Development of an In Vitro Model for the Multi-Parametric Quantification of the Cellular Interactions between Candida Yeasts and Phagocytes

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.

Vermamoeba vermiformis-Aspergillus fumigatus relationships and comparison with other phagocytic cells

“…At 15 min, 30 min, 1 h, 2 h and 4 h of incubation, the culture supernatant was discarded, and the wells were washed twice in PAS in order to remove non-adherent conidia. To determine the rate of internalized conidia, we added trypan blue, which is unable to penetrate viable phagocytes, to quench the fluorescence of the noninternalized conidia (Scott and Woods 2000;Dementhon et al 2012). Each assay was repeated three times.…”

Host-Pathogen Interaction and Signaling Molecule Secretion Are Modified in thedpp3Knockout Mutant of Candida lusitaniae

“…We previously developed an experimental model to infect J774 cell line macrophages with yeasts in RPMI medium (28). This model was used for in vitro study of the different parameters of the interaction between yeast strains and macrophages.…”

“…Macrophage infection with yeasts. Macrophages were infected as previously described (28) in RPMI medium. A multiplicity of infection (MOI) of 1 macrophage to 5 yeasts (1M:5Y) was used in all macrophage infection assays.…”

“…Fluorimetry and flow cytometry assays were conducted as previously described (28). Fluorimetry was used to determine the ratio of fungal phagocytosis, and flow cytometry helped to determine the ratio of macrophages engaged in phagocytosis, as well as the macrophage and yeast mortality rates.…”

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

“…Phagocytosis has been studied since the discovery of phagocytes by Metchnikoff in 1800 1 . Over the years, a variety of methods have been utilized to examine this important process essential for innate immune defense against invading bacterial, viral, fungal and parasitic pathogens [2][3][4][5] . Previous methods of quantification utilized microscopy and stereological techniques in order to visualize cells that are phagocytosing, which were then quantified by counting of internalized particles (manually or with use of software) [6][7][8] .…”