This work was supported by NIH grants AI56168, AI71142, AI87541 and AI100631 to MDP. Maurizio Del Poeta is Burroughs Wellcome Investigator in Infectious Diseases.

1. Cholesterol Depletion of J774A.1 Cells with M&#946;CD
<ol>
	<li>In a sterile biosafety cabinet, seed 105 J774A.1 macrophage-like cells per well on a 96-well cell culture plate in 200 &#956;l of Dulbecco&#8217;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Incubate at 37 &#176;C and 5% CO2 O/N.</li>
	<li>Remove media from the cell monolayer and wash the cells twice with 1x phosphate buffered saline (PBS) that has been filtered or autoclaved.</li>
	<li>Add 200 &#956;l of M&#946;CD solution at the desired concentration (10 mM or 30 mM in PBS) or 1x PBS as a control and incubate for 30 min at 37 &#176;C with shaking. Remove supernatant and reserve at RT for quantitative analysis with commercially available kit immediately following the procedure.</li>
	<li>Wash the cells two to three times with 1x PBS or serum free DMEM and continue with infection or lyse cells by pipetting two to three times with deionized H2O for analysis with thin layer chromatography or with a kit. 
	NOTE: Following cholesterol depletion a commercially available cholesterol quantification kit can be used. See materials section for details. Follow manufacturer&#8217;s instructions as written.</li>
</ol>
2. Observation of Cholesterol Content by Thin Layer Chromatography (TLC)
<ol>
	<li>Wash a TLC tank twice with acetone and once with a solution of petroleum ether:diethyl ether:acetic acid (65:30:1 by volume). Saturate the tank with the petroleum ether:diethyl ether:acetic acid (65:30:1 by volume) solution and leave O/N. 
	NOTE: Organic solvents should always be used under a fume hood to prevent inhalation of vapor. Gloves and lab coat should be worn at all times. Acetic acid is a strong acid and should be used with caution.</li>
	<li>In a sterile biosafety cabinet, seed 106 J774A.1 macrophage-like cells per well on a 6-well cell culture plate in a volume of 5 ml of warm DMEM supplemented with 10% FBS and 1% P/S. Incubate at 37 &#176;C, 5% CO2 O/N.</li>
	<li>Deplete macrophages of cholesterol by following steps 1.2 - 1.4, substituting 1 ml for 200 &#956;l where applicable to account for the larger well size.</li>
	<li>Add 500 &#956;l of Trypsin-EDTA to each well, incubate for 3 min at 37 &#176;C, and gently scrape cells with a cell scraper.</li>
	<li>Transfer into a microfuge tube and add an additional 500 &#956;l of warm DMEM supplemented with 10% FBS and 1% P/S.</li>
	<li>Spin the cells for 5 min at 300 x g and remove the supernatant.</li>
	<li>Add an additional 500 &#956;l of warm DMEM supplemented with 10% FBS and 1% P/S to the cell pellet and resuspend carefully by pipetting up and down.</li>
	<li>Remove 10 &#956;l of cells and count cells on a hemocytometer. Normalize the cell concentrations from each sample and add an equal number of cells to glass tubes. 
	NOTE: At this step, one may choose to combine cells from the same treatment groups to obtain a more concentrated final lipid extract.</li>
	<li>Centrifuge cells at 300 x g for 5 min at RT and remove media.</li>
	<li>Add 2 ml of methanol and vortex. Then, add 1 ml of chloroform and vortex. Check the phase status to make sure the solution in monophasic. 
	NOTE: Tubes can be stored at 4 &#176;C O/N.</li>
	<li>Centrifuge at 1,700 x g for 10 min at RT and transfer the supernatant to a new tube</li>
	<li>Add an additional 1 ml of chloroform followed by 1 ml of dH2O. Vortex twice for 30 sec. Centrifuge at 1,700 x g for 5 min at RT.</li>
