This study aimed to develop an improved in vitro model to finely quantify the cellular interactions between Candida yeasts and phagocytes. The method we developed is the first one that allows a semi-automated multi-parameter analysis for the simultaneous monitoring of two interacting populations in a host-pathogen context. The method was validated with three different Candida species and two types of phagocytes, the J774 murine macrophage cell line and human neutrophils.
Yeasts were labeled with CFW, a fluorochrome that binds cell wall of live and dead fungal cells. CFW shows a relative insensitivity of its fluorescence to pH, which is an advantage in a phagocytic assay as the phagolysosomal acidification can modify the fluorescence of an internalized yeast 
. The CFW was added in the culture medium at the beginning of the infection process to allow the continuous labeling of newly replicated yeasts. An increase in CFW fluorescence over time was interpreted as an increase in the fungal biomass, independently of the morphology and size of the cells. As the CFW can not enter viable macrophages, the multiplication of yeasts within macrophages was not assessed, unless yeasts were released in the medium. Phagocytes were specifically labeled with calcein-AM and anti-CD16-APC antibodies. Calcein-AM is a fluorogenic esterase substrate, used as a viability probe that reflects both enzymatic activity, which is required to convert calcein-AM to a fluorescent product, and cellular membrane integrity, which is required for intracellular retention of the fluorescence. Calcein-AM was chosen because cell viability studies showed it was quite well retained by living cells and released during cytolysis, with low pH sensitivity 
. Anti-CD16-APC antibodies specifically labeled the membrane of phagocytes.
To avoid the counting with the microscope, we chose flow cytometry to analyse the population of phagocytes. This method allows a rapid analysis of a large number of cells, and a simultaneous measure of two parameters at the individual cell level: the percentage of phagocytes engaged in phagocytosis of yeasts (either bound on the phagocyte membrane or internalized), and the survival of that same population of infected phagocytes, compared to their survival in absence of yeasts. To differentiate the CFW-labeled yeasts that were free or simply bound to the macrophage membrane from those ingested, we used the trypan blue to quench the CFW fluorescence of extra-macrophagic yeasts, leaving the internalized yeasts fluorescent.
From a strict analytical point of view, our model overcomes a bias in the interpretation of the data frequently generated when the percentage of phagocytosing macrophages and the percentage of internalized fungal biomass are analyzed separately. For example, our data showed that the percentage of phagocytosing macrophages was lower for C. glabrata, which could be interpreted as a lower recognition efficiency, but the percentage of internalized fungal biomass was actually higher for C. glabrata than for the other Candida species. Also, a lower percentage of internalized fungal cells does not necessarily reflect a lower internalized fungal biomass, as extra-macrophagic fungal cells may divide over time, and increase total fungal biomass. Thus, our model takes into account not only the percentage of phagocytosing macrophages, along with the macrophage survival, but also the percentage of total fungal biomass internalized.
When we compared the three Candida
species, we observed differences in the proportion of macrophages engaged in phagocytosis, in the survival rate of macrophages and in the efficiency of the macrophages to internalize the fungal biomass. We established that macrophages were more efficient to phagocyte C. glabrata
, as indicated by the lowest number of macrophages engaged in phagocytosis, the lowest macrophage cell death, and the highest proportion of the fungal biomass internalized. C. albicans
was the more aggressive species as it triggered the highest number of macrophages engaged in phagocytosis, the highest macrophage cell death and the lowest proportion of internalized fungal biomass. We found that C. lusitaniae
was more efficiently taken up than C. albicans
, and in smaller amounts than C. glabrata
. The differences in the aggressiveness of the Candida
species toward the phagocytes highlighted in our work correlate with already published studies, especially those showing that C. glabrata
was considerably less virulent than C. albicans
using a mouse model 
, and in vitro
competition experiments showing that J774 macrophages display strong preferences for phagocytosis of C. glabrata
compared to C. albicans
The differences in the interaction of the three Candida
species with the macrophages may be in part attributed to a different cell wall composition, whose role in the recognition by the phagocytes has been widely described 
. It has been reported that the cell wall of C. glabrata
harbors 50% more mannose and three times less chitin than C. albicans
. As the mannans are exposed on the external face of the cell wall, they are likely to be more easily recognized by the receptors (Mannose Receptor, TLR4, Dectin-2, Galectine-3) of the phagocytes, whose activation initiates phagocytosis 
. Furthermore, the cell wall composition can vary depending on the developmental state of the fungal cells 
. Candida albicans
shows a differential expression of cell wall proteins in yeast and hyphae 
, including particular glycosyltransferases that modify the glycan components, and therefore the PAMPs (Pathogen-Associated Molecular Patterns) exposed on the cell surface. Also, hyphae of C. albicans
do not expose beta-glucans like yeast cells do in bud scars, and thus do not activate the Dectin-1 receptor, which is involved in phagocytosis and immune response processes 
. Beta-glucans of C. lusitaniae
may be similarly masked in the small chains of cells, thus explaining why under that morphology, it is less recognized by the macrophages.
