Fibroblast-Immune Cell Crosstalk and Collagen Dynamics in Lactating Sheep Mammary Gland Revealed by Single-Cell Sequencing
The mammary gland is essential for milk production in sheep, and postpartum mammary health is critical for ensuring the successful transition of multiparous ewes into subsequent reproductive cycles. As lactation declines, the mammary gland undergoes substantial tissue remodeling, characterized by the dynamic reorganization of the extracellular matrix (ECM), particularly collagen. This remodeling is crucial for maintaining the structural integrity and function of the gland. Notably, during lactation, the mammary gland is prone to sagging and collagen loss, which are significant factors affecting mammary health and milk quality. In this study, we employed single-cell RNA sequencing (scRNA-seq) to characterize the cellular and molecular interactions that regulate ECM remodeling in the lactating sheep mammary microenvironment, with a focus on the crosstalk between fibroblasts and immune cells. Our comparative analysis of the peak and late lactation phases revealed significant shifts in the proportion of immune cells, including a decrease proportion in NK cells and macrophages and an increase proportion in neutrophils, T cells, and B cells, reflecting a dynamically evolving immune landscape. We identified key ligand-receptor interactions between fibroblasts and immune cells, emphasizing the central role of fibroblasts in collagen synthesis. Notably, the fibroblast-specific upregulation of the branched-chain amino acid (BCAA) degradation pathway supports their contribution to collagen production. A cross-species analysis of fibroblasts from sheep and human mammary glands revealed a conserved expression profile of ECM-related genes, suggesting a translational relevance to mammary structure and function. These findings provide new insights into mammary remodeling, with broad implications for understanding fibroblast-immune cell interactions in the context of postpartum mammary health and improving milk quality. Overall design: Mammary gland tissues were obtained from four lactating ewes, comprising two biological replicates each from peak lactation (PL) and late lactation (LL) stages.