	<li>Weigh a glass tube on a sensitive balance and use a glass Pasteur pipette to transfer the lower phase into the glass tube of known weight.</li>
	<li>Dry down lipids in a centrifugal evaporator until dry (approximately 2 hr). Weigh tube with dried lipids and calculate the dry lipid weight. 
	NOTE: Dry lipids can be stored in -20 &#176;C until ready to perform TLC.</li>
	<li>Dilute dried lipids in enough chloroform to normalize the concentration of lipid (usually 20 - 50 &#181;l) and load 20 &#181;l of the diluted lipid on a silica TLC plate. Load 20 &#956;g of cholesterol diluted in 20 &#956;l of chloroform as a standard.</li>
	<li>Add dried TLC plate to the saturated TLC tank and allow solvent to migrate up to 1 cm before plate edge. Remove TLC plate from tank and allow it to dry about 5 min.</li>
	<li>Visualize lipids by placing in an iodine vapor tank to check migration. Remove and allow spots to fade for about 10 - 15 min under the hood. 
	NOTE: Iodine is an inhalation hazard. Always use under a fume hood.</li>
	<li>Prepare a solution for neutral lipid staining by combining 60 ml of methanol with 60 ml of deionized H2O, 4&#160;ml of sulfuric acid, and 630 mg of manganese chloride.</li>
	<li>Carefully and slowly dip the TLC plate into the neutral lipid staining solution in a tray and remove without sloughing off the silica layer. 
	NOTE: The neutral lipid staining solution can be reused several times and so it can be retrieved from the tray and placed back in a bottle for later use.</li>
	<li>Allow plate to dry under the hood at RT until all bubbles has disappeared. Heat the TLC plate on a heat block set to 160 &#176;C and char to the desired color. 
	NOTE: A densitometry program such as Vision Works LS can be used to quantify the charred bands to compare lipid samples.</li>
</ol>
3. Infection of Macrophages with C. neoformans (H99)
<ol>
	<li>In a sterile biosafety cabinet, seed 105 macrophage-like cells per well on a 96-well cell culture plate in 200&#160;&#956;l of DMEM supplemented with 10% FBS and 1% P/S. Incubate at 37 &#176;C, 5% CO2, 5% CO2 O/N. 
	NOTE: Infection can also be done in glass bottomed confocal dishes for easier imaging; all amounts remain the same.</li>
	<li>Grow a culture of C. neoformans (H99) by inoculating 10 ml of YNB with one colony obtained from a struck plate and incubating it O/N at 30 &#176;C with shaking.</li>
	<li>Wash and count C. neoformans (H99) cells.
	<ol>
		<li>Centrifuge C. neoformans O/N culture at 1,700 x g for 10 min at 4 &#176;C.</li>
		<li>Remove media and discard. Wash cells with 5 ml of 1x PBS. Centrifuge at 1,700 x g for 10 min at 4 &#176;C.</li>
		<li>Remove PBS and wash with 5 ml of filtered 1x PBS. Centrifuge at 1,700 x g for 10 min at 4 &#176;C. Repeat this step 2 more times.</li>
		<li>Remove PBS and resuspend in 5 ml of 1x PBS.</li>
		<li>Make a serial dilution in PBS to obtain a 1:500 dilution of the washed culture.
		<ol>
			<li>Add 100 &#956;l of the original sample to 900 &#956;l of 1x PBS to obtain a 1:10 dilution.</li>
			<li>Add 100 &#956;l of 1:10 diluted sample to 900 &#956;l of 1x PBS to obtain a 1:100 dilution.</li>
			<li>Add 200 &#956;l of the 1:100 diluted sample to 800 &#956;l of 1x PBS to obtain a 1:500 dilution</li>
		</ol>
		</li>
		<li>Take 10 &#956;l of 1:500 dilution and count on hemocytometer to calculate the number of cells.</li>
	</ol>
	</li>
	<li>Prepare working solution for activating macrophages and opsonizing C. neoformans.
	<ol>
		<li>Dilute LPS and IFN&#947; 100x from stock solutions by adding 10 &#956;l to 990 &#956;l. 
