Contribution of neutrophils to early airway disease in cystic fibrosis children

 描述(申请人提供)：囊性纤维化(CF)呼吸道疾病的特征包括支气管扩张、血液中的中性粒细胞(PMN)炎症、粘液阻塞和细菌感染。在具有里程碑意义的研究中，REST CF联盟显示，CF儿童的第一批支气管扩张症出现在小气道，并与PMN重新聚集和气道液中中性粒细胞弹性酶(NE)的高活性相一致。最近，我们发现慢性CF病患者的大部分气道中性粒细胞在存活时释放去甲肾上腺素，这是包括代谢激活、caspase-1活性和过度胞吐在内的重编程过程的一部分。在这里，我们显示类似的过度胞吐发生在CF儿童的气道PMN中。此外，我们还介绍了一种新的人小气道模型，在该模型中，体外暴露于顶端CF气道液的幼稚血液PMN被诱导迁移，并成为体内CF型气道液的高排泄性、重集性特征。我们的长期目标是开发旨在纠正PMN功能障碍的治疗方法，理想的情况是通过对早期CF疾病进行有针对性的干预。作为这一目标的一步，我们将对中性粒细胞在早期CF中的作用进行一项机制研究：(I)我们对CF中性粒细胞功能障碍的新认识；(Ii)我们新的体外模型；(Iii)即将在荷兰启动的独特的REST-CF研究。这一前瞻性队列研究仿照澳大利亚的REST-CF研究，包括12个月、36个月和60个月的计算机断层扫描(CT)和呼吸道液体分析，以密切跟踪早期CF疾病的发生和发展。我们的总体假设是，早期的CF病与呼吸道PMN重新编程相一致。我们的目标是：目标1-在活体内确定早期CF病中PMN重编程的程度。假设1A：慢性阻塞性肺疾病儿童的呼吸道含有重新编程的中性粒细胞。假设1B：临床测量的慢性阻塞性肺疾病儿童的呼吸道疾病进展与重新编程的PMN的更高负担有关。目的2体外研究CF气道液对中性粒细胞重编程的作用机制。假设2A：在我们的模型中，来自CF儿童的气道液诱导PMN重编程。假设2B：调节重新编程的气道PMN中的功能通路可以抑制它们的功能障碍和相关的疾病负担。为了利用母公司研究提供的非凡研究机会，我们召集了一组具有多学科专业知识的CF研究人员。这项拟议的辅助研究具有高度的时间敏感性，因为母研究很快就会开始，而这里包括的高含量分析(例如，流式细胞术、图像细胞术、多重定量聚合酶链式反应、靶向质谱仪)需要立即处理患者样本。我们期望这项研究能够更好地从机制上理解早期的CF疾病，并为新的靶向治疗提供强有力的概念验证数据，以帮助预防CF呼吸道炎症的恶性循环。

 DESCRIPTION (provided by applicant): Hallmarks of cystic fibrosis (CF) airway disease include bronchiectasis, inflammation by polymorphonuclear neutrophils (PMNs) from blood, obstruction by mucus, and infection by bacteria. In landmark studies, the AREST CF consortium showed that first signs of bronchiectasis in CF children occur in small airways and coincide with PMN recruitment and high activity of neutrophil elastase (NE) in the airway fluid. Recently, we showed that a large fraction of airway PMNs in chronic CF disease release NE while viable, as part of a reprogramming process that includes metabolic activation, caspase-1 activity and hyperexocytosis. Here, we show that similar hyperexocytosis occurs in airway PMNs from CF children. In addition, we introduce a new model of human small airway in which naive blood PMNs exposed to apical CF airway fluid in vitro are induced to transmigrate and become hyperexocytic, recapitulating features of CF airway PMNs in vivo. Our long-term goal is to develop therapies aimed at correcting PMN dysfunction, ideally via targeted intervention in early CF disease. As a step toward this objective, we will conduct a mechanistic study of PMN function in early CF leveraging: (i) our renewed understanding of PMN dysfunction in CF; (ii) our new in vitro model, and (iii) the unique AREST-CF study about to be initiated in the Netherlands. This prospective cohort is modeled after the Australian AREST-CF study, including computed tomography (CT) and airway fluid analysis at 12, 36 and 60 months to closely follow the onset and progression of early CF disease. Our overall hypothesis is that early CF disease coincides with airway PMN reprogramming. Our aims are to: AIM 1- Determine the extent of PMN reprogramming in early CF disease, in vivo. Hypothesis 1A: Airways in CF children contain reprogrammed PMNs. Hypothesis 1B: Progression of airway disease measured clinically in CF children associates with higher burden of reprogrammed PMNs. AIM 2- Identify mechanisms of PMN reprogramming by CF airway fluid, in vitro. Hypothesis 2A: Airway fluid from CF children induces PMN reprogramming in our model. Hypothesis 2B: Modulation of functional pathways in reprogrammed airway PMNs can inhibit their dysfunction and associated disease burden. To leverage the extraordinary research opportunity afforded by the parent study, we assembled a group of CF researchers with multidisciplinary expertise. This proposed ancillary study is highly time-sensitive, since the parent study will start shortly, and high-content assays included here (e.g., flow cytometry, image cytometry, multiplexed qPCR, targeted mass spectrometry) require immediate processing of patient samples. We expect this study to yield better mechanistic understanding of early CF disease and strong proof- of-concept data on new targeted therapies to help prevent the vicious cycle of CF airway inflammation.