This research was supported in part by NIH awards 5T32AI07506, 5R01AI033774, 5R37AI033142, 5R01AI052733.

All animal work was done in accordance with regulations and guidelines of the Institute for Animal Studies at Albert Einstein College of Medicine.
1. Growth Conditions of C. neoformans H99 (Serotype A)
<ol>
	<li>Grow individual C. neoformans (Cn) colonies on Sabouraud agar plates.</li>
	<li>One day prior to the experiment, select one colony and inoculate 10 ml of Sabouraud broth.</li>
	<li>Allow culture to grow overnight at 37 &#176;C shaking at a speed of 250 rpm.</li>
</ol>
2. Isolation and Preparation of Primary Bone Macrophages from C57/Bl6 Mice
<ol>
	<li>Euthanize mice by placing animal in a CO2 chamber until no longer breathing. As a second measure, cervically dislocate the neck before beginning dissection. Sterilize entire body with 70% ethyl alcohol.</li>
	<li>Remove both femurs and tibia and place bones in sterile DMEM.</li>
	<li>Remove excess tissue and muscle using delicate wipes until bones are completely clean.</li>
	<li>Place only intact bones in a Petri dish containing 70% ethyl alcohol and incubate for 3 min to kill associated microorganisms. Remove bones and place in Petri dish with sterile DMEM.</li>
	<li>Carefully cut ends of bones. Hold bone over an empty 50 ml conical tube placed on ice and carefully flush 10 ml of cold DMEM using a 25 G needle through each bone. 
	NOTE: Each mouse will yield 4 bones (2 femurs, 2 tibias). Therefore one mouse should yield approximately 40 ml of cell suspension.</li>
	<li>Centrifuge cell suspension at room temperature for 10 min at 650 x g.</li>
	<li>During this centrifugation, prepare and filter sterilize bone marrow macrophage feeding media which consists of DMEM supplemented with 20% L929, 10% fetal calf serum, 10% NCTC-109, 1% Pen-Strep, 1% HEPES, 1% L-Glutamine, 1% non-essential amino acids, 0.1% 2-Mercaptoethanol.</li>
	<li>Resuspend resulting pellet with 10 ml of feeding media. Pass cell suspension through a 70 &#181;m cell strainer to disrupt cell clumps and remove large debris.</li>
	<li>Plate 1 ml of cell suspension into 10 ml of feeding media in Petri dishes that are specified for tissue culture use (for a total of 10 plates).</li>
	<li>Allow cells to adhere at 37 &#176;C with 10% CO2. Add 5 ml of fresh feeding media on day 3. On day 7, completely replace the feeding media and macrophages are ready to be used.</li>
	<li>The day prior to the experiment, detach macrophages from plate by removing feeding media and add 5ml of a gentle cell stripping reagent to plate.</li>
	<li>Incubate for 5-10 min at 37&#160;&#176;C and remove macrophages with gentle pipetting.</li>
	<li>Pellet cells by centrifugation at 650 x g for 10 min.</li>
	<li>Resuspend pellet in 1 ml of feeding media. Using a 1:20 dilution, calculate the concentration of macrophages by pipetting 10 &#181;l of cell suspension onto a haemocytometer.</li>
	<li>Using 14 mm glass bottomed Petri dishes, plate a concentration of 1 x 105 macrophages directly onto the inner well which holds a maximum of 200 &#181;l, so take caution to not exceed this volume as shown in Figure 4.</li>
	<li>Allow the cells to adhere to the glass Petri dish by placing the dishes in a 37&#160;&#176;C incubator for 1 hr.</li>
	<li>Add 1-2 ml of feeding media supplemented with LPS at 1 mg/ml and IFNg at 500 u/ml and allow cells to incubate overnight.</li>
</ol>
3. Co-incubation of Primary Murine Macrophages and C. neoformans
<ol>
	<li>On the day of experiment, remove 1 ml of overnight inoculated culture and pellet yeast cells by centrifugation for 5 min at 420 x g.</li>
	<li>Wash cells 3 times with sterile PBS to remove media and extracellular cryptococcal products like the polysaccharide glucuronoxylomannan (GXM) at the speed and time mentioned above.</li>
	<li>Resuspend cell pellet in 1 ml of sterile PBS and make a 1:100 dilution. Pipette 10 &#956;l of the dilution onto a haemocytometer and count cells. Add yeast cells at a MOI of 1:5. For example, if there are 1 x 105 macrophages/well, make a cell suspension of 5 x 106 /ml of Cn and add 100 &#181;l of this solution for a total amount of 5 x 105 Cn.</li>
	<li>Add calculated volume of cryptococcal cell suspension to 1 ml of feeding media with the mAb 18B7 antibody at a concentration of 10 &#956;g/ml. Incubate cell suspension at room temperature for 5 min.</li>
	<li>Remove feeding media from glass bottomed Petri dish and wash 1x with sterile PBS. Add 100 &#956;l of opsonized Cn directly to well.</li>
	<li>Allow cells to incubate for 2 hr at 37 &#176;C with 10% CO2.</li>
	<li>After co-incubation, check by microscope that macrophages have internalized Cn. To be considered internalized, cryptococcal cells should clearly be seen within the confines of the macrophage.</li>
	<li>Wash Petri dish 3x with sterile PBS to remove any and all extracellular Cn.</li>
	<li>Add 1-2 ml of feeding media without the addition of mAb 18B7, and set up microscope.</li>
</ol>
4. Visualizing Non-lytic Exocytosis in Real-time Utilizing Digital Light Microscopy
NOTE: To perform these experiments, a microscope that comes with an attached incubating chamber that includes CO2 delivery and a heating unit is required.
<ol>
	<li>Prior to the experiment, pre-warm the incubating chamber for at least 30 min to 37 &#176;C. Ensure that the CO2 unit reads 5% at the start of the experiment.</li>
	<li>Place glass bottomed Petri dish on microscope platform, cover with CO2 lid, and close all doors to ensure no heat escapes.</li>
	<li>Using the 10X objective with the microscope in phase contrast, focus on a clear field of infected macrophages. Based on the experiment, determine the objective used by how many macrophages need to be studied.</li>
	<li>Set up microscope software to take an image every 4 min for a 24 hr period.</li>
	<li>After 24 hr, the experiment will have reached completion and a total of 361 images will have been collected. Export these images as .jpegs at a 5% compression. Using Image J software, view images as a Visual Stack at 5 frames per sec. If the movie needs to be viewed for publication or a presentation, save it as either an .avi or .mov file depending on which format works best for the viewer.</li>
</ol>