		NOTE: LPS and IFN&#947; are used to enhance phagocytic uptake but are not required for phagocytosis. If there is an interest in fungicidal activity of macrophages, perform activation O/N at 37 &#176;C with shaking prior to infection.</li>
		<li>Per sample combine 7.5 &#956;l of diluted LPS, 1.25 &#956;l of diluted IFN&#947;, 1.25 &#956;l of GXM antibody and the volume of the C. neoformans culture that gives 1.25 x 105 cells. Bring volume up to 250 &#956;l multiplied by number of samples with DMEM supplemented with 10% FBS and 1% P/S.</li>
		<li>Vortex and incubate solution for 20 min at 37 &#176;C with shaking. 
		NOTE: Cholesterol depletion (steps 1.2 - 1.4) can be done concurrently with the opsonization step. Be sure to treat macrophages prior to combining the working solution, as the opsonized cells should optimally be used no longer than 20 min following the step 3.4.3 incubation.</li>
	</ol>
	</li>
	<li>Infect Macrophages
	<ol>
		<li>Wash macrophages twice with serum free DMEM and add 200 &#956;l of opsonized C. neoformans working solution to each well.</li>
		<li>Incubate for 2 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Fix and Stain Cells
	<ol>
		<li>Remove media and wash cells 2 times with DMEM.</li>
		<li>Air dry the cell monolayer for 10 min and add 200 &#956;l of ice cold methanol to fix the cells.</li>
		<li>Incubate for 15 min at RT and remove any remaining methanol.</li>
		<li>Add 200 &#956;l of 10x Giemsa and incubate for 5 min at RT.</li>
		<li>Wash 2 - 3 times with deionized water and dry O/N with cap off. 
		NOTE: Imaging can be done the next day or up to a week following staining.</li>
	</ol>
	</li>
	<li>Visualize and Count
	<ol>
		<li>Using a microscope, count 300 cells per data point (if there are 2 of the same treatment count 150 per well) and note the number of infected macrophages and number of engulfed Cryptococcus cells. 
		NOTE: To ensure even sampling per data point use 2 plates per condition, choose 3 non-overlapping areas per plate and count 50 cells per area.</li>
		<li>Calculate phagocytic index by multiplying the percentage of infected macrophages by the mean number of C. neoformans per macrophage. Normalize phagocytic index by expressing values as a percentage of the 1x PBS treated control. After several trials calculate the mean value and the standard deviation of the mean to determine trends in phagocytic index. Use student t-test to determine significance.</li>
		<li>Take micrographs of cells at 1,000X or 400X magnification.</li>
	</ol>
	</li>
</ol>
4. Trypan Blue Assay
<ol>
	<li>In a sterile biosafety cabinet, seed 106 macrophage-like cells per well on a 6-well cell culture plate in a volume of 5 ml of Dulbecco&#8217;s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Incubate at 37 &#176;C, 5% CO2 O/N.</li>
	<li>Deplete macrophages of cholesterol by following steps 1.2 - 1.4 substituting 2 ml for 200 &#956;l where applicable to account for the larger well size. 
	NOTE: Be sure to include a control treated with 1x PBS as well as a control that is scraped prior to any treatment.</li>
	<li>Add 500 &#956;l of 1x PBS to each well and gently scrape cells with a cell scraper. Transfer into a microfuge tube and suspend the cells by gently pipetting up and down.</li>
	<li>Remove 10 &#956;l of cells and stain with 1 &#956;l of 4% Trypan blue.</li>
	<li>Count cells on a hemocytometer and calculate viability using the following equation: % viability = [1 - (Blue cells / total cells)] x 100. Normalize values to the control that was untreated. After several trials calculate the mean value and the standard deviation to determine trends in viability. Use student t-test to determine significance.</li>
</ol>

