Quantificação de crescimento intracelular dentro de macrófagos é um rápido e confiável método para avaliar a virulência de Leishmania parasitas

The overall goal of this experiment is to assess the virulence of various Leishmania strains through the infection of murine bone marrow-derived macrophages in vitro, and the subsequent quantification of parasite intracellular growth kinetics. This method can help us answer key questions about the factors from both the host's macrophage and the pathogen that determine the virulence of the infective Leishmania parasites. The main advantage of this technique is that it is a fast, economic, and reliable method that require significantly fewer animals, compared to in vivo studies.

The implications of this technique extend toward the therapy of Leishmaniasis, as it can be used to assess the infectivity of mutated Leishmania strains, or the efficacy of therapeutic compounds. Visual demonstration of this method is critical, as the bone marrow-derived macrophage isolation and intracellular parasite quantification steps are difficult to learn from written protocols alone. Begin by securing a four to six week old female mouse to a dissection board, and disinfecting the animal with 70%ethanol.

Use scissors to make a one centimeter incision near one hip joint and cut the skin all the way around the joint. Pull the skin from the leg toward the ankle to carefully deglove the limb, and cut off the foot at or below the ankle joint to leave the tibia in tact. To locate the hip joint, manipulate the femur to identify the point of rotation, and use the scissors to sever the leg above the hip joint, leaving the proximal head of the femur in tact.

Remove as much muscle and connective tissue from the leg as possible, and place the cleaned bones in Petri dish containing sterile RPMI supplemented with antibiotics on ice. After harvesting the second set of leg bones, use sterilized forceps and a number ten blade to remove any remaining muscle and connective tissue. Transfer the cleaned bones to an empty Petri dish lid, and use the scalpel to remove both ends of each tibia to access the marrow.

Using forceps, place one tibia over the mouth of a 50 milliliter conical tube on ice, and insert the tip of a 25 gauge needle attached to a 10 milliliter syringe filled with mediums supplemented with antibiotics into one end of the tibia. Flush approximately five to 10 milliliters of medium through the bone into the conical tube until all of the red bone marrow tissue has been harvested and the bone appears white. When all of the marrow has been collected, centrifuge the pooled bone marrow, and re-suspend the bone marrow cell palette in five milliliters of bone marrow-derived macrophage medium.

Transfer the bone marrow cell suspension into a 175 square centimeter cell culture flask containing 60 milliliters of sterile bone marrow-derived macrophage medium, and rinse the tube two times with medium from the culture flask to ensure maximal cell recovery. After the second wash, transfer a 30 milliliter aliquot of cells into a second 175 square centimeter cell culture flask, and close each flask with a filter cap. Then, incubate both cultures overnight at 37 degrees Celsius and 5%carbon dioxide to allow separate of the resident macrophages from any contaminating fibroblasts or other adherent cells.

The next morning, transfer 10 milliliters of undifferentiated bone marrow cell-containing supernatant from each flask into individual 100 by 15 millimeter non-tissue culture treated polystyrene Petri dishes, and return the cells to the cell culture incubator for two to three more days. Then, feed the cultures with five milliliters of fresh 37 degree Celsius bone marrow-derived macrophage medium per dish, and return the plates to the incubator. On day seven or eight after culture, use an aspirating tip fitted to a vacuum line to transfer four autoclave sterilized 12 millimeter glass cover slips to the bottom of each well of a six well culture plate without overlapping.

Prepare identical plates for each time points of the experiment. Next, wash the bone marrow-derived macrophage cultures two times with fresh 37 degree Celsius PBS and five milliliters of PBS without calcium and magnesium supplemented with one millimolar EDTA to each plate. After five minutes at 37 degrees Celsius, pool the detached cells in a 50 milliliter conical tube, rinsing each plate two times with an additional five milliliters of PBS without calcium and magnesium.

After centrifugation, re-suspend the macrophages in five to 10 milliliters of fresh bone marrow-derived macrophage medium on ice. After counting, dilute the cells to five time 10 to the fifth per milliliter concentration in fresh bone marrow-derived macrophage medium, and add two milliliters of cells per well to the six well plate. Then, incubate the cells overnight in the cell culture incubator.

To infect the cells with L.amazonesis, dilute the parasite suspension to the appropriate experimental multiplicity of infection, and add 50 to 100 microliters of parasites to each well of all six well plates, including all time points of the experiment. Incubate the bone marrow-derived macrophages for the appropriate infection period and temperature. Then, wash the wells three times with two milliliters of fresh 37 degree Celsius PBS without calcium or magnesium per well, and fix the initial time point samples with 1.5 to 2 milliliters of 2%paraformaldehyde for 10 minutes.

For the plates corresponding to the later time points, add two milliliters of fresh bone marrow-derived macrophage medium to each well, and return the plates to the appropriate incubator. Then, fix and wash the cells for each of the remaining time point, as just demonstrated, and store the samples at four degrees Celsius in fresh PBS. To stain the cells with DAPI, replace the supernatant with 1.5 milliliters of fresh PBS without calcium and magnesium, supplemented with 0.1%non-ionic detergent for 10 minutes at room temperature, followed by three two milliliter washes with PBS without calcium or magnesium.

Next, stain the cells with two micrograms per milliliter of DAPI in one milliliter of PBS for one hour at room temperature. After three washes, place the cover slips cell side down onto individual glass microscope slide mounted with a commercially anti-fade mounting reagent. Then, to quantify the number of infected cells, use the 100x emersion oil objective of a fluorescence microscope and a manual counter to identify the number of smaller amastigote nuclei clustered around each large DAPI stained nucleus.

Leishmania has two infective forms, metacyclic promastigotes, which differentiate from procyclic promastigotes at the stationary phase of culture, and amastigote, the intracellular infective form found within the vertebrate host. Here, axenically differentiated amastigotes can be visualized within a bone marrow-derived macrophage one hour after the initial infection and before the formation of parasitophorous vacuoles via phagolysosomal fusion. At 48 hours, distinct large parasitophorous vacuoles harboring multiple parasites can be observed, characteristic of a virulent Leishmania infection.

Non-virulent log phase promastigotes, in contrast, are unable to initiate parasitophorous vacuole development, fail to replicate, and are eventually killed within host macrophages. In this experiment, a steady increase in the number of intracellular wild type metacyclic Leishmania parasites was observed up to 72 hours after infection, while little to no intracellular growth for mutant parasites with a impaired mitochondrial iron import was measured, suggesting that mitochondrial activity is required for parasite growth within macrophages. Macrophage infections carried out with wild type and mutant axenic amastigotes showed an intracellular growth pattern similar to that observed with metacyclic promastigotes, but with an even higher wild type parasite infectivity than seen from metacyclic promastigotes.

It is important to note that there is an initial 24 hour delay for metacyclic promastigote infections, representing the time required for internalized metacyclic promastigotes to first differentiate into amastigotes before beginning to replicate. Once mastered, this technique can be completed in 15 days if it is performed properly. While attempting this procedure, it's important to use the appropriate mouse strains and to perform careful quantification of both the macrophage and parasite cells to ensure consistent MOIs across all experiments.

After watching this video, you should have a good understanding of how to culture bone marrow-derived macrophages and to infect them with Leishmania for quantification of their intracellular parasite growth, following nuclear staining with DAPI. Don't forget that working with Leismania can be extremely hazardous, and that precautions, such as using a biological safety cabinet, and personal protective equipment, and avoiding sharp instruments should always be taken while performing this procedure.