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Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry
Susann Hartung1, Christopher Rauh2, Sarah Böttcher3
1Infections in Hematology and Oncology, Leibniz Institute for Infection Biology and Natural Product Research; Department for Hematology and Medical Oncology, Jena University Hospital; susann.hartung@leibniz-hki.de.
Abstract:
Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.
Insights
This study presents a rapid flow cytometry method to measure how human immune cells, monocytes and neutrophils, engulf Aspergillus fumigatus spores. This technique aids in evaluating immune response to fungal infections.
Area of Science:
- Medical Mycology
- Immunology
- Cell Biology
Background:
- Invasive pulmonary infections by Aspergillus fumigatus are a significant risk for immunocompromised individuals.
- Phagocytosis by innate immune cells, specifically monocytes and neutrophils, is crucial for clearing inhaled fungal spores from the lungs.
Purpose of the Study:
- To develop a fast and reliable flow cytometry protocol for quantifying phagocytosis of Aspergillus fumigatus conidia by human leukocytes.
- To enable simultaneous analysis of monocyte and neutrophil phagocytic activity from a single sample.
Main Methods:
- Utilized fluorescein isothiocyanate (FITC)-labeled Aspergillus fumigatus conidia for co-incubation with human leukocytes.
- Employed flow cytometry with anti-FITC antibody counterstaining to differentiate between internalized and cell-adherent conidia.
- Enabled assessment of phagocytosis percentages in both monocytes and neutrophils.
Main Results:
- The developed protocol provides a rapid and reliable measurement of fungal spore phagocytosis.
- The method allows for the discrimination of internalized versus adherent conidia.
- The assay can be combined with the analysis of other cell markers and is applicable to other fungal or bacterial pathogens.
Conclusions:
- This flow cytometry protocol offers an efficient tool for evaluating the phagocytic capacity of human leukocytes against Aspergillus fumigatus.
- It serves as a valuable method for microbiologists to assess pathogen virulence and for immunologists to study host defense mechanisms against fungal infections.
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