<ol>
	<li>Voelz, K., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcal+Interactions+With+the+Host+Immune+System.">Cryptococcal Interactions With the Host Immune System.</a> Eukaryotic cell. 9 (6), 835-846 (2010).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+interactions+with+macrophages:+evasion+and+manipulation+of+the+phagosome+by+a+fungal+pathogen.">Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.</a> Cellular microbiology. 15 (3), 403-411 (2012).</li><li>Sabiiti, W., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+infection+by+the+human+fungal+pathogen+Cryptococcus+neoformans.">Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans.</a> Future microbiology. 7 (11), 1297-1313 (2012).</li><li>Bliska, J. B., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pathogenic+bacteria+and+fungi+&#8212;+a+case+of+convergent+evolution.">Intracellular pathogenic bacteria and fungi &#8212; a case of convergent evolution.</a> Nature Reviews Microbiology. 7, 165-171 (2008).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+spread+and+massive+vacuole+formation+after+Cryptococcus+neoformans+infection+of+murine+macrophages.">Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.</a> BMC immunology. 8 (1), 16 (2007).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell-to-cell+spread+of+a+pathogenic+yeast.">Direct cell-to-cell spread of a pathogenic yeast.</a> BMC immunology. 8, 15 (2007).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+extrusion+and+host-cell+survival+after+Cryptococcus+neoformans+phagocytosis+by+macrophages.">Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.</a> Curr Biol. 16 (21), 2161-2165 (2006).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expulsion+of+live+pathogenic+yeast+by+macrophages.">Expulsion of live pathogenic yeast by macrophages.</a> Curr Biol. 16 (21), 2156-2160 (2006).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+fungal+pathogen+Cryptococcus+neoformans+escapes+macrophages+by+a+phagosome+emptying+mechanism+that+is+inhibited+by+Arp2/3+complex-mediated+actin+polymerisation.">The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation.</a> PLoS pathogens. 6 (8), (2010).</li><li>Carnell, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+polymerization+driven+by+WASH+causes+V-ATPase+retrieval+and+vesicle+neutralization+before+exocytosis.">Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.</a> The Journal of cell biology. 193 (5), 831-839 (2011).</li><li>Voelz, K., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+regulates+the+outcome+of+intracellular+macrophage+parasitism+by+Cryptococcus+neoformans.">Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans.</a> Infection and immunity. 77 (8), 3450-3457 (2009).</li><li>Bain, M. J., Lewis, E. L., Okai, B., Quinn, J., Gow, A. R. N., Erwig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lytic+expulsion/exocytosis+of+Candida+albicans+from+macrophages.">Non-lytic expulsion/exocytosis of Candida albicans from macrophages.</a> Fungal genetics and biology. 49 (9), 677-678 (2012).</li></ol>

