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.
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.
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 receptors4,5. Classical surface receptors, such as CR1, CR3, mannose-fucose, fibronectin, toll-like and scavenger receptors, mediate parasite attachment to macrophages6,7,8. 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 formation15,16 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 pathways19,20. 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.
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
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.
3. Assessment of Leishmania binding to macrophages
4. Assessment of Leishmania phagocytosis by macrophages
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.
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.
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.
8. Statistical analysis
NOTE: For data analysis and graphics, use a statistical analysis program.
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 show that both L. braziliensis-LCL and L. braziliensis-DL similarly bind to macrophages (Figure 1). Also, no differences were observed regarding L. braziliensis-LCL and L. braziliensis-DL phagocytosis by host cells (Figure 2). Finally, we compared the recruitment of LC3 to the PVs induced by L. braziliensis-LCL or L. braziliensis-DL in infected cells. After 30 min, 4 and 12 h of infection, we observed similar percentages of LC3 decorated PVs in L. braziliensis-LCL and L. braziliensis-DL-infected macrophages (Figure 3). These representative results showed that L. braziliensis-LCL and L. braziliensis-DL similarly interact with macrophages during binding, phagocytosis, and biogenesis of PVs, concerning the LC3 recruitment.
Microscopic images representing THP-1 cells efficiently transfected with PLC-GFP, Rab5-GFP, Rab7-GFP plasmids are shown in Figure 4.
Figure 1. Evaluation of L. braziliensis-LCL and L. braziliensis-DL binding to human macrophages. Human monocyte-derived macrophages were infected with L. braziliensis-LCL- or L. braziliensis-DL. After 10 min at 4 °C, the binding was assessed by confocal microscopy. (A) Confocal microscopy images of L. braziliensis-LCL or L. braziliensis-DL (labeled with CMTPX, red) binding to macrophages (labeled with phalloidin, green). For confocal microscopy, cell nuclei were labeled with DAPI (blue). Arrows depict Leishmania-macrophage binding. (B) Percentage of Leishmania binding to the macrophages. A total of 30 cells per group were analyzed. Data represent each replicate of one experiment performed in quintuplicate (unpaired t test, p > 0.05). Please click here to view a larger version of this figure.
Figure 2. Evaluation of L. braziliensis-LCL and L. braziliensis-DL phagocytosis by human macrophages. Human monocyte-derived macrophages were incubated with L. braziliensis-LCL or L. braziliensis-DL for 10 min at 4 °C followed by additional 1 h at 37 °C. Cells were then analyzed by fluorescence microscopy by counting a total of 400 cells. (A) Confocal microscopy images of human macrophages infected by L. braziliensis-LCL or L. braziliensis-DL. For confocal microscopy, cell nuclei were labeled with DAPI (blue). Arrows depict Leishmania parasites nuclei.(B) Percentage of Leishmania phagocytosis. Circles represent data from each replicate of one experiment performed in triplicate (unpaired t test, p > 0.05). Please click here to view a larger version of this figure.
Figure 3. Assessment of LC3 recruitment to PVs induced by L. braziliensis-LCL or L. braziliensis-DL in macrophages. Human monocyte-derived macrophages were infected and then stained with anti-LC3 antibody for 30 min, 4 and 12 h. (A) Confocal microscopy images of L. braziliensis-LCL or L. braziliensis-DL-infected macrophages labeled with anti-LC3 followed by the secondary anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (green). For confocal microscopy, cell nuclei were labeled with DAPI (blue); (B) Percentage of L. braziliensis-LCL or L. braziliensis-DL-induced PVs decorated with LC3-II. A total of 30 cells per group were analyzed. The circles correspond to each randomly selected field analyzed (unpaired t test, p > 0.05). Please click here to view a larger version of this figure.
Figure 4. THP-1 cells expressing PLC, Rab5 or Rab7. After differentiating into macrophages, THP-1 cells were subjected to nucleofection with each gene of interest coupled to GFP fluorescent probes: PLC, Rab5 and Rab7. Subsequently, these cells were fixed, had the nucleus stained with DAPI (blue) and were observed under a confocal microscope using a 63x/1.4 objective. Please click here to view a larger version of this figure.
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 mechanisms, including immunolabeling and the expression of fluorescent-labeled proteins, allows us to analyze the location of several proteins, as well as to simultaneously evaluate cell morphology. The advantages offered by these methods make them the best tools to monitor pathogen-host cell interaction34.
