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Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy
Published on: July 9, 2014
Assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy
Melissa J Grimm1, Anthony C D'Auria1, Brahm H Segal2
1Department of Medicine, Roswell Park Cancer Institute.
Abstract:
The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.
Insights
This study shows that NADPH oxidase in alveolar macrophages is crucial for controlling Aspergillus fungal growth after inhalation. This research details a method to assess this macrophage immune function against fungal spores.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- The lungs are constantly exposed to microbes, with alveolar macrophages acting as the first immune responders to inhaled fungi like Aspergillus.
- Pathogen recognition receptors on immune cells trigger inflammatory responses upon detecting microbial motifs.
- Phagocyte NADPH oxidase generates reactive oxygen intermediates essential for host defense, but its role in macrophages is not fully understood.
Purpose of the Study:
- To investigate the specific role of NADPH oxidase in macrophages during host defense against Aspergillus fumigatus.
- To establish and describe a method for evaluating the capacity of mouse alveolar macrophages to inhibit fungal growth.
Main Methods:
- Mouse alveolar macrophages (AMs) were stained in vivo.
- Ten days post-staining, AMs were isolated using bronchoalveolar lavage (BAL).
- Isolated AMs were cultured, exposed to green fluorescent protein (GFP)-expressing A. fumigatus spores, and analyzed via confocal microscopy to quantify fungal growth control.
Main Results:
- NADPH oxidase in alveolar macrophages was found to effectively control the growth of phagocytosed A. fumigatus spores.
- The study successfully established a method to assess this macrophage-mediated antifungal activity.
Conclusions:
- NADPH oxidase plays a significant role in the innate immune defense of alveolar macrophages against Aspergillus fumigatus.
- The developed methodology provides a valuable tool for studying macrophage function in controlling fungal infections.
