Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Podsumowanie

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Streszczenie

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Wprowadzenie

Candida species are the leading cause of life-threatening invasive fungal infections in immunocompromised patients¹. Candida glabrata, an emerging nosocomial pathogen, is the second or third most frequently isolated Candida species from Intensive Care Unit patients depending upon the geographical location^1-3. Phylogenetically, C. glabrata, a haploid budding yeast, is more closely related to the non pathogenic model yeast Saccharomyces cerevisiae than to pathogenic Candida spp. including C. albicans⁴. Consistent with this, C. glabrata lacks some key fungal virulence traits including mating, secreted proteolytic activity and morphological plasticity^4-5.

Although C. glabrata does not form hyphae, it can survive and replicate in murine and human macrophages^6-8 suggesting that it has developed unique pathogenesis mechanisms. Limited information is available about the strategies that C. glabrata employs to survive nutrient-poor intracellular macrophage environment and counteract oxidative and nonoxidative host responses mounted by host immune cells⁵. A pertinent macrophage model system is a prerequisite to delineate the interaction of C. glabrata with host phagocytic cells via functional genomic and proteomic approaches. Peripheral blood mononuclear cells (PBMCs) and bone marrow-derived macrophages (BMDMs) of human and murine origin, respectively, have earlier been used to study the interaction of C. glabrata with host immune cells^7,9. However, difficulty in obtaining PBMCs and BMDMs, their limited life span and intrinsic variation among different mammalian donors restrict the utilization of these cells as versatile model systems.

Here, we describe a method for establishment of an in vitro system to study the intracellular behavior of C. glabrata cells in macrophages derived from human monocytic cell line THP-1. The overall goal of this protocol was to enact a simple, inexpensive, quick, and reproducible cell culture model system that can be easily manipulated to study different aspects of host-fungal pathogen interaction.

THP-1 cells have previously been used to decipher the host immune response against a wide range of pathogens including bacteria, viruses, and fungi^10-12. Monocytic THP-1 cells are easy to maintain and can be differentiated, upon phorbol ester treatment, to macrophages which mimic monocyte-derived macrophages of human and express appropriate macrophage markers¹³. The main advantages of THP-1 macrophage model system are the ease-of-use and the lack of sophisticated equipment requirement.

The protocol presented here is easily adaptable to study the interaction of other human fungal pathogens with host immune cells. The current procedure can also be employed to identify virulence factors for the pathogen of interest using high throughput mutant screens. This proof-of-concept was exemplified by the successful use of THP-1 culture model system to identify a set of 56 genes that are required for survival of C. glabrata in human macrophages⁸.

Protokół

It is recommended to perform C. glabrata infection experiments in a laboratory with biosafety containment level 2 (BSL-2).

1. Preparation of THP-1 macrophage monolayer.
2. Preparation of C. glabrata cell suspension.
3. Infection of THP-1 macrophages with C. glabrata cells.
4. Measurement of phagocytosis rate and intracellular replication via colony forming unit assay.
5. Monitoring of intracellular replication using confocal laser scanning microscopy.

1. Preparation of THP-1 Macrophage Monolayer

1. THP-1 is a human monocytic cell line derived from the peripheral blood of a 1-year-old male child suffering from acute monocytic leukemia¹⁴. THP-1, a suspension cell line, is routinely maintained at 37 °C in humidified 5% CO₂ in RPMI-1640 culture medium supplemented with 10% FBS (fetal bovine serum). Treatment with phorbol 12-myristate 13-acetate (PMA, a phorbol ester) results in terminal differentiation of THP-1 monocytic cells into macrophages. Importantly, PMA treatment does not affect cell viability and differentiated cells do not divide.
2. Seed approximately 1.5 x 10⁶ THP-1 monocytes in 100 mm culture dishes in RPMI-1640 complete medium (RPMI-1640 medium supplemented with 10% serum, 2 mM glutamine and 1x antibiotics; 10 ml/dish) and let cells grow at 37 °C in 5% CO₂ for 2 days.
3. Once THP-1 cells have reached a density of 8 x 10⁵ cells/ml, transfer the culture dish to the cell culture laminar flow hood and gently swirl it to resuspend settled cells.
4. Pipette the cell suspension out, place into a 15 ml sterile tube and centrifuge at 1,000 rpm (130 x g) for 4 min. Discard supernatant and suspend THP-1 cell pellet in 5 ml fresh, prewarmed, RPMI-1640 complete medium.
5. Enumerate cells with a hemocytometer and dilute the cell suspension to a final density of 10⁶ cells/ml with prewarmed RPMI-1640 complete medium
6. Add 1 µl of 160 mM PMA stock (phorbol 12-myristate 13-acetate solution prepared in dimethyl sulfoxide) to 10 ml THP-1 cell suspension (final concentration 16 nM) and mix well. Dispense 1 ml cell suspension into each well of a 24-well tissue culture plate and incubate plate in the incubator maintained at 37 °C with 5% CO₂ for 12 hr.
7. Remove old medium, add 1 ml prewarmed RPMI-1640 complete medium and allow cells to recover for 12 hr at 37°C in 5% CO₂.
8. Observe cells under an inverted microscope to ensure that oval-shaped THP-1 monocytic cells in suspension have been differentiated into flattened, spindle-shaped and adherent  macrophages. These differentiated cells are now ready to conduct C. glabrata infection studies.