The authors declare that we have no competing financial interests or other conflicts of interest.

Here we have described a method by which fungal-macrophage interactions can be recorded and analyzed in real time over a 24 hr period. Our lab has used this protocol to study many of the temporal aspects of non-lytic exocytosis and will continue to use real-time microscopy to ascertain the morphological components that surround this process.
For this technique to yield ideal results, special attention should be given to certain steps in the protocol. The cell density that is plated the night before is important because too many cells can create a movie that is not usable as the viewing of the movement and interactions of individual cells is hindered, while too few cells may yield data that is not significant. The goal is to achieve an evenly spaced field of macrophages such that individual macrophages can be followed for the duration of the infection period. In that same vein, washing away the extracellular Cn is a critical step as extracellular Cn will divide over time which could obscure the field and lead to a loss of visibility of the macrophages. Depending on the length of the movie, the frame can shift out of focus which makes images unusable to study and therefore, the recording needs to be checked at various points during the infection.
As outlined, there are various ways this technique can be modified to fit within the parameters of other experiments. Many different fluorescent dyes and markers can be used to highlight membrane and phagosomal dynamics but one should be cautious of photo bleaching and the affect this may have on the fitness of the cells. A variety of cell types can also be used such as J774.1 macrophage-like cells, monocytes or amoebas in conjunction with other fungal strains to study the differences that may occur. We suggest using an objective of 10X because our experiments require the data collection of a large quantity of cells, however one could use a higher objective to study just a few cells. The timing that each image is taken can also be adjusted. Taking an image every 4 min for 24 hr yields a movie that runs roughly 1 minute in length when all frames have been compressed. Increasing either aspect (the duration of infection or the time between frames) increases the amount of files that need be stored which may be an issue for some laboratories.
Non-lytic exocytosis is an incredibly dynamic process that occurs rapidly and at various time intervals during infection. Therefore, trying to capture macrophages undergoing this interaction using other methods such as transmission and scanning electron microscopy can be extremely rare. Due to the nature of how samples are fixed and processed, one can&#8217;t be certain that what is being viewed is definitively non-lytic exocytosis versus other cellular processes such as lysis or apoptosis. Also, a hallmark of non-lytic exocytosis is that host and pathogen remain viable afterwards which is something that cannot be deduced from fixed images since these methods can only produce snapshots of a process at a given moment.
While this protocol can provide countless images of cellular processes, there are some limitations in using this technique to study host pathogen interactions. The low objective we use to study a large amount of macrophages at one time does not allow for a high enough resolution image to visualize the interaction between the phagosome and the cellular membrane, which is not a well understood aspect of non-lytic exocytosis. Once all the images have been recorded, the post analysis of following many macrophages can be very tedious and time-consuming. Macrophages, even those adhered to glass, can be very motile and will move in and out of the frame very quickly. This can make it hard to follow individual macrophages from start to finish, especially those that are near the periphery of the frame.
Despite these limitations, this is protocol can be used in a variety of ways to study the many different aspects of host-pathogen interactions.

