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PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis

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 granulo...

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Autores principales: Rafiq, Muhammad, Rivieccio, Flora, Zimmermann, Ann-Kathrin, Visser, Corissa, Bruch, Alexander, Krüger, Thomas, González Rojas, Katherine, Kniemeyer, Olaf, Blango, Matthew G., Brakhage, Axel A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8730815/
https://www.ncbi.nlm.nih.gov/pubmed/34986319
http://dx.doi.org/10.1128/msphere.00940-21
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author Rafiq, Muhammad
Rivieccio, Flora
Zimmermann, Ann-Kathrin
Visser, Corissa
Bruch, Alexander
Krüger, Thomas
González Rojas, Katherine
Kniemeyer, Olaf
Blango, Matthew G.
Brakhage, Axel A.
author_facet Rafiq, Muhammad
Rivieccio, Flora
Zimmermann, Ann-Kathrin
Visser, Corissa
Bruch, Alexander
Krüger, Thomas
González Rojas, Katherine
Kniemeyer, Olaf
Blango, Matthew G.
Brakhage, Axel A.
author_sort Rafiq, Muhammad
collection PubMed
description 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 drawback 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 show 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 liquid chromatography-tandem mass spectrometry (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. IMPORTANCE Polymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remain very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory.
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spelling pubmed-87308152022-01-14 PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis Rafiq, Muhammad Rivieccio, Flora Zimmermann, Ann-Kathrin Visser, Corissa Bruch, Alexander Krüger, Thomas González Rojas, Katherine Kniemeyer, Olaf Blango, Matthew G. Brakhage, Axel A. mSphere Research Article 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 drawback 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 show 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 liquid chromatography-tandem mass spectrometry (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. IMPORTANCE Polymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remain very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory. American Society for Microbiology 2022-01-05 /pmc/articles/PMC8730815/ /pubmed/34986319 http://dx.doi.org/10.1128/msphere.00940-21 Text en Copyright © 2022 Rafiq et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Rafiq, Muhammad
Rivieccio, Flora
Zimmermann, Ann-Kathrin
Visser, Corissa
Bruch, Alexander
Krüger, Thomas
González Rojas, Katherine
Kniemeyer, Olaf
Blango, Matthew G.
Brakhage, Axel A.
PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis
title PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis
title_full PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis
title_fullStr PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis
title_full_unstemmed PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis
title_short PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis
title_sort plb-985 neutrophil-like cells as a model to study aspergillus fumigatus pathogenesis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8730815/
https://www.ncbi.nlm.nih.gov/pubmed/34986319
http://dx.doi.org/10.1128/msphere.00940-21
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