Brunel Shan F, Bain Jude M, King Jill, Heung Lena J, Kasahara Shinji, Hohl Tobias M, Warris Adilia
Aberdeen Fungal Group, MRC Centre for Medical Mycology, Institute of Medical Sciences, University of Aberdeen.
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, US.
J Vis Exp. 2017 Apr 19(122):55444. doi: 10.3791/55444.
Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
烟曲霉是一种机会性真菌病原体，可在免疫功能低下的宿主中引起侵袭性感染，病死率很高。针对烟曲霉的免疫反应研究一直受到限制，因为缺乏用于在体外测量特定免疫细胞抗真菌活性的一致且可靠的检测方法。本文描述了一种新方法，使用荧光曲霉报告子（FLARE）分生孢子来评估来自人类供体的原代单核细胞和中性粒细胞对烟曲霉的抗真菌活性。这些分生孢子含有一个基因编码的dsRed报告子，由活的FLARE分生孢子组成性表达，并在外部用Alexa Fluor 633标记，Alexa Fluor 633在吞噬溶酶体内抗降解，从而能够区分活的和死的烟曲霉分生孢子。随后使用视频显微镜和流式细胞术来观察分生孢子与天然免疫细胞之间的相互作用，评估杀真菌活性，同时还提供有关吞噬细胞迁移、吞噬作用和真菌生长抑制的大量信息。这项新技术已经为原代免疫细胞与烟曲霉的宿主 - 病原体相互作用提供了令人兴奋的新见解。需要注意的是，进行该检测所需的实验室设置，包括必要的显微镜和流式细胞术设备，以及处理人类供体血液和基因工程改造真菌的能力。然而，该检测能够产生大量数据，并能揭示抗真菌反应的详细见解。该方案已成功用于研究原代免疫细胞与烟曲霉的宿主 - 病原体相互作用。需要注意的是，进行该检测所需的实验室设置，包括必要的显微镜和流式细胞术设备，以及处理人类供体血液和基因工程改造真菌的能力。然而，该检测能够产生大量数据，并能揭示抗真菌反应的详细见解。该方案已成功用于研究原代免疫细胞与烟曲霉的宿主 - 病原体相互作用。
