Name: assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy
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Description: Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

This work was supported by the National Institute of Allergy and Infectious Disease Grant R01AI079253 (to B.H.S.), and by the National Cancer Institute Cancer Center Support Grant CA016056 to Roswell Park Cancer Institute.

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. Waiting 10 days after administration allows for clearance of all labeled cells from circulation leaving only alveolar macrophage stably labeled with PKH268,9. PKH26 is a red fluorochrome that has excitation (551 nm) and emission (567 nm) characteristics compatible with rhodamine or phycoerythrin detection systems. Any labeling procedure can introduce artifact, including loss of viability. However, since we use the same approach for labeling of macrophages in different mouse genotypes (e.g., WT vs. NADPH oxidase-deficient), the effects of these artifacts would be uniform.
<ol>
	<li>Dilute stock solution of PKH26 (1 x 10-3 M in ethanol) 1:5 in diluent C provided by manufacturer.</li>
	<li>Load 100 &#181;l of diluted PKH26 into a 1/2 ml insulin syringe (29 G x 1/2&#8221;) for each mouse.</li>
	<li>Administer to mice by i.v. injection (either tail vein or retro orbital) at least 10 days prior to harvest.</li>
</ol>
2. Harvesting Alveolar Macrophages by Bronchoalveolar Lavage (BAL)
The number of mice to use for an experiment can vary. A typical yield from an unstimulated mouse is in the range of 3-5 x 105 alveolar macrophages, however this number can vary greatly based on factors such as size of the mouse, experience of the person performing the lavage, and the number of instillations of lavage fluid and the volume withdrawn from lungs. In this protocol lung lavage is performed with a closed thoracic cavity which along with repeated 1 ml lavages helps to increase cell yield.
<ol>
	<li>Euthanize mouse by administering a lethal dose of avertin anesthetic (12.5 mg) intraperitoneally (i.p.) using aseptic conditions to keep the solution sterile.</li>
	<li>Spray mouse thoroughly with 70% ethanol.</li>
	<li>Remove skin from the cranial end of animal exposing the thorax (Figure 1A).
	<ol>
		<li>Make a small cut in skin just beneath the diaphragm.</li>
		<li>Insert finger into the cut and pull skin away from the cranial end of the animal (Figure 1A).</li>
		<li>Push up slightly on the head, extending the ventral side of the neck (Figure 1A).</li>
	</ol>
	</li>
	<li>Cut a small hole through the rib cage into the right side of the thorax to deflate the lungs and also allow visualization of lungs during the following steps.</li>
	<li>Locate the trachea, and carefully dissect away surrounding tissue.
	<ol>
		<li>Remove the salivary glands, sternohyoid muscles and larynx, etc. with curved forceps.</li>
		<li>Carefully lift with a curved forceps and cut away with scissors the thin tissue covering the trachea (adventitia) revealing the underlying ringed cartilage structure.</li>
	</ol>
	</li>
	<li>Insert catheter into trachea.
	<ol>
		<li>Using curved forceps, place a suture behind (dorsal to) the trachea and pull a 10 cm length through the opening.</li>
		<li>Starting above the cricoid (the thickened ring of cartilage) carefully guide a 22 G catheter down the trachea stopping where the trachea disappears behind the sternum.</li>
		<li>Firmly tie suture around trachea and catheter for a snug fit. Remove needle from catheter.</li>
	</ol>
	</li>
	<li>Assemble 4-way stopcock.
	<ol>
		<li>Fill one 6 ml syringe with DPBS, 1% FBS and attach to 4-way stopcock as indicated in Figure 1B.</li>
		<li>Attach an empty 6 ml syringe at the indicated position on the 4-way stopcock (Figure 1B).</li>
		<li>Attach open port on the 4-way stopcock to catheter (Figure 1B). Be careful to not dislodge catheter from trachea.</li>
	</ol>
	</li>
	<li>Collect lavage fluid from lungs.
	 Note: Investigators may opt to use lower volumes of lavage fluid to minimize the possibility of damage to the tissue. It is important that the same approach be consistently applied in all mouse groups.
	<ol>
		<li>Turn the handle on the stopcock so the empty syringe is closed and slowly inject ~1 ml DPBS, 1% FBS to inflate lungs. Observe lung inflation through the hole cut in step 2.4.</li>
		<li>Be careful NOT to overinflate the lungs.</li>
		<li>Turn the handle on the stopcock so that the full syringe is closed, and carefully withdraw the fluid from the lungs.</li>
		<li>Observe the lungs deflate through the hole cut in step 1.4. The catheter may need to be moved slightly back or forth to get a good draw. Take special precaution to keep the catheter within the trachea.</li>
		<li>Repeat steps 2.8.1-2.8.4 5-6x until all the DPBS, 1% FBS has been injected and withdrawn from the lungs. Note: recovery of lung fluid will not be 100% of injected fluid.</li>
	</ol>
	</li>
	<li>Empty syringe with lavage fluid into a 50 ml conical tube. Keep cells at room temperature.</li>
