Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Summary

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

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 defense^1-3, 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.

Introduction

Alveolar macrophages are the first-line phagocytic cells that encounter inhaled microbes. Macrophages recognize Aspergillus motifs by pathogen recognition receptors, ingest and limit the growth of inhaled spores (conidia), and initiate inflammatory responses. The phagocyte NADPH oxidase converts molecular oxygen to superoxide anion and downstream reactive oxygen intermediates (ROIs). Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe bacterial and fungal infections and by excessive inflammatory responses.

Although NADPH oxidase is critical for neutrophil-mediated host defense

Protocol

All procedures performed on animals in this study were approved by the Animal Care and Use Committee at Roswell Park Cancer Institute and complied with all state, federal, and National Institutes of Health regulations.

1. In vivo Macrophage Labeling

Note: PHK26 is a lipophilic dye that will be taken up by phagocytic cells both in circulation and in the tissues when administered intravenously (i.v.). PKH26 was used for in vivo labeling to minimize the amount of post-harvest manipulation of the macrophage. PKH-26 has the advantage of stably accumulating in alveolar macrophages over prolonged periods....

Results

Collecting alveolar macrophage by BAL from unstimulated mice will result in a > 95% pure population based on cytology. The 3 and 7 hr (Figure 2) time points precede the hyphal stage of fungal growth and allow for a comparison of phagocytic efficiency of conidia among genotypes. The 14 hr time point is where genotypes can be compared with regard to the ability to inhibit the transition of phagocytosed conidia to the tissue-invasive hyphal stage (Figure 3). Intact macrophage can be iden.......

Discussion

Using this approach for ex vivo analysis of the antifungal activity of macrophages together with in vivo fungal challenge, we previously demonstrated that NADPH oxidase in macrophages plays an important role in defense against A. fumigatus⁴. The use of isolated alveolar macrophages enables us to focus on their antifungal activity in the absence of other immune cells (e.g., recruited neutrophils) that defend against Aspergillus in vivo.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling	Sigma	PKH26GL-1KT
2,2,2 Tribromoethanol	Sigma	T48402	Used to make AvertinAnesthetic
2-methyl-2-butanol	Sigma	A-1685	Used to make AvertinAnesthetic
Dulbecco's Phosphate Buffered Saline (without calcium and magnesium)	Corning	21-031-CV
NH4Cl	Sigma	A0171	Used to make ACK red cell lysis buffer
KHCO3	Sigma	P91444	Used to make ACK red cell lysis buffer
EDTA	Sigma	E6758	Used to make ACK red cell lysis buffer
22 G x 1.00 inch i.v. catheter	BD	381523	Insyte Autoguard Winged
Insulin syringes
6 ml Syringes
4-way stopcock	Fisher Scientific	50-700-077
Suture thread
50 ml conical centrifuge tubes
gauze sponges (4 inch square)
RPMI 1640 with L-glutamine	Corning	10-040-CV
Fetal Bovine Serum	Hyclone	SH30396.03	heat inactivated
Hema 3 Stain Set	Fisher Scientific	22-122-911
Cytoseal 60	Thermo Scientific	8310-4
Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants	BD	297252
Aspergillus fumigatous strain expressing green fluorescent protein			provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada
100 μm Cell strainers	BD Falcon	64753-00
Scissors
Forceps
Biological Safety Cabinet Class II
Sorval ST40 Centrifuge with swing bucket rotor
Leica DM1000 light microscope
Hemocytometer
Cytospin 2 centrifuge	Thermo Scientific
Cell culture incubator			37 °C, 5%  CO2
22 x 22 mm micro cover glass	VWR	48366-227	glass coverslips
6-well Cell culture plates	Corning	3506
10% Neutral Buffered Formalin	VWR	BDH0502-1LP
Vectashield mounting medium with DAPI	Vector Laboratories	H-1200
Leica TCS SP2 system with a laser point scanner			Mounted on DMIRE2 fluorescence microscope