Temporal Evolution of Fibroblast Responses Following Salivary Gland Ductal Ligation Injury

Extracellular matrix remodeling is a natural response to injury but, excessive extracellular matrix accumulation or fibrosis, is a causative factor in hundreds of diseases that limit organ function and impede regenerative responses. Fibrosis is closely related to inflammation, both of which occur in the salivary glands of patients treated with radiation for head and neck cancers and in patients suffering from the autoimmune condition, Sjögren's Disease. Despite the known involvement of fibrosis in disease and the inhibitory effects of fibrosis on tissue regeneration, the mechanisms through which fibrosis develops in the salivary gland are poorly understood. Stromal fibroblasts are the primary matrix-producing cells and are known to drive both fibrosis and inflammation. To define the signals produced by fibroblasts in response to injury, we induced a temporary obstructive injury though ligation of the primary submandibular and sublingual salivary gland ducts and then performed single-cell RNA sequencing and pathway analysis at timepoints immediately following the injury. Using bioinformatic approaches, we identified three unique fibroblast groups that dynamically respond to the injury. We characterized the changes in matrisomal and inflammatory gene expression over a 7-day time course and identified the group 2 fibroblasts to be the primary fibrogenic cells, although all groups produced ECM genes. Understanding how fibroblasts respond at early and late injury timepoints along with defining signaling pathways regulated by fibroblasts could lead to a better understanding of the contribution of fibroblast to acute injury responses to facilitate development of therapeutics that minimize fibrosis and promote regenerative gland responses in chronic disease states. Overall design: Submandibular and sublingual mouse salivary glands were dissociated into single cells followed by two rounds of magnetic activated cell sorting (MACs) to deplete for epithelial cells expressing EpCAM and red blood cells expressing Ter119. The samples then went through one round of dead cell removal using Miltenyi dead cell removal kit.