Identification of novel genes as being important for neutrophil functions

描述(申请人提供)：中性粒细胞是宿主先天免疫反应中高度特化的细胞，在清除细菌感染和宿主炎症反应中发挥关键作用。这些细胞的缺陷或过度活动可导致危及生命或衰弱的疾病，包括败血症、缺血/再灌注损伤、类风湿性关节炎和慢性阻塞性肺疾病(COPD)。此外，中性粒细胞是运动最快的哺乳动物细胞，具有明显的细胞极化和定向迁移机制，是研究细胞极化和定向迁移机制的良好模型。尽管已经做了大量的工作，但中性粒细胞生物学的许多方面仍然不清楚。此外，我们的基因表达分析揭示了大量在中性粒细胞中高表达的蛋白质，但对它们在中性粒细胞生物学中的作用知之甚少。与巨噬细胞等其他吞噬细胞不同，原代分化的中性粒细胞不能在培养中长时间生长，也不适合许多体外操作。大多数功能丧失研究依赖于小鼠的靶向基因失活或使用中性粒细胞样细胞，这些细胞在许多方面与初级中性粒细胞不同，不能用于体内研究。为了加速中性粒细胞的研究，我们开发了一种系统，允许在小鼠的原代中性粒细胞中高表达shRNAs。该方法适用于许多体内和体外检测，以确定靶蛋白在中性粒细胞生物学的各个方面的重要性。在这个探索性的R21提案中，我们计划筛选在中性粒细胞生物学或任何生物学中没有明显作用的靶点。我们的研究将有助于识别在先天性免疫和炎症反应中发挥关键作用的新蛋白质，并为中性粒细胞生物学提供新的线索，从而迅速扩大对中性粒细胞如何受到调节并承担其细胞功能的理解。因为我们建议研究的许多靶点都有在其他组织和细胞类型中表达的同系物，所以我们提议的研究可能会对中性粒细胞以外的细胞产生潜在影响。

DESCRIPTION (provided by applicant): Neutrophils are highly specialized cells in the host innate immune response that play a pivotal role in the clearance of bacterial infections and host inflammatory responses. Defects or overactivity in these cells can lead to life-threatening or debilitating diseases including sepsis, ischemia/reperfusion injury, rheumatoid arthritis, and chronic obstructive pulmonary disease (COPD). In addition, neutrophils are an excellent model for studying the mechanisms of cell polarization and directional cell migration for they are the fastest moving mammalian cells with overt cell polarity. Although a vast amount of work has been done, there are still many aspects of neutrophil biology that remain unclear. In addition, our gene expression analysis revealed a large number of proteins that are highly expressed in neutrophils, but with little knowledge about their roles in neutrophil biology. Unlike other phagocytes such as macrophages, primary differentiated neutrophils cannot be grown in culture for long and are not amenable to many of the in vitro manipulations. The majority of loss of function studies relies on targeted gene inactivation in mice or use neutrophil-like cells that difer from primary neutrophils in many regards and cannot be used for in vivo studies. To accelerate neutrophil research, we have developed a system that allows high expression of shRNAs in primary neutrophils in mice. The approach is amenable for a number of in vivo and in vitro assays for determining the importance of target proteins on various aspects of neutrophil biology. In this exploratory R21 proposal, we plan to screen targets that have no clear role in neutrophil biology or any biology. Our study will lead to identifying new proteins that play key roles in innate immunity and the inflammatory response and shed new lights into neutrophil biology, thus rapidly expanding the understanding of how neutrophils are regulated and undertake their cellular functions. Because many of the targets we propose to work on have homologs that are expressed in other tissues and cell type, our proposed studies may have potential impacts beyond neutrophils.