wall in the early recognition process. Furthermore, the amount of internalized fungal biomass was significantly increased for UV- killed cells compared to live cells (between 30% and nearly 100% depending on the species were engulfed at T 30 min). One may suppose that live cells produce a signal molecule (which would be inactivated by UV treatment) that interferes with the recognition by the macrophages. Moreover, the inactivation of the yeast cells confirmed some inter-species differences already noticed with live cells in terms of macrophage recognition: the J774 macrophages were the most efficient at taking up C. glabrata dead cells and the least efficient at taking up C. albicans dead cells. Live C. glabrata and C. lusitaniae cells triggered a slightly increased macrophage mortality compared to when inactivated (compare Figure 3A and Figure 4A and B). These data suggest a little contribution of fungal active mechanisms for both C. glabrata and C. lusitaniae in the macrophage killing. In contrast, live C. albicans cells triggered a much higher macrophage mortality (approximately 85% of macrophage killing after 5 hours and nearly 100% after 24 hours) than when inactivated (20% maximum). These data suggest that the macrophage cell death induced by C. albicans involves fungal active mechanisms. Using C. Lusitaniae and C. Glabrata Cells in Excess Increased the Macrophage Cell Death and Escape from Phagocytosis Challenging the macrophages with an excess of yeasts at a MOI of 1M:5Y affected macrophage survival. As expected, C. albicans was more aggressive than the other two Candida species at T 5 h. Interestingly, the macrophages infected with an excess of C. glabrata or C. lusitaniae showed an increased mortality compared to a MOI of 1M:1Y at T 24 h (48% vs. 22% in C. glabrata infections and 74% vs. 32% in C. lusitaniae infections; Figure 3 and Figure 5A). Despite the fact that the macrophages were equally engaged in phagocytosis with C. glabrata and C. lusitaniae (approximately 80% of live macrophages for both species at T 5 h and T 24 h), the proportion of internalized biomass differed significantly (88% of C. glabrata were taken up at T 5 h vs. 42% for C. lusitaniae (Figure 5A), again indicating that the macrophages were less efficient at engulfing C. lusitaniae compared to C. glabrata. At T 24 h, only 9% of the C. glabrata biomass and 7% of the C. lusitaniae biomass were within the macrophages. Together with the increase in macro- phage killing, this observation suggested that both C. glabrata and C. lusitaniae escaped the macrophages after 24 hours of infection at a MOI of 1M:5Y, causing phagocyte death, and multiplied outside the macrophages. We confirmed by observation under the microscope and video-microscopy that the three Candida species were capable of escaping from macrophage phagocytosis (Figure 6 and Movies S1-S3). At the beginning of the infection, C. albicans yeasts were phagocytosed by macrophages. After 30 minutes, yeasts started to form hyphae even inside macrophages. Hyphae disrupted the phagocyte membrane after 2–3 hours and allowed yeast to escape phagolysis (Movie S1). We observed that C. glabrata cells were massively phagocytosed by the J774 macrophages, but survived and multiplied inside, leading to phagocyte burst (Movie S2). Interestingly, we observed that C. lusitaniae cells were phagocytosed at the beginning of the infection, but after 2 hours, started to form chains of cells that were no longer recognized by phagocytes: macrophages got in contact with yeast chains but rarely engulfed them, which allowed yeasts to multiply extracel- lularly (Movie S3). Quantification of Candida - Phagocytes Interaction When Macrophages are used in Excess, the Killing of Macrophages by C. Albicans is Delayed and C. Glabrata and C. Lusitaniae are More Efficiently Taken Up When used in excess at a MOI of 5M:1Y, the macrophages were able to better survive C. albicans infection at T 5 h than at a MOI of 1M:1Y (66% vs. 13% survival; Figure 3 and Figure 5B), but after 24 hours, 98% of the phagocytes were killed and the entire fungal biomass was extra-macrophagic. Compared to a MOI of 1M:1Y, when the phagocytes were used in excess, smaller percentages of macrophages were engaged in phagocytosis with the yeast cells and higher proportions of fungal biomass were ingested, independently of the Candida species used. Only 1% of the live macrophages took up approximately 80% of the C. glabrata and C. lusitaniae biomass at T 30 min (Figure 5B). Candida glabrata remained fully contained until T 24 h, whereas C. lusitaniae multiplied outside of the macrophages, as observed with the microscope. These results showed that when macrophages are used in excess with respect to yeasts, they more efficiently resist C. albicans induced-cell death during the early stages of interaction and, as expected, engulf more fungal biomass, irrespective of the Candida species used. C. Albicans Triggers a Higher Neutrophil Cell Death than C. Glabrata and C. Lusitaniae We analyzed the interaction of the three Candida species with human neutrophils at different MOIs, using the same procedure as described above for the macrophages, except that quenching experiments could not be realized for a technical reason: the neutrophils were loosely adherent to plastic, compared with the macrophages, and were lost during the washes that were required for the trypan blue quenching. We did not try further to artificially make the neutrophils adhere to the bottom of the well using gelatin or collagen in order to avoid the interference of an external molecule with the Candida-phagocyte interaction. Furthermore, the determination of the percentage of internalized yeast cells is a less discriminatory parameter with neutrophils because a non-negligi- ble portion of the neutrophils are able to capture and destroy pathogens extracellularly owing to the formation of NETs (Neutrophils Extracellular Traps) [21]. We first measured the survival of the neutrophils and their association with the live stationary phase fungal cells by flow cytometry over a 5-hour infection period with a MOI of 1N:1Y (Figure 7). After 30 minutes, 20% of the neutrophils died in the presence of C. lusitaniae and C. glabrata, whereas 40% died in the presence of C. albicans. A longer infection time did not significantly change the killing of neutrophils when infected by C. lusitaniae and C. glabrata, but dramatically increased the killing by C. albicans (more than 90% of the neutrophils were killed at T 5 h). The association between the neutrophils and the three Candida species was similar during the infection and reached 50% at T 5 h. Increasing the number of yeasts over that of the neutrophils (MOI 1N:5Y) resulted in an approximate 10% increase in neutrophil cell death for each Candida species in the early stages of the infection (Figure 7), as approximately 30% the of neutrophils were killed by C. glabrata and C. lusitaniae, whereas 50% were killed by C. albicans. Nearly all of the neutrophils were killed by C. albicans within a 5-hour period. Furthermore, as expected, the neutrophils were more associated with the fungal cells at a MOI of 1N:5Y compared to a MOI of 1N:1Y during the infection. In the experiments conducted at a MOI of 5N:1Y, it is noteworthy that the neutrophils survived infection by C. albicans as well as the other species at T 30 min, and 36% were still alive at T 5 h. Also, as expected, the neutrophils were less associated with PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e32621