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.


Introduction
Aspergillus fumigatus is an opportunistic fungal pathogen and the most common fungal cause of invasive lung infections in the immunocompromised host 1,2 . Understanding how host immune cells recognize and eliminate Aspergillus is an important area of fungal research, however, the currently available fungicidal assays have significant limitations. Commonly used methods of measuring fungicidal activity include colorimetric assays and determination of colony forming units 3,4 . However, such methods provide information on fungal viability at a single time point rather than viewing the dynamic interaction between host and pathogen. Accordingly, these methods cannot take into account whether a fungus has been killed or simply (temporarily) restricted in growth or metabolic activity. We have developed a method capable of observing fungicidal activity directly whilst simultaneously capturing detailed information regarding phagocytosis, phagocyte migration and inhibition of fungal growth.
Previously, a protocol was published using real-time imaging as a means to measure phagocytosis of Candida albicans by a mouse macrophage cell line 5 . This concept has now been developed further to address the knowledge gap in our understanding of Aspergillus interactions with immune cells by utilizing FLuorescent Aspergillus REporter (FLARE) conidia 6,7,8

Protocol
The protocol for obtaining neutrophils and peripheral blood mononuclear cells (PBMC) is based on previously published methods 9 . The use of blood samples from healthy volunteers has been approved by the human research ethics committee of the University of Aberdeen. Before starting make sure all the ethical approvals are in place for drawing blood from healthy volunteers and/or patients for the purpose of the described experiment. Keep cells on ice where possible to increase their survival.

A. fumigatus Culture and Conditions
1. Prepare glucose minimal agar medium as described 10 .
1. Adjust media to pH 6.5 using 1 M NaOH and autoclave at 120 °C for 20 min. 2. Allow to cool to 65 °C. 3. Working in a sterile flow hood, pour 30 mL of cooled media into a T75 flask and allow to cool with the neck of the flask resting on a 25 mL pipette to create an agar slope.

A. fumigatus Labeling and Preparation
Note: When performing this assay for the first time, prepare the parental Af293-A. fumigatus strain. Take samples of both strains in microcentrifuge tubes and label only one of each strain as described below. This will allow one to have control conidia with no color and conidia with a single color, either dsRed or AF633, to test the setup and equipment. 6. Centrifuge for 10 min at 9,300 x g and carefully remove the supernatant. Resuspend the conidial pellet in 1 mL of 100 mM Tris-HCl pH 8.0 for 1 h to deactivate free-floating biotin. 7. Centrifuge for 10 min at 9,300 x g and carefully remove the supernatant. Wash the pellet twice with 1 mL of sterile PBS and resuspend the pellet in 1 mL of PBS. 8. Dissolve 1 mg of Streptavidin-AF633 in 0.5 mL of PBS to make a 2 mg/mL stock solution. Add 10 µL of 2 mg/mL streptavidin-AF633 per 1 mL of conidial suspension and incubate for 40 min at room temperature on a rocker covered in aluminum foil. The remaining Streptavidin-AF633 can be frozen in aliquots for use in subsequent experiments. 9. Centrifuge for 10 min at 9,300 x g and carefully remove supernatant. Resuspend the conidial pellet in 1 mL of PBS and count the conidia using a hemocytometer at a 1:1,000 dilution (1:100 and then 1:10). Adjust the conidial concentration to 3.6 x 10 6 /mL with CO 2 independent media, wrap in foil and store at 4 °C. NOTE: The conidia are now labeled with AF633 and will be referred to as FLARE conidia. 10. Optional: If stimulation with swollen conidia is required, after counting conidia (step 2.9), dilute conidia to 7.2 x 10 6 /mL in yeast nitrogen base (YNB) medium and add 500 µL conidial suspension to 4.5 mL YNB medium in an autoclaved 50 mL Erlenmeyer flask. Cover and place the flask in a shaking incubator (200 rpm at 37 °C) for 6 h.
1. Transfer the medium to a 15 mL tube. Centrifuge at 805 x g for 10 min at room temperature and wash once in PBS. Centrifuge again and resuspend pellet in 1 mL CO 2 independent medium, giving 3.6 x 10 6 conidia/mL.
Note: Conidia frequently clump after they become swollen making accurate counting difficult, it is advised to calculate with the counted numbers of resting conidia used.

Flow Cytometry
1. Add 200 µL of 1.2 x 10 6 /mL FLARE conidia to the cells in the 24-well plate, and then add 100 µL of 20 µg/mL voriconazole. Add 300 µL of RPMI and incubate for 16 h at 37 °C with 5% CO 2 . NOTE: Voriconazole is added to prevent conidial germination. 2. Add 25 µL of a 1 mM Calcofluor-white stock solution to each well for a final concentration of 25 µM and incubate for 10 min at 37 °C with 5% CO 2 . 3. Centrifuge at 582 x g for 10 min. Remove the supernatant and wash twice with PBS. 4. To harvest adherent cells (monocytes), add 1 mL of PBS with 3 mM EDTA to each well and incubate for 10 min at 37 °C with 5% CO 2 . Gently wash cells off the plate by pipetting and collect cells in a tube. 5. Wash once in FACS Buffer (2% fetal calf serum to PBS), and then resuspend in FACS Buffer for FACS analysis.
1. For FACS analysis, first adjust the compensation parameters of the flow cytometer and set positive gates using single-colored compensation tubes, including conidia with no color: dsRed+ conidia, AF633+ conidia, and Calcofluor White+ conidia (generated as in 4.2). 2. Set free conidia gate based on forward and side scatter using conidia with no color. Set cell gate on forward and side scatter by excluding the free conidia gate. 3. Analyze sample tube by recording 10,000 events.
NOTE: Cells associated with live conidia will be dsRed+ and AF633+. Cells associated with dead conidia will be AF633+ only. Bystander cells that have not engaged conidia will be dsRed-and AF633-. The conidia-engaged cell populations are further analyzed for Calcofluor White staining on the Pacific Blue channel. Conidia that remain outside of cells will be Calcofluor White+, and conidia that have been internalized will be Calcofluor White-.

