We thank Gon&#231;alo 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.

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
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Centrifuge the tubes at 252 &#215; g for 30 min at 24 &#176;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.</li>
		<li>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.</li>
		<li>Wash cells once and centrifuge at 190 &#215; g for 10 min at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend pellet in 1 mL of complete RPMI medium.</li>
		<li>Count the cells and plate 2 &#215; 106 cells in 500 mL of Roswell Park Memorial Institute (RPMI) supplemented with 25 mM N-[2-hydroxyethyl] piperazine-N&#8242;-[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 &#176;C under 5% CO2 in a 24-well plate to allow monocytes to differentiate into macrophages by adhesion.</li>
	</ol>
	</li>
	<li>THP-1 cultures
	<ol>
		<li>Grow THP-1 cell line at a concentration of 2 &#215; 105 cells in 10 mL of complete RPMI medium in 75 cm2 culture flask.</li>
		<li>Maintain cell cultures in an incubator at 37 &#176;C under 5% CO2 for 7 days.</li>
		<li>Centrifuge cells at 720 &#215; g for 10 min at 4 &#176;C and resuspend the pellet in complete RPMI medium.</li>
		<li>Count cells in a Neubauer chamber.</li>
		<li>Plate cells on 13 mm glass coverslips at a concentration of 2 &#215; 105 cells per well in 500 &#956;L of complete RPMI medium containing 100 nM phorbol myristate acetate (PMA) at 37 &#176;C under 5% CO2 to allow differentiation of THP1 cells into macrophages.</li>
		<li>After three days, wash twice cells with 0.9% NaCl solution to remove medium containing PMA.</li>
		<li>Incubate differentiated THP-1 cells in PMA-free complete RPMI medium at 37 &#176;C under 5% CO2 for an additional 2 days before starting experimentation.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Grow Leishmania spp. promastigotes at 1 x 105 parasites per 1,000 &#956;L of medium in a cell culture flask containing 5 mL of Schneider&#39;s medium supplemented with 50 &#956;g/mL gentamicin and 10% FBS.</li>
	<li>After incubating parasite axenic cultures in a biochemical oxygen demand (B.O.D.) at 24 &#176;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.</li>
	<li>Upon reaching the stationary phase of growth, incubate the parasites in 4 mL of 0.9% NaCl solution with 1 &#956;M CMTPX for 15 min at 37 &#176;C under 5% CO2 avoiding contact with light.</li>
	<li>Add FBS at a 1:1 proportion and incubate parasite suspension for an additional 1 min.</li>
	<li>Wash parasites thrice with PBS, followed by centrifugation at 1,781 &#215; g for 10 min.</li>
	<li>Ressuspend parasite pellet in 1,000 &#956;L of RPMI complete medium.</li>
	<li>Count parasites in a Neubauer chamber.</li>
</ol>
3. Assessment of Leishmania binding to macrophages
<ol>
	<li>Seed 2 &#215; 105 THP-1 cells or human monocyte-derived macrophages in 500 &#956;L of complete RPMI medium per well on a 24-well plate with 13 mm glass coverslips.</li>
	<li>Cultivate cells at 37 &#176;C under 5% CO2 for 24 h.</li>
	<li>Wash the cells twice with 0.9% NaCl solution and incubate in complete RPMI medium at 4&#160;&#176;C for 10 min.</li>
	<li>Add stationary phase promastigotes as described by A. L. Petersen22 at a 10:1 ratio to well plates, and then centrifuge at 720 &#215; g for 5 min under 4 &#176;C.</li>
	<li>Incubate at 4 &#176;C for 5 min.</li>
	<li>Wash the cells twice with 0.9% NaCl solution to remove any non-internalized promastigotes.</li>
	<li>Fix the cells in 4% paraformaldehyde for 15 min at room temperature.</li>
	<li>Incubate the coverslips with 15 mM NH4Cl for 15 min at room temperature.</li>
	<li>Wash thrice with PBS 0.15% bovine serum albumin (BSA). Incubate with blocking solution (3% BSA in PBS) for 1 h at room temperature.</li>
	<li>Wash thrice with PBS and then permeabilize with 0.15% PBS-Saponin for 15 min at room temperature.</li>
	<li>Add phalloidin (diluted 1:1,200) for 1 h at room temperature and protect from light.</li>
	<li>Mount coverslips using mounting media.</li>
	<li>Acquire images via a confocal fluorescence microscope using a 63&#215;/1.4 objective.</li>
</ol>
4. Assessment of Leishmania phagocytosis by macrophages
<ol>
	<li>Seed 2 &#215; 105 THP-1 cells or human monocyte-derived macrophages in 500 &#956;L of complete RPMI medium per well on a 24-well plate with 13 mm glass coverslips.</li>
	<li>Cultivate cells for 24 h at 37 &#176;C under 5% CO2.</li>
