Genetic and metabolic regulation of macrophage activation at steady state

项目摘要 从根本上讲，了解细胞是如何关闭并保持其静止状态与它是如何 已被激活。这项拟议研究的长期目标是确定细胞内在过程如何控制 并调节体内巨噬细胞的激活状态。因为调节失调和无端激活 免疫系统会引起一系列与不适当的炎症、破译 正常调节免疫激活的分子网络是解决这些健康问题的关键。 挑战。这将导致需要新的知识和技术来利用 巨噬细胞用于疾病预防和治疗。我们正在利用高度集中的 易处理的脊椎动物模型系统Danio Rerio，用于精细的遗传操作，高通量筛选， 以及体内成像来剖析内在代谢信号与巨噬细胞之间的复杂关系 激活。 该提案包括一系列项目，这些项目共同定义了基本的负面监管机构及其 保持天然免疫系统的功能，以维持巨噬细胞的正常平衡。 我们项目的开始基础源于新陈代谢和免疫信号的不断涌现 巨噬细胞的免疫激活途径相交，并在一种 斑马鱼细胞内结节样受体(NLR)。这种新的NLR中的一个基因失活，nlrc3l，导致 巨噬细胞的无端激活可能是由于代谢失调。该提案旨在界定 Nlrc3l的分子相互作用网络来理解这一非常重要的机制 巨噬细胞在正常生物条件下处于检查状态。我们正在采取高度集成的方法， 多水平--使用差异转录组学、蛋白质组学和代谢组学来通知候选基因 以及构成nlrc3l的可能的相互作用和效应器的通路，并使用 基因突变和生化研究。该提案还将利用前向基因筛查的力量来 发现与nlrc3l类似的其他基因，阻止在稳定状态下作用的巨噬细胞激活。 或者是全新的路径。我们设计了一种创新的筛查方法来评估巨噬细胞 使用活细胞报告器激活标记IRG1。最后，该提案将考察其影响 斑马鱼巨噬细胞激活中脂肪和葡萄糖代谢途径的遗传分析和应用 化学筛选。这项工作将受益于与专家小组的合作，将我们的发现扩展到 老鼠和人类的模型。综上所述，这些项目为理解 先天性免疫系统如何受到控制的遗传和新陈代谢基础，并将影响 我的实验室的方向远远超出了米拉五年的资助。

Project Summary Understanding how a cell is switched off and maintains its quiescence is fundamentally as important as how it is activated. The long-term goal of the proposed research is to determine how cell-intrinsic processes control and modulate activation states of macrophages in vivo. Because dysregulation and unprovoked activation of the immune system cause a host of human diseases associated with inappropriate inflammation, deciphering the molecular networks regulating immune activation normally is critical for addressing these health challenges. This will lead to new knowledge and technologies needed to harness the properties of macrophages for disease prevention and treatment. We are leveraging the unique advantages of the highly tractable vertebrate model system, Danio rerio, for exquisite genetic manipulations, high throughput screening, and in vivo imaging to dissect the complex relationship between intrinsic metabolic signaling and macrophage activation. The proposal encompasses a series of projects that collectively define essential negative regulators and their functions for keeping the innate immune system in check to maintain a normal equilibrium in macrophages. The starting basis of our projects stems from emerging evidence that metabolic and immune signaling pathways intersect to shape immune activation in macrophages, and a discovery of a null mutation in an intracellular NOD-like receptor (NLR) in zebrafish. A gene inactivation in this novel NLR, nlrc3l, causes unprovoked macrophage activation possibly due to metabolic dysregulation. The proposal seeks to define the network of molecular interactions of nlrc3l to understand this very important mechanism that keeps macrophages in check under normal biological conditions. We are taking a highly integrated approach at multiple levels-- using differential transcriptomics, proteomics, and metabolomics to inform candidate genes and pathways that constitute possible interactors and effectors of nlrc3l, and validating interactions using genetic mutants and biochemical studies. The proposal will also use the power of a forward genetic screen to discover additional genes akin to nlrc3l that prevent macrophage activation at steady state that act in the same or completely new pathways. We designed an innovative assay for the screen to assess macrophage activation using a live-cell reporter for an activation marker irg1. Finally, the proposal will examine the influence of lipid and glucose metabolic pathways on macrophage activation in zebrafish using genetic analyses and chemical screening. This work will benefit from collaboration with an expert group in extending our findings to mouse and human models. Taken together, these projects provide the important foundation for understanding the genetic and metabolic basis of how the innate immune system is kept in check, and will impact the direction of my lab far beyond the 5 years of MIRA funding.