This work was supported by the following Brazilian financing institutions: Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado da Bahia (FAPESB) with grants RED0011/2012 and RED008/2014. U.R.S., J.O.C., and M.E.S.M. acknowledge the scholarship granted by Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) and FAPESB, respectively.

Ethical considerations and human subjects 
All experiments with humans described in this study were conducted according to the Declaration of Helsinki and Brazilian Federal laws and approved by the State University of Santa Cruz&#39;s Ethics Committee (project identification code: 550.382/ 2014).
Human peripheral blood was collected from healthy volunteers from Ilh&#233;us city, Bahia, Brazil, not exposed to occupational activities related to the studied fungus. Individua...

Fungal Clearance Efficiency of Macrophages

All the authors declare no conflicts of interest.

For several fungal pathogens including Aspergillus fumigatus, Cryptococcus, Candida albicans,&#160;and others, conidial or yeast phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events in macrophage-pathogen interactions, as well as for the determination of the time of death of the internalized conidia14,39,40. Phagocytosis is the key process in the T...

The Trichoderma genus (Order: Hypocreales, Family: Hypocreaceae) is composed of ubiquitous, saprophytic fungi that are parasites of other fungal species and are capable of producing a range of commercially useful enzymes1. These fungal species are used for the production of heterologous proteins2, the production of cellulose3, ethanol, beer, wine, and paper4, in the textile industry5, food industry6, and in agriculture as biological control agents7,8. In addition to the industrial interest in these fungal species, the increasing number of infections in humans has given some Trichoderma species the status of opportunistic pathogens9.
Trichoderma spp. grow rapidly in culture, with initially white and cottony colonies that turn greenish yellow to dark green10. They are adapted to live in a wide range of pH and temperature conditions, and the opportunistic species are able to survive at physiological pH and temperatures and, thus, colonize different human tissues11,12,13. Importantly, the rise in the infection rate of Trichoderma spp. may be associated with virulence factors, and these are not well studied. In addition, studies focusing on understanding the immune response against opportunistic Trichoderma species are still rare.
During an infection, along with neutrophils, macrophages represent the line of defense responsible for phagocytosis, and, thus, prevent the growth and colonization of pathogens in different tissues. Using pattern recognition receptors, such as Toll-like receptors and C-type lectin receptors, macrophages phagocytose fungi and process them into phagolysosomes, thus promoting a respiratory burst, the release of pro-inflammatory cytokines, and the destruction of the phagocytosed microorganisms14. The mechanism of phagocytosis, however, can be affected and evaded by different microbial strategies, such as the size and shape of the fungal cells; the presence of capsules that hinder phagocytosis; decreasing the number of phagocytosis-inducing receptors; the remodeling of the structure of actin fibers in the cytoplasm; hindering the formation of pseudopodia; and phagosome or phagolysosome escape after the phagocytosis process14.
Many pathogens, including Cryptococcus neoformans, use macrophages as a niche to survive in the host, disseminate, and induce infection15. The phagocytosis and clearance assay is used to evaluate the immune response against pathogens and to identify the microbial strategies employed to evade the innate immune system15,16,17. This type of technique can also be used to examine the differential kinetics of phagocytosis, delayed phagosome acidification, and oxidative burst that result in reduced fungal killing18.
Different methods can be used to evaluate phagocytosis, fungal survival, and the evasion of the phagosome maturation process. These include fluorescence microscopy, which is used to observe phagocytosis, the cellular location, and the molecules produced during phagocytosis19; flow cytometry, which provides quantitative data on phagocytosis and is used to evaluate the different markers involved in the process20,21; intravital microscopy, which is used to assess microbial capture and phagosome maturation22; antibody-mediated phagocytosis, which is used to assess the specificity of the phagocytosis process for a pathogen23; and others24,25,26,27.
The protocol presented here employs a common, low-cost, and direct method using an optical microscope and plate growth assay to assess the phagocytosis and killing of fungal conidia. This protocol will provide the readers with step-by-step instructions for performing the phagocytosis and clearance assay using human peripheral blood mononuclear-derived macrophages exposed to T. stromaticum. PBMCs were used because Trichoderma conidia are applied as a biocontrol against phytopathogens and a biofertilizer for plant crops worldwide and have caused several human infections, called Trichodermosis. Besides that, there are only two previous works focusing on the interaction between Trichoderma conidia and the human immune system, in which we examined neutrophils28 and autophagy in macrophages29. This article shows first how the phagocytosis of the conidia of T. stromaticum by PBMC-derived macrophages can be studied, and then how the viability of the engulfed conidia can be assessed using simple microscopy-based techniques. This protocol may further facilitate investigations on macrophage-associated immune response or immune system modulation-related mechanisms.

