Fast and Quantitative Evaluation of Human Leukocyte Interaction with <i>Aspergillus fumigatus</i> Conidia by Flow Cytometry

Hartung, Susann; Rauh, Christopher; Hoang, Thi Ngoc Mai; Jahreis, Susanne; Wagner, Kathleen; Macheleidt, Juliane; Brakhage, Axel A.; Rummler, Silke; Hochhaus, Andreas; Lilienfeld‐Toal, Marie von

doi:10.1002/cyto.a.23653

Cited by 7 publications

(4 citation statements)

References 27 publications

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“…A mutant strain (pksP) lacking the DHN-melanin layer on the surface of conidia was phagocytosed more efficiently than the wild-type strain, in agreement with all the published data showing the protective role of DHN-melanin against immune cells (43). The observed phagocytosis rate was similar to primary neutrophils, which internalized around 40% of wild-type conidia after 2 h, reaching a maximum of 50% after 4 or 12 h (44,45). Measuring the release of inflammatory cytokines from dPLBs revealed that IL-8, but not IL-1β, increased over time following infection with opsonized conidia.…”

Section: Discussionsupporting

confidence: 88%

PLB-985 neutrophil-like cells as a model to studyAspergillus fumigatuspathogenesis

Rafiq

Rivieccio

Zimmermann

et al. 2021

Preprint

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Fungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance mean that additional research efforts and resources must be allocated to advancing our understanding of fungal pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous defense mechanisms, including production of antimicrobial extracellular vesicles. A major draw-back to work with neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We find that differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles produced during infection by employing a size-exclusion chromatography-based approach. Advanced LC-MS/MS proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis.

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Section: Discussionsupporting

confidence: 88%

PLB-985 neutrophil-like cells as a model to studyAspergillus fumigatuspathogenesis

Rafiq

Rivieccio

Zimmermann

et al. 2021

Preprint

Self Cite

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“…The lack of morphological characterization also results in unresolved spore clusters and the lack of properly identification of spores that are adherent to macrophages (Kraibooj et al, 2014). As a consequence, it is impossible to determine the precise phagocytosis measures solely based on cytometry data (Hartung et al, 2018). In contrast, microscopy-based methods provide comprehensive details about the phagocytosis process from both the host view and the pathogen view.…”

Section: Discussionmentioning

confidence: 99%

The geographical region of origin determines the phagocytic vulnerability of Lichtheimia strains

Hassan

Cseresnyés

Al-Zaben

et al. 2019

Environmental Microbiology

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Summary Mucormycoses are life‐threatening infections that affect patients suffering from immune deficiencies. We performed phagocytosis assays confronting various strains of Lichtheimia species with alveolar macrophages, which form the first line of defence of the innate immune system. To investigate 17 strains from four different continents in a comparative fashion, transmitted light and confocal fluorescence microscopy was applied in combination with automated image analysis. This interdisciplinary approach enabled the objective and quantitative processing of the big volume of image data. Applying machine‐learning supported methods, a spontaneous clustering of the strains was revealed in the space of phagocytic measures. This clustering was not driven by measures of fungal morphology but rather by the geographical origin of the fungal strains. Our study illustrates the crucial contribution of machine‐learning supported automated image analysis to the qualitative discovery and quantitative comparison of major factors affecting host–pathogen interactions. We found that the phagocytic vulnerability of Lichtheimia species depends on their geographical origin, where strains within each geographic region behaved similarly, but strongly differed amongst the regions. Based on this clustering, we were able to also classify clinical isolates with regard to their potential geographical origin.

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“…This method is very time-consuming since for each condition serial dilutions of lysed neutrophils are needed. Therefore, we and others have developed several approaches to determine phagocytosis of bacteria by neutrophils (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), many of them in multi-well plates. Here, we describe a powerful flow cytometry method to quantify phagocytosis of S. aureus by freshly isolated human neutrophils in vitro.…”

Section: Introductionmentioning

confidence: 99%

Use of Flow Cytometry to Evaluate Phagocytosis of Staphylococcus aureus by Human Neutrophils

et al. 2021

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Neutrophils play a key role in the human immune response to Staphylococcus aureus infections. These professional phagocytes rapidly migrate to the site of infection to engulf bacteria and destroy them via specialized intracellular killing mechanisms. Here we describe a robust and relatively high-throughput flow cytometry assay to quantify phagocytosis of S. aureus by human neutrophils. We show that effective phagocytic uptake of S. aureus is greatly enhanced by opsonization, i.e. the tagging of microbial surfaces with plasma-derived host proteins like antibodies and complement. Our rapid assay to monitor phagocytosis can be used to study neutrophil deficiencies and bacterial evasion, but also provides a powerful tool to assess the opsonic capacity of antibodies, either in the context of natural immune responses or immune therapies.

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Fast and Quantitative Evaluation of Human Leukocyte Interaction with Aspergillus fumigatus Conidia by Flow Cytometry

Cited by 7 publications

References 27 publications

PLB-985 neutrophil-like cells as a model to studyAspergillus fumigatuspathogenesis

PLB-985 neutrophil-like cells as a model to studyAspergillus fumigatuspathogenesis

The geographical region of origin determines the phagocytic vulnerability of Lichtheimia strains

Use of Flow Cytometry to Evaluate Phagocytosis of Staphylococcus aureus by Human Neutrophils

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