The Role of Lung Megakaryocytes in Airway Disease after Neonatal Hyperoxia

早产儿出生后暴露在氧气(O2)中，导致长期发育 对肺部的影响。大约70%的极早产婴儿(胎龄29周)会 增加了肺部发病率和/或儿童早期喘息障碍，尽管许多人没有 诊断为肺发育不良(BPD)。这些婴儿特别容易患呼吸道感染。 呼吸道病毒感染后的高反应性(AHR)，其机制尚不清楚。在这里，我们利用 基于一项新发现的低剂量高氧小鼠模型和独特的儿科人类组织信息库 新生儿氧气增加了肺巨核细胞(MKs)的丰度，MKs是一种未被研究的偏向髓系细胞 对免疫调节功能的影响。呼吸道病毒感染后，肺巨噬细胞释放促纤维化细胞因子，如 作为一种关键的炎症调节因子和转化生长因子β的激活剂，血栓反应蛋白-1(TSP-1) 1(转化生长因子β1)，导致纤维化。我们假设新生儿高氧血症通过增加肺组织中的 肺巨噬细胞的募集和感染后易导致巨噬细胞释放促纤维化因子(如TSP-1)。目标 1将确定出生后高氧肺环境如何影响肺MK招募和播种，包括 不同发育年龄和MK耗竭对肺MK人群的影响。目标2将决定 应用体外细胞因子检测新生儿高氧对肺MK转录组激活前后的影响 和RNAseq.实验还将通过比较流感病毒来确定AHR是否依赖MK或TSP-1 MK缺失小鼠对TSP-1基因缺失转基因小鼠的感染模型目标3将 确定新生儿氧气如何影响骨髓MK池，包括其对血小板生成的影响。 本建议是为医学博士安德鲁·戴拉格颁发的为期五年的指导性研究奖和培训计划，以 在小鼠和获得的人类组织中研究氧气诱导的呼吸道功能障碍的机制。Dr。 戴拉格是罗切斯特大学医学中心的儿科学(新生儿学)助理教授。这个 这里的研究建立在戴拉格博士作为一名临床新生儿专家和一名对 O2损伤和高氧后气道高反应性(AHR)。作为他职业发展计划的一部分，戴拉格博士将 通过四(4)个职业目标获得专业知识：1)在调查中增加知识和技术技能 利用体内平移模型进行肺损伤后的发育和修复，2)生物信息学中的靶向训练 分析，包括转录，3)开发将活体实验室发现应用于人类的专业知识 组织和临床人类疾病，以及4)发展必要的技能来领导有效的翻译研究 程序。戴拉格博士将通过应用流式细胞术、免疫组织化学、 转录学，以及针对老鼠和人类组织的有针对性的生物信息学培训。在完成这项工作后 在职业发展奖上，戴拉格博士将询问早年氧气如何驱动AHR的一个机械角色， 加深我们对新生儿氧气暴露如何导致长期肺部发病率的理解。

Former preterm infants are exposed to oxygen (O2) after birth which results in long-term developmental impacts on the lung. Approximately 70% of infants born extremely prematurely (<29 weeks’ gestational age) will have increased pulmonary morbidity and/or early childhood wheezing disorders even though many are not diagnosed with Bronchopulmonary Dysplasia (BPD). These infants are especially vulnerable to airway hyperreactivity (AHR) after respiratory viral infections through poorly understood mechanisms. Herein, we utilize a low-dose hyperoxia mouse model and a unique pediatric human tissue repository grounded on a new discovery that neonatal O2 increases the abundance of lung megakaryocytes (MKs), an understudied myeloid cell biased toward immunomodulatory functions. After respiratory viral infection, lung MKs release profibrotic cytokines such as Thrombospondin-1 (TSP-1), a critical inflammatory regulator and activator of transforming growth factor beta 1 (TGFβ1) that drives fibrosis. We hypothesize that neonatal hyperoxia primes the lung for AHR by increasing the recruitment of lung MKs and predisposing MKs to release pro-fibrotic factors (e.g. TSP-1) after infection. Aim 1 will determine how the hyperoxic lung environment after birth effects lung MK recruitment and seeding including how O2 at different developmental ages and MK depletion affect the lung MK population. Aim 2 will determine how neonatal hyperoxia effects lung MK transcriptome before and after activation using in vitro cytokine assays and RNAseq. Experiments will also determine if AHR is MK or TSP-1 dependent by comparing Influenza infection models of MK-depleted mice to transgenic mice with the TSP-1 gene deleted from MKs. Aim 3 will determine how neonatal O2 effects the bone marrow MK pool, including its effects on platelet production. This proposal is a five-year mentored research award and training plan for Dr. Andrew Dylag, MD to investigate oxygen-induced mechanisms of airway dysfunction in both mice and procured human tissues. Dr. Dylag is an Assistant Professor of Pediatrics (Neonatology) at the University of Rochester Medical Center. The research herein builds on Dr. Dylag’s experience as a clinical neonatologist and a basic scientist interested in O2 injury and post-hyperoxia airway hyperreactivity (AHR). As part of his career development plan, Dr. Dylag will attain expertise through four (4) career aims: 1) Increase knowledge and technical skills in the investigation of lung development and repair after injury using translational in vivo models, 2) Targeted training in bioinformatics analysis including transcriptomics, 3) Develop expertise in applying in vivo laboratory discoveries to human tissues and clinical human disease, and 4) Develop the necessary skills to lead an effective translational research program. Dr. Dylag will attain his stated goals by applying new skills in flow cytometry, immunohistochemistry, transcriptomics, and targeted bioinformatics training to a mouse and human tissues. At the completion of this career development award, Dr. Dylag will have interrogated one mechanistic role of how early life O2 drives AHR, advancing our understanding of how neonatal O2 exposures drive longer-term pulmonary morbidity.