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		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Corning</td>
			<td>CLS431470</td>
			<td>15 mL centrifuge tubes, polypropylene, conical bottom with lid, individually sterile</td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Cell Culture Plate</td>
			<td>Kasvi</td>
			<td>K12-024</td>
			<td>Made of polystyrene with alphanumeric identification; The Cell Culture Plate is DNase, RNase and pyrogen-free and free of cytotoxic substances; Sterilized by gamma radiation;</td>
		</tr>
		<tr>
			<td>Cell culture CO2 incubator</td>
			<td>Sanyo</td>
			<td>303082</td>
			<td>A CO2 incubator serves to create and control conditions similar to a human body, thus allowing the in vitro growth and proliferation of different cell types.</td>
		</tr>
		<tr>
			<td>Centrifuge Microtube (eppendorf type) 1.5 mL</td>
			<td>Capp</td>
			<td>5101500</td>
			<td>Made from polypropylene, with a cap attached to the tube for opening and closing with just one hand. It has a polished interior against protein adhesion and for sample visibility, being free of DNase, RNase and Pyrogens</td>
		</tr>
		<tr>
			<td>Circular coverslip 15 mm</td>
			<td>Olen</td>
			<td>K5-0015</td>
			<td>Circular coverslips are used for microscopy techniques in cell culture. Made of super transparent translucent glass; with thickness of 0.13 mm</td>
		</tr>
		<tr>
			<td>Class II Type B2 (Total Exhaust) Biosafety Cabinets</td>
			<td>Esco Lifesciences group</td>
			<td>2010274</td>
			<td>Airstream Class II Type B2 Biosafety Cabinets (AB2) provide product, operator and environmental protection and are suitable for work with trace amounts of toxic chemicals and agents assigned to biological safety levels I, II or III. In a Class II Type B2 cabinet, all inflow and downflow air is exhausted after HEPA/ULPA filtration to the external environment without recirculation across the work surface.</td>
		</tr>
		<tr>
			<td>Dextrose Potato Agar medium</td>
			<td>Merck</td>
			<td>145</td>
			<td>Potato Dextrose Agar is used in the cultivation and enumeration of yeasts and fungi</td>
		</tr>
		<tr>
			<td>EDTA vacuum blood collection tube</td>
			<td>FirstLab</td>
			<td>FL5-1109L</td>
			<td>EDTA is the recommended anticoagulant for hematology routines as it is the best anticoagulant for preserving cell morphology.</td>
		</tr>
		<tr>
			<td>Entellan</td>
			<td>Merck</td>
			<td>1.07961&#160;</td>
			<td>Fixative agent; Entellan is a waterless mounting medium for permanent mounting for microscopy.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A2720801</td>
			<td>Fetal bovine serum (FBS) is a&#160;universal growth supplement of cell and tissue culture media. FBS is a natural cocktail of most of the factors required for cell attachment, growth, and proliferation, effective for most types of human and animal (including insect) cells.</td>
		</tr>
		<tr>
			<td>Flaticon</td>
			<td></td>
			<td></td>
			<td>&#160;database of images</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Merck</td>
			<td>24900988</td>
			<td>The cryoprotectant agent glycerol is used for freezing cells and spores</td>
		</tr>
		<tr>
			<td>Histopaque-1077</td>
			<td></td>
			<td></td>
			<td>polysucrose solution</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td></td>
			<td></td>
			<td>&#160;Image analysis software</td>
		</tr>
		<tr>
			<td>Microscopy slides</td>
			<td>Precision</td>
			<td>7105</td>
			<td>Slide for Microscopy 26 x 76 mm Matte Lapped Thickness 1.0 to 1.2 mm. Made of special optical glass and packaged with silk paper divider with high quality transparency free of imperfections</td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Prism</td>
			<td>C1801</td>
			<td>The Prism Mini Centrifuge was designed to be extremely compact with an exceptionally small footprint. Includes 2 interchangeable quick-release rotors that spin up to 6000 rpm. An electronic brake provides quick deceleration and the self-opening lid allows easy access to the sample, reducing handling time.</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Kasvi</td>
			<td>K5-0011</td>
			<td>The Neubauer Counting Chamber is used for counting cells or other suspended particles.</td>
		</tr>
		<tr>
			<td>Panoptic fast&#160;</td>
			<td>Laborclin</td>
			<td>620529</td>
			<td>Laborclin&#39;s&#160; panoptic fast c is a kit for quick staining in hematology</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin Solution - 10,000U</td>
			<td>LGC- Biotechnology&#160;</td>
			<td>BR3011001</td>
			<td>antibiotic is used in order to avoid possible contamination by manipulation external to the laminar flow.</td>
		</tr>
		<tr>
			<td>Petri dish 90 x 15 mm Smooth</td>
			<td>Cralplast</td>
			<td>18248</td>
			<td>Disposable Petri dish; Made of highly transparent polystyrene (PS); flat bottom; Smooth;Size: 90 x 15 mm.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>thermo fisher Scientific</td>
			<td>10010001</td>
			<td>PBS is a water-based saline solution with a simple formulation. It is isotonic and non-toxic to most cells. It includes sodium chloride and phosphate buffer and is formulated to prevent osmotic shock while maintaining the water balance of living cells.</td>
		</tr>
		<tr>
			<td>Pipette Pasteur 3 mL Sterile</td>
			<td>Accumax</td>
			<td>AP-3-B-S</td>
			<td>STERILE ACCUMAX PASTEUR 3 ML PIPETTE with 3 mL capacity, made of transparent low-density polyethylene (LDPE) and individually sterile</td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>TS-HM16R</td>
			<td>The Thermo Scientific Heraeus Megafuge 16R Refrigerated Centrifuge is a refrigerated centrifuge with the user-friendly control panel makes it easy to pre-set the speed, RCF value, running time,&#160;temperature, and running profile. The Megafuge 16R can reach maximum speeds of&#160;15,200 RPM and maximum RCF of 25,830 x g.</td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>Merck</td>
			<td>MFCD00217820</td>
			<td>HEPES Modification, with L-glutamine and 25 mM HEPES, without sodium bicarbonate, powder, suitable for cell culture</td>
		</tr>
		<tr>
			<td>The single channel micropipettes</td>
			<td>Eppendorf</td>
			<td>Z683809</td>
			<td>Single-channel micropipettes are used to accurately transfer and measure very small amounts of liquids.</td>
		</tr>
		<tr>
			<td>Tip for Micropipettor</td>
			<td>Corning</td>
			<td>4894</td>
			<td>Capacity of 10 &#181;L and 1,000 &#181;L Autoclavable</td>
		</tr>
		<tr>
			<td>Triocular inverted microscope</td>
			<td>LABOMED</td>
			<td>VU-7125500</td>
			<td>It allows you to observe cells inside tubes and bottles, without having to open them, thus avoiding contamination problems.</td>
		</tr>
	</tbody>
</table>

