Table 3_Temporal evolution of fibroblast responses following salivary gland ductal ligation injury.xlsx

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, regenerative responses, and can interfere with regenerative therapies. 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 autoimmune conditions, such as Sjögren's Disease. Despite the known involvement of fibrosis in disease and the inhibitory effects of fibrosis on tissue regeneration, the mechanisms through which extracellular matrix is elaborated 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 temporal responses of fibroblasts 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 one group of fibroblasts to be the primary injury-responsive fibrogenic cell type. Understanding how fibroblasts respond at the early and later 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 the development of therapeutics that minimize fibrosis and promote regenerative gland responses in chronic disease states.

细胞外基质重塑（extracellular matrix remodeling）是机体对损伤的自然应答，但过量的细胞外基质蓄积，即纤维化（fibrosis），是数百种疾病的致病因素——这些疾病会限制器官功能、阻碍组织再生反应，还会干扰再生治疗的实施。纤维化与炎症反应密切相关，二者均会出现在头颈部癌症放疗患者的唾液腺，以及干燥综合征（Sjögren's Disease）这类自身免疫病患者的唾液腺中。尽管已知纤维化参与疾病进程且会抑制组织再生，但唾液腺中细胞外基质生成的具体机制仍鲜为人知。基质成纤维细胞（Stromal fibroblasts）是主要的基质产生细胞，且已知其可同时驱动纤维化与炎症反应。为明确成纤维细胞对损伤的时序应答特征，我们通过结扎下颌下腺与舌下腺的主导管构建暂时性阻塞性损伤模型，并在损伤后即刻的多个时间点开展单细胞RNA测序（single-cell RNA sequencing）与通路分析。借助生物信息学（bioinformatic）方法，我们鉴定出三个可动态响应损伤的独特成纤维细胞亚群。我们对7天时间进程中的基质蛋白组（matrisomal）与炎症基因表达变化进行了系统表征，并确定其中一个成纤维细胞亚群为主要的损伤响应性致纤维化细胞类型。阐明成纤维细胞在损伤早期与晚期的应答机制，同时明确成纤维细胞调控的信号通路，将有助于更好地理解成纤维细胞对急性损伤应答的贡献，从而助力开发能够减轻纤维化、促进慢性疾病状态下腺体再生反应的治疗手段。