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The authors have nothing to disclose.

In working with this protocol it is important to obtain accurate cell counts when plating mammalian cells and opsonizing C. neoformans cells. This minimizes variation between trials and ensures an accurate 1:1 target to effector ratio throughout the study. It is also critical to coordinate the timing of the cholesterol depletion and infection to prevent the opsonized yeast cells or treated macrophage cells from resting at RT in between the procedures. Long waiting periods could lead to loss of antibody opsonizat...

Phagocytosis is a process by which extracellular entities are internalized by host cells. It is a key weapon in the immune system&#8217;s arsenal to defend against pathogens, but the process may often be subverted by pathogens to allow for internalization and spreading throughout the body1. Phagocytosis is mediated by several signaling events that result in attachment and engulfment via rearrangements of the host cell&#8217;s cytoskeleton. &#8216;Professional&#8217; phagocytes are able to recognize and bind to opsonins on the surface of the invading pathogen to signal for attachment and the formation of lamellipodia, which engulf the pathogen and form a phagosome2. Among the so-called &#8216;professional&#8217; phagocytes are macrophages. Macrophages are highly specialized cells that carry out protective functions that include seeking out and eliminating disease causing agents, repairing damaged tissues, and mediating inflammation, most of these through the process of phagocytosis1,2.
Cryptococcus neoformans is a species of pathogenic yeast that causes a serious disease known as Cryptococcosis. Cryptococcus spores are inhaled by the host and result in a pulmonary infection that is usually asymptomatic. It is thought that exposure is extremely prevalent; a sample of 61 children from the Pediatric Infectious Diseases Clinic at the Bronx-Lebanon Hospital Center found that all those surveyed had antibodies to&#160;the cryptococcal polysaccharide glucuronoxylomannan and other studies have shown prevalence in both human immunodeficiency virus (HIV) uninfected and infected adults3,4. Alveolar macrophages are the first line of response to the pulmonary infection and in most cases successfully clear the pathogen. However, in immunocompromised individuals (e.g., HIV and AIDS patients) the yeast is able to survive within the macrophages. In these cases, the macrophages can serve as a niche for the replication of the pathogen and may facilitate its dissemination to the central nervous system (CNS) where the disease becomes fatal5&#8211;8. It is thought that macrophages may even deliver the yeast directly into the meninges, helping the yeast to cross the blood brain barrier via the &#8220;Trojan horse&#8221; model3,9&#8211;11. Thus, it is important to understand the process of phagocytosis and the factors that affect it, especially in cryptococcal infections.
Previous work in other pathogen systems point to cholesterol and lipid rafts formed by cholesterol as having an important role to play in phagocytosis12&#8211;15. Cholesterol is the most abundant lipid species in mammalian cells and comprises 25 - 50% of the mammalian cell membrane16. It has been found to play a role in modulating the biophysical properties of membranes by changing their rigidity17. Cholesterol and sphingolipids together form lipid microdomains within the membrane known as lipid rafts. Lipid rafts have been found to be involved in the formation of caveolae, as well as providing an isolated domain for certain types of signaling16&#8211;18. Due to their small size, it is difficult to study lipid rafts in vivo. One useful way to study the role of lipid rafts is to alter their constituents. Methyl-&#946;-cyclodextrin (M&#946;CD) is a compound that has been found to deplete cholesterol from mammalian membranes and is commonly used to study the role of lipid rafts18.
In this protocol, we present a method to deplete cholesterol from host cell membranes and quantify the effect of the depletion on the ability of the host cells to phagocytose C. neoformans in vitro. This procedure makes use of cell culture techniques on an immortalized macrophage like cell line (J774A.1) as a model for infection. Cholesterol depletion was accomplished by exposure to M&#946;CD, which has a hydrophobic core specific to the size of sterols and is able to act as a sink for cholesterol to draw it out of the membrane19. Cholesterol depletion was measured quantitatively using a commercially available kit and qualitatively using a modified Bligh-Dyer lipid extraction followed by thin layer chromatography (TLC)20. Phagocytosis was measured by infecting the cell line with a culture of opsonized yeast mixed with a cocktail of interferon-&#947; and lipopolysaccharide for activating the macrophages. Cryptococcus was opsonized using a glucuronoxylomannan (GXM) antibody21&#8211;23. Staining and microscopy experiments allowed for visualization of the cells and calculation of the phagocytic index to assess the degree of phagocytosis. Taken together, this protocol describes a basic method that integrates the alteration of lipid composition with a physiological process.

