Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Our research is focused on understanding the interaction between the Trichoderma fungi and the mammalian immune system. How do some Trichoderma species cause disease? How can they modulate the immune system, and how can this modulation impact the development of the disease?

To answer this question, we use in vitro randomized models. We show that Trichoderma species'modulates macrophages and neutrophils by targeting receptors involved in the phagocytosis process. These receptors include toll-like receptors and interleukin receptors.

When there is a decrease in the phagocyte capacity of immune cells, susceptibility to pathogens, especially intracellular pathogens, are increased. Fungi have many invasion mechanism to survive in the host. These include avoiding phagocytosis and surviving inside macrophage.

Pathogen species use macrophage as Trojan horses to survive inseminating the host. Here, we propose to know if Trichoderma can survive inside macrophages and for how long they survive. To begin, plate 10 microliters of the mother stock suspension of Trichoderma stromaticum on the PDA plate.

Incubate the culture at 28 degrees Celsius in the dark for 7 to 10 days until the conidia are observed and the culture turns green to obtain the fresh F1 generation. Wash the culture by adding three to five milliliters of PBS to the plate, then collect and again dispense the PBS over the culture to gradually remove the conidia. Repeat this process until the suspension turns dark green.

Transfer the recovered suspension from the plate into a sterile 15-milliliter tube. Centrifuge the suspension at 1, 160 G for five minutes at 12 degrees Celsius and resuspend the pellet in five milliliters of PBS. Suspend the final pellet in two to three milliliters of PBS and count the conidia in a Neubauer chamber.

Transfer the blood collected from the donor to a 50-milliliter tube and add PBS to obtain a final volume of 35 milliliters. Transfer the blood slowly along the wall of the tube containing the polysucrose solution to form a biphasic suspension. Immediately centrifuge the suspension at 1, 600 G for 30 minutes at 24 degrees Celsius.

After centrifugation, observe the formation of a white ring containing the PBMCs and collect it by aspirating gently in a 15-milliliter tube. Then, add PBS to the tube containing the PBMCs to obtain a final volume of 10 milliliters. Homogenize the suspension and centrifuge at 1, 600 G for 30 minutes at 24 degrees Celsius.

After centrifugation, wash the cells three times at 1, 600 G for five minutes with 10 milliliters of PBS before resuspending the pellet in two milliliters of R10 medium. To treat human-derived macrophages with Trichoderma stromaticum conidia, using sterile tweezers, add a round glass cover slip to each well of a 24-well plate. Count the PBMC suspension isolated from human blood and adjust the volume to plate 8 times 10 to 5th cells per well to a final volume of one milliliter with R10 medium.

After plating, use an inverted microscope. Observe the cell morphology. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide for seven days, replacing the medium with fresh R10 medium every two days for the differentiation of the monocytes into macrophages.

After seven days, observe the cell morphology using an inverted microscope. Then, remove the medium from each well and wash the cells with one milliliter of PBS to remove debris and non-adherent cells. Next, prepare T.stromaticum conidia suspension at a final concentration of 8 times 10 to the 4th conidia per milliliter in R10 medium.

Add one milliliter of suspension to each well to obtain a multiplicity of infection of 1:10. Challenge the macrophages for three hours at 37 degrees Celsius under 5%carbon dioxide. After incubation, remove the medium and wash the cells with PBS to remove the non-phagocytized conidia.

Then, add one milliliter of R10 medium to each well and incubate the plate at different times. After the corresponding incubation, remove the medium and add one milliliter of PBS to that well. Then, using tweezers and a needle, remove the round glass cover slips from the wells.

Next, stain the cover slips using the components of the staining kit. Once the cover slips are dry, fix them on a glass slide using a mounting agent For conidial viability assay, challenge the macrophages with T.stromaticum conidia and incubate the plate for different times at 37 degrees Celsius under 5%carbon dioxide. After each incubation, wash the cells twice with PBS to remove the un-phagocytosed conidia.

Then, add 0.5 milliliters of sterile distilled water and incubate for 30 minutes. After confirming the cells are lysed, collect the suspension into a 1.5-milliliter tube. Centrifuge the suspension at 6, 000 times G for five minutes at room temperature and discard the supernatant carefully before resuspending the pellet in 10 microliters of sterile distilled water.

Inoculate 10 microliters of the suspension onto the PDA plate and incubate a 28 degree Celsius in the dark. At the beginning of the phagocytosis process, free conidia, conidia attached to the outer membrane of macrophages, or completely internalized by macrophages were observed. With increased phagocytosis time, free and conidia attached to the outer membrane were completely internalized by the macrophages.

The conidia were also observed in an upper plane compared to the macrophages, and more than one conidium was found in the same macrophage. After long periods, phagocytosed conidia germinate inside the macrophages, and free conidia germinate in the R10 medium, forming hyphae at 37 degrees Celsius. The phagocytosed conidia remained viable even after 120 hours of interaction with the macrophages.

After phagocytosis, growth was monitored in terms of the time required for the culture to occupy the entire plate and to form colored green conidia.