Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Shan F. Brunel; Jude M. Bain; Jill King; Lena J. Heung; Shinji  Kasahara; Tobias M. Hohl; Adilia Warris

doi:10.3791/55444

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Shan F. Brunel, Jude M. Bain, Jill King, Lena J. Heung, Shinji Kasahara, Tobias M. Hohl, Adilia Warris

Memorial Sloan-Kettering Cancer Center

Research output: Contribution to journal › Article › peer-review

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Abstract

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labelled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus.It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Original language	English
Article number	e55444
Journal	Journal of visualized experiments : JoVE
Volume	122
Early online date	23 Mar 2017
DOIs	https://doi.org/10.3791/55444
Publication status	Published - 19 Apr 2017

Bibliographical note

This work has been supported by the MRC and the University of Aberdeen (MRC Centre for Medical Mycology) and by the Wellcome Trust Strategic Award - Medical Mycology Fungal Immunology (SFB, JMB, JK, AW). A special thank you is given to the support from the Chloe fund. TMH is supported by a grant from the National institute of health (NIH, code RO1 093808) and the Memorial Sloan Kettering Cancer Center is supported by NIH grant P30 CA008748. Additionally, TMH is an investigator in the Pathogenesis of Infectious Disease supported by the Burroughs Wellcome Fund. We wish to thank the Aberdeen Microscopy and Histology Facility for their help and support.

Keywords

Aspergillus fumigatus
live cell imaging
antifungal activity
FLARE
human leucocytes

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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title = "Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus",

abstract = "Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labelled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus.It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.",

keywords = "Aspergillus fumigatus, live cell imaging, antifungal activity, FLARE, human leucocytes",

author = "Brunel, {Shan F.} and Bain, {Jude M.} and Jill King and Heung, {Lena J.} and Shinji Kasahara and Hohl, {Tobias M.} and Adilia Warris",

note = "This work has been supported by the MRC and the University of Aberdeen (MRC Centre for Medical Mycology) and by the Wellcome Trust Strategic Award - Medical Mycology Fungal Immunology (SFB, JMB, JK, AW). A special thank you is given to the support from the Chloe fund. TMH is supported by a grant from the National institute of health (NIH, code RO1 093808) and the Memorial Sloan Kettering Cancer Center is supported by NIH grant P30 CA008748. Additionally, TMH is an investigator in the Pathogenesis of Infectious Disease supported by the Burroughs Wellcome Fund. We wish to thank the Aberdeen Microscopy and Histology Facility for their help and support.",

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AU - Bain, Jude M.

AU - King, Jill

AU - Heung, Lena J.

AU - Kasahara, Shinji

AU - Hohl, Tobias M.

AU - Warris, Adilia

N1 - This work has been supported by the MRC and the University of Aberdeen (MRC Centre for Medical Mycology) and by the Wellcome Trust Strategic Award - Medical Mycology Fungal Immunology (SFB, JMB, JK, AW). A special thank you is given to the support from the Chloe fund. TMH is supported by a grant from the National institute of health (NIH, code RO1 093808) and the Memorial Sloan Kettering Cancer Center is supported by NIH grant P30 CA008748. Additionally, TMH is an investigator in the Pathogenesis of Infectious Disease supported by the Burroughs Wellcome Fund. We wish to thank the Aberdeen Microscopy and Histology Facility for their help and support.

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N2 - Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labelled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus.It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

AB - Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labelled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus.It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

KW - Aspergillus fumigatus

KW - live cell imaging

KW - antifungal activity

KW - FLARE

KW - human leucocytes

U2 - 10.3791/55444

DO - 10.3791/55444

M3 - Article

VL - 122

JO - Journal of visualized experiments : JoVE

JF - Journal of visualized experiments : JoVE

M1 - e55444

ER -

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Abstract

Bibliographical note

Keywords

UN SDGs

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