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 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.
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.
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. Individuals with reported health medical conditions or using medication were excluded. All subjects voluntarily agreed to participate and signed an informed consent form before their inclusion in this study.
1. Preparation of the reagents and solutions
NOTE: Prepare the following reagents and solutions before proceeding. See the Table of Materials (TOM) for the reagent and material supplier information.
2. Trichoderma stromaticum culture and processing
3. Peripheral blood mononuclear cell (PBMC) isolation
NOTE: The PBMCs are obtained by the density barrier method using a polysucrose solution of density 1.077 g/mL30.
4. Phagocytosis kinetics
NOTE: The human peripheral blood mononuclear-derived macrophages are treated with T. stromaticum conidia at a multiplicity of infection (MOI) of 1:10 or only R10 medium as a control. The multiplicity of infection (MOI) represents the ratio of the infectious agents added to the infection targets and is presented as absolute numbers: MOI = agents:targets31. Here, we use 1 T. stromaticum conidia (agent) for 10 macrophages (targets). Then, the cells are incubated at 37 °C and 5% CO2 for different periods of time (3 h, 24 h, 48 h, 72 h, 96 h, and 120 h). Next, a round glass coverslip is used to visualize the adherent macrophages involved in the phagocytosis process under a microscope. An experimental design figure is provided (Figure 1).
NOTE: The use of 24-well plates is suggested.
Figure 1: Schematic representation of the phagocytosis and conidial viability assay using human peripheral blood mononuclear-derived macrophages. (1–10) Phagocytosis assay: The T. stromaticum must be grown in PDA, and the conidia are recovered by washing the plate with PBS. The PBMCs should be isolated by the density barrier method using the described protocol, cultured in 24-well plates with sterile round glass coverslips for 7 days for differentiation into macrophages, and then treated with an MOI of 1:10 with different time intervals. The round glass coverslips are removed and stained with the staining kit, and the results are analyzed using light microscopy. (1–8,11–13) Conidia viability assay: After isolation, the PBMCs are cultured in 24-well plates without round glass coverslips for 7 days for differentiation into macrophages and then treated with an MOI of 1:10 for different time intervals. The cells must be lysed by incubation with distilled water; the suspension is then centrifuged, and then the resuspended pellet is plated in PDA to analyze the growth kinetics of Trichoderma. This figure was designed using a database of images. Please click here to view a larger version of this figure.
5. Conidial viability assay after phagocytosis
NOTE: An experimental design figure is provided (Figure 1).
NOTE: Use 24-well plates.
6. Statistical analysis
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.
Figure 2: Phagocytosis of T. stromaticum conidia by human peripheral blood mononuclear-derived macrophages. Representative results of phagocytosis kinetics (A) under 40x and (B) 100x objectives showing PBMC-derived macrophages containing T. stromaticum conidia. (C) T. stromaticum conidia free and attached to the outer membrane; (D) T. stromaticum conidia completely internalized by macrophages; (E) T. stromaticum conidia in an upper plane; (F) multiple phagocytosis; and (G) conidia germination inside (H) and outside of the macrophages. Arrows: yellow, free conidia; white, conidia attached to the outer membrane; black, completely internalized conidia; red, conidia germination inside the macrophages. Dashed arrows: black, multiple phagocytosis; red, conidia germinating in the R10 medium outside of the macrophages; white, conidia in the upper plane. The scale bar indicates 20 µm. Please click here to view a larger version of this figure.
Figure 2 shows the representative results of the phagocytosis kinetics. In the beginning of the phagocytosis process, free T. stromaticum conidia can be observed, as well as conidia attached to the outer membrane of macrophages or completely internalized by them (Figure 2A–C). Fewer conidia can be observed free or attached to the outer membrane of the macrophages with increasing phagocytosis time.
Notably, in this work, conidia could be found free and attached to the outer membrane (Figure 2C) at the beginning of the phagocytosis process, completely internalized by the macrophages (Figure 2D), or in an upper plane compared to the macrophages (Figure 2E) but not phagocytosed, and more than one conidium could be found in the same macrophage (Figure 2F). After long periods of time (96 h), phagocytosed conidia could germinate inside the macrophages (Figure 2G), and free conidia could germinate in the R10 medium to form hyphae at 37 °C (Figure 2H).
After phagocytosis, it was possible to observe that T. stromaticum conidia remained viable even after 120 h of interaction with the human PBMC-derived macrophages. The representative results show the culture of recovered conidia (Figure 3A) and represent the time (in days) required for the culture to occupy the entire culture plate (Figure 3B) and to form colored green conidia (Figure 3C). Only discrete T. stromaticum conidia were able to evade the phagosome and be found free in the cytoplasm of the human peripheral blood mononuclear-derived macrophages29. The ability of Trichoderma to decrease the production of reactive oxygen and nitrogen species may also contribute to the survival of conidia inside the phagosome and explain the viability after phagocytosis28,29,36. So far, we have shown that the phagocytosed conidia remain viable and are capable of growing in the PDA medium even after 120h. Other microorganisms have also been reported to survive after engulfment by macrophages, such as Aspergillus27,37 and Cryptococcus38.