2. Preparation of C. glabrata Cell Suspension

C. glabrata wild-type strain Bg2 will be used to infect THP-1 macrophages. C. glabrata cells are routinely cultured in liquid YPD (Yeast extract (1%)-Peptone (2%)-Dextrose (2%)) medium. Solid YPD medium is prepared by adding 2% agar before medium autoclaving.

1. To prepare a culture of C. glabrata, pick a single colony up from the agar plate with a sterile, disposable loop and inoculate into 10 ml YPD  liquid medium and incubate for 14-16 hr with shaking (200 rpm) at 30 °C.
2. Centrifuge this culture (1 ml) at 4,000 rpm (1,800 x g) for 5 min in a table top microcentrifuge, wash cells thrice with sterile PBS (phosphate-buffered saline, pH 7.4) and suspend cell pellet in 1 ml PBS.
3. Measure OD₆₀₀ of C. glabrata cell suspension and dilute with an appropriate volume of sterile PBS to get a density of 2 x 10⁶ cells/ml (OD₆₀₀ = 0.1). Alternatively, the desired cell density can be achieved by counting yeast cells using a hemocytometer.

3. Infection of THP-1 Macrophages with C. glabrata Cells

1. Add 50 µl C. glabrata cell suspension to THP-1 macrophages (10⁶ cells seeded per well of a 24-well culture plate) to obtain a MOI (multiplicity of infection) of 0.1 and incubate plate at 37 °C with 5% CO₂.
2. To determine viable yeast counts, dilute 50 μl C. glabrata cell suspension 100-fold in sterile PBS and plate 100 μl on YPD solid medium. Incubate plates at 30 °C and count manually the number of yeast colonies appearing after 1-2 days. These numbers will, henceforth, be referred as 0 hr C. glabrata CFUs (colony forming units).
3. After 2 hr coculturing of THP-1 cells with C. glabrata cells, discard culture medium by inverting 24-well tissue culture plate in a reservoir and wash gently thrice with prewarmed sterile PBS to remove nonphagocytosed extracellular C. glabrata cells. Gentle washes with PBS are absolutely essential to accomplish complete removal of all extracellular yeast without causing any damage to the THP-1 macrophage monolayer.

4. Measurement of Phagocytosis Rate and Intracellular Replication via Colony Forming Unit Assay

1. Lyse C. glabrata infected THP-1 macrophages in 1 ml sterile H₂O for 2 min, scrape gently to remove the macrophage debris from the well and collect cell lysate in a microcentrifuge tube. Microscopic verification of complete macrophage lysis is pivotal for the recovery of all internalized yeast.
2. Prepare a 100-fold dilution of lysate in sterile PBS, plate 100 μl on YPD solid medium and incubate plates at 30 °C.
3. After 1-2 days, count manually the number of C. glabrata colonies that appeared on YPD medium, multiply with dilution factor (2 hr CFUs) and calculate phagocytosis rate which refers to the percent of C. glabrata cells that are ingested by THP-1 macrophages after 2 hr coincubation  using the following formula.
                          % phagocytosis = [(CFUs at 2 h)/(CFUs at 0 h)] X 100
4. To measure the rate of intracellular replication of C. glabrata cells in THP-1 macrophages, collect intracellular yeast at different time points post infection i.e. 4, 6, 8, 10, 12, and 24 hr, by repeating steps 3.3-4.3.
5. Calculate fold survival/replication of C. glabrata cells in THP-1 macrophages by dividing total CFUs at any given time point with those at 2 hr (phagocytosed yeast).