	<li>Wash cells twice in 0.9% NaCl solution and incubate in complete RPMI medium in 24-well plate at 4 &#176;C for 10 min.</li>
	<li>Add stationary phase Leishmania spp. as described by A. L. Petersen22 at a 10:1 (parasite:host cell) ratio, and then centrifuge at 720 &#215; g for 10 min under 4 &#176;C.</li>
	<li>Incubate cells at 4 &#176;C for 5 min.</li>
	<li>Wash the cells twice with 0.9% NaCl solution to remove any non-internalized promastigotes.</li>
	<li>Incubate the cells in supplemented RPMI medium at 37 &#176;C for 1 h.</li>
	<li>Fix the cells with 4% paraformaldehyde for 15 min.</li>
	<li>Mount coverslips using preferred mounting media.</li>
	<li>Count no less than 400 cells in random fields under a fluorescence microscope using a 100&#215;/1.4 objective.</li>
</ol>
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.
<ol>
	<li>To investigate the biogenesis of Leishmania-induced PV, transfect THP1 cells with PLC26,27, Akt26,27, Rab 528,29,30 or Rab 728,29,31 plasmids. 
	&#8203;NOTE: This methodology can be used to transfect THP-1 cells with other genes than those listed above.</li>
	<li>Seed THP-1 cells at 1.5 x 107 in 75 cm&#178; tissue culture flasks containing 10 mL complete RPMI medium supplemented with 100 ng/mL PMA and 50 &#181;M 2-mercaptoethanol for 48 h.</li>
	<li>Wash cells once in 0.9% NaCl solution.</li>
	<li>Detach cells using a non-enzymatic cell dissociation solution and centrifuge (250 &#215; g) for 5 min at room temperature.</li>
	<li>Resuspend THP-1 cells in 1 mL of RPMI medium and perform counts in a Neubauer chamber.</li>
	<li>Centrifuge THP-1 cells again at 250 &#215; g for 10 min at room temperature. Discard the supernatant.</li>
	<li>Resuspend 2 &#215; 106 cells in 100 &#181;L of Nucleofector solution and incubate with 0.5 &#181;g of the plasmid coding for the protein of interest, tagged with a fluorescent protein.</li>
	<li>Transfer the suspension containing THP-1 cells and nucleic acid to the Nucleofector cuvette.</li>
	<li>Transfect THP1 cells using Nucleofector Program Y-001.</li>
	<li>Recover the transfected cells (2x106) and seed in 500 &#956;L RPMI medium on 24-well plates with 13 mm glass coverslips (4 wells/transfection).</li>
	<li>Incubate THP-1 cells in complete RPMI medium at 37 &#176;C for 0.5, 2, 4, 6, 12 and 24 h.</li>
	<li>Repeat steps 3.13 and 3.13.</li>
</ol>
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.
<ol>
	<li>Seed 2 &#215; 105 THP-1 cells or human monocyte-derived macrophages in 500 &#956;L complete RPMI medium on a 24-well plate with 13 mm glass coverslips.</li>
	<li>Cultivate cells for 24 h at 37 &#176;C under 5% CO2.</li>
	<li>Wash cells twice in 0.9% NaCl solution and incubate in complete RPMI medium.</li>
	<li>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 &#215; g for 5 min under 4 &#176;C.</li>
	<li>Incubate at 37 &#176;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.
	<ol>
		<li>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 &#176;C until labeling.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Wash the cells thrice with 0.9% NaCl solution at room temperature.</li>
	<li>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.</li>
	<li>Wash the cells thrice with 0.9% NaCl solution at room temperature.</li>
	<li>Mount coverslips using preferred mounting media.</li>
	<li>Acquire images via confocal fluorescence microscope using a 63x/1.4 objective.</li>
</ol>
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.
<ol>
	<li>Leave the 13 mm glass coverslips at room temperature and protect them from the light at least 30 min before the acquisition.</li>
	<li>Clean the coverslips with an absorbent tissue.</li>
	<li>Add a drop of immersion oil to the objective and add the slide.</li>
	<li>Move the objective up until the oil touches the slide.</li>
	<li>Observe and adjust the focus on the microscope and choose the option 63x objective with oil.</li>
	<li>Open the Leica program and adjust the lasers in the 488, 552, and 405 wavelengths.</li>
	<li>Select the image resolution 1,024 x 1,024.</li>
	<li>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 &#956;m for slice thickness to get confocal images with good resolutions.</li>
	<li>Wait for the image acquisition, and then select the option &#34;Maximum Projection&#34; in the Leica tools.</li>
	<li>Save the experiment.</li>
	<li>Export the lif or tiff format images to a computer and open the FIJI program.</li>
