Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Podsumowanie

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Streszczenie

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).

Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Wprowadzenie

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania, resulting in a broad spectrum of clinical manifestations in the vertebrate host, including cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis¹. The World Health Organization (WHO) estimates that over one billion people are at risk, with more than one million new cases reported per year².

Leishmania spp. are obligate intracellular protozoans that survive inside host cells, including monocytes, macrophages and dendritic cells³. Leishmania-macrophage interaction is a complex process that involves multiple host cell receptors and parasite ligands either through direct interaction or by opsonization involving complement receptors^4,5. Classical surface receptors, such as CR1, CR3, mannose-fucose, fibronectin, toll-like and scavenger receptors, mediate parasite attachment to macrophages^6,7,8. These receptors recognize molecules on the surface of Leishmania, including the 63 kDa glycoprotein (gp63) and glycolipid lipophosphoglycan (LPG)⁹. These are the most abundant molecules on the surface of promastigotes and play an essential role in the subversion of host immune response, favoring the establishment of parasite infection in mammalian cells¹⁰. After parasite surface ligands bind to macrophage receptors, F-actin accumulates on mammalian cell surfaces, surrounding parasites as they are phagocytosed. Subsequently, this leads to the formation of a parasite-induced compartment termed parasitophorous vacuole (PV), which presents phagolysosomal features¹¹. Once inside these phagolysosomes, parasites undergo several alterations essential to survival and multiplication³.

The biogenesis of PVs is a highly regulated membrane trafficking process critical to the intracellular survival of this pathogen¹². The formation of this compartment results from sequential fusion events between phagosomes and compartments of the host endocytic pathway. Classical cell biology studies have revealed that the maturation of PVs involves the acquisition of monomeric G protein Rab 5 and Rab 7 proteins, which are mainly associated with early and late endosome maturation, respectively¹³. In addition, these compartments acquire lysosome-associated membrane proteins 1 and 2 (LAMP 1, LAMP 2), the principal protein constituents of the lysosomal membrane and microtubule-associated protein 1A/1B-light chain 3 (LC3), an autophagosome marker¹⁴. Despite apparent similarities, the kinetics of PV formation^15,16 and the morphology of these compartments vary depending on Leishmania species. For example, infection caused by L. mexicana or L. amazonensis induces the formation of large compartments containing a great number of parasites¹⁷. By contrast, other species, such as L. braziliensis and L. infantum, form smaller vacuoles that normally contain only one or two parasites in each vacuole¹⁸.

Despite this knowledge surrounding host cell-Leishmania interaction, the initial events triggered by contact between host receptors and parasite ligands have not been fully elucidated. These events are known to be determinants of the outcome of parasite infection and are dependent on parasite species, the type of host cell receptors recruited to recognize parasites and the activation of macrophage signaling pathways^19,20. Therefore, it is essential to identify the molecules involved in the biogenesis of Leishmania-induced PVs and determine the role(s) played by these molecules in infection establishment and outcome. Here, we describe a method of monitoring early events occurring during the phagocytosis of Leishmania, including binding, internalization, phagosome formation and maturation. This work could aid in clarifying the participation of PLC, Akt, Rab5, Rab7 and LC3 in the formation of PVs induced by different Leishmania species. Importantly, this protocol can be used to investigate the participation of other proteins involved in PV maturation. Future studies will expand the knowledge surrounding mechanisms involved in Leishmania-host cell interaction and contribute to the design of novel chemotherapeutic strategies.

Protokół

Cells were obtained from healthy donors following the approval of procedures by the National Research Ethics Committees (ID: 94648218.8.0000.0040).

