The study of recycling endosomes in innate immune cells

我的研究项目主要集中在先天免疫细胞中细胞因子运输的机制。细胞因子向细胞表面的运输涉及再循环内体（RE），这是用于将蛋白质货物分选和运输到质膜和从质膜分选和运输的必要隔室。最近，已经发现RE组成性地将细胞因子、肿瘤坏死因子-α（TNF）从高尔基体运输到巨噬细胞中的细胞膜。然而，RE在粒细胞中细胞因子的运输和释放中的存在和功能知之甚少。粒细胞是体内最丰富的循环先天免疫细胞，并具有从血液中迁移到组织中的能力。在组织浸润时，粒细胞释放大量的细胞因子，其具有强效的促炎作用。因此，粒细胞是强大的分泌细胞，在免疫中起着双刃剑的作用。此外，粒细胞具有强大的蛋白质运输系统，可作为研究细胞因子分泌的完美模型。目标 * 该计划的长期目标是确定先天免疫细胞中细胞因子运输的途径。关于先天免疫细胞中细胞因子运输的文献很少，我们有强有力的证据表明这些细胞中通过RE进行细胞因子运输。具体的短期目标是：*1。表征粒细胞中推定的RE。* 2.通过RE和分泌颗粒确定细胞因子转运途径以用于胞吐作用。* 3.表征调节细胞因子从快速和缓慢再循环的RE隔室释放的信号传导途径。*我们建议解决的信号分子GTPases和SNARE的蛋白质货物，包括细胞因子从中性粒细胞的释放的要求。我们将首先描述粒细胞中的RE，确定细胞因子如何通过GTP酶和SNARE进行运输，并确定RE与细胞膜融合所需的调节分子。我们还将评估最近描述的快速和缓慢再循环的RE隔间对先天免疫细胞中细胞因子运输的贡献。感兴趣的细胞因子是有效的免疫调节性TNF，其在细菌脂多糖（LPS）刺激中性粒细胞期间大量分泌。使用高分辨率成像，我们将绘制在中性粒细胞分泌细胞因子期间GTP酶、SNARE、颗粒和RE相关蛋白的精细分布。*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新的、未发现的膜运输区室。了解RE在先天免疫细胞中的功能将有助于阐明蛋白质货物，特别是细胞因子的新运输途径，这些蛋白质货物具有重要的免疫调节功能。

My research program is focused on the mechanisms of cytokine trafficking in innate immune cells. Trafficking of cytokines to the cell surface involves recycling endosomes (REs), an essential compartment for sorting and transportation of protein cargo to and from the plasma membrane. Recently, REs have been discovered to constitutively traffic the cytokine, tumour necrosis factor-alpha (TNF), from the Golgi to the cell membrane in macrophages. However, the presence and function of REs in trafficking and release of cytokines in granulocytes is poorly understood. Granulocytes are the most abundant circulating innate immune cells in the body, and have the ability to transmigrate into tissues from the blood. Upon tissue infiltration, granulocytes release copious quantities of cytokines, which have potent proinflammatory effects. Thus, granulocytes are powerful secretory cells that function as a double-edged sword in immunity. In addition, granulocytes have robust protein trafficking systems that serve as perfect models for studying the secretion of cytokines.***Objectives*** The long-term objective of this program is to determine pathways of cytokine trafficking in innate immune cells. There is a paucity of literature on cytokine trafficking in innate immune cells, and we have strong evidence for cytokine trafficking via REs in these cells. The specific short-term objectives are to:***1. Characterize the putative REs in granulocytes.***2. Determine the cytokine trafficking pathway via REs and secretory granules for exocytosis.***3. Characterize signaling pathways that regulate release of cytokines from rapidly and slowly recycled compartments of REs.*** We propose to resolve the requirements for the signaling molecules GTPases and SNAREs in the release of protein cargo including cytokines from neutrophils. We will first characterize REs in granulocytes, determine how cytokines are trafficked via GTPases and SNAREs, and identify regulatory molecules required for RE fusion with cell membranes. We will also assess the contribution of recently described rapidly and slowly recycled compartments of REs to cytokine trafficking in innate immune cells. The cytokine of interest is the potent immunomodulatory TNF which is secreted in abundance during stimulation of neutrophils by bacterial lipopolysaccharide (LPS). Using high resolution imaging, we will map the fine distribution of GTPases, SNAREs, granule, and RE-associated proteins during neutrophil secretion of cytokines.*** The findings arising from this proposal will reveal new, undiscovered membrane trafficking compartments 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.**