	<li>Pellet cells by centrifuging at 300 x g for 5 min at room temperature and pour off supernatant. This can be saved for cytokine analysis if desired.</li>
	<li>Resuspend cells in 1-5 ml RPMI 1640 with 10% FBS and count on a hemocytometer. In the unstimulated mouse, &#62; 95% of cells harvested by BAL are expected to be macrophages, which can be confirmed by cytology.</li>
</ol>
3. Culturing and Harvesting Fungus
The Aspergillus fumigatus used in this protocol expresses GFP throughout all stages of its life cycle: spores, germlings and hyphae. This fungal strain was provided by Dr. Margo Moore, Simon Fraser University, Burnaby, BC, Canada.
<ol>
	<li>Plate conidia of an Aspergillus fumigatus strain expressing GFP on Sabouraud brain heart infusion (BHI) agar slants with Chloramphenicol (0.05 g/L) and Gentamicin (0.5 g/L).</li>
	<li>Incubate in the dark loosely capped for 7 to 10 days at room temperature.</li>
	<li>Harvest conidia by washing slant with 10 ml of 0.01% Tween 20 in DPBS. Lightly scrape the fungus with the stripette to loosen. Rinse the slant several times to increase yield.</li>
	<li>Pass conidial suspension through a 100 &#181;m cell strainer into a 50 ml conical.</li>
	<li>Pellet conidia by centrifuging at 400 x g for 10 min.</li>
	<li>Resuspend conidia in 50 ml DPBS and count using a hemocytometer.</li>
	<li>Wash twice with DPBS and dilute to desired concentration.</li>
</ol>
4. Fungal Challenge and Confocal Microscopy
To investigate the role of macrophage NADPH oxidase in host defense, the ability of isolated alveolar macrophages from mice of three genotypes to control the growth of phagocytosed Aspergillus spores was evaluated. The genotypes used in this study include; 1) wild-type mice, 2) Ncf1*/* Mn- mice with a naturally occurring Ncf1 mutation (Ncf1 encodes for p47phox, a required component of the phagocyte NADPH oxidase) leaving them NADPH oxidase deficient, and 3) Ncf1*/* Mn+ transgenic mice (MN denotes the transgene harboring wildtype Ncf1 under the control of the human CD68 promoter) where NADPH oxidase activity has been reconstituted in the monocyte/macrophage population10,11.
Confocal microscopy is best suited for this assay due to the presence and growth of non-phagocytosed conidia on the slides. The confocal microscope will allow for visualization of individual planes within the Z-axis, and enable differentiation between fungus growing around rather than inside macrophage. All confocal images are acquired using a 63X oil immersion lens. Z-stacks are acquired with an electronic zoom factor of 2.5. The thickness of a Z-stack is determined using DAPI and bright field images to locate the top and bottom of the field. Z-stacks ranged from 14 to 24 &#181;m with individual slices ~1 &#181;m thick. For quantification of macrophages, single scans of 1X electronic zoom were performed on 10 fields per genotype. Intact macrophages were identified based on PKH26 and DAPI staining of the membrane and nucleus respectively. Conidia were identified based on FITC expression and bright field image.
<ol>
	<li>Prepare sterile coverslips and plates
	<ol>
		<li>Under a Biological Safety Cabinet, soak 22 x 22 mm glass coverslips in 70% ethanol for 5-10 min.</li>
		<li>Rinse coverslips in sterile PBS.</li>
		<li>Using sterile forceps, gently place a single coverslip in the bottom of each well of a sterile singly packed 6-well plate.</li>
	</ol>
	</li>
	<li>Seed ~1 x 106 harvested PKH26 labeled alveolar macrophages suspended in 300 &#181;l RPMI 1640, 10% FBS onto each coverslip. The exact number may vary depending on the harvest yield.</li>
	<li>Allow macrophage to adhere by incubating at 37 &#176;C with 5% CO2 overnight.</li>
	<li>The following morning, add ~1 x 107 GFP-expressing A. fumigatus conidia suspended in 100 &#181;l pre-warmed (37 &#176;C) RPMI 1640, 10% FBS.</li>
	<li>Return plate to 37 &#176;C incubator with 5% CO2.</li>
	<li>Cells should be fixed for a minimum of 2 hr at room temperature or overnight at 4 &#176;C. At 3, 7, and 14 hr fix cells by adding an equal volume of 2% formaldehyde to each well (final concentration 1% formaldehyde). Place plates for the early time points at 4 &#176;C overnight. For the final time point (14 hr), fix cells at room temperature for 2 hr.</li>
	<li>After fixation, gently remove coverslips using forceps and rinse in DPBS.</li>
	<li>Remove excess fluid by placing the edge of the coverslip on a folded Kimwipe.</li>
	<li>Apply 6 &#181;l fluorescence mounting medium with DAPI to a glass microscope slide.</li>
	<li>Lay one edge of the coverslip on the slide and gently drop with cell side down into the mounting medium. Mounting medium will spread and form an even layer beneath the coverslip. There is no need to press on the coverslip.</li>
	<li>Seal edges of coverslip with clear nail polish and allow to air dry.</li>
	<li>Store prepared slides in a slide box (protected from light) at room temperature until ready to analyze by confocal microscopy.</li>
</ol>

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The authors have nothing to disclose.

