Deciphering the role of p16INK4A+ fibroblasts in lung fibrosis

解读 p16INK4A 成纤维细胞在肺纤维化中的作用

• 批准号: 10559515
• 负责人: Tien Peng
• 金额: $ 55.69万
• 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: 2022-02-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10559515
• 关键词: 3-Dimensional; Acceleration; Address; Adoptive Cell Transfers; Adoptive Transfer; Aging; Alveolus; Area; Biological Assay; Biological Markers; Biology; CDKN2A gene; Cell Cycle Arrest; Cell Line; Cell Separation; Cells; Characteristics; Chemicals; Chronic lung disease; Cicatrix; Collagen; Cues; Data; Disease; Disease model; Drug Screening; Elderly; Engineering; Epithelium; Exhibits; Fibroblasts; Fibrosis; Genetic; Genetic Models; Human; Image; In Vitro; Induction of Apoptosis; Inflammatory; Injury; Knowledge; Ligands; Lung; Maintenance; Metaplasia; Methodology; Modeling; Morphology; Mus; Organ; Organoids; Outcome; Pathogenesis; Pathologic; Pathology; Phenotype; Physiological; Process; Pulmonary Fibrosis; Regenerative response; Reporter; Reporting; Risk Factors; Role; Testing; Therapeutic; Time; Tissues; Traction; Transplantation; Tumor Suppressor Proteins; Uncertainty; Validation; age related; aged; cell behavior; cell type; combinatorial; defined contribution; epithelial stem cell; fibrotic lung; fibrotic lung disease; functional decline; genetic approach; high throughput screening; idiopathic pulmonary fibrosis; in vivo; lung injury; migration; novel; progenitor; programs; prospective; rational design; regenerative cell; response; screening; senescence; single cell analysis; single-cell RNA sequencing; stem cells; therapeutic target; tool; tumorigenesis; wound healing

Project Summary/Abstract Aging is one of the biggest risk factors for many chronic lung diseases, including idiopathic pulmonary fibrosis (IPF). However, uncertainties remain as to how age-related processes contribute to the pathogenesis of IPF, particularly in deciphering the cell types that might play a role in both aging and fibrotic transformation. Senescence, a cellular state characterized by an irreversible cell cycle arrest and other morphologic transformations, is thought to be a cellular phenotype that becomes increasingly prevalent in aged organs, contributing to the decline in function over time. p16INK4A, a tumor suppressor, is one of the best-studied biomarkers of senescent cells in vitro and in vivo. Numerous genetic models have been built to remove p16INK4A+ cells from tissues, demonstrating a functional role for these cells in promoting age-related pathologies such as lung fibrosis. Despite the knowledge gained from strategies that remove p16INK4A+ cells, deletion of these cells precludes the identification and functional characterization of p16INK4A+ cells in vivo, leaving a large gap in our understanding of how senescent cells might behave in tissues rather than in a culture dish. To address this, we constructed a novel genetic reporter of senescence by engineering an amplified fluorescent tag driven by expression of p16INK4A (mouse referred to as INK4A H2B-GFP Reporter-In-Tandem, or INKBRITE) to isolate and characterize p16INK4A+ cells in vivo and ex vivo. To our surprise, we were able to identify p16INK4A+ fibroblasts in young and healthy lungs that contribute to scar-forming cells in areas of fibrotic remodeling. Furthermore, p16INK4A+ cells are able to alter the epithelial progenitor microenvironment and alter stem cell regenerative outcomes. This led us to the hypothesis that p16INK4A+ fibroblasts are capable of contributing to fibrosis by forming scars and inducing an pathologic epithelial response, and the INKBRITE reporter could be leveraged as a screening platform to identify compounds that more precisely target p16INK4A+ fibroblasts in vivo. Through the use of single cell RNA sequencing, adoptive cell transfers, lineage tracing, high-content imaging, and various methodologies outlined in this proposal, we aim to better define the cellular contribution of p16INK4A+ fibroblasts to fibrotic transformation, and define agents that might better remove it from tissues.

项目概要/摘要 衰老是许多慢性肺部疾病（包括特发性肺纤维化）的最大危险因素之一 （IPF）。然而，年龄相关过程如何影响 IPF 的发病机制仍存在不确定性， 特别是在破译可能在衰老和纤维化转化中发挥作用的细胞类型方面。 衰老，一种以不可逆的细胞周期停滞和其他形态学特征为特征的细胞状态 转化，被认为是一种在衰老器官中变得越来越普遍的细胞表型， 随着时间的推移，导致功能衰退。 p16INK4A 是一种肿瘤抑制因子，是研究最深入的肿瘤抑制因子之一 衰老细胞的体外和体内生物标志物。已经建立了许多遗传模型来去除 p16INK4A+ 来自组织的细胞，证明了这些细胞在促进与年龄相关的病理学方面的功能作用，例如 肺纤维化。尽管从去除 p16INK4A+ 细胞的策略中获得了知识，但删除这些细胞 妨碍了体内 p16INK4A+ 细胞的鉴定和功能表征，在我们的研究中留下了很大的空白 了解衰老细胞在组织中而不是在培养皿中的行为方式。为了解决这个问题，我们 通过设计由驱动的放大荧光标签构建了一种新型的衰老基因报告基因 p16INK4A（小鼠称为 INK4A H2B-GFP Reporter-In-Tandem，或 INKBRITE）的表达以分离和 体内和离体表征 p16INK4A+ 细胞。令我们惊讶的是，我们能够在 年轻健康的肺部有助于纤维化重塑区域的疤痕形成细胞。此外， p16INK4A+ 细胞能够改变上皮祖细胞微环境并改变干细胞再生 结果。这使我们得出这样的假设：p16INK4A+ 成纤维细胞能够通过以下方式促进纤维化： 形成疤痕并诱导病理性上皮反应，INKBRITE 报告基因可用作 一个筛选平台，用于识别更精确地靶向体内 p16INK4A+ 成纤维细胞的化合物。通过 使用单细胞 RNA 测序、过继细胞转移、谱系追踪、高内涵成像和各种 根据本提案中概述的方法，我们的目标是更好地定义 p16INK4A+ 成纤维细胞的细胞贡献 纤维化转化，并确定可以更好地将其从组织中去除的药物。