1. Cell cultures

1. Human monocyte-derived macrophages
   NOTE: To obtain human monocyte-derived macrophages for in vitro differentiation into macrophages, collect blood from healthy donors and purify peripheral blood mononuclear cells (PBMC) as described by D. English and B. R. Andersen²¹.
   1. After collecting peripheral blood (50 mL), pour it into a heparinized tube and then dilute the blood 1:1 in a phosphate buffer solution (PBS) at room temperature. Gently place diluted heparinized blood on top of previously distributed density gradient medium.
   2. Centrifuge the tubes at 252 × g for 30 min at 24 °C to avoid hemolysis.
      NOTE: Set centrifuge break-off to avoid mixing of gradient layers. After centrifugation, discontinuous gradient layers are formed from the bottom to the top: erythrocytes, density gradient medium, PBMC ring and plasma.
   3. Transfer the PBMC ring, located between the density gradient medium and plasma layers, to a new tube and fill with PBS to wash out excess density gradient medium.
   4. Wash cells once and centrifuge at 190 × g for 10 min at 4 °C.
   5. Discard the supernatant and resuspend pellet in 1 mL of complete RPMI medium.
   6. Count the cells and plate 2 × 10⁶ cells in 500 mL of Roswell Park Memorial Institute (RPMI) supplemented with 25 mM N-[2-hydroxyethyl] piperazine-N′-[2-ethane sulfonic acid] (HEPES), 2 g/L sodium bicarbonate, 2 mM glutamine, 20 g/mL ciprofloxacin and 10% inactivated Fetal Bovine Serum (FBS) (complete RPMI medium) for 7 days at 37 °C under 5% CO₂ in a 24-well plate to allow monocytes to differentiate into macrophages by adhesion.
2. THP-1 cultures
   1. Grow THP-1 cell line at a concentration of 2 × 10⁵ cells in 10 mL of complete RPMI medium in 75 cm² culture flask.
   2. Maintain cell cultures in an incubator at 37 °C under 5% CO₂ for 7 days.
   3. Centrifuge cells at 720 × g for 10 min at 4 °C and resuspend the pellet in complete RPMI medium.
   4. Count cells in a Neubauer chamber.
   5. Plate cells on 13 mm glass coverslips at a concentration of 2 × 10⁵ cells per well in 500 μL of complete RPMI medium containing 100 nM phorbol myristate acetate (PMA) at 37 °C under 5% CO₂ to allow differentiation of THP1 cells into macrophages.
   6. After three days, wash twice cells with 0.9% NaCl solution to remove medium containing PMA.
   7. Incubate differentiated THP-1 cells in PMA-free complete RPMI medium at 37 °C under 5% CO₂ for an additional 2 days before starting experimentation.

2. Parasite cultures and CellTracker Red staining

NOTE: To visualize parasites through fluorescence microscopy, perform staining using CellTracker Red fluorescent dye (CMTPX). Alternatively, other markers, including carboxyfluorescein can be used in accordance with manufacturer instructions or promastigotes constitutively expressing GFP, RFP, or other fluorescent reporter genes. Parasites used to infect cells are those at stationary phase of growth obtained from a promastigote axenic culture of no more than 7 passages.

1. Grow Leishmania spp. promastigotes at 1 x 10⁵ parasites per 1,000 μL of medium in a cell culture flask containing 5 mL of Schneider's medium supplemented with 50 μg/mL gentamicin and 10% FBS.
2. After incubating parasite axenic cultures in a biochemical oxygen demand (B.O.D.) at 24 °C, perform daily counting in a Neubauer chamber. Check for parasite form (thin, elongated) and mobility for 5 days. Parasites are considered in stationary phase of growth when two consecutive counts with 8 hours of interval display similar amounts.
3. Upon reaching the stationary phase of growth, incubate the parasites in 4 mL of 0.9% NaCl solution with 1 μM CMTPX for 15 min at 37 °C under 5% CO₂ avoiding contact with light.
4. Add FBS at a 1:1 proportion and incubate parasite suspension for an additional 1 min.
5. Wash parasites thrice with PBS, followed by centrifugation at 1,781 × g for 10 min.
6. Ressuspend parasite pellet in 1,000 μL of RPMI complete medium.
7. Count parasites in a Neubauer chamber.

