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

Summary

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.

Abstract

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 fungal clearance efficiency of macrophages.

Introduction

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 agents^7,8.....

Protocol

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's Ethics Committee (project identification code: 550.382/ 2014).

Human peripheral blood was collected from healthy volunteers from Ilhéus city, Bahia, Brazil, not exposed to occupational activities related to the studied fungus. Individua.......

Discussion

For several fungal pathogens including Aspergillus fumigatus, Cryptococcus, Candida albicans, 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 conidia^14,39,40. Phagocytosis is the key process in the T.......

Materials

Name	Company	Catalog Number	Comments
15 mL centrifuge tubes	Corning	CLS431470	15 mL centrifuge tubes, polypropylene, conical bottom with lid, individually sterile
24-Well Flat Bottom Cell Culture Plate	Kasvi	K12-024	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;
Cell culture CO2 incubator	Sanyo	303082	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.
Centrifuge Microtube (eppendorf type) 1.5 mL	Capp	5101500	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
Circular coverslip 15 mm	Olen	K5-0015	Circular coverslips are used for microscopy techniques in cell culture. Made of super transparent translucent glass; with thickness of 0.13 mm
Class II Type B2 (Total Exhaust) Biosafety Cabinets	Esco Lifesciences group	2010274	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.
Dextrose Potato Agar medium	Merck	145	Potato Dextrose Agar is used in the cultivation and enumeration of yeasts and fungi
EDTA vacuum blood collection tube	FirstLab	FL5-1109L	EDTA is the recommended anticoagulant for hematology routines as it is the best anticoagulant for preserving cell morphology.
Entellan	Merck	1.07961	Fixative agent; Entellan is a waterless mounting medium for permanent mounting for microscopy.
Fetal Bovine Serum	Gibco	A2720801	Fetal bovine serum (FBS) is a 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.
Flaticon			database of images
Glycerol	Merck	24900988	The cryoprotectant agent glycerol is used for freezing cells and spores
Histopaque-1077			polysucrose solution
Image J			Image analysis software
Microscopy slides	Precision	7105	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
Mini centrifuge	Prism	C1801	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.
Neubauer chamber	Kasvi	K5-0011	The Neubauer Counting Chamber is used for counting cells or other suspended particles.
Panoptic fast	Laborclin	620529	Laborclin's  panoptic fast c is a kit for quick staining in hematology
Penicillin/Streptomycin Solution - 10,000U	LGC- Biotechnology	BR3011001	antibiotic is used in order to avoid possible contamination by manipulation external to the laminar flow.
Petri dish 90 x 15 mm Smooth	Cralplast	18248	Disposable Petri dish; Made of highly transparent polystyrene (PS); flat bottom; Smooth;Size: 90 x 15 mm.
Phosphate buffered saline (PBS)	thermo fisher Scientific	10010001	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.
Pipette Pasteur 3 mL Sterile	Accumax	AP-3-B-S	STERILE ACCUMAX PASTEUR 3 ML PIPETTE with 3 mL capacity, made of transparent low-density polyethylene (LDPE) and individually sterile
Refrigerated Centrifuge	Thermo Scientific	TS-HM16R	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, temperature, and running profile. The Megafuge 16R can reach maximum speeds of 15,200 RPM and maximum RCF of 25,830 x g.
RPMI-1640 Medium	Merck	MFCD00217820	HEPES Modification, with L-glutamine and 25 mM HEPES, without sodium bicarbonate, powder, suitable for cell culture
The single channel micropipettes	Eppendorf	Z683809	Single-channel micropipettes are used to accurately transfer and measure very small amounts of liquids.
Tip for Micropipettor	Corning	4894	Capacity of 10 µL and 1,000 µL Autoclavable
Triocular inverted microscope	LABOMED	VU-7125500	It allows you to observe cells inside tubes and bottles, without having to open them, thus avoiding contamination problems.