Transcriptional Regulation of Migrating Neutrophils during Pneumonia

项目摘要/摘要 肺炎仍然是发病率和死亡率的一个重要原因，最近已成为人们关注的焦点。 在新冠肺炎大流行期间。肺部在免疫学上是独一无二的，遍及肺部的中性粒细胞 准备好应对潜在的病原体挑战，而不会对周围组织造成伤害。中断 这种平衡导致了肺炎的许多严重并发症，但我们缺乏针对 中性粒细胞介导的免疫功能障碍。我们的初步数据表明，中性粒细胞经历了 肺炎期间它们从循环到肺泡腔的转录重塑。一个子集 随着中性粒细胞从血管系统转移到间质，促炎症基因的表达上调，而 当细胞从间质移动到肺泡时，与新陈代谢和脱颗粒有关的基因上调 太空。维持适当的中性粒细胞功能和发育需要严格的基因调控 变化，但这种变化背后的具体途径构成了一个重大的知识鸿沟。的目标是 这项提议是为了描绘中性粒细胞在迁移过程中所经历的转录和功能变化。 在肺炎期间从循环到感染部位。我们的中心假设是，在 肺炎，适当的中性粒细胞功能是通过被禁止的对特定的反应实现的 环境刺激，导致由它们的相互作用历史定义的异质种群。 目的1：验证肺炎部位特异性中性粒细胞反应是通过调谐实现的假设 环境刺激对中性粒细胞转录组和功能的影响。在体外和体内使用互补 我们将确定迁移、病原体相互作用和细胞因子信号转导对 中性粒细胞基因程序和功能。结果将包括预测的转录和功能变化 根据我们的初步研究。这些发现随后将在肺炎的活体模型中得到证实，采用 外源性添加标记的中性粒细胞。目的2：检验假设在进入牙槽间隙的过程中， 中性粒细胞分化为肺特有的亚群，具有转录和功能。 异质性。使用体内细胞标记、流式细胞仪辅助细胞分选和单细胞 RNA测序，我们将跟踪中性粒细胞转录组从循环到肺泡间隙的调制 单细胞级别。目的3：核因子κB-RelA是中性粒细胞中的一种中心转录因子，推动a 在迁移到肺泡腔期间的促炎转录程序。利用体外互补技术 在体内功能丧失模型中，我们将确定中性粒细胞NFκB-RelA在肺炎中的作用 结果中性粒细胞转录组和中性粒细胞功能。这些研究的结果将产生积极的影响 影响肺炎生物学领域，加深我们对中性粒细胞功能的了解，并为 为未来以宿主为导向的中性粒细胞肺炎治疗药物的开发奠定基础。

Project Summary/Abstract Pneumonia remains a significant cause of morbidity and mortality, which has come into sharp focus recently during the COVID-19 pandemic. The lung is immunologically unique, and neutrophils traversing the lungs must be ready to respond to potential pathogen challenges without causing injury to surrounding tissues. Disruptions of this balance lead to many severe complications of pneumonia, but we lack therapeutics directed toward neutrophil-mediated immune dysfunction. Our preliminary data demonstrate that neutrophils undergo transcriptional remodeling as they travel from the circulation to the alveolar space during pneumonia. A subset of pro-inflammatory genes is upregulated as neutrophils move from the vasculature to the interstitium, while genes related to metabolism and degranulation are upregulated as cells move from interstitium to the alveolar space. Maintenance of appropriate neutrophil function and development requires tight regulation of gene changes, yet the specific pathways underlying such changes constitute a major knowledge gap. The goal of this proposal is to delineate the transcriptional and functional changes neutrophils undergo as they migrate from the circulation to the site of infection during pneumonia. Our central hypothesis is that during pneumonia, appropriate neutrophil function is achieved by proscribed responses to specific environmental stimuli, resulting in a heterogenous population defined by their interaction history. Aim 1: Test the hypothesis that location-specific neutrophil responses in pneumonia are achieved by the tuning of neutrophil transcriptome and function by environmental stimuli. Using complementary in vitro and in vivo models we will determine the contributions of migration, pathogen interaction and cytokine signaling to neutrophil gene programs and function. Outcomes will include transcriptional and functional changes predicted by our preliminary studies. These findings will then be confirmed in an in vivo model of pneumonia, employing exogenously added labeled neutrophils. Aim 2: Test the hypothesis that during transit to the alveolar space, neutrophils differentiate into subpopulations unique to the lung, with transcriptional and functional heterogeneity. Using a combination of in vivo cell labeling, flow cytometry assisted cell sorting, and single cell RNA sequencing, we will track modulations in the neutrophil transcriptome from circulation to alveolar space at a single cell level. Aim 3: NFκB-RelA is a central transcription factor in neutrophils, driving the upregulation of a pro-inflammatory transcriptional program during migration to the alveolar space. Using complementary in vitro and in vivo loss-of function models we will determine the effect of neutrophil NFκB-RelA on pneumonia outcomes neutrophil transcriptome and neutrophil function. The results of these studies will have a positive impact the field of pneumonia biology furthering our knowledge of neutrophil function and laying the groundwork for future development of novel host-directed neutrophil-based pneumonia therapeutics.