Vesicular trafficking mechanisms regulating granulocyte function

项目概述：细胞内囊泡转运对于中性粒细胞生理学的各个方面都是必不可少的， 这种机制的缺陷会导致人类疾病。在中性粒细胞中，囊泡运输与 胞吐作用、吞噬作用、信号传导和NET产生的过程;然而， 调节不同中性粒细胞分泌细胞器的动员需要进一步阐明。我们有 确定了中性粒细胞颗粒运输的几个关键调节因子，包括小的GTTRab 27 a及其 效应器JFC 1和Munc 13 -4。我们现在已经确定WASH是一种调节细胞骨架的中性粒细胞因子 重构、囊泡运输和吞噬体成熟。此外，我们还率先开发了 的系统生物学方法来分析囊泡动力学和肌动蛋白重塑粒细胞。最后我们 已经确定了一种新的内体晚期成熟机制，该机制涉及内体与核蛋白之间的相互作用。 钙传感器Munc 13 -4和晚期内体SNARE蛋白syntaxin 7（STX 7），以调节TLR 9信号传导 和下游中性粒细胞功能。在这里，我们使用创新的定量方法来阐明 与胞吐作用、吞噬作用、晚期内体成熟相关的调节囊泡转运的机制 和中性粒细胞中的NET产生。我们还建议使用我们新设计的小分子抑制剂， Rab 27 a-JFC 1和Munc 13 -4-STX 7结合，以研究囊泡转运机制并阐明 使用全身性炎症和感染的体内模型研究疾病中的中性粒细胞功能。的中心目标 这项授权是为了阐明控制嗜中性粒细胞促炎过程的囊泡转运机制， 开发干扰这些过程的转化方法，并为其使用提供临床前验证 以减轻全身炎症。由于中性粒细胞活化失调对宿主有害， 中性粒细胞分泌蛋白质在内皮细胞损伤中起重要作用， 内毒素血症、败血症和无菌性炎症，这些研究具有重要的生理意义， 潜在的临床应用。我们推测Rab 27 a对囊泡转运的差异调节 并且其效应物是决定特异性中性粒细胞功能和对损伤的反应的基本机制。 我们还提出，特定囊泡转运途径的小分子调节剂将阻止某些细胞的增殖。 在全身炎症过程中中性粒细胞活化的有害后果。为了验证我们的假设， 提出以下具体目标：1）定义差异调节囊泡运输的机制， 在吞噬和NETosis过程中，中性粒细胞颗粒亚群的肌动蛋白依赖性推进和阻断; 2） 通过核内体TLR信号转导建立线粒体核酸传感的分子机制， 中性粒细胞; 3）开发机制和翻译方法，以减少嗜中性粒细胞介导的系统性 炎症而不影响先天免疫反应。拟议的研究将阐明 调节中性粒细胞囊泡运输的机制，并导致治疗炎症性疾病的新策略。

Project Summary: Intracellular vesicular transport is essential for all aspects of neutrophil physiology and defects in this mechanism leads to disease in humans. In neutrophils, vesicular trafficking is associated with the processes of exocytosis, phagocytosis, signaling and NETs production; however, the molecular mechanisms that regulate mobilization of the different neutrophil secretory organelles require further elucidation. We have identified several key regulators of neutrophil granule trafficking, including the small GTPase Rab27a and its effectors JFC1 and Munc13-4. We have now identified WASH as a neutrophil factor that regulates cytoskeleton remodeling, vesicular trafficking and phagosomal maturation. Furthermore, we have pioneered the development of systems biology approaches to analyze vesicular dynamics and actin remodeling in granulocytes. Finally, we have identified a novel mechanism of late endosomal maturation that involves the interaction between the calcium sensor Munc13-4 and the late endosomal SNARE protein syntaxin 7 (STX7), to regulate TLR9 signaling and downstream neutrophil functions. Here, we use innovative quantitative methods to elucidate the mechanisms regulating vesicular transport associated with exocytosis, phagocytosis, late endosomal maturation and NETs production in neutrophils. We also propose to use our newly designed small-molecule inhibitors of Rab27a-JFC1 and Munc13-4-STX7 binding to investigate mechanisms of vesicular transport and to elucidate neutrophil function in disease using in vivo models of systemic inflammation and infection. The central goal of this grant is to elucidate the vesicular transport mechanisms that govern neutrophil pro-inflammatory processes, develop translational approaches to interfere with these processes and provide preclinical validation for their use to attenuate systemic inflammation. Since dysregulated neutrophil activation is injurious to the host and neutrophil secretory proteins play fundamental roles in the damage to the endothelium associated with endotoxemia, sepsis and sterile inflammation, these studies have important physiological significance and potential clinical applications. We hypothesize that the differential regulation of vesicular transport by Rab27a and its effectors is an essential mechanism to determine specific neutrophil functions and responses to insult. We also propose that small-molecule modulators of specific vesicular transport pathways will prevent some of the deleterious consequences of neutrophil activation during systemic inflammation. To test our hypotheses we propose the following Specific Aims: 1) Define the mechanisms that differentially regulate vesicular trafficking, actin-dependent propulsion and blockage of neutrophil granule subsets during phagocytosis and NETosis; 2) Establish the molecular mechanisms of mitochondrial nucleic acid-sensing by endosomal TLR signaling in neutrophils; 3) Develop mechanistic and translational approaches to decrease neutrophil-mediated systemic inflammation without affecting the innate immune response. The proposed research will elucidate the molecular mechanisms regulating neutrophil vesicular transport and lead to new strategies to treat inflammatory diseases.