viability assay of trichoderma stromaticum conidia inside human peripheral blood mononuclear-derived macrophages

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the&#160;fungal clearance efficiency of macrophages.

Author Spotlight: Unraveling the Interplay Between &lt;em&gt;Trichoderma stromaticum&lt;/em&gt; and the Mammalian Immune System

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

Our research is focused on understand 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 lectin receptors. When there is a decrease in phagocyte capacity of immune cells, susceptibility to pathogens, especially intracellular pathogens does increase.Ongoing, have many invasion mechanism to survive in the host. This include avoiding phagocytosis and survive inside macrophage. Pathogenic species use macrophage as Trojan horses to survive in disseminating the host.Here, we propose that to know if Trichoderma can survive inside macrophage, and for how long they survive.

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. We used the phagocytosis of T. stromaticum conidia to assess the viability of the conidia after phagocytosis, since the evasion of phagocytosis and the escape of phagosomes are mechanisms of fungal virulence. Researchers should perform these techniques as one of the first assays when investigating a species of clinical interest.
<p class="jove_c...

Watch this Scientific Journal Video about Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System at JoVE.com

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia.

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial ...

viability-assay-trichoderma-stromaticum-conidia-inside-human

Research

JoVE Journal

Immunology and Infection

Culturing Trichoderma stromaticum and Obtaining Conidia for Phagocytosis Assay

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 seven 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.Resuspend the final pellet in two to three milliliters of PBS and count the conidia in a Neubauer chamber.

Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Blood and Differentiation of PBMCs into Macrophages for Phagocytosis Assay

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 PBMC's, 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 in 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.

Phagocytosis Kinetics and Trichoderma stromaticum Conidial Viability Assay Using Human Peripheral Blood Mononuclear-Derived Macrophages

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 eight times 10 to fifth 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 eight times 10 to the fourth conidia per milliliter in R10 medium. Add one milliliter of suspension to each well to obtain a multiplicity of infection of one to 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 nonphagocetized 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 unphagocytosed 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 at 28 degrees 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.

421 Views.  State University of Santa Cruz - UESC.  The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia. 

Fungal Clearance Efficiency of Macrophages

Summary