Dissection of Macrophage Antifungal Activity against Aspergillus fumigatus

巨噬细胞抗烟曲霉抗真菌活性的剖析

基本信息

批准号：
8584085
负责人：
TOBIAS M HOHL
金额：
$ 20.77万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-07 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8584085
关键词：
Accounting Alveolar Macrophages Antifungal Agents Aspergillosis Aspergillus Aspergillus fumigatus Autoimmunity Biological Biological Assay Breathing Candidate Disease Gene Cells Data Defense Mechanisms Disease Dissection Experimental Designs Filament Fluorescence Gene Silencing Germination Goals Human Hyphae Imagery Immune In Vitro Individual Industrial fungicide Infection Injury Knowledge Leukocytes Lung Malignant Neoplasms Measures Mediating Medical Technology Metalloproteases Methods Modeling Molds Molecular Morbidity - disease rate Mus Mycoses Myelogenous NADPH Oxidase Neutrophil Infiltration Opportunistic Infections Organ failure Patients Process RNA Interference Reporter Reproduction spores Respiratory Tract Infections Role Side Site Sorting - Cell Movement Staging Testing Therapeutic Tissues Tube Work base cell killing cell type cohort cytotoxic fighting fungus immune activation improved in vitro activity in vivo innate immune function innovation killings macrophage mortality neutrophil novel prevent public health relevance research study respiratory small hairpin RNA transmission process uptake

项目摘要

DESCRIPTION (provided by applicant): Airborne conidia (spores) represent infectious propagules that are responsible for transmission of major human mycoses. Humans inhale ubiquitous Aspergillus fumigatus conidia on a daily basis. Conidial germination into tissue-invasive hyphae leads to invasive aspergillosis (IA), a devastating cause of infectious morbidity and mortality in patients with impaired respiratory innate immune function. Although conidial engulfment and killing are hallmarks of alveolar macrophage (AM) function during respiratory infection, antifungal effector mechanisms of these cells remain poorly defined, since assays that measure alveolar macrophage conidiacidial activity in the lung have been difficult to achieve. To dissect AM conidiacidal activity, we developed a fluorescent Aspergillus reporter (FLARE) strain that, upon conidial uptake, tags AMs with a fluorescent signature. Conidial killing induces a change in the fluorescence signature, enabling us to observe and quantify cell type-specific conidial killing in the lung and test tube. We harness the FLARE strain to demonstrate that AMs employ distinct conidiacidal mechanisms and implicate matrix metalloprotease 12 (MMP12) in this process. With this approach, we examine a model of AM function that integrates matrix metalloprotease 12 as a significant cytotoxic effector mechanism to achieve fungicidal activity. The hypothesis that underlies this proposal is that AM-specific factors - that include MMP12 - control A. fumigatus conidial germination at the earliest stages of respiratory fungal infection. The aims will (1) investigate MMP12 as a major effector of AM conidiacidal activity and (2) integrate alternate AM conidiacidal mechanisms using a functional RNAi-based approach. The experimental design incorporates both a candidate gene (MMP12) and a systematic discovery approach to dissect AM antifungal activity. These studies will define matrix metalloproteolytic activity as a potential novel antifungal effector mechanism and will identify candidate genes that regulate the full spectrum of AM conidiacidal activity.

描述（由申请人提供）：空气中的分生孢子（孢子）代表传染性繁殖体，负责传播主要的人类真菌病。人类每天吸入无处不在的烟曲霉分生孢子。分生孢子萌发为组织侵袭性菌丝会导致侵袭性曲霉病 (IA)，这是呼吸道先天免疫功能受损患者感染发病和死亡的毁灭性原因。尽管分生孢子吞噬和杀灭是呼吸道感染期间肺泡巨噬细胞（AM）功能的标志，但这些细胞的抗真菌效应机制仍不清楚，因为测量肺中肺泡巨噬细胞分生孢子活性的测定很难实现。为了剖析 AM 分生孢子活性，我们开发了一种荧光曲霉报告基因 (FLARE) 菌株，该菌株在分生孢子摄取后，用荧光信号标记 AM。分生孢子杀灭引起荧光特征的变化，使我们能够观察和量化肺和试管中细胞类型特异性分生孢子杀灭。我们利用 FLARE 菌株来证明 AMs 采用不同的分生孢子机制，并在此过程中暗示基质金属蛋白酶 12 (MMP12)。通过这种方法，我们研究了 AM 功能模型，该模型将基质金属蛋白酶 12 整合为重要的细胞毒性效应机制，以实现杀菌活性。这一提议的假设是 AM 特异性因子（包括 MMP12）在呼吸道真菌感染的最早阶段控制烟曲霉分生孢子的萌发。目标将 (1) 研究 MMP12 作为 AM 分生孢子活性的主要效应子，以及 (2) 使用基于功能性 RNAi 的方法整合替代的 AM 分生孢子机制。实验设计结合了候选基因 (MMP12) 和系统发现方法来剖析 AM 抗真菌活性。这些研究将基质金属蛋白水解活性定义为一种潜在的新型抗真菌效应机制，并将鉴定调节全谱 AM 分生孢子活性的候选基因。