Name: בידוד תאי דם חד-גרעיניים היקפיים (PBMCs) מדם אנושי והתמיינות של PBMCs למקרופאגים לבדיקת פגוציטוזה
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

יעילות פינוי פטרייתי של מקרופאגים

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.

מחבר זרקור: חשיפת יחסי הגומלין בין Trichoderma stromaticum לבין מערכת החיסון של יונקים

בדיקת כדאיות של Trichoderma stromaticum conidia בתוך מקרופאגים חד-גרעיניים בדם היקפי אנושי

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

בידוד תאי דם חד-גרעיניים היקפיים (PBMCs) מדם אנושי והתמיינות של PBMCs למקרופאגים לבדיקת פגוציטוזה

להעביר את הדם שנאסף מהתורם לצינור 50 מיליליטר ולהוסיף PBS כדי לקבל נפח סופי של 35 מיליליטר. מעבירים את הדם באיטיות לאורך דופן הצינור המכיל את תמיסת הרב-סוכרוז ליצירת השעיה דו-פאזית. מיד, צנטריפוגה את המתלה ב 1, 600 G במשך 30 דקות ב 24 מעלות צלזיוס.לאחר הצנטריפוגה, התבוננו בהיווצרות טבעת לבנה המכילה את PBMC, ואספו אותה על ידי שאיפה עדינה בצינור 15 מיליליטר. לאחר מכן, הוסף PBS לצינור המכיל את PBMCs כדי לקבל נפח סופי של 10 מיליליטר. הומוגניזציה המתלה בצנטריפוגה ב 1, 600 G במשך 30 דקות ב 24 מעלות צלזיוס.לאחר צנטריפוגה, לשטוף את התאים שלוש פעמים ב 1, 600 גרם במשך חמש דקות עם 10 מיליליטר של PBS. לפני השעיית הגלולה בשני מיליליטר של R10 בינוני.

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 ...