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

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<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 Trichoderma stromaticum 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.
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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

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

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Abstract

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