Development of an In Vitro Model for the Multi- Parametric Quantification of the Cellular Interactions between Candida Yeasts and Phagocytes Karine Dementhon1,2., Sofiane El-Kirat-Chatel1,2., Thierry Noe¨ l1,2* 1 Univ. Bordeaux, Microbiologie Fondamentale et Pathoge´nicite´, UMR 5234, Bordeaux, France, 2 CNRS, Microbiologie Fondamentale et Pathoge´nicite´, UMR 5234, Bordeaux, France Abstract We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes. Citation: Dementhon K, El-Kirat-Chatel S, Noe¨l T (2012) Development of an In Vitro Model for the Multi-Parametric Quantification of the Cellular Interactions between Candida Yeasts and Phagocytes. PLoS ONE 7(3): e32621. doi:10.1371/journal.pone.0032621 Editor: Dana Davis, University of Minnesota, United States of America Received July 8, 2011; Accepted February 2, 2012; Published March 30, 2012 Copyright: 2012 Dementhon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Sofiane El-Kirat-Chatel PhD was supported by the French Ministe`re de l’Enseignement Supe´rieur et de la Recherche. This work was supported by grants from the University of Bordeaux and the Centre National de la Recherche Scientifique. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: thierry.noel@u-bordeaux2.fr . These authors contributed equally to this work. Introduction Deep and invasive fungal infections caused by Candida species are increasing among immunocompromised individuals. Candida albicans is the species most often involved, but emerging non- albicans species, such as C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii and C. lusitaniae are becoming increasingly frequent and are responsible for nearly half of candidiasis cases [1]. When infecting the host, the fungal cells are confronted with innate immune cells, essentially macrophages and neutrophils. Given the evidence that phagocytosis of fungal cells is the first step in the control of infection, developing a cellular model allowing an accurate analysis of the overall interaction involving different Candida species and phagocytes, appears to be of great interest in that way it constitutes an alternative method to in vivo experiments to evaluate virulence of Candida strains. The main objective of this work was to develop a simple and reproducible method for the simultaneous monitoring of the kinetics for phagocyte association to yeasts, phagocyte survival at the individual cell level, and for fungal cells uptake by phagocytes over a 24-hour infection. The second objective was to make the method sensitive enough to detect small variations during the yeast-phagocyte interaction, and amenable to high-throughput screening of banks of mutants. An accurate evaluation of phagocytosis requires 1) to analyze the phagocyte association to yeast cells and the phagocyte survival simultaneously 2) to distinguish between yeasts that had been internalized by phagocytes from those unphagocytosed, and to measure the uptake of fungal cells by phagocytes while taking into account extracellular yeast multiplication during the infection process. A critical step is to label yeasts during the infection process. Most of the studies already reported used FITC (Fluorescein isothiocyanate, a dye that covalently binds amino acids of proteins [2]) to separately label yeasts before infection. However, the use of FITC made the phagocytosis analysis limited to early time points [3], because yeast replication led to fluorescence decrease, or restricted to fixed cells stained with antibodies coupled with FITC for longer incubation time points. Instead, we chose CalcoFluor White (CFW) which specifically labels fungal cell walls [4]: when added to the culture medium, it allowed the continuous labeling of yeasts, even those that are newly generated by replication along the 24-hour infection. Phagocytes were double-stained with calcein, a marker of PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e32621