Titan cells confer protection from phagocytosis in Cryptococcus neoformans infections

Abstract

The human fungal pathogen Cryptococcus neoformans produces an enlarged "titan" cell morphology when exposed to the host pulmonary environment. Titan cells exhibit traits that promote survival in the host. Previous studies showed that titan cells are not phagocytosed and that increased titan cell production in the lungs results in reduced phagocytosis of cryptococcal cells by host immune cells. Here, the effect of titan cell production on host-pathogen interactions during early stages of pulmonary cryptococcosis was explored. The relationship between titan cell production and phagocytosis was found to be nonlinear; moderate increases in titan cell production resulted in profound decreases in phagocytosis, with significant differences occurring within the first 24 h of the infection. Not only were titan cells themselves protected from phagocytosis, but titan cell formation also conferred protection from phagocytosis to normal-size cryptococcal cells. Large particles introduced into the lungs were not phagocytosed, suggesting the large size of titan cells protects against phagocytosis. The presence of large particles was unable to protect smaller particles from phagocytosis, revealing that titan cell size alone is not sufficient to provide the observed cross-protection of normal-size cryptococcal cells. These data suggest that titan cells play a critical role in establishment of the pulmonary infection by promoting the survival of the entire population of cryptococcal cells.

Fig 1

Large size cannot provide cross-protection from phagocytosis. Mice were intranasally infected with 3.5 × 10⁶ large 30-μm or small 10-μm inert beads. At 3 days postinoculation, lungs were lavaged and samples were fixed and examined by microscopy to determine phagocytosis of the beads. (A) Individual infections with either 30-μm inert beads or 10-μm inert beads. (B) Coinfections with a 1:1 ratio of 30-μm and 10-μm beads. Greater than 100 beads were counted per sample. Error bars represent standard deviations for 3 mice per treatment.

Fig 2

The relationship between titan cell production and phagocytosis is nonlinear. (A) Two hypothesized relationships between titan cell production and phagocytosis include (i) a linear relationship in which titan cells are protected from phagocytosis by size alone (dotted line) and (ii) a nonlinear relationship in which small amounts of titan cell production result in a global decrease in phagocytosis (solid line). (B) Mice were intranasally inoculated with 5 × 10⁶ cells of the wild-type or mutant cryptococcal strains that exhibit altered titan cell formation. At 3 days postinfection, lungs were lavaged, and the resulting BAL samples were fixed and examined by microscopy for titan cell production and phagocytosis. Black triangles, gray diamonds, white squares, and black circles indicate infections with the gpr4Δ gpr5Δ, gpr5Δ, wild-type, and otc1Δ strains, respectively. r² values were calculated based on linear regression and exponential decay curves. Four to five mice were inoculated per treatment, and >2,000 cells were examined per mouse.

Fig 3

Titan cell production reduces phagocytosis by 24 h postinfection. Mice were intranasally inoculated with 5 × 10⁶ cells of the wild-type (WT), gpr4Δ gpr5Δ, or otc1Δ strains. At 24, 48, or 72 h postinfection, lungs were lavaged, and the resulting samples were fixed immediately. Samples were examined for titan cell production (A) and phagocytosis (B). Error bars represent standard deviations from three to five mice per strain. Greater than 300 cells per mouse were examined.

Fig 4

Nascent cells exhibit reduced phagocytosis. Mice were intranasally infected with 5 × 10⁶ cells of Alexa Fluor 488-labeled wild-type (WT) cells, Alexa Fluor 488-labeled otc1Δ cells, or Alexa Fluor 594-labeled gpr5Δ cells. At 3 days postinfection, lungs were lavaged and the samples were fixed and examined by microscopy. Titan cell formation (A) and phagocytosis (B) were quantified in unstained and stained populations of cells in each lavage sample. Error bars indicate standard deviations from three to five mice per strain and >2,000 cells per mouse.

Fig 5

Titan cell production provides cross-protection from phagocytosis to normal-size cryptococcal cells. Mice were intranasally inoculated with 5 × 10⁶ cells of Alexa Fluor 488-stained wild-type (WT) cells, Alexa Fluor 594-stained gpr5Δ cells, or an approximate 1:1 ratio of wild-type and gpr5Δ cells. At 3 days postinfection, lungs were lavaged, and the BAL samples were fixed and examined by microscopy for titan cell production, phagocytosis, and fluorescence. Dotted lines indicate half of the phagocytosis observed in the individual infections. The “total coinfection” bar indicates the total amount of phagocytosis observed during the coinfection with wild-type cells indicated in dark gray and gpr5Δ cells indicated in light gray. Error bars represent standard deviations from three to five mice per treatment with >500 cells examined per mouse.