Loss of Fas-signaling in pro-fibrotic fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis. Loss of Fas-signaling in pro-fibrotic fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible fibrotic disease of the distal lung alveoli that culminates in respiratory failure and reduced lifespan. Unlike normal lung repair in response to injury, IPF is associated with the accumulation and persistence of fibroblasts and myofibroblasts and continued production of collagen and other extracellular matrix (ECM) components. Prior in vitro studies have led to the hypothesis that the development of resistance to Fas-induced apoptosis by lung fibroblasts and myofibroblasts contibributes to their accumulation in the distal lung tissues of IPF patients. Here, we test this hypothesis in vivo in the resolving model of bleomycin-induced pulmonary fibrosis in mice. Using genetic loss-of-function approaches to inhibit Fas signaling in fibroblasts, novel flow cytometry strategies to quantify lung fibroblast subsets and transcriptional profiling of lung fibroblasts by bulk and single cell RNA-sequencing, we show that Fas is necessary for lung fibroblast apoptosis during homeostatic resolution of bleomycin-induced pulmonary fibrosis in vivo. Furthermore, we show that loss of Fas signaling leads to the persistence and continued pro-fibrotic functions of lung fibroblasts. Our studies provide novel insights into the mechanisms that contribute to fibroblast survival, persistence and continued ECM deposition in the context of IPF and how failure to undergo Fas-induced apoptosis prevents fibrosis resolution. Overall design: Bulk RNA-seq,: Naïve(4 reps) + 3wk_Fas-- (4 reps) + 3wk_Fas++ (4 reps) + 6wk_Fas-- (4 reps) + 6wk_Fas++ (4 reps) scRNA-seq: paired Naive, 3wk_Fas--, 3wk_Fas++, 6wk_Fas--, 6wk_Fas++

特发性肺纤维化（Idiopathic Pulmonary Fibrosis, IPF）是一种进展性、不可逆的远端肺肺泡纤维化疾病，最终可引发呼吸衰竭并缩短患者寿命。与机体针对损伤的正常肺修复过程不同，IPF的病理特征为成纤维细胞与肌成纤维细胞蓄积且持续存在，同时胶原蛋白及其他细胞外基质（extracellular matrix, ECM）组分持续合成。
既往体外研究提出假说：肺成纤维细胞与肌成纤维细胞对Fas诱导的细胞凋亡产生抗性，这一机制参与了IPF患者远端肺组织中上述细胞的蓄积过程。本研究在博莱霉素诱导的小鼠肺纤维化消退模型中，对该假说进行体内验证。研究人员通过基因功能缺失手段抑制成纤维细胞中的Fas信号通路，结合新型流式细胞术策略量化肺成纤维细胞亚群，并通过批量RNA测序（bulk RNA-sequencing）与单细胞RNA测序（single cell RNA-sequencing）对肺成纤维细胞开展转录组分析，结果证实：在体内博莱霉素诱导的肺纤维化稳态消退过程中，Fas对肺成纤维细胞的细胞凋亡是必需的。此外，研究发现Fas信号通路缺失会导致肺成纤维细胞持续存活并维持促纤维化功能。本研究为IPF背景下成纤维细胞存活、持续蓄积及细胞外基质持续沉积的相关机制，以及Fas诱导的细胞凋亡缺陷如何阻碍纤维化消退，提供了全新的研究视角。
整体实验设计：
批量RNA测序（bulk RNA-seq）分组：未造模对照组（Naïve，4次生物学重复）、造模3周Fas基因敲低组（3wk_Fas--，4次生物学重复）、造模3周Fas基因过表达组（3wk_Fas++，4次生物学重复）、造模6周Fas基因敲低组（6wk_Fas--，4次生物学重复）、造模6周Fas基因过表达组（6wk_Fas++，4次生物学重复）
单细胞RNA测序（scRNA-seq）分组：配对的未造模对照组、造模3周Fas敲低组、造模3周Fas过表达组、造模6周Fas敲低组、造模6周Fas过表达组