Manipulation of Phagocytosis in Macrophage

描述(申请人提供)：这项研究的长期目标是了解激活的巨噬细胞的微生物机制，并设计出治疗感染的方法。这些研究的实验模型是小鼠巨噬细胞体外感染单核细胞增生性李斯特氏菌(Lm)。在非激活的J774或骨髓来源的巨噬细胞中，细菌通过吞噬作用内化后不久就从空泡逃逸到细胞质中。用干扰素和脂多糖激活的巨噬细胞通过阻止内化细菌从液泡中逃逸来抑制其生长。激活的巨噬细胞的快速逃逸及其直接抑制为鉴定激活的巨噬细胞内的基本杀菌化学物质提供了一个功能环境。有待检验的假设是，通过改变膜转运和向液泡输送抑制穿孔的活性氧和活性氮中间体，可以抑制LM逃脱到激活的巨噬细胞的细胞质中；这些活动是通过GTP酶Rab5a和rac2的控制募集来协调的。这项研究项目将通过应用定量显微镜方法测量细胞内化学成分，确定激活如何改变巨噬细胞的空泡室。激活的和非激活的巨噬细胞的空泡和吞噬小体成熟的时间将使用活的LM感染的巨噬细胞的时间推移比率荧光显微镜来量化。各种荧光细胞器标记物与含有野生型或突变型LM的空泡或含有调理红细胞的吞噬小体的关联将被量化。标志物将包括柑橘碱(YFP的变种)与肌动蛋白、Rab5a、Rab7、LAMP-1、3个磷脂酰肌醇结合结构域、rac1、rac2、诱导型一氧化氮合酶(INOS)和吞噬细胞氧化酶复合体的组成部分gp47Phox的嵌合体。荧光显微镜将被用来定位胆固醇。被LM穿孔的空泡将使用新的方法来检测细菌逃逸到细胞质中。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。为了识别信号复合体，将使用荧光共振能量转移(FRET)显微镜来定位激活的rac1和rac2，并检测这些蛋白与空泡上的iNOS、gp47Phox和Rab GTP酶之间的相互作用。FRET化学计量学将用于测量吞噬小体和LM液泡上荧光嵌合体的相对浓度。Rab5a和rac2对激活巨噬细胞吞噬小体成熟和防止LM逃逸的贡献将在表达这些分子的突变形式的细胞中进行测量，包括显性阴性或固有活性的rac1、rac2和Rab5a，以及通过小的抑制性RNA去除rac2的细胞。随后对LM从空泡中逃逸的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.