5. Monitoring of Intracellular Replication Using Confocal Laser Scanning Microscopy

1. Following the steps described in Section 1, seed PMA-treated 5 x 10⁵ THP-1 cells in each well of two four-well chamber slides and incubate at 37 °C with 5% CO₂ for 12 hr.
2. Replace old medium with prewarmed RPMI-1640 complete medium and allow cells to recover from PMA treatment for 12 hr.
3. Prepare GFP (green fluorescent protein)-tagged C. glabrata cell suspension as described in Section 2 and infect to THP-1 macrophages to a MOI of 1. If C. glabrata strains expressing GFP are not available, yeast cells labeled with FITC (fluorescein isothiocyanate) can  be used to monitor early events post infection viz. phagocytosis rate and phagolysosomal maturation at 2 hr.
4. Incubate slides for 2 hr at 37 °C with 5% CO₂, invert them carefully to discard medium and wash 3x with sterile, prewarmed PBS.
5. Add 500 µl prewarmed RPMI-1640 complete medium to each chamber of one slide and incubate it at 37 °C with 5% CO₂ for 22 hr.
6. To fix 2 hr C. glabrata-infected THP-1 macrophages, add 500 µl of 3.7% formaldehyde (prepared in PBS) to each chamber of the other slide and incubate it at room temperature for 20 min in the dark.
7. Wash slide thrice with PBS, add 500 µl Triton-X (0.7%), and incubate at room temperature for 5 min in dark.
8. Wash slide 3x with PBS, remove chamber from culture slide and air dry it for 3-5 min in the dark.
9. Carefully mount a coverslip using Vectashield mounting medium containing DAPI (4',6-diamidino-2-phenylindole) on slide avoiding bubble formation. Remove gently the excess liquid with Kimwipe and seal coverslip edges with fingernail polish. Store slide in the dark at 4°C until use.
10. Repeat steps 5.4 and 5.6-5.9 to process THP-1 cells 24 hr post infection.
11. Image cells using a laser scanning confocal microscope (60X oil-immersion objective, excitation at 405 nm and 488 nm for DAPI and GFP stain, respectively).

Wyniki

Infection analyses of PMA-treated THP-1 macrophages with C. glabrata wild type (wt) cells revealed that wt cells were phagocytosed by macrophages at a rate of 55-65% after 2 hr coincubation. Further, C. glabrata cells were able to resist killing by THP-1 macrophages and underwent a moderate 5- to 7-fold increase in CFUs after 24 hr of coculturing with THP-1 macrophages⁸.  Intracellular replication of wild type cells, transformed with GFP-expressing plasmid, in THP-1 mac...

Dyskusje

Innate immune system plays an important role in the control of opportunistic fungal infections. Macrophages contribute to antifungal defense by ingestion and destruction of the fungal pathogen. Thus, elucidation of factors that are required for survival and/or counteracting the antimicrobial functions of macrophages will advance our understanding of fungal virulence strategies. In this context, we have established an in vitro cell culture model system using macrophages derived from a human monocytic cell line TH...

Materiały

Name	Company	Catalog Number	Comments
THP-1	American Type Culture Collection	TIB 202	Human acute monocytic leukemia cell line
RPMI-1640	Hyclone	SH30096.01	For maintaining THP-1 cells
Phorbol 12-myristate 13-acetate	Sigma-Aldrich	P 8139	Caution: Hazardous
YPD	BD-Difco	242710	For growing Candida glabrata cells
Formaldehyde	Sigma-Aldrich	F8775	For fixation of C. glabrata-infected THP-1 macrophages
Phosphate buffered saline (PBS)			Buffer (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl,  pH 7.4) for washes
Saline-sodium citrate (SSC)			Buffer (3 M NaCl, 0.3 M sodium citrate for 20x concentration) for washes
Prehybridization buffer			Buffer (50% formamide, 5x Denhardt’s solution, 5x SSC, 1% SDS) for hybridization
VECTASHIELD mounting medium	Vector Labs	H-1200	For mounting slides for confocal microscopy
32P-labeled α-dCTP	JONAKI-BARC	LCP-102	For radiolabeling of signature tags
100 mm tissue culture dishes	Corning	430167	To culture THP-1 cells
24-well tissue culture plate	Corning	3527	To perform C. glabrata infection studies in THP-1 macrophages
4-chamber tissue culture-treated glass slide	BD Falcon	REF354104	To image C. glabrata-infected THP-1 macrophages
Hemocytometer	Rohem India		For enumeration of cells
Table top microcentrifuge	Beckman Coulter	Microfuge 18	For spinning down cells in microtubes
Table top centrifuge	Remi	R-8C	For spinning down cells in 15 ml tubes
Spectrophotometer	Amersham Biosciences	Ultraspec 10	To monitor absorbance of yeast cells
Plate incubator	Labtech	Refrigerated	To grow C. glabrata cells
Shaker incubator	New Brunswick	Innova 43	To grow C. glabrata cells
Water jacketed CO2  incubator	Thermo Electron Corporation	Forma series 2	To culture THP-1 cells
Confocal microscope	Carl Ziess	Ziess LSM 510 meta	To observe C. glabrata-infected THP-1 macrophages
Compound microscope	Olympus	CKX 41	To observe C. glabrata and THP-1 macrophages
PCR machine	BioRad	DNA Engine	To amplify unique tags from input and output genomic DNA
Hybridization oven	Labnet	Problot 12S	For hybridization
PhosphorImager	Fujifilm	FLA-9000	For scanning hybridized membranes
Thermomixer	Eppendorf	Thermomixer Comfort	For denaturation of radiolabeled signature tags
Gel documentation unit	Alphainnontech	Alphaimager	To visualize ethidium bromide-stained DNA in agarose gels