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

Summary

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.

Abstract

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 (IFNγ) 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.

Introduction

Phagocytosis is a process by which extracellular entities are internalized by host cells. It is a key weapon in the immune system’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’s cytoskeleton. ‘Professional’ 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 ‘professional’ 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 phagocytosis^1,2.

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 the cryptococcal polysaccharide glucuronoxylomannan and other studies have shown prevalence in both human immunodeficiency virus (HIV) uninfected and infected adults^3,4. 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 fatal^5–8. 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 “Trojan horse” model^3,9–11. 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 phagocytosis^12–15. 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 signaling^16–18. 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-β-cyclodextrin (MβCD) 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 MβCD, 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-γ and lipopolysaccharide for activating the macrophages. Cryptococcus was opsonized using a glucuronoxylomannan (GXM) antibody^21–23. 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.

Protocol

1. Cholesterol Depletion of J774A.1 Cells with MβCD

1. In a sterile biosafety cabinet, seed 10⁵ J774A.1 macrophage-like cells per well on a 96-well cell culture plate in 200 μl of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Incubate at 37 °C and 5% CO₂ O/N.
2. Remove media from the cell monolayer and wash the cells twice with 1x phosphate buffered saline (PBS) that has been filtered or autoclaved.
3. Add 200 μl of MβCD 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 °C with shaking. Remove supernatant and reserve at RT for quantitative analysis with commercially available kit immediately following the procedure.
4. 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’s instructions as written.

2. Observation of Cholesterol Content by Thin Layer Chromatography (TLC)

1. 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.
2. 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 °C, 5% CO₂ O/N.
3. Deplete macrophages of cholesterol by following steps 1.2 - 1.4, substituting 1 ml for 200 μl where applicable to account for the larger well size.
4. Add 500 μl of Trypsin-EDTA to each well, incubate for 3 min at 37 °C, and gently scrape cells with a cell scraper.
5. Transfer into a microfuge tube and add an additional 500 μl of warm DMEM supplemented with 10% FBS and 1% P/S.
6. Spin the cells for 5 min at 300 x g and remove the supernatant.
7. Add an additional 500 μl of warm DMEM supplemented with 10% FBS and 1% P/S to the cell pellet and resuspend carefully by pipetting up and down.
8. Remove 10 μl 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.
9. Centrifuge cells at 300 x g for 5 min at RT and remove media.
10. 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 °C O/N.
11. Centrifuge at 1,700 x g for 10 min at RT and transfer the supernatant to a new tube
12. 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.
13. 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.
14. 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 °C until ready to perform TLC.
15. Dilute dried lipids in enough chloroform to normalize the concentration of lipid (usually 20 - 50 µl) and load 20 µl of the diluted lipid on a silica TLC plate. Load 20 μg of cholesterol diluted in 20 μl of chloroform as a standard.
16. 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.
17. 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.
18. Prepare a solution for neutral lipid staining by combining 60 ml of methanol with 60 ml of deionized H₂O, 4 ml of sulfuric acid, and 630 mg of manganese chloride.
19. 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.
20. 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 °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.

3. Infection of Macrophages with C. neoformans (H99)

1. In a sterile biosafety cabinet, seed 10⁵ macrophage-like cells per well on a 96-well cell culture plate in 200 μl of DMEM supplemented with 10% FBS and 1% P/S. Incubate at 37 °C, 5% CO_2, 5% CO₂ O/N.
   NOTE: Infection can also be done in glass bottomed confocal dishes for easier imaging; all amounts remain the same.
2. 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 °C with shaking.
3. Wash and count C. neoformans (H99) cells.
   1. Centrifuge C. neoformans O/N culture at 1,700 x g for 10 min at 4 °C.
   2. Remove media and discard. Wash cells with 5 ml of 1x PBS. Centrifuge at 1,700 x g for 10 min at 4 °C.
   3. Remove PBS and wash with 5 ml of filtered 1x PBS. Centrifuge at 1,700 x g for 10 min at 4 °C. Repeat this step 2 more times.
   4. Remove PBS and resuspend in 5 ml of 1x PBS.
   5. Make a serial dilution in PBS to obtain a 1:500 dilution of the washed culture.
      1. Add 100 μl of the original sample to 900 μl of 1x PBS to obtain a 1:10 dilution.
      2. Add 100 μl of 1:10 diluted sample to 900 μl of 1x PBS to obtain a 1:100 dilution.
      3. Add 200 μl of the 1:100 diluted sample to 800 μl of 1x PBS to obtain a 1:500 dilution
   6. Take 10 μl of 1:500 dilution and count on hemocytometer to calculate the number of cells.
4. Prepare working solution for activating macrophages and opsonizing C. neoformans.
   1. Dilute LPS and IFNγ 100x from stock solutions by adding 10 μl to 990 μl.
      NOTE: LPS and IFNγ 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 °C with shaking prior to infection.
   2. Per sample combine 7.5 μl of diluted LPS, 1.25 μl of diluted IFNγ, 1.25 μl of GXM antibody and the volume of the C. neoformans culture that gives 1.25 x 10⁵ cells. Bring volume up to 250 μl multiplied by number of samples with DMEM supplemented with 10% FBS and 1% P/S.
   3. Vortex and incubate solution for 20 min at 37 °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.
5. Infect Macrophages
   1. Wash macrophages twice with serum free DMEM and add 200 μl of opsonized C. neoformans working solution to each well.
   2. Incubate for 2 hr at 37 °C.
6. Fix and Stain Cells
   1. Remove media and wash cells 2 times with DMEM.
   2. Air dry the cell monolayer for 10 min and add 200 μl of ice cold methanol to fix the cells.
   3. Incubate for 15 min at RT and remove any remaining methanol.
   4. Add 200 μl of 10x Giemsa and incubate for 5 min at RT.
   5. 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.
7. Visualize and Count
   1. 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.
   2. 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.
   3. Take micrographs of cells at 1,000X or 400X magnification.

4. Trypan Blue Assay

1. 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’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Incubate at 37 °C, 5% CO₂ O/N.
2. Deplete macrophages of cholesterol by following steps 1.2 - 1.4 substituting 2 ml for 200 μl 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.
3. Add 500 μl 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.
4. Remove 10 μl of cells and stain with 1 μl of 4% Trypan blue.
5. 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.

Results

Cholesterol Depletion

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

Discussion

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...

Materials

List of materials used in this article

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