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.
Keywords: Neutrophil extracellular trap, Candida albicans, Neutrophil, Fungicidal activity, Micrococcal nuclease
Background
Candida albicans is an opportunistic fungal pathogen that resides as a commensal on mucosal surface and the skin in most humans. Environmental changes in temperature, nutrition, or the presence of serum induces its transformation from yeast form to hyphae. Candida infection is one of the top leading causes of overall healthcare-associated bloodstream infections in medical centers as well as regional hospitals. Mortality among patients with invasive candidiasis is as high as 40% even after receiving antifungal therapy (Brown et al., 2012; Chen et al., 2014; Kullberg and Arendrup, 2015). Patients with neutropenia and neutrophil dysfunction are at high risk for invasive candidiasis, suggesting the importance of neutrophil anti-Candida functions in host defense (Antachopoulos et al., 2007; Horn et al., 2009; Lionakis and Netea, 2013). Our work showed that mouse neutrophils utilize two independent mechanisms, one NADPH oxidase-dependent and the other NADPH oxidase-independent, to kill C. albicans. NADPH oxidase-dependent killing of opsonized C. albicans requires phagocytosis mediated by CR3, and NADPH oxidase-independent killing of unopsonized C albicans is through NET formation mediated by dectin-2 (Wu et al., 2017 and 2019). MNase is an enzyme that digests NET. To demonstrate the effect of NET, it is necessary to compare the killing ability of neutrophils that is treated with MNase to that treated with heat-inactivated MNase. Difference between the two treatments denotes killing by NET and not by other factors (Wu et al., 2019). Neutrophils are phagocytic. Taking up microorganisms through receptors triggers robust ROS production that kills the engulfed organism. To assay for NADPH oxidase-dependent killing of opsonized C. albicans, one of the important steps is to wash off un-engulfed microorganisms after allowing time for phagocytosis to take place (Vonk et al., 2012; Wu et al., 2017). In contrast, since NET kills microorganisms extracellularly, it is critical not to wash off un-engulfed organisms to assay for NET-mediated killing of C. albicans. Moreover, instead of lysing neutrophils by ddH2O at high pH (pH 11.0) to release ingested microorganisms, NET-forming neutrophils and fungal hyphal elements are detached from the wells by mini cell scraper and DNA digesting enzyme MNase (Wu et al., 2017 and 2019). The method described here were published in 2019 (Wu et al., 2019) to observe killing of C. albicans by NET. This method can be adapted to quantify NET-mediated killing of other biofilm-forming organisms.
Materials and Reagents
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Acknowledgments
AcknowledgmentThis work was supported by Academia Sinica thematic project AS-105-TP-B08 to BWH and the Ministry of Science and Technology research grants 104-2320-B-002-052-MY2 and 107-2321-B-002-053-MY3 to BWH and SYW, respectively. This protocol was adapted from a publication by Vonk et al. (2012) and modified according to protocols that quantified the viability of biofilm-forming microorganisms in other studies ( Morici et al., 2016; Mohammed et al., 2017).
Competing interests
NO financial competing interests.
Ethics
Mouse study was carried out in strict accordance with the recommendations in the Guidebook for the Care and Use of Laboratory Animals, The Third Edition published by The Chinese-Taipei Society of Laboratory Animal Sciences in 2007. All animal procedures and experimental protocols were approved by AAALAC-accredited facility, the Committee on the Ethics of Animal Experiments of the National Taiwan University College of Medicine (Permit Number: 20140304, 20140533 and 20180013).
References
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