3. Assessment of Leishmania binding to macrophages

1. Seed 2 × 10⁵ THP-1 cells or human monocyte-derived macrophages in 500 μL of complete RPMI medium per well on a 24-well plate with 13 mm glass coverslips.
2. Cultivate cells at 37 °C under 5% CO₂ for 24 h.
3. Wash the cells twice with 0.9% NaCl solution and incubate in complete RPMI medium at 4 °C for 10 min.
4. Add stationary phase promastigotes as described by A. L. Petersen²² at a 10:1 ratio to well plates, and then centrifuge at 720 × g for 5 min under 4 °C.
5. Incubate at 4 °C for 5 min.
6. Wash the cells twice with 0.9% NaCl solution to remove any non-internalized promastigotes.
7. Fix the cells in 4% paraformaldehyde for 15 min at room temperature.
8. Incubate the coverslips with 15 mM NH₄Cl for 15 min at room temperature.
9. Wash thrice with PBS 0.15% bovine serum albumin (BSA). Incubate with blocking solution (3% BSA in PBS) for 1 h at room temperature.
10. Wash thrice with PBS and then permeabilize with 0.15% PBS-Saponin for 15 min at room temperature.
11. Add phalloidin (diluted 1:1,200) for 1 h at room temperature and protect from light.
12. Mount coverslips using mounting media.
13. Acquire images via a confocal fluorescence microscope using a 63×/1.4 objective.

4. Assessment of Leishmania phagocytosis by macrophages

1. Seed 2 × 10⁵ THP-1 cells or human monocyte-derived macrophages in 500 μL of complete RPMI medium per well on a 24-well plate with 13 mm glass coverslips.
2. Cultivate cells for 24 h at 37 °C under 5% CO₂.
3. Wash cells twice in 0.9% NaCl solution and incubate in complete RPMI medium in 24-well plate at 4 °C for 10 min.
4. Add stationary phase Leishmania spp. as described by A. L. Petersen²² at a 10:1 (parasite:host cell) ratio, and then centrifuge at 720 × g for 10 min under 4 °C.
5. Incubate cells at 4 °C for 5 min.
6. Wash the cells twice with 0.9% NaCl solution to remove any non-internalized promastigotes.
7. Incubate the cells in supplemented RPMI medium at 37 °C for 1 h.
8. Fix the cells with 4% paraformaldehyde for 15 min.
9. Mount coverslips using preferred mounting media.
10. Count no less than 400 cells in random fields under a fluorescence microscope using a 100×/1.4 objective.

5. Evaluation of Leishmania -induced vacuole maturation

NOTE: THP-1 cell transfection should be performed as described by M. B. Maess, B. Wittig and S. Lorkowski ²³. Here we summarize this protocol, with minimal modifications. Nucleofection is a specific transfection method that requires a nucleofector. As an alternative method, cells can be transfected using lipofectamine²⁴ and lentivirus transduction²⁵.

1. To investigate the biogenesis of Leishmania-induced PV, transfect THP1 cells with PLC^26,27, Akt^26,27, Rab 5^28,29,30 or Rab 7^28,29,31 plasmids.
   ​NOTE: This methodology can be used to transfect THP-1 cells with other genes than those listed above.
2. Seed THP-1 cells at 1.5 x 10⁷ in 75 cm² tissue culture flasks containing 10 mL complete RPMI medium supplemented with 100 ng/mL PMA and 50 µM 2-mercaptoethanol for 48 h.
3. Wash cells once in 0.9% NaCl solution.
4. Detach cells using a non-enzymatic cell dissociation solution and centrifuge (250 × g) for 5 min at room temperature.
5. Resuspend THP-1 cells in 1 mL of RPMI medium and perform counts in a Neubauer chamber.
6. Centrifuge THP-1 cells again at 250 × g for 10 min at room temperature. Discard the supernatant.
7. Resuspend 2 × 10⁶ cells in 100 µL of Nucleofector solution and incubate with 0.5 µg of the plasmid coding for the protein of interest, tagged with a fluorescent protein.
8. Transfer the suspension containing THP-1 cells and nucleic acid to the Nucleofector cuvette.
9. Transfect THP1 cells using Nucleofector Program Y-001.
10. Recover the transfected cells (2x10⁶) and seed in 500 μL RPMI medium on 24-well plates with 13 mm glass coverslips (4 wells/transfection).
11. Incubate THP-1 cells in complete RPMI medium at 37 °C for 0.5, 2, 4, 6, 12 and 24 h.
12. Repeat steps 3.13 and 3.13.

