Reprogramming of type 2 alveolar epithelial cells in idiopathic pulmonary fibrosis and regulation by TGFb1.

特发性肺纤维化中 2 型肺泡上皮细胞的重编程及 TGFb1 的调节。

• 批准号: 10821591
• 负责人: Max Louis Cohen
• 金额: $ 4.04万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10821591
• 关键词: Abnormal Cell; Abnormal Epithelial Cell; Adult; Affect; Alveolar; Antibodies; Attenuated; Automobile Driving; Basal Cell; Biochemical Markers; Biopsy; Biopsy Specimen; Cell Differentiation process; Cell Line; Cell Reprogramming; Cells; Cessation of life; Clinical Trials; Coculture Techniques; Critical Care; Cyst; Data; Dependence; Development; Diagnosis; Diagnostic; Disease; Ectopic Expression; Embryo; Epigallocatechin Gallate; Epithelial Cells; Epithelium; Extracellular Matrix; Fellowship; Fibroblasts; Fibrosis; Gene Expression; Gene Expression Profiling; Genes; Human; Immunohistochemistry; In Vitro; Institution; Knowledge; Lung; MUC5B gene; Medicine; Mesenchymal; Mesenchyme; Messenger RNA; Modeling; Monitor; Nature; Operative Surgical Procedures; Organoids; Overlapping Genes; Pathogenesis; Pathway Analysis; Pathway interactions; Patients; Phase; Population; RNA; Regulation; Research; Resources; Role; Sampling; Schedule; Secretory Cell; Signal Pathway; Signal Transduction; Slice; Source; Structure of parenchyma of lung; Surface; System; Testing; Time; Tissues; Transforming Growth Factor beta; Transforming Growth Factors; Transitional Cell; Type II Epithelial Receptor Cell; Undifferentiated; WNT Signaling Pathway; airway epithelium; alveolar epithelium; career; cell type; clinical diagnosis; diagnostic signature; fibrotic interstitial lung disease; fibrotic lung; idiopathic pulmonary fibrosis; in vitro Model; in vivo; inhibitor; insight; lung development; migration; notch protein; programs; repaired; response; self-renewal; single-cell RNA sequencing; surfactant; targeted treatment; tissue culture; transcriptome sequencing; transcriptomics; transdifferentiation

Idiopathic Pulmonary Fibrosis (IPF) is the most common fibrotic interstitial lung disease among adults. The cause of IPF is not fully understood, and it is frequently progressive, often leading to death within several years of diagnosis. In IPF, there is loss of alveolar epithelial cells, including type 1 cells (AEC1s), which line the alveolar airspace surface, and type 2 (AEC2s), which secrete surfactant, self-renew, and give rise to AEC1s and development of honeycomb cysts. These cysts are lined by "bronchiolized" epithelium, so-called because of expression of airway and secretory cell markers such as p63, KRT5, KRT17, and MUC5B. The origin of the cells lining these cysts is not understood, but have generally been thought to be the result of migration of airway epithelial cells (basal and/or club), in a failed attempt at alveolar repair. Recent single-cell RNA (sc-RNA) sequencing studies have uncovered widespread AEC2 and other epithelial cell abnormalities in end-stage IPF tissue, such as intermediate/transitional cell states and ectopic expression of genes associated with airway cells (such as KRT5+ AEC2s). Our lab has recently shown that AEC2s are capable of reprogramming into KRT5+ basal-cell like cells in in vitro organoid cultures. These suggest a new hypothesis that the bronchiolized epithelium lining honeycomb cysts may actually be derived from reprogrammed AEC2s. This study seeks to characterize whether the epithelial abnormalities present in end-stage IPF tissue are also present earlier in the IPF disease course and to determine the role of TGFβ1 in regulating the aforementioned reprogramming. Samples from patients undergoing surgical biopsy for the purpose of clinical diagnosis will be analyzed by sc-RNA sequencing, to characterize the AEC2 and other epithelial cell populations and reconstruct estimated lineages, especially surrounding the induction of the basal-cell differentiation master- regulator Sox2 within AEC2s. These samples will be compared to normal and end-stage IPF tissue, in order to test the hypothesis that AEC2 reprogramming is an early feature of IPF. In addition, diagnostic biopsy samples will be obtained from patients who took epigallocatechin gallate (EGCG) for two weeks prior to biopsy. EGCG is a mesenchyme-specific inhibitor of TGFβ signaling under study in humans and will therefore allow us to examine the hypothesis that AEC2 reprogramming abnormalities seen in diagnostic biopsies can be reversed by TGFβ blockade. Finally, organoid co-cultures and precision-cut lung slices cultures will be used to examine the contribution of important signaling pathways, such as TGFβ, Wnt, and Notch, in driving AEC2 reprograming towards a SPC-/KRT5+ basal-cell like state. Knowledge of the mechanisms driving AEC2 reprograming in IPF may provide fundamental insight into the cause of this disorder and contribute to the development of targeted therapies for this incurable and frequently fatal disease. This proposed project will be performed as part of the research phase of Pulmonary & Critical Care Medicine Fellowship at UCSF and utilizes comprehensive institutional support and resources, in order to prepare the applicant for an independent research career.

