Lung fibrosis mediated by telomere dysfunction in alveolar type II cells

II型肺泡细胞端粒功能障碍介导的肺纤维化

• 批准号: 10200132
• 负责人: Paul j WOLTERS
• 金额: $ 59.27万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-05 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200132
• 关键词: Affect; Aftercare; Age; Air; Alveolar Macrophages; Biological; Biological Assay; CCL2 gene; Cells; Coculture Techniques; Data; Dendritic Cells; Dichloromethylene Diphosphonate; Elements; Epithelial; Epithelial Cells; Fibroblasts; Fibrosis; Functional disorder; Future; Growth Factor; Harvest; Histopathology; Human; Hydroxyproline; Hyperplasia; In Vitro; Integrins; Knowledge; Link; Lung; Lung diseases; Measures; Mediating; Mediator of activation protein; Mesenchymal; Messenger RNA; Methods; Modeling; Molecular; Molecular Abnormality; Mus; Particulate; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Phenotype; Prevalence; Proteins; Proteome; Pulmonary Fibrosis; Smoking; Stains; Telomere Shortening; Testing; Time; Transforming Growth Factor beta; Up-Regulation; age related; alveolar epithelium; alveolar type II cell; beta-Galactosidase; cell type; chemokine; defined contribution; experimental study; genetic approach; idiopathic pulmonary fibrosis; improved; in vivo; insight; integrin beta6; macrophage; mast cell; men; response; senescence; telomere; transcriptome sequencing

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder whose prevalence increases with age. IPF alveolar type II cells (AECII cell) have shortened telomeres and express markers of senescence such as senescence associated beta-galactosidase (SA-βgal). Although short telomeres and a senescent phenotype are always found in IPF AECII cells, there is little data linking these abnormalities, to molecular mediators facilitating the lung remodeling found in IPF patients. This lack of insight is due to the absence of a model of lung remodeling mediated by telomere dysfunction. This proposal seeks to overcome these knowledge gaps by using a model of lung fibrosis mediated by telomere dysfunction isolated to alveolar type II cells (TRF1SC mice) to examine whether telomere dysfunction in AECII cells is a molecular switch that drives pathologic abnormalities found in IPF. Aim 1. To establish the contribution of macrophages to lung remodeling in TRF1SC mice. This aim uses pharmacologic (clodronate) and genetic approaches to deplete alveolar macrophages in TRF1SC mice and define the contribution of macrophage expansion to lung fibrosis. It explores whether AECII cell expression of the chemokine CCL2 mediates macrophage expansion by crossing CCL2F/F mice to TRF1SC mice. At key time-points lungs will be harvested from clodronate treated or CCL2F/F/TRF1SC mice and measures of remodeling and fibrosis quantified and compared to relevant controls. Aim 2. To establish the profibrotic molecular mediators generated by AECII cells in TRF1SC mice. The AECII cell-specific, TGFβ activating, integrin αvβ6, will be quantified at key time-points and correlated with active TGFβ levels. Capacity for senescent AECII cells to activate TGFβ will be quantified in the MLE TGFβ activation assay +/- β6 blocking Ab. Contribution of β6–mediated of TGFβ activation to lung fibrosis will be tested by crossing β6 deficient mice to TRF1SC mice and measures of lung remodeling quantified. RNAseq will identify pro-fibrotic elements of the senescence associated secretory proteome in TRF1SC mice, and establish whether they change over time. Aim 3. To establish that senescence programming can be leveraged to identify and target profibrotic AECII cells. SA-βgal staining will be used to isolate senescent AECII cells from IPF lungs. Measures of the capacity for senescent IPF AECIIs to activate TGFβ will be compared between SA-βgal+ to SA-βgal- control AECII cells in the presence and absence of β6 blocking Ab. Profibrotic pathways will identified in senescent IPF AECIIs by comparing mRNA levels between the senescent SA-βgal+ IPF AECII cells and normal controls. AECII cells isolated from IPF or TRF1SC mice will be treated with a senolytic drug (ABT263) and expression of senolytic markers and profibrotic mediators compared between treated and untreated cells. To test effect of senolysis on lung remodeling in vivo, TRF1SC mice will be treated with ABT263 and at various time-points after treatment, measures of senescence and remodeling compared with vehicle treated controls.

特发性肺纤维化（IPF）是一种进行性肺部疾病，其患病率随年龄增加而增加。 IPF肺泡II型细胞（AECII细胞）具有缩短的端粒并表达衰老标志物，如 衰老相关β-半乳糖苷酶（SA-βgal）。虽然短端粒和衰老 表型总是在IPF AECII细胞中发现，几乎没有数据将这些异常与 在IPF患者中发现的促进肺重塑的分子介质。这种缺乏洞察力是由于 缺乏由端粒功能障碍介导的肺重塑模型。这项建议旨在 通过使用分离的端粒功能障碍介导的肺纤维化模型， 肺泡II型细胞（TRF 1 SC小鼠），以检查AECII细胞中的端粒功能障碍是否是一种分子机制， 导致IPF病理异常的开关。目标1。确定的贡献 巨噬细胞对TRF 1 SC小鼠肺重塑的影响。这一目标使用药理学（氯膦酸盐）和遗传学 在TRF 1 SC小鼠中耗尽肺泡巨噬细胞并确定巨噬细胞的贡献的方法 扩展到肺纤维化。它探讨了AECII细胞表达趋化因子CCL 2是否介导了 通过将CCL 2F/F小鼠与TRF 1 SC小鼠杂交来扩增巨噬细胞。在关键时间点，将采集肺 从氯膦酸盐处理的或CCL 2F/F/TRF 1 SC小鼠和重塑和纤维化的测量定量， 与相关对照相比。目标2.建立促纤维化分子介质， TRF 1 SC小鼠中的AECII细胞。AECII细胞特异性、TGFβ活化、整合素αvβ6将在关键时间点定量。 时间点，并与活性TGFβ水平相关。衰老的AECII细胞激活TGFβ的能力将是 在MLE TGFβ活化测定+/- β6阻断Ab中定量。β6介导的TGFβ的贡献 将通过将β6缺陷小鼠与TRF 1 SC小鼠杂交来测试肺纤维化的活化，并测量肺纤维化的程度。 重塑量化RNAseq将鉴定衰老相关的分泌性纤维化的促纤维化元件。 TRF 1 SC小鼠中的蛋白质组，并确定它们是否随时间变化。目标3.证明 可以利用衰老编程来鉴定和靶向促纤维化AECII细胞。SA-βgal 将使用染色从IPF肺分离衰老AECII细胞。衰老能力的测量 将比较IPF AECII激活TGFβ的SA-βgal+与SA-βgal-对照AECII细胞， 存在和不存在β6阻断Ab。通过以下方法在衰老IPF AECII中识别促纤维化途径： 比较衰老的SA-βgal+ IPF AECII细胞和正常对照之间的mRNA水平。AECII细胞 将用衰老清除药物（ABT 263）处理从IPF或TRF 1 SC小鼠分离的抗衰老蛋白， 标记物和促纤维化介质之间的比较。为了测试senolysis对 体内肺重塑，TRF 1 SC小鼠将用ABT 263处理，并在处理后的不同时间点， 与媒介物处理的对照相比的衰老和重塑的测量。