Live Cell Video Microscopy
Note: The choice of microscope will depend upon what is available locally, but the microscope setup will need to include an inverted stage, an environmental chamber heated to 37 °C and appropriate excitation/emission filters for dsRed (532/561 nm) and AF633 (633 nm). FLARE conidia do not work with the 488 nm laser in lieu of the 532/561 nm laser for dsRed. When performing this experiment for this first time, use the control conidia with no color and single color to test for background fluorescence and bleed-through between the dsRed and AF633 channels.
1. Turn on the microscope heater prior to the experiment and allow sufficient time for the environmental control chamber to warm to 37 °C. The time taken for chamber temperature to stabilize will vary for different microscope setups. 2. Turn on the microscope and computer, and load the imaging software. Mount the imaging dish on the microscope stage and adjust the focus to find the human neutrophils or monocytes. 3. For dsRed and AF633, set the laser power to 10% and exposure time to 1 s in the acquisition settings. 4. Remove the imaging slide and add 100 µL of 3 x 10 6 /mL FLARE conidial suspension to the appropriate wells (total volume 300 µL/well). Record the time that FLARE conidia are added to the dish. 5. Return the slide to the stage and adjust camera sensitivity for dsRed and AF633 to clearly see the conidia but not overexpose the image. Set up a stage points list if multi-point acquisition is required. 6. Commence imaging when all points are in focus, the channels are optimized and the cycle time and duration is set. Cycle time and duration of imaging depend on the experimental question. The goal is to keep the cycle time as short as possible (≤2 min) with 1 min allowing for in depth analysis of uptake dynamics.

Representative Results
Representative results are shown following the described protocol. Figure 1 illustrates a representative 6 h live-cell video with human neutrophils and FLARE conidia. dsRed (red) is expressed by the conidia themselves while they have been labeled with AF633 (Magenta).

Figure 2
is an image from a single time point from a similar movie as displayed in Figure 1, illustrating the difference between live and dead FLARE conidia. Dead conidia are distinguished from live conidia by losing their dsRed (red) signal while maintaining a bright AF633 (magenta) fluorescence. A. fumigatus conidia often survive within phagocytes for over 6 h. Therefore, to effectively quantify killing, flow cytometry plots are shown for a 16 h incubation period in Figure 3. This data was obtained with an alveolar macrophage cell line as an example but can be performed with any cell type. In Figure 4 the migration is shown in the first hour of stimulation for human neutrophils as well as their velocity and directional movement. Figure 5 shows the percentage of neutrophils and monocytes that have ingested 1, 2, 3 or more conidia while Figure 6 displays the number of conidia that germinate into hyphae.

Discussion
This method describes an innovative in vitro method to study the antifungal activity of human primary phagocytes against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia, live cell imaging and flow cytometry. Previous studies have demonstrated the added value of using FLARE conidia both in vivo in murine experimental fungal infection and in vitro assessment of antifungal immunity 6,7 . Combining FLARE conidia with this next generation live cell imaging technique allows for a setup to measure viability of individual conidia during dynamic interactions in vitro.
The described method has the potential to investigate the specific roles and importance of certain cellular processes of immune cells on their antifungal responses. In addition, antifungal responses of phagocytes from specific patients suffering from defined single component defects in their immune system can be assessed in greater detail. Very early and initial antifungal responses can be studied in great detail over 6 h of continuous imaging. This allows for even subtle differences to be detected, such as the velocity of phagocyte migration towards the fungus, internalization rates, dynamics of fungicidal events 12 , which would be undistinguishable using conventional phagocytosis methods.
Any cell type could be used with this method; the cell types used here were selected because these phagocytes constitute the first and second lines of defense in the human airways against A. fumigatus 13 . Interestingly, very clear differences can be observed between how monocytes and neutrophils engage with resting and swollen conidia and hyphae. Monocytes hardly migrate to conidia, while neutrophils display much more migratory activity. Additional fluorescent markers such as live-dead markers on immune cells can be included in the assay to provide insights into phagocyte survival following interactions with Aspergillus. Interesting potential future applications for this technology include the co-culture of different host cell types, the consequences for antifungal responses (e.g., macrophages on an epithelial monolayer, monocyte-derived dendritic cells together with neutrophils), and the real-time measurement of reactive oxygen species production or neutrophil extracellular trap formation following stimulation with Aspergillus.
A few points need to be noted to use this protocol, foremost the laboratory setup required: a microscope with an inverted stage, an environmental chamber stable at 37 °C, and excitation/emission filters for dsRed (532/561 nm) and AF633 (633 nm). The ability of the microscope to keep the cells in focus and retain the oil immersion (particularly relevant during multi-point acquisition) should be monitored periodically due to the long duration of imaging. For quantification of fungicidal activity flow cytometry facilities are required. Additionally, appropriate institutional and ethical approvals need to be in place to work with healthy donor and patient derived blood samples and genetically manipulated fungi. It is strongly recommended to use fresh blood samples (use on the same day) to increase cell viability and preserve functionality. Ideally, isolation of cells is performed immediately after drawing the blood samples and neutrophils are imaged immediately after isolation.
In conclusion, a next generation live cell imaging technique to assess in great detail phagocyte antifungal activity against A. fumigatus is described. This technology can provide a unique insight into individual cell-fungal interactions in early innate immune defenses against Aspergillus.

Disclosures
The authors have nothing to disclose.