<table><tbody><tr><td>Class II type A2 Biosafety Cabinet</td><td>Labconco</td><td>3460009</td><td></td></tr><tr><td>J774A.1 cell line</td><td>ATCC</td><td>TIB-67</td><td>Arrives Frozen. See ATCC instructions for culturing.</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Gibco</td><td>11995-065</td><td>Store at 4 &amp;deg;C and warm to 37 &amp;deg;C prior to use</td></tr><tr><td>HI Fetal Bovine Serum Performance Plus</td><td>Gibco</td><td>10082-147</td><td>Keep frozen at -20 &amp;deg;C and thaw before adding to DMEM</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Gibco</td><td>15140-122</td><td>Used to suplement DMEM</td></tr><tr><td>Isotemp Cell culture incubator</td><td>Fisher Scientific</td><td></td><td>Model # 3530</td></tr><tr><td>96-Well culture dish</td><td>Corning Inc. Costar</td><td>3595</td><td></td></tr><tr><td>10x Phosphate Buffered Saline</td><td>Fisher Scientific BioReagents</td><td>BP3994</td><td>Dilute to 1x and filter or autoclave prior to use.</td></tr><tr><td>Methyl-&amp;beta;-cyclodextrin</td><td>Sigma Life Science</td><td>C4555-10G</td><td>Dissolve in 1x PBS to make solutions of 10 mM and 30 mM concentrations</td></tr><tr><td>Orbital shaker</td><td>Labline</td><td></td><td></td></tr><tr><td>Amplex Red Cholesterol Assay Kit</td><td>Life Technologies Molecular Probes</td><td>A12216</td><td>All reagents for Cholesterol Assay are contained within the kit. Follow Manufacturer instructions.</td></tr><tr><td>96-Well Black Assay plate</td><td>Corning Inc. Costar</td><td>3603</td><td></td></tr><tr><td>FilterMax microplate reader</td><td>Molecular Devices</td><td></td><td>Model F5</td></tr><tr><td>TLC chamber</td><td>Sigma-Aldrich</td><td>Z126195-1EA</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>650498-4L</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860-2L-R</td><td></td></tr><tr><td>TLC Paper</td><td>Whatman</td><td>3030917</td><td>Cut down to size needed for TLC tank</td></tr><tr><td>Fume hood</td><td></td><td></td><td>Any fume hood that complies with AIHA/ANSI Standards</td></tr><tr><td>6-Well plate</td><td>Corning Inc. Costar</td><td>3506</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Cell scraper</td><td>Corning Inc. Costar</td><td>3010</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>1490</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td></td><td>Model Alegra x-30R</td></tr><tr><td>Votex mixer</td><td>Fisher Scientific</td><td>12-812</td><td></td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td></td><td>Model # MS104S meaures down to 0.1 mg</td></tr><tr><td>Glass Pasteur pipette</td><td>Fisherbrand</td><td>13-678-20A</td><td></td></tr><tr><td>Cholesterol</td><td>Avanti Polar Lipids</td><td>700000</td><td></td></tr><tr><td>SpeedVac concentrator</td><td>Thermo Scientific</td><td></td><td>Model # SPD2010</td></tr><tr><td>Petroleum ether</td><td>Fisher Scientific</td><td>E139-1</td><td></td></tr><tr><td>Diethyl ether</td><td>Sigma-Aldrich</td><td>309966</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td></td></tr><tr><td>TLC Silica Gel 60 with concentrating zone</td><td>Analytical Chromatograhy Millipore</td><td>1.11845.0001</td><td></td></tr><tr><td>Iodine chips</td><td>Sigma-Aldrich</td><td>376558-50G</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>320501</td><td></td></tr><tr><td>Manganese chloride</td><td>Sigma-Aldrich</td><td>244589</td><td></td></tr><tr><td>UVP EC3 Imaging System</td><td>Ultra-Violet Products Ltd.</td><td></td><td>Use the Vision Works LS software for densitometry analysis</td></tr><tr><td>Glass bottom confocal dish</td><td>MatTek</td><td>P35G-1.5-10C</td><td>www.glassbottomdishes.com</td></tr><tr><td>Cryptococcus neoformans (H99)</td><td></td><td></td><td>Obtained from Duke University Medical Center</td></tr><tr><td>YNB</td><td>BD</td><td>239210</td><td>See manufacturer for preparation instructions. Use a glucose concentration of 20 g/L.</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma</td><td>L4391-1MG</td><td>Dissolve in 1x PBS to make 1 mg/ml stock. Store at -20 &amp;deg;C.</td></tr><tr><td>Interferon gamma</td><td>Sigma</td><td>I4777</td><td>Dissolve in 1x PBS to make 0.1 mg/ml stock solution</td></tr><tr><td>Glucuronoxylomannan antibody (anti-GXM)</td><td></td><td></td><td>Gift from Arturo Casadevall&#039;s Lab concentration is 1.98 mg/ml</td></tr><tr><td>Giemsa</td><td>MP Biomedicals</td><td>194591</td><td>Dissolve 0.8 g of Giemsa in 25 ml of glycerol and heat to 60 &amp;deg;C for 1 hr. Add 25 ml of methanol to the solution and allow to age at room temperature for at least 1 month.</td></tr><tr><td>Microscope</td><td>Zeiss</td><td></td><td>Observer.D1 microscope with AxioCam MRm for taking images</td></tr></tbody></table>

