Manipulation of Phagocytosis in Macrophage

描述（由申请人提供）：本研究的长期目标是了解活化巨噬细胞的微生物机制，并设计治疗感染的方法。这些研究的实验模型是单核增生李斯特菌（Listeria monocytogenes, Lm）体外感染小鼠巨噬细胞。在未激活的J774或骨髓来源的巨噬细胞中，细菌在被吞噬后不久就从液泡中逃逸到细胞质中。被干扰素+脂多糖激活的巨噬细胞通过阻止细菌从液泡中逃逸来抑制内化细菌的生长。活化的巨噬细胞的快速逃逸及其直接抑制为鉴定活化的巨噬细胞内必需的杀微生物化学物质提供了功能设置。需要验证的假设是：Lm逃逸到活化的巨噬细胞的细胞质中，通过改变膜运输和将抑制穿孔的活性氧和活性氮中间体传递到液泡中来抑制；并且这些活动是通过控制GTPases Rab5a和Rac2的募集来协调的。本研究项目将通过应用定量显微方法测量细胞内化学，确定活化如何改变巨噬细胞空泡区室。在活化和非活化的巨噬细胞中，液泡和吞噬体成熟的时间将使用活的、lm感染的巨噬细胞的延时、比例荧光显微镜进行量化。各种荧光细胞器标记与含有野生型或突变型Lm的液泡或含有调理红细胞的吞噬体的关联将被量化。标记物将包括黄嘌呤（YFP的一种变体）与肌动蛋白、Rab5a、Rab7、lamp - 1,3磷酸肌苷结合域、Rac1、Rac2、诱导型一氧化氮合酶（iNOS）和gp47 phox（吞噬细胞氧化酶复合物的一种成分）的嵌合体。荧光显微镜将用于定位胆固醇。由Lm穿孔的液泡将被用于检测细菌逃逸到细胞质中的新方法。液泡中活性氧中间体（ROI）和活性氮中间体（RNI）的产生与液泡成熟、穿孔和细菌逃逸有关。为了识别信号复合物，荧光共振能量转移（FRET）显微镜将用于定位活化的Rac1和Rac2，并检测这些蛋白与液泡上的iNOS、gp47phox和Rab gtpase之间的相互作用。FRET化学计量学将用于测量在吞噬体和Lm液泡上的荧光嵌合体的相对浓度。在活化的巨噬细胞中，Rab5a和Rac2对吞噬体成熟和阻止Lm逃逸的贡献将在表达这些分子突变形式的细胞中进行测量，包括显性阴性或构成活性的Rac1， Rac2和Rab5a，以及通过小抑制rna耗尽Rac2的细胞中。随后对液泡中Lm逸出效率的影响将被量化。

DESCRIPTION (provided by applicant): The long range goals of this research are to understand the microbial mechanisms of activated macrophages and to devise methods for therapeutic manipulation of infections. The experimental model for these studies is in vitro infection of murine macrophages with the bacterium Listeria monocytogenes (Lm). In non-activated, J774 or bone marrow-derived macrophages, bacteria escape from vacuoles into cytoplasm shortly after they are internalized by phagocytosis. Macrophages activated with interferon-( plus lipopolysaccharide inhibit growth of internalized bacteria by preventing their escape from the vacuole. The rapid escape and its direct inhibition by the activated macrophage provide a functional setting for identification of essential microbicidal chemistries inside activated macrophages. The hypotheses to be tested are that escape of Lm into cytoplasm of activated macrophages is inhibited by altered membrane trafficking and delivery into the vacuole of reactive oxygen and reactive nitrogen intermediates that inhibit perforation; and that these activities are coordinated by the controlled recruitment of the GTPases Rab5a and Rac2. This research project will determine how activation changes macrophage vacuolar compartments, by applying quantitative microscopic methods for measuring intracellular chemistries. The timing of vacuole and phagosome maturation in activated and non-activated macrophages will be quantified using time-lapse, ratiometric fluorescence microscopy of live, Lm-infected macrophages. The association of various fluorescent organelle markers with vacuoles containing wild-type or mutant Lm or with phagosomes containing opsonized erythrocytes will be quantified. Markers will include chimeras of citrine (a variant of YFP) plus actin, Rab5a, Rab7, LAMP-1, 3 phosphoinositide-binding domains, Rac1, Rac2, inducible nitric oxide synthase (iNOS), and gp47 phox, a component of the phagocyte oxidase complex. Fluorescence microscopy will be used to localize cholesterol. Vacuoles perforated by Lm will be identified using new methods for detecting bacterial escape into cytoplasm. Reactive oxygen intermediate (ROI) and reactive nitrogen intermediate (RNI) generation in vacuoles will be localized relative to vacuole maturation, perforation and bacterial escape. To identify signaling complexes, fluorescence resonance energy transfer (FRET) microscopy will be used to localize activated Rac1 and Rac2, and to detect interactions between those proteins and iNOS, gp47phox and Rab GTPases on vacuoles. FRET stoichiometry will be applied to measure relative concentrations of fluorescent chimeras on phagosomes and Lm vacuoles. The contributions of Rab5a and Rac2 to phagosome maturation and prevention of Lm escape in activated macrophages will be measured in cells expressing mutant forms of those molecules, including dominant negative or constitutively active Rac1, Rac2 and Rab5a, and in cells depleted of Rac2 via small inhibitory RNAs. Consequent effects on the efficiency of Lm escape from vacuoles will be quantified.