal signaling in skin, and, in SCN, multiple myelin proteins and regulators of neuronal development. Extensive comparisons with available databases revealed that various proteins associated with distinct skin diseases and pain exhibited significant abundance changes in dependence on age and/or sex. Taken together, our study uncovers hitherto unknown sex and age differences at the level of proteins and protein networks. Overall, we provide a unique proteome resource that facilitates mechanistic insights into somatosensory and skin biology, and integrates age and sex as biological variables – a prerequisite for successful preclinical studies in mouse disease models.

Tissue-resident macrophages are essential to protect from pathogen invasion and maintain organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and defined their expression signature. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor SpiC with these cells. Single-cell RNA sequencing showed that the macrophages in the adult thymus are composed of two populations distinguished by the expression of Timd4 and Cx3cr1. Remarkably, Timd4⁺ cells were located in the cortex, while Cx3cr1⁺ macrophages were restricted to the medulla and the cortico-medullary junction. Using shield chimeras, transplantation of embryonic thymuses, and genetic fate mapping, we found that the two populations have distinct origins. Timd4⁺ thymic macrophages are of embryonic origin, while Cx3cr1⁺ macrophages are derived from adult hematopoietic stem cells. Aging has a profound effect on the macrophages in the thymus. Timd4⁺ cells underwent gradual attrition, while Cx3cr1⁺ cells slowly accumulated with age and, in older mice, were the dominant macrophage population in the thymus. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.