Quantification of Candida - Phagocytes Interaction percentage is indicated on the left side of the horizontal bar. The white area of the bar indicates the percentage of non-phagocytosing macrophages, the shaded tones area indicates the percentage of phagocytosing macrophages. The right part shows the fluorometry analysis of the fungal population: the increase in the biomass compared to T 30 min is given as a multiplication factor on the right side of the horizontal bar. The white area of the bar represents the percentage of extra-macrophagic yeasts, the grey area shows the percentage of fungal biomass internalized within the J774 macrophages (i.e the uptake of fungal biomass). Interactions of J774 macrophages with stationary phase (A) or exponential phase (B) living yeasts at 1M:1Y MOI. Note that macrophage killing and the part of phagocytosing macrophages were higher with C. albicans than with C. glabrata and C. lusitaniae (T 5 h and T 24 h), and that C. albicans uptake was lower compared to the two other species. Each condition was performed in quintuplet (flow cytometry experiments) or in triplicate (fluorescence quenching experiments) per experiment. Each bar is the average of three independent experiments 6 standard error. See also Figure S5 which details how this diagram was built. The scale bars on the light microscopy panels represent 10 mm for the upper and middle panels and 5 mm for the lower panel. doi:10.1371/journal.pone.0032621.g003 rather than stationary phase, 10% more macrophages were recruited for phagocytosing 20% less fungal cells (see the data obtained for T 5 h for example). Altogether, these results suggested that the C. glabrata yeasts derived from exponential phase were less efficiently recognized by the phagocytes. Globally, C. lusitaniae behaved similarly to C. glabrata with however some interesting differences of interaction with macro- phages. Survival of macrophages infected with C. lusitaniae was generally 10% lower than with C. glabrata, except at T 24 h when using exponential phase yeast cells, which resulted in a marked decrease of macrophage survival down to 42% (Figure 3B, left bars). Phagocytosis of C. lusitaniae also required a higher number of macrophages when compared to C. glabrata: 65% of the live macrophages were engaged in phagocytosis of C. lusitaniae stationary yeast cells, and 50–55% for exponential yeast cells. The proportion of engulfed yeast cells was generally lower with C. lusitaniae, and the proportion of free extra-macrophagic yeasts was higher than with C. glabrata. We verified with the microscope that C. lusitaniae was never cleared off the medium by the macrophages (Figure S6). Yeasts divided actively outside leading to an increase of the fungal biomass at T 24 h (x 4.3 with stationary phase cells, and x 3 with exponential phase cells, of which 94% were extra- macrophagic). These results indicated that the C. lusitaniae cells were less efficiently internalized by the J774 macrophages than the C. glabrata cells. By comparing the data obtained at T 5 h and T 24 h with stationary yeast cells of C. lusitaniae, it is interesting to note that the increase of intra-cellular fluorescence (Figure 3A, right bars) was not correlated with an increase of the number of phagocytosing macrophages which remains constant (left bars); this suggested that the macrophages that had already ingested fungal cells were more susceptible to engulf other yeast cells than na¨ıve macrophages. The analysis of the data obtained in the same conditions with C. lusitaniae exponential yeast cells (Figure 3B) led to a completely different conclusion: the 30% decrease in macrophage survival from T 5 h to T 24 h can be set in parallel with a considerable decrease of the engulfed fungal biomass (from 38% to 6%, meanwhile the biomass was only multiplied by x 2), suggesting that part of the internalized C. lusitaniae had escaped macrophages. By assaying the glucose available in the culture medium during the infection process (Method S1 and Table S1), we verified that macrophage cell death observed during infection was neither due to any nutritional starvation nor to a release of a toxic compound in the supernatant by one of the cell types. These results indicated that J774 macrophage mortality was directly related to the phagocytosis of the Candida cells. A relationship could be drawn between the glucose consumption and the outcome of the interaction at T 5 h: when yeast cells were in the majority engulfed, the glucose consumption was similar to that of the macrophages alone, whereas when the yeast cells were extra- macrophagic, the consumption of glucose resembled that of the yeasts alone. When infected with UV-killed yeasts, macrophages used 2.5 times more glucose than uninfected macrophages. This reflects the metabolic cost of phagocytosis of dead fungal cells. On the other hand, we investigated the capacity of the macrophages to kill the different species of Candida. The survival of the yeast cells was determined following 5 and 24 hours of incubation with phagocytic cells (Method S2 and Figure S7). 100% of the C. albicans cells survived, while 20% and 40% of the C. glabrata and C. lusitaniae cells were killed within 24 hours of interaction with the macrophages, respectively. In conclusion, the J774 macrophages were less efficient in phagocytosing C. albicans compared to C. glabrata and C. lusitaniae. Our results also suggested that the exponential phases of C. lusitaniae and C. glabrata escaped more easily from macrophage phagocytosis than the stationary phase cells. Even if the growth- dependent physiological state of the Candida cells influenced the interaction with the macrophages at some levels (macrophage survival or the efficiency of the fungal biomass uptake), the global profile of the interaction within a Candida species was similar for the exponential and stationary phases. Thus, for technical convenience, we chose to infect the phagocytes with stationary cultures in the subsequent experiments. Our choice to work with cells in the stationary phase was reinforced by a study that showed that the transcriptional profile of yeast cells in vivo during an infection episode was related to the stationary phase in the laboratory [19]. Interaction of Inactivated Yeast Cells with Macrophages We first inactivated yeasts with heat treatment. When we used heat-killed (HK) yeasts, we observed about 20% of macrophages cell death at T 24 h when compared to uninfected macrophages controls, independently of the species used (Figure 4A). The J774 macrophages were the most efficient at taking up C. glabrata dead cells (16% of phagocytosing macrophages internalized 51% of the fungal biomass at T 24 h) and the least efficient at taking up C. albicans dead cells (43% of phagocytosing macrophages internal- ized 45% of the fungal biomass). The comparison of live and inactivated cells in terms of the efficiency of phagocytosis by the macrophages is only relevant at T 30 min, before yeasts multiplication takes place. The amounts of macrophages phago- cytosing HK yeasts were smaller than with the live cells, independently of the species used. On the other hand, the fungal biomass internalized was similar for HK yeasts and live yeasts (20 to 55% depending on the species). The decrease in the amount of macrophages engaged in phagocytosis could be attributed to the loss of the fungal metabolic activity and/or to the cell wall alterations resulting from heat treatment. To determine if the manner of killing yeasts had an effect on the interaction with the J774 macrophages, we also infected phagocytes with UV-killed yeasts (Figure 4B). UV-killed cells lose their ability to replicate but retain an intact cell wall [20]. The amounts of macrophages engaged in phagocytosis with UV-killed yeasts were higher than when heat-killed yeasts were used, and similar to the live cells, suggesting a predominant role of the cell PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e32621