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Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages
Published on: December 10, 2013
GATA1-deficient human pluripotent stem cells generate neutrophils with improved antifungal immunity that is mediated
Andrew S Wagner1, Frances M Smith1, David A Bennin2
1Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
Abstract:
Neutrophils are critical for host defense against fungi. However, the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here, we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation, antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal β-glucan, including an upregulation of the pentose phosphate pathway (PPP), similar to primary human neutrophils in vitro. Furthermore, we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively, these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response.
Insights
Genetically modified human stem cell-derived neutrophils (iNeutrophils) show enhanced antifungal activity against Aspergillus fumigatus. Deleting GATA1 improves iNeutrophil function, offering a new model for studying fungal immunity.
Area of Science:
- Immunology
- Stem Cell Biology
- Mycology
Background:
- Neutrophils are crucial for fighting fungal infections.
- Primary neutrophils have limitations for research due to short lifespan and genetic modification challenges.
- Human induced pluripotent stem cell-derived neutrophils (iNeutrophils) offer a tractable model.
Purpose of the Study:
- To investigate the role of GATA1 in neutrophil antifungal activity.
- To characterize iNeutrophils for studying neutrophil-fungal interactions.
- To explore the metabolic and functional changes in modified iNeutrophils.
Main Methods:
- Generation of GATA1 knockout (KO) human iPSCs and differentiation into iNeutrophils.
- Assessment of iNeutrophil maturation, receptor expression, and effector functions.
- Analysis of metabolic shifts, including the pentose phosphate pathway (PPP), upon fungal stimulation.
- Evaluation of the role of CD18 in GATA1-KO iNeutrophil antifungal activity.
Main Results:
- GATA1-KO iNeutrophils exhibited enhanced maturation and antifungal activity against Aspergillus fumigatus.
- These modified iNeutrophils showed increased expression of pattern recognition receptors and improved effector functions.
- iNeutrophils displayed metabolic reprogramming upon fungal β-glucan stimulation, including PPP upregulation.
- CD18 deletion impaired GATA1-KO iNeutrophil killing of A. fumigatus but did not affect PPP upregulation.
Conclusions:
- iNeutrophils provide a valuable in vitro system for studying human neutrophil antifungal immunity.
- GATA1 deletion enhances iNeutrophil antifungal capabilities, highlighting its regulatory role.
- CD18 plays a role in iNeutrophil-mediated fungal killing, while metabolic pathways like PPP are regulated independently.
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