Airway epithelial cell and lymphocyte interactions in chronic lung allograft dysfunction pathogenesis

肺移植后长期存活的主要障碍是肺功能的进行性丧失， 称为慢性同种异体肺移植功能障碍(Clad)，其病理基础是小气道内的缩窄性纤维化。 霍尔马克。超过一半的肺移植受者在移植后4年内受到CLAD的影响，并否定了许多 与移植相关的生活质量和功能改善。皮特、多伦多和加州大学旧金山分校 移植项目对来自肺移植的小气道刷毛进行了精细化的转录分析 受体作为一种新的技术来了解包膜部位的基因表达变化 病理学的发展。我们已经发表了与clad相关的基因表达变化，并在我们的 中锋。这项建议利用这一创新的方法来理解包裹体的发病机制。 我们的初步数据显示，在呼吸道刷检中，早期的缺氧途径上调，包括 利用低氧条件下的呼吸道上皮细胞培养和 呼吸道刷状转录本的通径分析。这种低氧信号可能反映了紊乱 与肺移植相关的微血管形成、无支气管循环和血管炎症。在……里面 收集T淋巴细胞，我们还观察到肿瘤坏死因子超家族(TNFSF)基因上调， 它们是淋巴细胞靶标细胞凋亡的主要驱动因素。我们的数据显示气道俱乐部有优先的细胞凋亡。 细胞，小气道的保护者和祖细胞，与上调的肿瘤坏死因子相关的凋亡相关- 诱导配体(TRAIL)表达。我们的单细胞在呼吸道刷检和支气管肺泡中的研究 灌洗液(BAL)显示这些途径在上皮细胞和淋巴样细胞类型之间的分离。基于 这些数据，我们假设呼吸道低氧先于TNFSF基因表达和T细胞介导的气道 导致CLAD发病机制的俱乐部细胞凋亡。为了检验这一假设，我们将生成平行队列 跨三个中心研究CLAD和对照的批量和单细胞转录本，允许 基因表达签名的严格交叉验证。我们将用细胞培养来补充这些研究- 基于确定驱动这些基因表达变化的机制的方法。在目标1中，我们将量化 低氧相关转录物在呼吸道刷状细胞中与COLAD相关，并确定低氧如何促进 淋巴细胞性炎症。在目标2中，我们将确定TNFSF共刺激的细胞来源和动力学 使用呼吸道刷子和BAL液包裹的分子表达。在目标3中，我们将调查线索是否 优先诱导俱乐部细胞凋亡。三项大型肺移植翻译研究的协同效应 拥有世界级跨机构生物统计基础设施的项目提供了一个独特的机会 严格处理这一假设。慢性粒细胞白血病细胞特异性基因表达的时程特征 通过这项研究将产生的发展迫切需要作为替代生物标记物来支持 靶向治疗的临床试验，并开创一种新的包裹性诊断方法。

The major barrier to long term survival following lung transplantation is a progressive loss of lung function, termed chronic lung allograft dysfunction (CLAD), for which constrictive fibrosis in small airways is a pathologic hallmark. CLAD affects over half of lung transplant recipients by 4 years post-transplant and negates much of the quality of life and functional improvements associated with transplantation. Pitt, Toronto, and UCSF Lung Transplant programs have refined transcriptional analysis of small airway brushings from lung transplant recipients as a novel technique to understand the gene expression changes at the anatomical site where CLAD pathology develops. We have published gene expression changes associated with CLAD validated across our centers. This proposal leverages this innovative approach to understand mechanisms of CLAD pathogenesis. Our preliminary data show an early upregulation of hypoxia pathways in airway brushings including genes that recruit and activate cytotoxic T lymphocytes using both airway epithelial cell culture in hypoxic conditions and pathway analysis of airway brush transcriptomes. This hypoxia signaling may reflect disordered microvasculature, absent bronchial circulation, and vascular inflammation associated with lung transplant. In recruited T lymphocytes, we also observed upregulation of tumor necrosis factor superfamily (TNFSF) genes, which are major drivers of apoptosis in lymphocyte targets. Our data show preferential apoptosis in airway club cells, the protectors and progenitors of small airways, in association with upregulated TNF-related apoptosis- inducing ligand (TRAIL) expression. Our single cell investigations in airway brushings and bronchoalveolar lavage (BAL) fluid show the segregation of these pathways across epithelial and lymphoid cell types. Based on these data, we hypothesize that airway hypoxia precedes TNFSF gene expression and T cell-mediated airway club cell apoptosis that drive CLAD pathogenesis. To test this hypothesis, we will generate parallel cohorts investigating bulk and single cell transcriptomes of CLAD versus controls across three centers, allowing for rigorous cross-validation of gene expression signatures. We will complement these studies with cell culture- based methods to determine mechanisms driving these gene expression changes. In Aim 1, we will quantify hypoxia-related transcripts in airway brush cells with respect to CLAD and determine how hypoxia can promote lymphocytic inflammation. In Aim 2, we will determine the cellular sources and kinetics of TNFSF co-stimulatory molecule expression in CLAD using airway brushes and BAL fluid. In Aim 3, we will investigate whether TRAIL preferentially induces club cell apoptosis. The synergy of three large lung-transplant translational research programs with world-class cross-institutional biostatistical infrastructures provides a unique opportunity to address this hypothesis rigorously. The cell-specific gene expression signatures over the time course of CLAD development that will be generated through this study are critically needed as surrogate biomarkers to support clinical trials of targeted therapies and to pioneer a novel approach to CLAD diagnosis.