Our experiments showed that the growth phase of the fungal cells is important for the host-pathogen interaction. When yeast cells were taken from the exponential phase, less C. lusitaniae
and C. glabrata
biomass was engulfed by the macrophages and they escaped more easily from macrophage than when taken from the stationary phase. We speculated that some cell wall components may be differentially exposed such as beta-1,2 mannosides, recognized by galectin 3 of macrophages, which are in higher amount in the cell wall of yeast from the stationary phase than from the exponential phase 
The experiments conducted with inactivated yeast cells allowed us to investigate the interaction independently from the morphological changes and from the metabolic activity of the yeast cells. Heat treatment suppresses metabolic activity but artificially increases β-glucan exposure, altering the outer layer of mannans, whereas UV treatment was shown to maintain an intact cell wall 
. We found that macrophages were more efficient to internalize UV-killed than heat-killed cells, emphasizing a predominant role of the cell wall (and likely of the mannans) in the recognition by the macrophages. Interestingly, UV-killed yeasts were more efficiently internalized by the macrophages than live cells, despite a similar amount of macrophages engaged in phagocytosis. We speculate that live fungal cells produce a signal molecule that negatively interferes with macrophage recognition.
Macrophage cell death was slightly higher when macrophages were infected by living C. glabrata
and C. lusitaniae
cells than when infected with dead yeasts: thus we speculated that the fungal metabolic activity of these two species did not significantly contribute to the observed macrophage killing. Up to 20% of the macrophages died in the presence of the inactivated yeast cells, irrespective of the species used. This suggests that the phagocytosis of inert yeast cells triggered macrophage killing via
a macrophage-dependent mechanism. It has already been reported that the uptake of yeast polysaccharides led to a macrophage cell death of 10–20% after 6 or 24 hours of incubation 
. In contrast, the ability of C. albicans
to kill macrophages was greatly reduced after the inactivating treatment. Our data confirm already published studies showing that the metabolic activity of C. albicans
, and the capacity to produce hyphae, largely contributed to macrophage killing.
In our work, the three Candida
species mostly survived to macrophage phagocytosis, according to three different mechanisms. As previously shown 
, our data confirmed that C. albicans
survived to phagolysis and rapidly produced hyphae from within the macrophages and thus escaped, killing the host cell and multiplying outside. In agreement with other work 
, we found that C. glabrata
mostly survived phagolysis and divided within the macrophages, which eventually burst and released the yeast cells. Beside its ability to form pseudo-hyphae, we found that C. lusitaniae
quickly formed small chains of cells, less efficiently recognized by the macrophages as confirmed by video-microscopy. Once phagocytosed, a proportion of the C. lusitaniae
cells were able to survive, multiply within the macrophages and escape. Thus the present data support the hypothesis that there may be a correlation between the morphology of the Candida
specie and the strategy used to escape from the macrophages.
Some mechanisms important for the survival and escape of yeasts from phagocytes have recently been described. For C. albicans,
beside the known yeast-to-hyphae transition 
, the contribution of other mechanisms allowing survival and resistance to phagocyte killing was reported: the inhibition of the phagosome maturation 
, the production of CO2
from arginine to induce germ tube formation 
, the trehalose biosynthetic pathway 
and the expression of Hyr1p, a GPI-anchored cell wall protein of the hyphal form 
, and degradation of host-derived reactive oxygen species (ROS) by fungal superoxyde dismutases 
. In C. glabrata,
and glycosylphosphatidylinositol-linked aspartyl proteases 
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 
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 
. It was also shown that phagocytosis by human neutrophils can be elicited solely by β-1,6-glucans 
. They kill pathogens intracellularly in the phagolysosome by a set of enzymes and anti-microbial molecules, and by the production of ROS 
. 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 
. On the other hand, the pathogens can trigger neutrophil cell death, either by inducing NETs release 
, 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) 
. 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 
, 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.