Name: Isolatie van perifere mononucleaire bloedcellen (PBMC's) uit menselijk bloed en differentiatie van PBMC's in macrofagen voor fagocytosetest
Uploaded: 2023-10-20T13:10:00
Description: Watch this Scientific Journal Video about Isolation of Peripheral Blood Mononuclear Cells PBMCs from Human Blood and Differentiation of PBMCs into Macrophages for Phagocytosis Assay at JoVE.com

isolation of peripheral blood mononuclear cells (pbmcs) from human blood and differentiation of pbmcs into macrophages for phagocytosis assay

Efficiëntie van schimmelklaring van macrofagen

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.

Auteur in de schijnwerpers: het ontrafelen van de wisselwerking tussen Trichoderma stromaticum en het immuunsysteem van zoogdieren

Levensvatbaarheidstest van Trichoderma stromaticum Conidia in menselijk perifeer bloed Mononucleair-afgeleide macrofagen

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.

Watch this Scientific Journal Video about Isolation of Peripheral Blood Mononuclear Cells PBMCs from Human Blood and Differentiation of PBMCs into Macrophages for Phagocytosis Assay at JoVE.com

Isolatie van perifere mononucleaire bloedcellen (PBMC's) uit menselijk bloed en differentiatie van PBMC's in macrofagen voor fagocytosetest

Breng het verzamelde bloed van de donor over naar een buis van 50 milliliter en voeg PBS toe om een uiteindelijk volume van 35 milliliter te verkrijgen. Breng het bloed langzaam over langs de wand van de buis met de polysucrose-oplossing om een bifasische suspensie te vormen. Centrifugeer de suspensie onmiddellijk bij 1.600 G gedurende 30 minuten bij 24 graden Celsius.Observeer na het centrifugeren de vorming van een witte ring met de PBMC's en verzamel deze door voorzichtig op te zuigen in een buis van 15 milliliter. Voeg vervolgens PBS toe aan de buis met de PBMC's om een eindvolume van 10 milliliter te verkrijgen. Homogeniseer de suspensie in centrifuge bij 1.600 G gedurende 30 minuten bij 24 graden Celsius.Was de cellen na het centrifugeren driemaal bij 1.600 G gedurende vijf minuten met 10 milliliter PBS. Voordat u de pellet opnieuw in suspensie brengt, in twee milliliter R10-medium.

Research

JoVE Journal

Immunology and Infection

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

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 ...

Culturing Trichoderma stromaticum and Obtaining Conidia for Phagocytosis Assay

Het kweken van Trichoderma stromaticum en het verkrijgen van conidia voor fagocytose-assay

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

Fagocytosekinetiek en Trichoderma stromaticum conidiale levensvatbaarheidstest met behulp van mononucleair afgeleide macrofagen van menselijk perifeer bloed