The mechanism associated with phagocytosis in Leishmania infection remain poorly understood. Here we describe methods to evaluate the early states of curing during Leishmania interaction to the host cells. This technique presents as the main advantage, allowing the study of different states of Leishmania phagocytosis and the participation of several proteins.
It is worth noting that this technique can also be extended to other types of studies, including infection by bacteria, yeasts, or be it engulfment by other types of cells. People trying this technique should take care of the exposure time of the cells to the Nucleofector solution. In addition, we suggest start testing transfection using a maximum of two plasmids.
Demonstrating the procedure will be Amanda Reboucas, a PhD student from our laboratory. To begin with, seed 0.2 million THP-1 cells, or human monocyte-derived macrophages in 500 microliters RPMI per well on a 24 well plate with 13 millimeter glass coverslips. Incubate for 24 hours at 37 degrees Celsius under 5%carbon dioxide, wash cells twice with 0.9%saline and incubate the cells in a Complete RPMI Medium.
Add stationary phase Leishmania species to the cells at a 10 parasite is to one host cell ratio, and then, centrifuge at 720 times g for 10 minutes at four degrees Celsius. After incubating the cells at four degrees Celsius for five minutes, wash the cells twice with 0.9%saline solution to remove any non-internalized promastigotes. Incubate the cells in supplemented RPMI Medium at 37 degrees Celsius for one hour, and then, fix the cells with 4%PFA for 15 minutes.
Next, mount coverslips using preferred mounting media. Then, count at least 400 cells in random fields under a fluorescence microscope. To investigate the Leishmania-induced PV biogenesis, start transfecting THP-1 cells with plasmid by seeding 15 million THP-1 cells in 75 square centimeters tissue culture flasks containing 10 milliliters of Complete RPMI Medium supplemented with 100 nanograms per milliliter PMA, and 50 micromolar to mercaptoethanol for 48 hours.
Then, wash the cells once in 0.9%saline solution and detach the cells using a non-enzymatic cell dissociation solution. Then, centrifuge at 250 times g for five minutes at room temperature. Next, resuspend the THP-1 cells in one milliliter of RPMI Medium and perform counts in a Neubauer chamber.
Centrifuge the cells again at 250 times g for 10 minutes at room temperature and discard the supernatant. Resuspend 2 million cells in 100 microliters of Nucleofector solution and incubate with 0.5 micrograms of the plasmid coating for the protein of interest, tagged with a fluorescent protein. Transfer the suspension containing THP-1 cells and nucleic acid to the Nucleofector cuvette.
Then, transfect the cells using Nucleofector program Y one. Recover the transfected cells and seed in 500 microliters of RPMI Medium on 24 well plates with 13 millimeter glass coverslips. Incubate the cells and Complete RPMI Medium at 37 degrees Celsius for 0.5, 2, 4, 6, 12, and 24 hour.
Leave the 13 millimeter glass coverslips at room temperature and protect them from the light at least 30 minutes before the acquisition. Clean the coverslips with absorbent tissue. Next, add a drop of immersion oil to the objective, and then add the slide.
Move the objective up until the oil touches the slide. Observe and adjust the focus on the microscope and choose option 63x objective with oil. Open the LIQA program and adjust the lasers in the 488, 552, and 405 wavelengths.
Select the image resolution as 1024 x 1024. After clicking on the live button, set the Z-stack and press the begin option. Wait for the image acquisition and then select the option, maximum projection, in the tools.
Save the experiment, and export the TIFF format images to a computer. The results showed that both Leishmania Braziliensis LCL, and Leishmania Braziliensis DL, similarly bind to macrophages. Also, no differences were observed regarding Leishmania Braziliensis LCL and Leishmania Braziliensis DL phagocytosis by host cells.
The recruitment of LC3 to the parasitophorous vacuole, or PV's, induced by Leishmania Braziliensis LCL or Leishmania Braziliensis DL, was compared in infected cells. After four hours of infection, similar percentages of LC3-decorated PV's in Leishmania Braziliensis LCL and Leishmania Braziliensis DL infected macrophages were observed. Thus, results showed that Leishmania Braziliensis LCL and Leishmania Braziliensis DL similarly interact with macrophages during binding, phagocytosis, and biogenesis of PV's concerning the LC3 recruitment.
Representative microscopic images demonstrated efficient transfection of THP-1 cells with PLC-GFP, Rab5-GFP, Rab7-GFP plasmids. People trying this protocol must pay attention to temperature incubation and protect the microscope samples from the light, all the types. Modulating the expression of the identified proteins precipitating Leishmania phagocytosis will demonstrate the importance of those proteins in Leishmania infection outcome.
We can extend this technique toward different types of pathology studies and mechanisms associated with parasite establishment and target identification for the development of therapeutic strategies.