The stages of a fungal infection between cryptococcal cells and macrophages are well documented 1-3. After yeast cells are ingested by macrophages, a variety of interactions may occur: the macrophage may lyse releasing its fungal load into the extracellular space, or it could control the infection by keeping the yeast cells within the confines of its cellular membrane 4. However, several years ago a new outcome was described independently by two groups: non-lytic exocytosis, a process in which a macrophage expunged some or all of the cryptococcal cells into the surrounding environment or a neighboring cell and both host and pathogen remain viable 5-8. Several studies have attempted to understand the molecular mechanisms behind this interaction but we still do not have a full grasp on what drives this phenomenon.
The capability to capture images in real-time and then subsequently analyze multiple infected macrophages allows one to answer many questions about the physical properties surrounding non-lytic exocytosis in regards to timing, spatial capacity, change in morphology and even stress of macrophages during a fungal infection. By allowing the cells to be housed in an environment that is comparable to that of an incubator, we can glimpse the dynamic nature of macrophage-fungal cell interactions. Researchers have used this technique to study various components of this process. The role of the phagosome in this process was made apparent using fluorescent staining and actin blocking agents 9, while another group used this method to show that non-lytic exocytosis was blocked in cells that have had the WASH nucleating protein domains deleted 10. The effect of cytokine signaling has also been ascertained using real-time microscopy 11. This process is not limited to C. neoformans as it has also been observed with Candida albicans, another fungal pathogen that has the capability to infect macrophages 12. These findings are examples of how this method can provide a wealth of information to an area we know so little about.

<table><tbody><tr><td>Sabouraud Dextrose Agar</td><td>BD</td><td>DF0109-17-1</td><td></td></tr><tr><td>Sabouraud Dextrose Broth</td><td>BD</td><td>DF0382-17-9</td><td></td></tr><tr><td>Dubelcco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Corning Cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Atlanta Biologicals</td><td>S12450</td><td></td></tr><tr><td>NCTC 109</td><td>Invitrogen</td><td>21340-039</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>HEPES Buffer</td><td>Corning Cellgro</td><td>25-060-Cl</td><td></td></tr><tr><td>Glutamax</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Corning Cellgro</td><td>25-025-Cl</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Invitrogen</td><td>21985-023</td><td>Toxic</td></tr><tr><td>3003 Tissue Culture Petri Dishes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>08772E</td><td></td></tr><tr><td>CellStripper</td><td>Corning Cellgro</td><td>25-056-Cl</td><td></td></tr><tr><td>Mat-tek Glass Bottom Culture Dishes</td><td>MatTek</td><td>P35GC-1.5-14-C</td><td></td></tr><tr><td>LPS</td><td>Sigma</td><td>L3137</td><td></td></tr><tr><td>Mouse IFN-g</td><td>Roche</td><td>NC 9222016</td><td></td></tr><tr><td>mAb 18B7</td><td>Non-commerical antibody produced in our lab</td><td></td><td></td></tr><tr><td>Axiovert 200M Microscope with Incubating Chamber</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

visualizing non-lytic exocytosis of cryptococcus neoformans from macrophages using digital light microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