	<li>Open the experiment and set the view stack with the hyper stack. Then select open files individually and stitch tiles.</li>
	<li>Select the free hands tool in the Fiji toolbar and trace the cell carefully by hand.</li>
	<li>Press the Analyze button and measure to visualize the fluorescence intensity.</li>
	<li>Repeat this process to each cell per group.</li>
	<li>Save the measurements and export them to a spreadsheet editor.</li>
	<li>Add this data to a statistical analysis program and do the statistical analysis.</li>
</ol>
8. Statistical analysis
NOTE: For data analysis and graphics, use a statistical analysis program.
<ol>
	<li>Open the program.</li>
	<li>Insert the obtained data and test the normality parameters.</li>
	<li>For data with normal distribution, use the Student t-test and for nonparametric tests, Mann-Whitney test.</li>
	<li>Consider data with a statistically significant difference when the p-value is less than 0.05.</li>
	<li>Prepare graphics representing the data, with central tendency measures (mean or median) and variation measures.</li>
</ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autophagic+pathway+is+actively+modulated+by+phase+II+Coxiella+burnetii+to+efficiently+replicate+in+the+host+cell.">The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell.</a> Cellular Microbiology. 9 (4), 891-909 (2007).</li><li>Vieira, O. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Rab5+and+Rab7+recruitment+to+phagosomes+by+phosphatidylinositol+3-kinase.">Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase.</a> Molecular and Cellular Biology. 23 (7), 2501-2514 (2003).</li><li>Roberts, R. L., Barbieri, M. A., Ullrich, J., Stahl, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+rab5+activation+in+endocytosis+and+phagocytosis.">Dynamics of rab5 activation in endocytosis and phagocytosis.</a> Journal of Leukocyte Biology. 68 (5), 627-632 (2000).</li><li>Vitelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+small+GTPase+Rab7+in+the+late+endocytic+pathway.">Role of the small GTPase Rab7 in the late endocytic pathway.</a> Journal of Biological Chemistry. 272 (7), 4391-4397 (1997).</li><li>Matte, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+major+Promastigotes+Evade+LC3-Associated+Phagocytosis+through+the+Action+of+GP63.">Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63.</a> PLoS Pathogens. 12 (6), 1005690 (2016).</li><li>Dias, B. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+Induction+Greatly+Enhances+Leishmania+major+Intracellular+Survival+Compared+to+Leishmania+amazonensis+in+CBA/j-Infected+Macrophages.">Autophagic Induction Greatly Enhances Leishmania major Intracellular Survival Compared to Leishmania amazonensis in CBA/j-Infected Macrophages.</a> Frontiers in Microbiology. 9, 1890 (2018).</li><li>Babcock, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+phagocytosis+by+confocal+microscopy.">Quantitation of phagocytosis by confocal microscopy.</a> Methods in Enzymology. 307, 319-328 (1999).</li><li>Sanderson, M. J., Smith, I., Parker, I., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Cold Spring Harbor Protocols. 2014 (10), 071795 (2014).</li><li>Lennartz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipases+and+phagocytosis:+the+role+of+phospholipid-derived+second+messengers+in+phagocytosis.">Phospholipases and phagocytosis: the role of phospholipid-derived second messengers in phagocytosis.</a> International Journal of Biochemistry &amp; Cell Biology. 31 (3-4), 415-430 (1999).</li><li>Rashidfarrokhi, A., Richina, V., Tafesse, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+Early+Stages+of+Phagocytosis.">Visualizing the Early Stages of Phagocytosis.</a> Journal of Visualized Experiments. (120), e54646 (2017).</li><li>Ramarao, N., Meyer, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+resists+phagocytosis+by+macrophages:+quantitative+assessment+by+confocal+microscopy+and+fluorescence-activated+cell+sorting.">Helicobacter pylori resists phagocytosis by macrophages: quantitative assessment by confocal microscopy and fluorescence-activated cell sorting.</a> Infection and Immunity. 69 (4), 2604-2611 (2001).</li><li>Bain, J., Gow, N. A., Erwig, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+into+host-fungal+pathogen+interactions+derived+from+live-cell+imaging.">Novel insights into host-fungal pathogen interactions derived from live-cell imaging.</a> Seminars in Immunopathology. 37 (2), 131-139 (2015).</li></ol>

