Single cell map of the adult female mouse urethra reveals epithelial and stromal macrophages with distinct functional identities

The epithelial lining of the urethra is an understudied mucosal barrier with emerging roles in antimicrobial defense during urinary tract infections. Here, we present a comprehensive cellular atlas of the adult female mouse urethra, focusing on epithelial and resident immune cells which act in concert at mucosal sites to direct host defense. Single cell RNA-sequencing revealed two distinct macrophage populations compartmentalized within the epithelium and stroma. Epithelial-associated macrophages display a highly dendritic morphology and populate the urethral lining in increasing numbers over the course of development. Epithelial-associated macrophages, express Cx3cr1, MHCII genes, Cd74 and Aif1/Iba-1, representing an activated macrophage type (Mac-Activated) and also express genes involved in antigen presentation and inflammatory response. In contrast, stromal macrophages express the scavenging receptors Mrc1/Cd206, Lyve1, Cd163 and Mgl2/Cd301b and display a signature enriched for endocytic function, vasculature development and tissue homeostasis (Mac-Endocytic). We identified epithelial cells in the urethral lining and associated glands expressing the monocyte chemoattractant genes Cx3cl1 and Cxcl17. Chemoattractant gene expression in the urethral epithelium follows a proximal-distal gradient which correlates with the increasing density of epithelial-associated macrophages expressing the receptor Cx3cr1, along the proximal-distal axis of the urethra. The study delineates spatially compartmentalized macrophage subsets in the urethra and implicate epithelial-derived chemokines in the establishment of macrophage positioning and functional specialization along the urethral axis. 5 µm sections mouse female urethra were prepared from FFPE tissue and mounted on a Xenium slide (10x Genomics). Manufacturer recommendations for sample processing and operation of the Xenium analyzer were followed for downstream sample preparation and data collection.