germ tube formation [42], the trehalose biosynthetic pathway [43,44] and the expression of Hyr1p, a GPI-anchored cell wall protein of the hyphal form [45], and degradation of host-derived reactive oxygen species (ROS) by fungal superoxyde dismutases [46]. In C. glabrata, autophagy [47] and glycosylphosphatidylino- sitol-linked aspartyl proteases [40] were shown to be crucial for survival within macrophages. The strategies employed by C. lusitaniae to survive and escape macrophage phagocytosis remain to be investigated at the molecular level. Neutrophils are thought to be the most efficient phagocytic cells to fight fungal cells [48] Recognition of yeast cells are mediated by specific PRR, in particular TLR 2, TLR 4 and dectin-1, which recognize the glucans and mannans of the fungal cell wall [29]. It was also shown that phagocytosis by human neutrophils can be elicited solely by b-1,6-glucans [49]. They kill pathogens in- tracellularly in the phagolysosome by a set of enzymes and anti- microbial molecules, and by the production of ROS [50–51]. Alternatively, they can kill pathogens extracellularly, through the degranulation and release of antimicrobial molecules, and the release of Neutrophil Extracellular Traps (NETs) from the dying neutrophils [21,52]. On the other hand, the pathogens can trigger neutrophil cell death, either by inducing NETs release [53], or by inducing the phagocytosis of the complement or IgG opsonized targets through the CR3 receptor, which leads to neutrophil apoptosis (Phagocytosis-Induced Cell Death or PICD) [54–55]. In our experiments, the neutrophils did not significantly differ in their association with the three Candida species. However, C. albicans triggered the highest neutrophil cell death, regardless of the MOI tested, while C. glabrata and C. lusitaniae were relatively inefficient to counteract an attack by the neutrophils. As the Candida cells were non-opsonized in our experiments, it is likely that the neutrophil cell death we observed was not PICD, but rather due to the release of NETs [21], as it could be observed in our experiments with propidium iodure staining (data not shown). The in vitro cellular model we developed in this study was proved to be sensitive enough to detect phenotypic differences not only between different Candida species, but also within a same Candida species, differences resulting from small variations in MOI, or from the developmental state of the yeast. The data obtained by our team (unpublished data) also showed that different mutants of C. lusitaniae had measurable differences in their interaction with macrophages and neutrophils. Finally, we believe that our model is suitable for large scale screening of banks of C. lusitaniae mutants and of other yeast species, with the goal to identify new fungal genes important for each step of the interaction with immune host cells: recognition and ingestion of yeasts by the phagocytes, survival of both cell types, and escape of yeasts from phagocytes. Materials and Methods Strains, Media and Growth Conditions Candida albicans SC5314, Candida glabrata ATCC 90030 and Candida lusitaniae CBS 6936 (ATCC 38533) were used as wild-type strains in all of the experiments. The Candida strains were grown in standard medium YPD (1% yeast extract, 2% bactopeptone, 2% glucose). The murine macrophage cell line J774A.1 (ATCC TIB- 67) was cultured in DMEM (Gibco) containing 10% decom- plemented fetal bovine serum (FBS, Gibco) and 1 mM sodium pyruvate, or in complete RPMI (cRPMI: RPMI-1640 (Sigma) without phenol red and supplemented with 10% decomplemented FBS, 1 mM sodium pyruvate and 2 g/L sodium bicarbonate) for the infection experiments, at 37uC in 5% CO2. For the preparation of the heat-killed yeasts, the cells were incubated for 30 min at 90uC. Quantification of Candida - Phagocytes Interaction Isolation and Purification of Human Neutrophils The neutrophils polymorphonuclear (PMN) cells were isolated and purified from fresh human blood (EFS Aquitaine Limousin, Bordeaux) as described [56]. Briefly, dextran allowed the sedimentation of red blood cells. A hypotonic lysis in the presence of ammonium chloride was used to remove any remaining red blood cells. The leucocytes were deposited onto a Percoll (Fluka, SIGMA) density gradient (1.075/1.095) and centrifuged at 30006g for 20 min at 4uC. The lower ring containing the PMN was collected, the PMNs were washed with PBS and resuspended in 0.5X cRPMI. The resulting population contained at least 95% neutrophils. Infection of Phagocytes with Yeasts J774 macrophages were plated in culture-treated white 96-well plates with clear well bottoms (Greiner Bio-one) in 200 ml of cRPMI and incubated overnight at 37uC in 5% CO2 to adhere. Triplicates or quintuplets of the wells were done in each plate and three plates were set up to perform a time course analysis of the infection over 24 hours (at T 30 min, T 5 h and T 24 h). To prevent variation over time by repeatedly sampling the content of a single plate, a separate plate was set up for each time point. We used 26105 macrophages per well for two multiplicities of infection (MOI 1M:1Y and 1M:5Y) and 36105 macrophages per well at a MOI of 5M:1Y. The loss of unadhered cells was lower than 2% of the initial population. Yeast cells were collected from overnight culture in YPD supplemented with 5 mg/ml Calcofluor White (CFW, Sigma) and adjusted to the required concentrations in cRPMI plus 5 mg/ml CFW. Because the size or the morphology of the cells in a culture can influence the value in the OD600 measurements, we established the correlation between OD600 and CFU for each strain of each yeast species. J774 macrophages were infected with 200 ml of CFW-labeled yeasts in 5 mg/ml CFW at the required concentration depending on the MOIs tested. As a control, the uninfected phagocytes were stained with 5 mg/ml CFW in cRPMI. PBS alone was used as a negative fluorescent control. Yeasts in cRPMI without phagocytic cells were included in the plate. We verified that 5 mg/ml CFW did not alter the growth or viability of phagocytes and yeast cells. The neutrophils were purified just before being infected with yeasts, as described for macrophages, except that the interactions were not followed beyond 5 hours since these phagocytes have a short life span (about 8 hours). Fluorometry and Quenching Analysis CFW fluorescence (lex 350 nm, lem 460 nm) was measured using a FluoStar Optima fluorimeter (BMG Labtech). The results were recorded in arbitrary units of fluorescence (AUF). The vital dye trypan blue (Sigma) was used to quench fluorescence in the spectral range of CFW. The fluorescence of yeasts either free in the medium or attached to the macrophage membrane was quenched. As trypan blue cannot enter viable cells, the unquenched fluorescence reflected the yeast cells that were internalized in viable macrophages. At each time point, the plate was centrifuged 5 min 22006g and the wells were washed with PBS to remove the unbound CFW. To measure the overall process of phagocytosis, the non-ingested yeasts were not removed during the assay. For the quenching experiments, each condition was duplicated in the plate: 200 ml of PBS was added to one well in order to determine the total fluorescence of the CFW-labeled yeasts (intra-macrophagic and extracellular), and 200 ml of trypan blue was added to another well at a final concentration of either 1 mg/ml for C. albicans or 250 mg/ml for both C. lusitaniae and C. glabrata in order to assess the fluorescence of the internalized CFW- PLoS ONE | www.plosone.org 13 March 2012 | Volume 7 | Issue 3 | e32621