The study of recycling endosomes in innate immune cells

先天免疫细胞回收内体的研究

• 批准号: RGPIN-2014-03601
• 负责人: Lacy, Paige
• 金额: $ 2.55万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=565971
• 关键词: study; recycling; endosomes; innate; immune

My research program is focused on studying the role of recycling endosomes (REs) in immune cells. Recycling endosomes (REs) are specialized secretory compartments that recycle cell surface receptors through the endocytic and exocytotic pathways. These secretory organelles have been well characterized in numerous cell types. Recently, REs were found to have multiple roles in protein trafficking beyond their originally characterized function in trafficking cell surface receptors. REs have been discovered to constitutively traffic the cytokine, tumour necrosis factor-alpha (TNF-alpha), from the Golgi to the cell membrane in macrophages, a type of innate immune cell. However, the presence of REs and their role in trafficking and release of cytokines in immune cells in general is poorly studied. Many immune cells, including neutrophils and eosinophils (granulocytes), are complex and highly granulated secretory cells capable of releasing a wide array of cytokines. Granulocytes are the most abundant circulating innate immune cell in the body, and are powerful secretory cells that function as a double-edged sword in immunity. These specialized secretory cells have the ability to transmigrate into tissues and release copious quantities of cytokines. Thus, immune cells have robust protein trafficking systems and serve as perfect models for studying RE function and exocytosis of cytokines. Objectives The long-term objective of this study is to determine the presence and function of REs, a novel secretory compartment, in cytokine trafficking in innate immune cells. There is a paucity of literature on RE function in innate immune cells, and yet there is strong evidence for RE existence and function in these cells. The specific short-term objectives are to: 1. Characterize TNF-alpha trafficking in REs in innate immune cells using tools and reagents for their detection. 2. Determine additional cytokines trafficked via REs for secretion in innate immune cells. 3. Characterize signaling pathways that regulate release of cytokines from REs in innate immune cells. Specifically, we will assess the contribution of the recently described rapidly and slowly recycled compartments of REs to cytokine trafficking in neutrophils, macrophages, and eosinophils. Rapid and slow recycling REs can be detected by distinct patterns of expression of specific Rab guanosine triphosphatases (GTPases). The cytokines of interest are numerous and include the potent immunomodulatory TNF-alpha, the neutrophil chemokine interleukin-8, and the recently characterized interleukin-33, all of which are secreted by innate immune cells. We will also determine whether R- and Q-SNAREs are required, and identify the specific SNAREs required for RE fusion with the cell membrane. We propose to resolve the requirements for various Rabs and SNAREs in the release of protein cargo including cytokines from neutrophils. Using high resolution imaging and ultrastructural studies, we will map the fine distribution of Rabs, SNAREs, granule and RE-associated proteins during neutrophil RE exocytosis. The findings arising from this proposal will reveal a new, undiscovered membrane trafficking compartment in important innate immune cells required to maintain immunity. Understanding the function of REs in innate immune cells will lead to the elucidation of novel trafficking pathways for protein cargo, particularly cytokines that serve an essential immunomodulatory function.

我的研究项目主要集中在研究免疫细胞中回收内体（RE）的作用。再循环内体（Recycling Endosomes，RE）是通过内吞和外吞途径再循环细胞表面受体的特化分泌隔室。这些分泌细胞器已在许多细胞类型中得到很好的表征。最近，发现RE在蛋白质运输中具有多种作用，超出了其最初在运输细胞表面受体中的特征功能。已经发现RE组成性地将细胞因子、肿瘤坏死因子-α（TNF-α）从高尔基体运输到巨噬细胞（一种先天免疫细胞）中的细胞膜。然而，RE的存在及其在免疫细胞中细胞因子的运输和释放中的作用通常研究得很少。许多免疫细胞，包括嗜中性粒细胞和嗜酸性粒细胞（粒细胞），是复杂的和高度颗粒化的分泌细胞，能够释放广泛的细胞因子。粒细胞是体内最丰富的循环先天免疫细胞，并且是功能强大的分泌细胞，在免疫中起着双刃剑的作用。这些特化的分泌细胞具有迁移到组织中并释放大量细胞因子的能力。因此，免疫细胞具有强大的蛋白质运输系统，并作为研究RE功能和细胞因子胞吐作用的理想模型。目的本研究的长期目标是确定RE（一种新型分泌区室）在先天免疫细胞中细胞因子运输中的存在和功能。关于RE在先天免疫细胞中的功能的文献很少，但有强有力的证据表明RE在这些细胞中存在和功能。具体的短期目标是：1。使用检测工具和试剂表征先天免疫细胞中RE的TNF-α运输。2.确定通过RE运输的用于在先天免疫细胞中分泌的额外细胞因子。3.表征调节先天免疫细胞中RE释放细胞因子的信号通路。具体来说，我们将评估最近描述的快速和缓慢回收的车厢RE细胞因子的中性粒细胞，巨噬细胞和嗜酸性粒细胞的运输的贡献。快速和缓慢的再循环RE可以通过特异性Rab鸟苷三磷酸酶（GTP酶）的不同表达模式来检测。感兴趣的细胞因子有很多，包括有效的免疫调节性TNF-α、中性粒细胞趋化因子白细胞介素-8和最近表征的白细胞介素-33，所有这些都由先天免疫细胞分泌。我们还将确定是否需要R-和Q-SNARE，并确定RE与细胞膜融合所需的特定SNARE。我们建议解决各种Rabs和SNARE在从中性粒细胞释放蛋白质货物（包括细胞因子）中的要求。使用高分辨率成像和超微结构研究，我们将绘制的Rabs，SNARE，颗粒和RE相关蛋白在中性粒细胞RE胞吐的精细分布。这项提议的发现将揭示维持免疫力所需的重要先天免疫细胞中一个新的、未发现的膜运输区室。了解RE在先天性免疫细胞中的功能将导致阐明蛋白质货物的新的运输途径，特别是具有重要免疫调节功能的细胞因子。