To better understand the phagocytic process involving different particles, it is crucial to analyze this highly dynamic process at the molecular level35. Confocal-fluorescence microscopy has been used for decades to this end and has been shown to be an excellent tool for quantifying phagocytosis through the determination of numbers of internalized particles, or the types of proteins known to be involved in early stages of host-pathogen interaction34. The present study proposed the use of confocal microscopy to analyze events occurring during the phagocytosis of L. braziliensis isolated from patients with different clinical forms (LCL and DL). This technique enables us to study cells expressing specific fluorescent proteins, including PLC, Akt, Rab 5, and Rab 7, and subsequently evaluate the participation of these proteins in the phagocytosis of Leishmania isolates to identify elements relevant to different infection outcomes.
The present study employed primary macrophages and THP1 cells to assess L. braziliensis phagocytosis at early stages of infection. The presently described protocol can also be used to study phagocytosis in Leishmania spp. by other phagocytes, including dendritic cells, monocytes, macrophage cell-lines, and neutrophils derived from human peripheral blood. During the parasite internalization process, a dynamic change in F-actin occurs at the cell membrane surface11. We then labeled proteins located in the cell membrane using a specific marker of phagocytosis36, such as fluorescent PLC, which allowed us to observe the binding stage of Leishmania to host cells, as shown in Figure 4. Staining parasites with fluorescent markers, such as CMTPX or CSFE, is also crucial to assess parasite binding to host cells by immunofluorescence. It is worth noting that this assay requires careful execution: i) wash coverslips gently using washing solutions at room temperature (25 °C), otherwise, samples can be damaged; ii) prepare reagent dilutions precisely; and iii) protect the samples from light34.
A confocal microscope configured to the optimal laser excitement wavelength is capable of obtaining a high-quality sample image. Labeled cells can be stored for weeks in the dark at 4 °C or frozen until the time of analysis. The use of confocal microscopy to evaluate phagocytosis is limited by prolonged times of exposure and high intensity laser beams, which can damage samples, and, in some cases, lead to high levels of background detection in images35,37.
In the present study, instead of using live imaging to follow the phagocytosis of Leishmania spp., we performed a kinetic study by fixing cells at several early times of infection (30 min, 4 h, and 12 h). It must be considered that live imaging offers some advantages, such as the potential to analyze the spatial and temporal dynamics of myriad cellular processes, including phagocytosis, and capturing details that are not observable in static images34. However, live imaging requires that cells be healthy throughout the entire experimentation process, including controlling temperature, pH and oxygen conditions in a microscopic chamber. It is important to note that this cannot be reliably performed at several laboratories around the world.
The nucleofection protocol described has demonstrated efficacy in the transfection of THP-1 cells, as previously reported by M. B. Maess, B. Wittig and S. Lorkowski 23. In this process, it is crucial to gently detach cells to avoid cell damage or loss in cell viability. Based on our experience, we recommend using a non-enzymatic cell dissociation solution to detach cells from plates prior to performing transfection. The authors of the original protocol23 state that the main limitations of this procedure are the need for cells to be in suspension during the nucleofection process, and the fact that inadequate detachment can cause stress. Despite these limitations, the protocol does allow for reliable transfection, reaching a 90% successful transfection rate without losing cell viability.
The characterization of PVs using a set of endocytic markers, including PLC, Akt, Rab5, and Rab7, is essential to improving our understanding of Leishmania phagocytosis. Identifying new proteins that participate in PV biogenesis and comprehensively characterizing these compartments can clarify differences in macrophage response during Leishmania spp. infection. The contribution of our results to the body of knowledge surrounding Leishmania infection outcome will undoubtedly advance our understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets. It is worth noting that this technique can also be extended to other types of studies, including infection by bacteria, yeasts or bead engulfment by many types of cells38,39.
The authors have nothing to disclose.
We thank Gonçalo Moniz Institute, Fiocruz Bahia, Brazil and the department of microscopy for assistance. This work was supported by INOVA-FIOCRUZ number 79700287000, P.S.T.V. holds a grant for productivity in research from CNPq (305235/2019-2). Plasmids were kindly provided by Mauricio Terebiznik, University of Toronto, CA. The authors would like to thank Andris K. Walter for English language revision and manuscript copyediting assistance.
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 |