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.

Cholesterol Depletion
Analysis of the supernatant reserved in step 1.3 of the protocol by following the manufacturer&#8217;s instructions in the Amplex Red Cholesterol Assay kit yields an elevated concentration of cholesterol in M&#946;CD treated sample as compared to the 1x PBS control. Depending on cell type and M&#946;CD concentration used cholesterol depletion may vary. For J774 treated with 10 mM M&#946;CD, a depletion of approximately 50% was observed. Depletion can be calculated using valu...

Watch this Scientific Journal Video about Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans at JoVE.com

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.

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 ...

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Immunology and Infection

12.7K Views.  Stony Brook University. 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. 

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

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

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 ...

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 ...

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.

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.

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

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

The Effect of Aspirin and Ibuprofen on the Phagocytic Ability of Macrophages

This video showcases a method to assess the impact of aspirin and ibuprofen, at their minimum inhibitory concentrations, on macrophage phagocytosis using pH-sensitive fluorescent dye-stained fungal cells. The drugs inhibit cyclooxygenase-2, decreasing prostaglandin formation and boosting macrophage phagocytosis, as observed through fluorescence measurements of pH-sensitive dye.

1. Safety considerations

	Personnel should use personal protective equipment (PPE).
	Handle the pathogen and macrophages in a Class II Biological safety ...

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Immunology

Immunodiagnostics

Cellular Immunodiagnostics

A Lactate Dehydrogenase Release Assay to Detect Cryptococcus neoformans Infection in Macrophages

To prepare fungal cells for a macrophage infection, collect and centrifuge C. neoformans cells from a mid-log phase culture, followed by three gentle washes with 1 milliliter of room temperature PBS under the same centrifuge conditions.
After the last wash, re-suspend the fungal cells at a 1.2 times 10 to the eighth cells per milliliter, of antibiotic-free cell culture medium concentration, and add 1 milliliter of fungal cell suspension to each of 4 wells of a 70% to 80% confluent 6-well macrophage culture plate. Then, place the plate in the cell culture incubator for three hours. Carefully tilt the plate to allow each well to be gently washed three times with 1 milliliter of PBS per well per wash.
To measure the infection proficiency after the last wash, add 1 milliliter of antibiotic-free medium to each well of the 6-well plate, and place the plate in the cell culture incubator. At the appropriate experimental time points, collect the supernatant from each well, and measure the amount of LDH in each well according to standard protocols. At the same time points, lyse the uninfected macrophage cells to determine the value for maximum cytotoxicity.
The cytotoxicity can then be calculated using the formula as indicated.

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

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