The images that this technique produces can be analyzed in a variety of ways to collect information surrounding what happens after cells have phagocytosed C. neoformans. As seen in Figure 1, there are approximately 100 macrophages that are either infected or uninfected. Each one of these macrophages gives the viewer an opportunity to study host-pathogen interactions on a very microscopic level. As Figures 2 and 3 show, there are different outcomes that can occur between a macrophage and yeast cell, and even between macrophage-macrophage. Macrophages can be scored as undergoing non-lytic exocytosis, cell-cell transfer, or any other cellular process that may occur. The timing of such events during the infection can also be ascertained based on the parameters that are set when the movie is being recorded.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52084/52084fig1highres.jpg" width="500" /> 
Figure 1: Field of Infected Macrophages. This figure shows an entire field of individually infected macrophages at the start of a 24 hr infection period. It should be noted that the field is free of extracellular Cn and more importantly, how clear it is which macrophages are infected (as indicated by a large arrowhead) and which contain no fungal cells (as indicated by a small arrow). Scale bar represents 50 &#956;m.
<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52084/Updated_Field_of_Macrophages.mov" target="_blank">Movie 1</a>: Visualization of Macrophage-C. neoformans 24 hr Infection Period. The initial frame of the movie shows many macrophages throughout the field with some that are uninfected and some that have phagocytized varying amounts of cryptococcal cells. As the movie progresses, the cells are all motile and can be seen moving about the field. We are able to follow each of these macrophages to see what kind of cellular process they undergo.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52084/52084fig2highres.jpg" width="500" /> 
Figure 2: Primary Murine Macrophage Undergoing Non-lytic Exocytosis. An infected macrophage (Panel A, indicated by a white arrowhead) is seen undergoing the beginning stages of non-lytic exocytosis. The phagosome starts to enlarge (panel B-C) and the fungal load starts to shift. Several Cn cells are then released into the extracellular space as can be seen in panels D-E. A second infected macrophage in Panel F, indicated by a white arrowhead, also undergoes a non-lytic exocytosis event (Panel G-H). At the end of the process, both macrophages are left empty (Panel I) as shown by the white arrowheads. Scale bar represents 25 &#956;m.
<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52084/Non-Lytic_Exocytosis.avi" target="_blank">Movie 2</a>: Visualizing Primary Murine Macrophage Undergoing Non-lytic Exocytosis. In this movie, it is clear that the cryptococcal cells are within the confines of an infected macrophage. The macrophage becomes agitated and a yeast cell can be seen budding within the phagosome. Cryptococcal cells are then quickly expunged into the surrounding environment. A second infected macrophage undergoes non-lytic exocytosis and more yeast cells are released into the extracellular space. The host cells remain viable after cryptococcal exocytosis as indicated by their ability to continue moving.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/52084/52084fig3highres.jpg" width="500" /> 
Figure 3: Primary Murine Macrophage Undergoing Cell-Cell Transfer. Another process that can be captured using this protocol is cell-cell transfer which occurs when cryptococcal cells are transferred from one macrophage to a neighboring macrophage. Two infected macrophages are in close proximity to each other indicated by white arrowheads (Panel A) and move even closer to initiate cell-cell transfer (Panel B). A cell-cell bridge indicated by the red arrowheads is formed between the two macrophages such that the cryptococcal cells are not exposed to the surrounding environment during the transfer (Panel C, E). After the transfer is complete, the cells break away (Panel D, F). Scale bar represents 25 &#956;m.
<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52084/Cell-Cell_Transfer.mov" target="_blank">Movie 3</a>: Visualizing Cell-Cell Transfer Between Infected Macrophages. On occasion, macrophages will interact in such a way that facilitates transfer of cryptococcal cell between host cells. The yeast cells are not exposed to the extracellular environment and the host cells remain viable. In this movie, two infected macrophages create cell-cell bridges on two separate occasions to shuttle cryptococcal cells from one macrophage to another.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/52084/52084fig4highres.jpg" width="500" /> 
Figure 4: Schematic showing preparation of cells.

Watch this Scientific Journal Video about Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy at JoVE.com

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

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Immunology and Infection

11.7K Views.  Albert Einstein College of Medicine. We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

Video: Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

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 ...

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.

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 ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

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 ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

Biology

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Summary

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Chapters in this video

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

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

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

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

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

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Visualizing the Early Stages of Phagocytosis

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis