The study of recycling endosomes in innate immune cells

我的研究重点是细胞因子在先天免疫细胞中的运输机制。细胞因子向细胞表面的运输涉及回收内小体(RES)，这是将蛋白质货物分拣到质膜和从质膜运输出去的基本隔间。最近，人们发现Res能将细胞因子肿瘤坏死因子-α从高尔基体运输到巨噬细胞的细胞膜上。然而，Res在粒细胞运输和释放细胞因子中的存在和功能还知之甚少。粒细胞是体内循环中最丰富的先天免疫细胞，具有从血液向组织中迁移的能力。在组织渗透时，粒细胞释放大量的细胞因子，这些细胞因子具有强大的促炎作用。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，是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细胞因子在先天免疫细胞中的运输途径。关于细胞因子在先天免疫细胞中的转运的文献很少，我们有强有力的证据证明细胞因子在这些细胞中通过RES进行转运。具体的短期目标是： 1.粒细胞中可能存在的RES的特征。 2.确定细胞因子通过RES和分泌颗粒转运的途径。 3.确定调节快速和缓慢循环的RE隔室释放细胞因子的信号通路。 我们建议解决中性粒细胞释放包括细胞因子在内的蛋白质货物时对信号分子、GTP酶和SNARs的要求。我们将首先确定粒细胞中RE的特征，确定细胞因子如何通过GTP酶和SNARs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循环的RE隔室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强大的免疫调节肿瘤坏死因子，它在细菌脂多糖(LPS)刺激中性粒细胞时大量分泌。使用高分辨率成像，我们将绘制GTP酶、SNARS、颗粒和RE相关蛋白在中性粒细胞分泌细胞因子过程中的精细分布。 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新的、未被发现的膜转运间隔。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运输途径，特别是细胞因子，这些途径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.