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Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes.

Dementhon K, El-Kirat-Chatel S, Noël T - PLoS ONE (2012)

Bottom Line: The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species.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.

View Article: PubMed Central - PubMed

Affiliation: Université Bordeaux, Microbiologie Fondamentale et Pathogénicité, 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.

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Analysis of the interactions involving the J774 macrophages and living yeast cells in stationary and exponential phases over 24-hour time course experiments.The left part of the diagram shows the flow cytometry analysis of the J774 macrophages: J774 survival 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 ± standard error. See also Figure S5 which details how this diagram was built. The scale bars on the light microscopy panels represent 10 µm for the upper and middle panels and 5 µm for the lower panel.
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pone-0032621-g003: Analysis of the interactions involving the J774 macrophages and living yeast cells in stationary and exponential phases over 24-hour time course experiments.The left part of the diagram shows the flow cytometry analysis of the J774 macrophages: J774 survival 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 ± standard error. See also Figure S5 which details how this diagram was built. The scale bars on the light microscopy panels represent 10 µm for the upper and middle panels and 5 µm for the lower panel.

Mentions: It is known that the yeast cell wall composition and transcript profile vary with the physiological state of development [17], notably when considering cells grown in the stationary vs. exponential phase. The J774 macrophages were infected with C. albicans, C. lusitaniae and C. glabrata blastospores from either the exponential or stationary phase at a MOI of 1M:1Y and the phenotypes of the interactions were compared over time. Results are represented in Figure 3 (see also Figure S5), showing in parallel the behavior of the macrophages (percentage of viable macrophages and percentage of phagocytosing macrophages), and the behavior of the yeast cells (increase of the total fungal biomass and percentage of engulfed fungal biomass) during the infection process. After 30 min of incubation, nearly 100% of the macrophages infected with the different yeast species were still alive. Occasionally, we observed a slight increase of the number of active macrophages when infected macrophages were compared to uninfected controls, probably due to an increased metabolism in response to the presence of yeasts. Overall, few macrophages were engaged in phagocytosis of yeast cells as early as 30 min (6 to 24% according to the yeast species).


Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes.

Dementhon K, El-Kirat-Chatel S, Noël T - PLoS ONE (2012)

Analysis of the interactions involving the J774 macrophages and living yeast cells in stationary and exponential phases over 24-hour time course experiments.The left part of the diagram shows the flow cytometry analysis of the J774 macrophages: J774 survival 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 ± standard error. See also Figure S5 which details how this diagram was built. The scale bars on the light microscopy panels represent 10 µm for the upper and middle panels and 5 µm for the lower panel.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316538&req=5

pone-0032621-g003: Analysis of the interactions involving the J774 macrophages and living yeast cells in stationary and exponential phases over 24-hour time course experiments.The left part of the diagram shows the flow cytometry analysis of the J774 macrophages: J774 survival 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 ± standard error. See also Figure S5 which details how this diagram was built. The scale bars on the light microscopy panels represent 10 µm for the upper and middle panels and 5 µm for the lower panel.
Mentions: It is known that the yeast cell wall composition and transcript profile vary with the physiological state of development [17], notably when considering cells grown in the stationary vs. exponential phase. The J774 macrophages were infected with C. albicans, C. lusitaniae and C. glabrata blastospores from either the exponential or stationary phase at a MOI of 1M:1Y and the phenotypes of the interactions were compared over time. Results are represented in Figure 3 (see also Figure S5), showing in parallel the behavior of the macrophages (percentage of viable macrophages and percentage of phagocytosing macrophages), and the behavior of the yeast cells (increase of the total fungal biomass and percentage of engulfed fungal biomass) during the infection process. After 30 min of incubation, nearly 100% of the macrophages infected with the different yeast species were still alive. Occasionally, we observed a slight increase of the number of active macrophages when infected macrophages were compared to uninfected controls, probably due to an increased metabolism in response to the presence of yeasts. Overall, few macrophages were engaged in phagocytosis of yeast cells as early as 30 min (6 to 24% according to the yeast species).

Bottom Line: The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species.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.

View Article: PubMed Central - PubMed

Affiliation: Université Bordeaux, Microbiologie Fondamentale et Pathogénicité, 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.

Show MeSH
Related in: MedlinePlus