The study of recycling endosomes in innate immune cells

我的研究计划集中在研究循环内含体(RES)在免疫细胞中的作用。循环内小体(Res)是一种特殊的分泌室，通过胞内和胞外途径回收细胞表面受体。这些分泌细胞器在许多细胞类型中都有很好的特征。最近，人们发现Res在蛋白质转运中具有多种功能，而不仅仅是在运输细胞表面受体方面发挥作用。在巨噬细胞中，Res被发现结构性地将细胞因子肿瘤坏死因子-α(TNF-α)从高尔基体运输到细胞膜，巨噬细胞是一种天然免疫细胞。然而，Res的存在及其在免疫细胞中运输和释放细胞因子中的作用一般研究得很少。许多免疫细胞，包括中性粒细胞和嗜酸性粒细胞(粒细胞)，是复杂的高度颗粒化的分泌细胞，能够释放大量的细胞因子。粒细胞是体内最丰富的循环先天免疫细胞，是一种强大的分泌细胞，在免疫中起着双刃剑的作用。这些特化的分泌细胞有能力转化到组织中，并释放大量的细胞因子。因此，免疫细胞具有强大的蛋白质运输系统，是研究RE功能和细胞因子胞吐的理想模型。目的本研究的长期目标是确定一种新的分泌室Res在先天免疫细胞的细胞因子转运中的存在和功能。关于稀土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稀土在这些细胞中的存在和功能。具体的短期目标是：1.使用检测工具和试剂来表征肿瘤坏死因子-α在先天免疫细胞中的转运。2.确定通过RES运输的其他细胞因子，以便在先天免疫细胞中分泌。3.研究调节天然免疫细胞释放细胞因子的信号通路。具体地说，我们将评估最近描述的快速和缓慢循环的RE隔室对中性粒细胞、巨噬细胞和嗜酸性粒细胞的细胞因子运输的贡献。通过Rab鸟苷三磷酸酶(GTP酶)的不同表达模式，可以检测到快速和缓慢的RE循环。感兴趣的细胞因子很多，包括强大的免疫调节肿瘤坏死因子-α，中性粒细胞趋化因子白介素8，以及最近表征的白介素33，所有这些都是由天然免疫细胞分泌的。我们还将确定是否需要R-和Q-圈套，并确定RE与细胞膜融合所需的特定圈套。我们建议解决各种RABS和陷阱在中性粒细胞释放包括细胞因子在内的蛋白质货物中的要求。利用高分辨率成像和超微结构研究，我们将绘制RABS、SNARS、颗粒和RE相关蛋白在中性粒细胞RE胞吐过程中的精细分布。这一提议的发现将揭示维持免疫所需的重要先天免疫细胞中一个新的、未被发现的膜运输隔间。了解Res在先天免疫细胞中的功能将有助于阐明蛋白质货物的新的运输途径，特别是服务于基本免疫调节功能的细胞因子。

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.