Figure 3: Clearance ability of human peripheral blood mononuclear-derived macrophages infected with T. stromaticum conidia. After a challenge with T. stromaticum conidia (3 h, 24 h, 48 h, 72 h, 96 h, or 120 h) the PBMC-derived macrophages were lysed, and the suspension was plated in PDA to evaluate the viability of the phagocytosed conidia. (A) Representative results of T. stromaticum growth after phagocytosis by PBMC-derived macrophages are shown. Monitoring (B) the T. stromaticum growth and (C) conidiation on different days for the positive control with T. stromaticum in PBS (see step 5.2.5), the control for the R10 medium (see step 5.1.2), and the conidia phagocytosed by macrophages for different time periods (3 h, 24 h, 48 h, 72 h, 96 h, 120 h). Representative results from four healthy donors. Mean ± standard deviation (SD). Kruskal-Wallis test. The symbol # indicates significance at p < 0.05, n = 4. Please click here to view a larger version of this figure.
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 conidia14,39,40. Phagocytosis is the key process in the Trichoderma-host interaction. Trichoderma genus modulate several phagocytosis pathways, including Dectin-1 and Dectin-2, Toll-like receptor 2 and Toll-like receptor 4, MHC, and NF-κB25,28,41,42,43. Trichoderma cause trichodermosis in compromised and non-immunocompromised individuals9. Importantly, several species of this genus are used in agriculture and are the potential source of Trichodermosis. Understanding the virulence factors of T. stromaticum and the mechanism of the immune response in the human body against these fungi is urgently needed, as occupational hazards caused by the Trichoderma species, including T.stromaticum, are rising alarmingly. The method presented here provides details for performing the phagocytosis and killing assay for studying the Trichoderma-macrophage interaction in human peripheral blood-derived macrophages; however, this method can also be used to study macrophages from different tissues, including alveolar, peritoneal, or lineage macrophages.
The first step in this protocol starts with a mother stock (M1) suspension to obtain fresh cultures of the fungus. Trichoderma cultures grow fast, as they usually take over the entire plate and begin to show green-colored conidia within 5 days. The temperature and light are crucial factors for Trichoderma growth, and alterations in these factors can affect the kinetics of Trichoderma and compromise the experiment44.
After washing the T. stromaticum culture, a final volume between 5-8 mL should be recovered. The color intensity of the suspension is proportional to the conidia concentration. The conidia suspension obtained can be used for in vitro and in vivo assays, such as for assessing phagocytosis26, neutrophil extracellular traps28, intranasal exposure36,41, intraperitoneal exposure26, intracutaneous infection45, and intravenous infection46. Previous research conducted with human macrophages has evaluated the autophagy induced by Trichoderma29 and the ability of the genus to inhibit the formation of extracellular traps in neutrophils28, which is a mechanism used to eliminate fungal pathogens and other microorganisms.
It is important to note that the culture medium used to maintain the cells can impact conidial germination (Figure 3G,H). If germination is not desired for a particular experiment, we recommend performing an initial screening assay with the cell culture medium to establish the growth kinetics of Trichoderma in that particular medium. This assay should be performed before starting the in vitro experiments with the desired cell lineage, and the experiment should be stopped after 72h if the medium is found not to be compatible. The germination of Trichoderma conidia (Figure 3G,H) can be observed from 96h inside and outside the macrophages.
As Trichoderma species present differences in morphology and the optimal temperature and pH for their growth, the results presented here may not be reproducible for all species of the genus, and this protocol may need adaptations when performed with other species. As an example, Trichoderma asperelloides conidia germinate more quickly in culture than those of T. stromaticum.
Previous documented protocols to evaluate phagocytosis and the killing of the pathogen using flow cytometry and fluorescence microscopy require expensive reagents, but they provide specific information about the phagocytosis process19. The protocol presented in this article requires only an optical microscope and plate growth to evaluate phagocytosis and provides comprehensive images of the phagocytosis process. Different protocols can be used to obtain differentiated macrophages and induce macrophage lysis. These protocols include the use of different times for macrophages differentiation instead of the 7 days used here26 or the use of 2.5% deoxycholate47 to lyse the macrophages instead of distilled water at 37 °C and 5% CO2 for 30 min, as used here in this protocol. These methods have been effectively used for different fungal species such as A. fumigatus27, A. nidulans18, and Cryptococcus neoformans15.
Some limitations of this protocol are related to the use of PBS that is not endotoxin-free, the use of round glass coverslips, and the use of human blood. We recommend testing the PBS (not endotoxin-free) on cells to ensure it cannot activate the monocytes and bias the experiment. The use of round glass coverslips is very useful for staining and counting the cells, but the cells may not spread evenly under the slide, as some cells migrate to the edge of the plate well, and this difference may have an impact on the estimated total cell number in the round glass coverslips. As we do not use immunofluorescence or similar techniques in this protocol, the presence of non-phagocytosed conidia in an upper plane compared to the macrophages may lead to the misinterpretation of the phagocytosis results. As we use blood from human donors, the final PBMC count varies between donors, and low donor cell counts may not be sufficient for all experiments.
In summary, these techniques can be used to measure the function of macrophages and the fungal clearance, specifically for filamentous fungi such as Trichoderma and other microbes, such as yeasts and bacteria. Furthermore, this protocol provides future directions for studying real variability in the immune response against conidia-forming fungi in human populations, which cannot be observed using isogenic animal models or cell lines.
The authors have nothing to disclose.
This work was supported by the following Brazilian financing institutions: Fundação de Amparo à 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 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and FAPESB, respectively.
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. |