The funders had no role in study design, data collection or analysis, the decision to publish, or preparation of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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&#160;&#176;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 &#176;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.

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.&#160;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.

<table><tbody><tr><td>2-mercaptoethanol</td><td>Thermo Fisher Scientific</td><td>21985023</td><td></td></tr><tr><td>AlexaFluor 488-conjugated goat anti-rabbit IgG</td><td>Thermo Fisher Scientific</td><td>Tem varios no site</td><td></td></tr><tr><td>anti-LC3 antibody</td><td>Novus Biologicals</td><td>NB600-1384</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Thermo Fisher Scientific</td><td>X</td><td></td></tr><tr><td>CellStripper</td><td>Corning</td><td>25-056-CI</td><td></td></tr><tr><td>CellTracker Red (CMTPX) Dye</td><td>Thermo Fisher Scientific</td><td>C34552</td><td></td></tr><tr><td>Centr&amp;iacute;fuga</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Ciprofloxacin</td><td>Isofarma</td><td>X</td><td></td></tr><tr><td>CO2 incubator</td><td>Thermo Fisher Scientific</td><td>X</td><td></td></tr><tr><td>Confocal fluorescence microscope (Leica SP8)</td><td>Leica</td><td>Leica SP8</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>Fluorescence microscope (Olympus Lx73)</td><td>Olympus</td><td>Olympus Lx73</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15750045</td><td></td></tr><tr><td>Glutamine</td><td>Thermo Fisher Scientific</td><td>35050-061</td><td></td></tr><tr><td>HEPES (N- 2-hydroxyethyl piperazine-N&amp;rsquo;-2-ethane-sulfonic acid)</td><td>Gibco</td><td>X</td><td></td></tr><tr><td>Histopaque</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>M-CSF</td><td>Peprotech</td><td>300-25</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>A9434</td><td></td></tr><tr><td>Normal goat serum</td><td>Sigma</td><td>NS02L</td><td></td></tr><tr><td>Nucleofector 2b Device</td><td>Lonza</td><td>AAB-1001</td><td></td></tr><tr><td>Nucleofector solution</td><td>Lonza</td><td>VPA-1007</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>Phalloidin</td><td>Invitrogen</td><td>A12379</td><td></td></tr><tr><td>Phorbol myristate acetate (PMA)</td><td>Sigma</td><td>P1585</td><td></td></tr><tr><td>Phosphate buffer solution (PBS)</td><td>Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>ProLong Gold Antifade kit</td><td>Life Technologies</td><td>P36931</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640 medium</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Saponin</td><td>Thermo Fisher Scientific</td><td>X</td><td></td></tr><tr><td>Schneider&#039;s Insect medium</td><td>Sigma</td><td>S0146</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>S8636</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X</td><td></td></tr></tbody></table>

investigating the phagocytosis of leishmania using confocal microscopy

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.

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&#39; 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&#160;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62459/62459fig01.jpg" /> 
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 &#176;C, the binding was assessed by confocal microscopy. (A)&#160;Confocal microscopy images of L. braziliensis-LCL or L. braziliensis-DL (labeled with CMTPX, red) binding to macrophages (labeled with phalloidin, green).&#160;For confocal microscopy, cell nuclei were labeled with DAPI (blue).&#160;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 &#62; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62459/62459fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62459/62459fig02.jpg" /> 
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&#160;for 10 min at 4 &#176;C followed by additional 1 h at 37 &#176;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 &#62; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62459/62459fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62459/62459fig03.jpg" /> 
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 &#62; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62459/62459fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62459/62459fig04.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62459/62459fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Investigating the Phagocytosis of Leishmania using Confocal Microscopy at JoVE.com

