Lung Remodeling Mediated by Telomere Dysfunction in Alveolar Type II Cells

特发性肺纤维化(IPF)是一种遗传介导的进行性肺部疾病。证据点 以端粒功能异常为核心的IPF发病机制。我们的知识很有限 肺泡2型(AT2)细胞端粒功能障碍与细胞分子的关系 重塑的纤维化肺的成分。试图在端粒的小鼠模型中阐明这种联系 小鼠固有的长端粒和使用额外的促纤维化刺激限制了功能障碍。 例如小剂量的博莱霉素，它可能会掩盖功能障碍的端粒对病理生物学的明显输入。 这项建议试图通过应用一种新的进行性肺纤维化驱动的模型来克服这些限制 单纯通过端粒功能障碍分离AT2细胞(TRF1SC小鼠)，研究肺纤维化的机制。这个 假说是在TRF1SC小鼠中发生的事件在发病机制中模拟早期上皮特异性事件 这一模型将为IPF中观察到的上皮细胞重构的起源提供新的见解。 为了验证这一假设，将探索以下目标：目标1建议使用TRF1SC/P53-/-小鼠 确定P53是否是进行性上皮转分化和重塑驱动的分子介质 TRF1SC小鼠AT2细胞端粒功能障碍。单细胞RNAseq(ScRNAseq)将用于定义 P53在TRF1SC AT2细胞端粒去帽后重编程中的作用及其他 促纤维化上皮细胞亚型。目的2建议对TRF1SC/TZAP-/-小鼠进行研究，以检验这一假设 TRF1SC小鼠出现纤维化的潜伏期需要端粒修剪。TRF1SC/TZAP-/-小鼠将得到治疗 用他莫昔芬治疗9个月，比较端粒磨损、上皮重塑和纤维化的指标 对照组：TRF1SC小鼠。ScRNAseq将用于确定端粒修剪对AT2细胞的贡献 在对照组、TRF1SC和TRF1SC/TZAP-/-小鼠中重新编程。正常AT2细胞的端粒修剪将是 通过研究老年TZAP-/-小鼠端粒长度的变化来进行研究。目标3建议培养IPF和 控制AT2细胞在存在和不存在hTERT过表达的器官系统中建立 是否增加端粒长度可恢复IPF肺上皮细胞的命运和功能。 比较的终点包括：端粒长度测量，通过共染色检测γH_2AX和 β-半乳糖苷酶和p16染色进行衰老重编程。AT2细胞转分化为AT1 细胞，并通过qPCR和免疫组织化学染色。HTERT过表达对其他肺间质纤维化上皮细胞的影响 将通过比较IPF上皮细胞混合群体的scRNAseq基因表达来研究亚型 在存在或不存在端粒酶逆转录酶的情况下培养。如果成功，这些研究将提供对 端粒功能障碍介导的细胞-细胞相互作用推动IPF中的肺纤维化。

Idiopathic pulmonary fibrosis (IPF) is a genetically mediated, progressive, lung disorder. Evidence points to dysfunctional epithelial telomeres as a core disease-initiating abnormality in IPF. There is limited knowledge of the relationship between alveolar type 2 (AT2) cell telomere dysfunction and the cellular and molecular elements of the remodeled fibrotic lung. Attempts to elucidate this connection in mouse models of telomere dysfunction have been limited by the inherently long telomeres of mice and the use additional fibrogenic stimuli such as low dose bleomycin, which may obscure the distinct input of dysfunctional telomeres to the pathobiology. This proposal seeks to overcome these limitations by applying a novel model of progressive lung fibrosis driven solely by telomere dysfunction isolated to AT2 cells (TRF1SC mice) to study mechanisms of lung fibrosis. The hypothesis is that events developing in TRF1SC mice mimic early epithelial-specific events in the pathogenesis of IPF and that this model will provide new insights into the origins of epithelial cell remodeling observed in IPF. To test this hypothesis, the following aims will be explored: Aim 1 proposes to use TRF1SC/p53-/- mice to establish whether p53 is a molecular mediator of progressive epithelial transdifferentiation and remodeling driven by AT2 cell telomere dysfunction in TRF1SC mice. Single cell RNAseq (scRNAseq) will be used to define the role of p53 on reprogramming of TRF1SC AT2 cells after telomere uncapping, and the appearance of other profibrotic epithelial cell subtypes. Aim 2 proposes to study TRF1SC/TZAP-/- mice to test the hypothesis that the latency of onset of fibrosis in TRF1SC mice requires telomere trimming. TRF1SC/TZAP-/- mice will be treated with tamoxifen for 9 months and measures of telomere attrition, epithelial remodeling, and fibrosis compared to control TRF1SC mice. scRNAseq will be used to define the contribution of telomere trimming to AT2 cell reprogramming in control, TRF1SC, and TRF1SC/TZAP-/- mice. Telomere trimming in normal AT2 cells will be investigated by studying changes in telomere lengths in aged TZAP-/- mice. Aim 3 proposes to culture IPF and control AT2 cells in the presence and absence of hTERT overexpression in an organoid system to establish whether augmenting telomere length restores epithelial cell fate and function of IPF lung epithelial cells. Comparative endpoints include: telomere length measures, DNA damage detected by co-staining for γH2AX and TRF2, senescence reprogramming by β-galactosidase and p16 staining. AT2 cell transdifferentiation into AT1 cells and by qPCR and immunostaining. The influence of hTERT overexpression on other IPF epithelial cell subtypes will be studied by comparing gene expression by scRNAseq of mixed populations of IPF epithelial cells cultured in the presence or absence of hTERT. If successful, these studies will provide an understanding into cell-cell interactions, mediated by telomere dysfunction, driving lung fibrosis in IPF.