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: 10444902
• 负责人: Max Louis Cohen
• 金额: $ 7.62万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10444902
• 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; Cells; Cessation of life; Clinical Trials; Coculture Techniques; Critical Care; Cyst; Data; Dependence; Development; Diagnosis; Diagnostic; Disease; Ectopic Expression; Embryo; Epigallocatechin Gallate; Epithelial; Epithelial Cells; Extracellular Matrix; Fellowship; Fibroblasts; Fibrosis; Gene Expression; Gene Expression Profiling; Genes; Human; Immunohistochemistry; In Vitro; Institutional support resources; 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; 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; base; 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; repaired; response; self-renewal; 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病 成年人。 IPF 的病因尚不完全清楚，并且通常是进行性的，通常会导致 诊断后几年内死亡。在 IPF 中，肺泡上皮细胞丢失， 包括排列在肺泡空腔表面的 1 型细胞 (AEC1s) 和排列在肺泡空腔表面的 2 型细胞 (AEC2s) 分泌表面活性剂，自我更新，并产生 AEC1 和蜂窝状囊肿的发育。这些 囊肿内衬有“细支气管化”上皮，之所以如此命名是因为表达了气道和 分泌细胞标记物，例如 p63、KRT5、KRT17 和 MUC5B。这些细胞的起源 囊肿尚不清楚，但通常认为是气道迁移的结果 上皮细胞（基底细胞和/或棒状细胞），肺泡修复尝试失败。最近的单细胞 RNA (sc-RNA) 测序研究发现广泛存在的 AEC2 和其他上皮细胞 终末期 IPF 组织异常，例如中间/过渡细胞状态和异位 与气道细胞相关的基因的表达（例如 KRT5+ AEC2）。我们的实验室最近表明 AEC2 能够在体外类器官中重编程为 KRT5+ 基底细胞样细胞 文化。这些提出了一个新的假设，即细支气管化上皮内衬蜂窝状囊肿 实际上可能源自重新编程的 AEC2。本研究旨在描述是否 终末期 IPF 组织中存在的上皮异常也存在于 IPF 早期 疾病过程并确定 TGFβ1 在调节上述重编程中的作用。 出于临床诊断目的而接受手术活检的患者的样本将被 通过 sc-RNA 测序进行分析，以表征 AEC2 和其他上皮细胞群 重建估计的谱系，特别是围绕基底细胞的诱导 AEC2 内的分化主调节器 Sox2。这些样本将与正常样本进行比较 终末期 IPF 组织，以检验 AEC2 重编程是早期特征的假设 的IPF。此外，诊断性活检样本将从以下患者身上获取： 表没食子儿茶素没食子酸酯（EGCG）在活检前两周。 EGCG是间充质特异性的 TGFβ信号传导抑制剂正在人体中进行研究，因此使我们能够检查 假设诊断活检中发现的 AEC2 重编程异常可以通过以下方法逆转 TGFβ 阻断。最后，类器官共培养和精确切割的肺切片培养将用于 检查重要信号通路（例如 TGFβ、Wnt 和 Notch）在驾驶中的贡献 AEC2 重编程为 SPC-/KRT5+ 基底细胞样状态。了解驱动机制 IPF 中的 AEC2 重编程可能为了解这种疾病的原因提供基本见解 有助于开发针对这种无法治愈且常常致命的靶向疗法 疾病。该拟议项目将作为肺与疾病研究阶段的一部分进行 加州大学旧金山分校的重症监护医学奖学金，利用全面的机构支持和 资源，以便为申请人的独立研究生涯做好准备。