6. Evaluation of the recruitment of LC3 to Leishmania spp. PVs

NOTE: The autophagic membrane marker LC3 can be used to investigate whether phagosomes present autophagic features. LC3 recruitment to Leishmania-induced PVs can be assessed during infection by immunolabelling cells with the anti-LC3 antibody, as previously described by C. Matte³² and B. R. S. Dias³³.

1. Seed 2 × 10⁵ THP-1 cells or human monocyte-derived macrophages in 500 μL complete RPMI medium on a 24-well plate with 13 mm glass coverslips.
2. Cultivate cells for 24 h at 37 °C under 5% CO₂.
3. Wash cells twice in 0.9% NaCl solution and incubate in complete RPMI medium.
4. Add stationary phase Leishmania spp. promastigotes as described by A. L. Petersen²² at a 10:1 (parasite: host cells) ratio and centrifuge cells at 720 × g for 5 min under 4 °C.
5. Incubate at 37 °C for 30 min or 4 h. Then wash twice and fix the cells to evaluate the LC3 recruitment to Leishmania-induced PV membranes at the early stages of infection.
   1. Alternatively, to assess LC3 recruitment to PV membranes at later stages of infection, wash twice another macrophage group at 4 h of infection to remove any non-internalized promastigotes. Incubate infected cells in complete RPMI medium for an additional 12 h and 24 h, to finally wash twice and fix.
      NOTE: Fixed cells can be kept in PBS or 0.9% NaCl solution at 4 °C until labeling.
6. Simultaneously block and permeabilize the fixed cells in 0.1% Triton X-100, 1% BSA, 20% normal goat serum, 6% non-fat dry milk, and 50% FBS for 20 min at room temperature.
7. Incubate the cells with anti-LC3 antibody (1: 200) diluted in PBS for 2 h at room temperature.
   NOTE: As a negative control of the immunostaining, a group of cells should be incubated with immunoglobulin G (IgG) from the animal of primary antibody origin in a concentration equivalent to that used for the primary antibody.
8. Wash the cells thrice with 0.9% NaCl solution at room temperature.
9. Incubate the cells with AlexaFluor 488-conjugated goat anti-rabbit IgG (1:500) or the preferred fluorescent-dye conjugated secondary antibodies for 1 h at room temperature.
10. Wash the cells thrice with 0.9% NaCl solution at room temperature.
11. Mount coverslips using preferred mounting media.
12. Acquire images via confocal fluorescence microscope using a 63x/1.4 objective.

7. Confocal microscopy acquisition and Fiji quantification

NOTE: Acquiring immunofluorescence images should be performed using a confocal laser scanning microscope. To reach a better resolution, use an oil-immersion 63x objective lens.

