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Neutrophil Extracellular Trap Killing Assay of Candida albicans
Sheng-Yang Wu1, Betty A Wu-Hsieh1
1Graduate Institute of Immunology, National Taiwan University College of Medicine, Taipei, Taiwan.
Abstract:
Fungal pathogen Candida albicans is one of the top leading causes of overall healthcare-associated bloodstream infections worldwide. Neutrophil is the major effector cell to clear C. albicans infection. Our study showed that mouse neutrophils utilize two independent mechanisms to kill C. albicans: one is CR3 downstream NADPH oxidase-dependent mechanism that kills opsonized C. albicans; the other one is dectin-2-mediated NADPH oxidase-independent neutrophil extracellular trap (NET) that kills unopsonized C. albicans. Neutrophil killing of opsonized C. albicans requires phagocytosing the organism and production of reactive oxygen species production (ROS). Most existing protocols that assay for neutrophil killing of C. albicans requires a washing step after allowing neutrophils to phagocytose the organism. By definition, NET kills organisms extracellularly. Therefore, it is important to skip the washing step and add an optimal ratio of neutrophils and C. albicans to the wells. To demonstrate the effect of NET, it is necessary to compare killing ability of neutrophils treated with micrococcal nuclease (MNase), an enzyme that digests NET, to that treated with heat-inactivated MNase. MNase is also applied to release NET-bound fungal elements for counting. This protocol can be applied to assay NET killing of other biofilm-forming organisms.
Insights
Mouse neutrophils kill Candida albicans via two pathways: NADPH oxidase for opsonized fungi and neutrophil extracellular traps (NETs) for unopsonized fungi. This study details a protocol to assay NET killing mechanisms.
Area of Science:
- Immunology
- Microbiology
- Infectious Diseases
Background:
- * *Candida albicans* is a leading cause of healthcare-associated bloodstream infections.
- * Neutrophils are critical for clearing *C. albicans* infections.
- * Existing assays for neutrophil killing may not accurately reflect NET activity.
Purpose of the Study:
- * To elucidate the distinct mechanisms by which neutrophils kill *C. albicans*.
- * To present a refined protocol for assessing neutrophil extracellular trap (NET)-mediated killing of *C. albicans*.
- * To highlight the importance of avoiding washing steps in NET-specific assays.
Main Methods:
- * Investigated two neutrophil-mediated killing mechanisms: CR3-dependent NADPH oxidase and dectin-2-mediated NET formation.
- * Assessed killing of opsonized and unopsonized *C. albicans* by neutrophils.
- * Utilized micrococcal nuclease (MNase) to confirm NET-dependent killing and facilitate fungal element release.
Main Results:
- * Mouse neutrophils employ independent pathways to kill *C. albicans*.
- * Opsonized *C. albicans* are killed via NADPH oxidase-dependent phagocytosis and reactive oxygen species (ROS) production.
- * Unopsonized *C. albicans* are killed via NADPH oxidase-independent NETs.
Conclusions:
- * Neutrophils possess dual mechanisms for *C. albicans* clearance.
- * NET formation is crucial for combating unopsonized *C. albicans*.
- * The described protocol accurately assays NET killing and can be adapted for other biofilm-forming pathogens.