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

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.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

investigating-the-phagocytosis-of-leishmania-using-confocal-microscopy

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.2K Views.  Gonçalo Moniz Institute. Het mechanisme geassocieerd met fagocytose bij Leishmania-infectie blijft slecht begrepen. Hier beschrijven we methoden om de vroege genezingstoestanden tijdens Leishmania-interactie met de gastheercellen te evalueren. Deze techniek presenteert als het belangrijkste voordeel, waardoor de studie van verschillende toestanden van Leishmania fagocytose en de deelname van verschillende eiwitten mogelijk is.Het is vermeldenswaard dat deze techniek ook kan worden uitgebreid naar andere soorte...

Video: Investigating the Phagocytosis of Leishmania using Confocal Microscopy

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

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 ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites' ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Here we study implications of Leishmania-host interaction by exploring Leishmania-infected dendritic cells migration. The differentiation and infection of dendritic cells, migration analysis, and the evaluation of adhesion complexes and actin dynamics are described. This method can be applied to other host cell migration studies when infected with Leishmania or other intracellular parasite species.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous ...

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106&#160;cells per well on 24-well plates in order to obtain 2 x 105&#160;macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These ...

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life cycle of Leishmania parasites consists of two alternating life stages (amastigotes and promastigotes), during which parasites reside within either arthropod vectors or vertebrate hosts, respectively. Notably, the complex interactions between Leishmania parasites and several cells of the immune system largely influence the outcome of infection. Importantly, although macrophages are known to be the main host niche for Leishmania replication, parasites are also phagocytosed by other innate immune cells, such as neutrophils and dendritic cells (DCs). 
DCs play a major role in bridging the innate and adaptive branches of immunity and thus orchestrate immune responses against a wide range of pathogens. The mechanisms by which Leishmania and DCs interact remain unclear and involve aspects of pathogen capture, the dynamics of DC maturation and activation, DC migration to draining lymph node (dLNs), and antigen presentation to T cells. Although a large body of studies support the notion that DCs play a dual role in modulating immune responses against Leishmania, the participation of these cells in susceptibility or resistance to Leishmania remains poorly understood. After infection, DCs undergo a maturation process associated with the upregulation of surface major histocompatibility complex (MHC) II, in addition to costimulatory molecules (namely, CD40, CD80, and CD86). 
Understanding the role of DCs in infection outcome is crucial to developing therapeutic and prophylactic strategies to modulate the immune response against Leishmania. This paper describes a method for the characterization of Leishmania-DC interaction. This detailed protocol provides guidance throughout the steps of DC differentiation, the characterization of cell surface molecules, and infection protocols, allowing scientists to investigate DC response to Leishmania infection and gain insight into the roles played by these cells in the course of infection.

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

Dendritic cells (DCs) are essential components of innate immunity against Leishmania infection. The mechanisms underlying the complex interaction between DCs and Leishmania remain poorly understood. Here, we describe methods to evaluate how Leishmania infection affects the immunobiological function of human DCs, such as migration-related and costimulatory molecule expression.

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life ...

In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease.
By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR.
After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR.&#160;Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.

Here we present a longitudinal evaluation of golden hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc by bioluminescent imaging and by PCR. Hamsters were evaluated 1 day post infection (1 dpi), 1 week after infection (8 dpi), and 3 weeks after infection (22 dpi) and euthanized at the 50th dpi and 8 months post infection.

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if ...

Biology

An In Vitro Assay to Quantify the Intracellular Growth of Parasite in Macrophages

Source: Sarkar, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57486">Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites.</a> J. Vis. Exp. (2018).
This video demonstrates an assay to quantify the intracellular growth of parasites in bone marrow-derived macrophages (BMMs). The macrophage cells are infected with the motile and non-motile invasive stages of Leishmania, metacyclic promastigotes and amastigotes, respectively. The parasites invade the macrophages, getting internalized into a parasitophorous vacuole and multiplying inside. The intracellular parasite growth is quantified using a microscope upon fluorescently staining the macrophage and parasite nuclei.

1. Plating BMMs on Coverslips for Infection 

	Prepare 6-well tissue culture plates by placing four (4) sterile 12 mm glass coverslips (autoclaved and ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

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