1. Leave the 13 mm glass coverslips at room temperature and protect them from the light at least 30 min before the acquisition.
2. Clean the coverslips with an absorbent tissue.
3. Add a drop of immersion oil to the objective and add the slide.
4. Move the objective up until the oil touches the slide.
5. Observe and adjust the focus on the microscope and choose the option 63x objective with oil.
6. Open the Leica program and adjust the lasers in the 488, 552, and 405 wavelengths.
7. Select the image resolution 1,024 x 1,024.
8. Click on the Live button, set the Z stack, and press the Begin option. Then, do it again and press the End button. We recommend 20 μm for slice thickness to get confocal images with good resolutions.
9. Wait for the image acquisition, and then select the option "Maximum Projection" in the Leica tools.
10. Save the experiment.
11. Export the lif or tiff format images to a computer and open the FIJI program.
12. Open the experiment and set the view stack with the hyper stack. Then select open files individually and stitch tiles.
13. Select the free hands tool in the Fiji toolbar and trace the cell carefully by hand.
14. Press the Analyze button and measure to visualize the fluorescence intensity.
15. Repeat this process to each cell per group.
16. Save the measurements and export them to a spreadsheet editor.
17. Add this data to a statistical analysis program and do the statistical analysis.

8. Statistical analysis

NOTE: For data analysis and graphics, use a statistical analysis program.

1. Open the program.
2. Insert the obtained data and test the normality parameters.
3. For data with normal distribution, use the Student t-test and for nonparametric tests, Mann-Whitney test.
4. Consider data with a statistically significant difference when the p-value is less than 0.05.
5. Prepare graphics representing the data, with central tendency measures (mean or median) and variation measures.

Wyniki

This report aims to evaluate the early events occurring during the phagocytosis of L. braziliensis isolated from patients presenting L. braziliensis-LCL or L. braziliensis-DL form of CL. Using confocal microscopy, we investigated the main events associated with parasites' phagocytosis: binding, internalization, and phagosome maturation. We first evaluated the L. braziliensis-LCL or L. braziliensis-DL binding and phagocytosis by human monocyte-derived macrophages. The data ...

Dyskusje

Leishmania-macrophage interaction is a complex process and involves several steps that can influence disease development⁵. To better understand the mechanisms involved in the interaction of unopsonized Leishmania and host cells, we have described a protocol that employs confocal fluorescence microscopy to assess phagocytosis from early to late stages of Leishmania infection. The use of fluorescence techniques involving two or more fluorophores to investigate cell biology...

Materiały

Name	Company	Catalog Number	Comments
2-mercaptoethanol	Thermo Fisher Scientific	21985023
AlexaFluor 488-conjugated goat anti-rabbit IgG	Thermo Fisher Scientific	Tem varios no site
anti-LC3 antibody	Novus Biologicals	NB600-1384
Bovine serum albumin (BSA)	Thermo Fisher Scientific	X
CellStripper	Corning	25-056-CI
CellTracker Red (CMTPX) Dye	Thermo Fisher Scientific	C34552
Centrífuga	Thermo Fisher Scientific
Ciprofloxacin	Isofarma	X
CO2 incubator	Thermo Fisher Scientific	X
Confocal fluorescence microscope (Leica SP8)	Leica	Leica SP8
Fetal Bovine Serum (FBS)	Gibco	10270106
Fluorescence microscope (Olympus Lx73)	Olympus	Olympus Lx73
Gentamicin	Gibco	15750045
Glutamine	Thermo Fisher Scientific	35050-061
HEPES (N- 2-hydroxyethyl piperazine-N’-2-ethane-sulfonic acid)	Gibco	X
Histopaque	Sigma	10771
M-CSF	Peprotech	300-25
NH4Cl	Sigma	A9434
Normal goat serum	Sigma	NS02L
Nucleofector 2b Device	Lonza	AAB-1001
Nucleofector solution	Lonza	VPA-1007
Paraformaldehyde	Sigma	158127
Phalloidin	Invitrogen	A12379
Phorbol myristate acetate (PMA)	Sigma	P1585
Phosphate buffer solution (PBS)	Thermo Fisher Scientific	10010023
ProLong Gold Antifade kit	Life Technologies	P36931
Roswell Park Memorial Institute (RPMI) 1640 medium	Gibco	11875-093
Saponin	Thermo Fisher Scientific	X
Schneider's Insect medium	Sigma	S0146
Sodium bicarbonate	Sigma	S5761
Sodium pyruvate	Sigma	S8636
Triton X-100	Sigma	X