Evidence of a role for monocytes in dissemination and brain invasion by Cryptococcus neoformans

Abstract

The pathogenesis of cryptococcosis, including the events leading to the production of meningoencephalitis, is still largely unknown. Evidence of a transcellular passage of Cryptococcus neoformans across the blood-brain barrier (BBB) and subsequent BBB disruption exists, but the paracellular passage of free yeasts and the role of monocytes in yeast dissemination and brain invasion (Trojan horse method) remain uncertain. We used our model of disseminated cryptococcosis, in which crossing of the BBB starts 6 h after intravenous inoculation, to study paracellular passage of the BBB. We prepared bone marrow-derived monocytes (BMDM) infected in vitro with C. neoformans (BMDM yeasts) and free yeasts and measured fungal loads in tissues. (i) Spleen and lung CFU were >2-fold higher in mice treated with BMDM yeasts than in those treated with free yeasts for 1 and 24 h (P < 0.05), while brain CFU were increased (3.9 times) only at 24 h (P < 0.05). (ii) By comparing the kinetics of brain invasion in naïve mice and in mice with preestablished cryptococcosis, we found that CFU were lower in the latter case, except at 6 h, when CFU from mice inoculated with BMDM yeasts were comparable to those measured in naïve mice and 2.5-fold higher than those in mice with preestablished cryptococcosis who were inoculated with free yeasts. (iii) Late phagocyte depletion obtained by clodronate injection reduced disease severity and lowered the fungal burden by 40% in all organs studied. These results provide evidence for Trojan horse crossing of the BBB by C. neoformans, together with mechanisms involving free yeasts, and overall for a role of phagocytes in fungal dissemination.

FIG. 1.

Representative in vitro phagocytosis of C. neoformans by BMDM. BMDM were incubated with FITC-labeled C. neoformans strain H99 and monoclonal antibody E1 (1.5 μg/10⁶ yeasts) for 90 min at a BMDM/yeast ratio of 3:1. (A) Epifluorescence microscopy. The bright FITC staining is partially quenched after phagocytosis, allowing the distinction between free yeasts (thin arrow), intracellular yeasts (arrowheads), and adherent yeasts (large arrow). Uninfected BMDM (black stars) are also visible. Magnification, ×1,000. (B to D) Flow cytometry analysis of CD11b-APC-labeled BMDM (B), FITC-labeled C. neoformans H99 (C), and BMDM-H99 inoculum prepared as described above (D). CD11b-APC-labeled BMDM are seen in gate R1, free FITC-labeled yeasts (FITC^high APC⁻ cells) are seen in R2, phagocytosed yeasts (FITC^low APC⁺ cells) are seen in R3, and BMDM-adhering yeasts or just internalized yeasts (FITC^{high/intermediate} APC⁺ cells) are seen in R4.

FIG. 2.

Effect of inoculation with yeasts internalized in monocytes on C. neoformans tissue burden. Mice were inoculated with 2 × 10⁴ BMDM-H99 or 2 × 10⁴ free H99 (free-H99) and sacrificed 1, 6, and 24 h after inoculation. Results are expressed as ratios of the mean CFU/g of organ for the experimental group (BMDM-H99) to that for the control group (free-H99) (data are means ± SD for three mice in each group). The dotted line denotes a ratio of 1. Two independent experiments included the three time points, and the last one included only the 1-h and 24-h study points. *, P < 0.05.

FIG. 3.

Effect of established cryptococcal infection on brain invasion by a second isolate of C. neoformans. Inoculation with 2 × 10⁴ free KN99α NAT or BMDM-KN99α NAT cells was done on day 0 to naïve mice (free-d0 or BMDM-d0 group) or mice injected 2 days before with 2 × 10⁶ KN99α cells (free-d2 or BMDM-d2 group), as summarized in the table. Mice were sacrificed 1, 6, and 24 h thereafter. Results are expressed as CFU/g of brain (data are means ± SD for three mice). Only CFU data resulting from KN99α NAT inoculation are presented.

FIG. 4.

Effects of delayed and sustained monocyte depletion on the course of cryptococcal infection. (A) Mice were infected with 10⁵ H99 yeasts and treated by daily i.v. infection with clodronate liposomes (depleted group) or with PBS (control group) for 4 days. Macroscopic examination of representative organs from mice in the control group showed multiple abscesses in the spleen (B) and hemorrhages in the brain (D), but mice in the depleted group showed no visible lesions in the spleen (C) or the brain (E). (F) Ratios of fungal loads measured in the spleens, lungs, and brains of depleted and control mice (data are means ± SD for three animals per group from three independent experiments).