特发性肺纤维化（IPF）是最常见的纤维化间质性肺疾病 成年人。 IPF的原因尚不完全理解，并且经常是渐进的，经常领导的 在诊断的几年内死亡。在IPF中，肺泡上皮细胞的损失， 包括1型单元（AEC1），其中肺泡空域表面和2型（AEC2S） 秘密表面活性剂，自我更新，并引起AEC1和蜂窝囊肿的发展。这些 囊肿由“支气管化”上皮衬里，由于气道表达和 P63，KRT5，KRT17和MUC5B等秘书细胞标记。这些细胞的起源 囊肿不了解，但通常被认为是气道迁移的结果 上皮细胞（基础和/或俱乐部），试图肺泡修复的尝试失败。最近的单细胞 RNA（SC-RNA）测序研究已经发现了宽度的AEC2和其他上皮细胞 末期IPF组织的异常，例如中间/过渡细胞状态和Ecopic 与气道细胞相关的基因（例如KRT5+ AEC2S）的表达。我们的实验室最近显示了 AEC2能够将其重新编程到KRT5+基本细胞中的体外细胞中 文化。这些表明了一个新的假设，即支气管化上皮衬里蜂窝状囊肿 实际上可能是从重编程的AEC2派生的。这项研究试图表征是否是否 IPF早期也存在于末期IPF组织中存在的上皮异常 疾病过程并确定TGFβ1在确定优先重新编程中的作用。 为了临床诊断而进行手术活检的患者的样本将是 通过SC-RNA测序分析，以表征AEC2和其他上皮细胞群体以及 重建估计的谱系，特别是围绕基本细胞的诱导 AEC2中的分化主调节器Sox2。这些样本将与正常和 末期IPF组织，以检验AEC2重编程的假设是早期特征 IPF。此外，还将从触摸的患者那里获得诊断活检样本 活检前两周，表藻酸酯食酸酯（EGCG）持续了两周。 EGCG是一种特定于间质的 在人类中研究的TGFβ信号传导的抑制剂，因此将使我们能够检查 假设诊断活检中看到的AEC2重编程异常可以通过 TGFβ阻滞。最后，Organoid共培养和精确切割的肺切片将用于 检查重要信号通路（例如TGFβ，Wnt和Notch）在驱动方面的贡献 AEC2对SPC-/KRT5+基本细胞状态进行重新编程。了解驾驶机制 IPF中的AEC2重编程可能会提供对这种疾病原因和 为这种无法治愈且经常致命的靶向疗法的发展做出贡献 疾病。该提出的项目将作为肺部研究阶段的一部分进行。 在UCSF的重症监护医学奖学金，并利用全面的机构支持和 资源，以准备申请人为独立研究职业做好准备。