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.,&#160;recruited neutrophils) that defend against Aspergillus in vivo.
<p class="jo...

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 defense1-3, the importance of NADPH oxidase in macrophages is not well defined. Prior studies have shown that alveolar macrophages ingest and kill Aspergillus spores, whereas neutrophils principally target the hyphal stage5. However, there have been conflicting results as to the role of NADPH oxidase in macrophage in controlling the growth of A. fumigatus spores6,7.
The goal of this method was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus spores. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. To most closely model the host response to inhaled fungi, we used alveolar macrophages harvested by bronchoalveolar lavage from unstimulated mice immediately following sacrifice. The use of isolated alveolar macrophages enabled us to focus on their antifungal activity in the absence of other immune cells (e.g.,&#160;recruited neutrophils) that defend against Aspergillus in vivo. In this method, alveolar macrophages were stained in vivo prior to harvest so as to minimize the amount of post-harvest manipulation.
Another advantage to this protocol is the use of a GFP-expressing A. fumigatus strain. This fungus expresses GFP from the conidial stage through the hyphal stage and has no need for further staining. To obtain images with greater detail, we chose confocal microscopy which enables the reconstruction of three-dimensional structures from the obtained images. Three-dimensional reconstruction gives the ability to differentiate between hyphae growing around rather than in or through a macrophage. Conidia can also be precisely distinguished as intracellular versus extracellular.

<table><tbody><tr><td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td><td>Sigma</td><td>PKH26GL-1KT</td><td></td></tr><tr><td>2,2,2 Tribromoethanol</td><td>Sigma</td><td>T48402</td><td>Used to make AvertinAnesthetic</td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma</td><td>A-1685</td><td>Used to make AvertinAnesthetic</td></tr><tr><td>Dulbecco&amp;#039;s Phosphate Buffered Saline (without calcium and magnesium)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>A0171</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>P91444</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>22 G x 1.00 inch i.v. catheter</td><td>BD</td><td>381523</td><td>Insyte Autoguard Winged</td></tr><tr><td>Insulin syringes</td><td></td><td></td><td></td></tr><tr><td>6 ml Syringes</td><td></td><td></td><td></td></tr><tr><td>4-way stopcock</td><td>Fisher Scientific</td><td>50-700-077</td><td></td></tr><tr><td>Suture thread</td><td></td><td></td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>gauze sponges (4 inch square)</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 with L-glutamine</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30396.03</td><td>heat inactivated</td></tr><tr><td>Hema 3 Stain Set</td><td>Fisher Scientific</td><td>22-122-911</td><td></td></tr><tr><td>Cytoseal 60</td><td>Thermo Scientific</td><td>8310-4</td><td></td></tr><tr><td>Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td><td>BD</td><td>297252</td><td></td></tr><tr><td>&lt;em&gt;Aspergillus fumigatous&lt;/em&gt; strain expressing green fluorescent protein</td><td></td><td></td><td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td></tr><tr><td>100 &amp;mu;m Cell strainers</td><td>BD Falcon</td><td>64753-00</td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Biological Safety Cabinet Class II</td><td></td><td></td><td></td></tr><tr><td>Sorval ST40 Centrifuge with swing bucket rotor</td><td></td><td></td><td></td></tr><tr><td>Leica DM1000 light microscope</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Cytospin 2 centrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td></td><td></td><td>37 &amp;deg;C, 5%&amp;nbsp; CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>22 x 22 mm micro cover glass</td><td>VWR</td><td>48366-227</td><td>glass coverslips</td></tr><tr><td>6-well Cell culture plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VWR</td><td>BDH0502-1LP</td><td></td></tr><tr><td>Vectashield mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Leica TCS SP2 system with a laser point scanner</td><td></td><td></td><td>Mounted on DMIRE2 fluorescence microscope</td></tr></tbody></table>

assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy

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.

Collecting alveolar macrophage by BAL from unstimulated mice will result in a &#62; 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 ide...

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

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

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

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Immunology and Infection

11.4K Views.  Roswell Park Cancer Institute. A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

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

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Bioengineering

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

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.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Medicine

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled&#160;conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

Fungal Clearance Efficiency of Macrophages

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the&#160;fungal clearance efficiency of macrophages.

Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia.

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial ...

Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells

Source: Brunel, S. F. et al., <a href="https://app.jove.com/v/55444">Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus.</a>J. Vis. Exp. (2017)
This video demonstrates live cell imaging for the analysis of antifungal activity in human neutrophils using fluorescent Aspergillus reporter conidia. Initially, both red and magenta fluorescence are detected within neutrophils, while red fluorescence decreases over time, indicating the degradation of conidia and confirming the antifungal activity of neutrophils.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

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

Summary

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Chapters in this video

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells