Trojan Horse Transit Contributes to Blood-Brain Barrier Crossing of a Eukaryotic Pathogen

Visualization of Trojan horse crossing. (A) Experimental design for visualization of both sides of the permeable membrane. (B) A field of view showing loaded macrophages associated with the endothelial monolayer (top row) and the bottom of the porous membrane with loaded macrophages alone (bottom row). Both mammalian cell types were stained with CellMask plasma membrane dye (blue); THPs were further stained with DFFDA from the flow sorting (green) and contained mCherry-stained fungi (red). Scale bar, 10 μm. (C) Merged images from a larger field of view, again showing all cell types on the top of the membrane (left), whereas only loaded phagocytes are visible on the bottom (right). Scale bar, 20 μm. Images are representative of multiple fields from two independent experiments, each with three independent time points. The fields shown are from a 1-h time point. We observed loaded macrophages associated with the bottom of the membrane most frequently early in the incubation period (more at 1 h than at 2 to 3 h; not shown); this may represent a time-dependent loss of phagocyte viability or ability to initiate transit. See Movie S1 for another example.

Differential regulation of free and internalized fungal crossing by immune mediators. (A to C) Transmigration of free and internalized fungi in the presence of various chemoattractants, with matched controls. (A) fMLP, N-formyl-methionine-leucyl-phenylalanine peptide (100 nM). DMSO, dimethyl sulfoxide. *, P < 0.0001 (by Sidak’s multiple-comparison test). (B) MCP-1 (also known as CCL2), monocyte chemoattractant protein-1 (100 ng/ml). *, P < 0.03 (by Sidak’s multiple-comparison test). (C) INO, inositol (1 mM). *, P < 0.05 (by Sidak’s multiple-comparison test). (D and E) Transmigration assays using BBB pretreated with TNF-α (10 ng/ml) or IFN-β (1 ng/ml) for 24 h before addition of loaded THPs (*, P < 0.0003 [for comparisons between TNF-α and other treatments by Tukey’s multiple-comparison test]) (D) or of free fungi (*, P < 0.0001 [for comparisons between each compound and PBS by Tukey’s multiple-comparison test]) (E). (F and G) TEER values of model BBBs treated with 10 ng/ml TNF-α (F) or 1 ng/ml IFN-β (G) for 24 h prior to initiation of the study (t = 0 h), at which point the medium was replaced with fresh medium without cytokines (*, P < 0.01; #, P < 0.0002 [all compared to PBS at the same time point by Dunnett’s multiple-comparison test]).

Loaded macrophages provide an alternative route for mutant cryptococci to gain access into the brain. (A) The roles of Cps1 and Ure1 in BBB crossing. HA (green ovals on the surface of the fungi [red]) made by Cps1 is recognized by the endothelial cell (EC) surface receptor CD44 (blue shapes), which triggers endocytosis of the fungal cells. The accumulation of ammonia generated by Ure1 may damage cellular junction proteins, facilitating fungal brain entry. (B and C) Transmigration of free or internalized fungi, comparing wild-type and either cps1Δ (B), or ure1Δ (C) mutants. Means plus SEM are plotted. * denotes P < 0.002 and P < 0.0001 in panels B and C, respectively (both determined by Sidak’s multiple-comparison test).

Visualization of Trojan horse crossing by real-time and electron microscopy. (A) hCMEC monolayers were grown on glass or 0.4% PA pads were grown to confluence and fixed, and adherens junctions were stained with anti-VE-cadherin antibody. Nuclei were stained with propidium iodide. (B and C) Two examples of loaded primary human monocytes crossing endothelia, from Movie S2 (B) and Movie S3 (C); see text for details. (D) TEM of a loaded monocyte in the process of transendothelial migration (left), with a corresponding drawing to identify structures (right). N, nucleus; V, vacuole; F, fungal cell; B, brain endothelial cell.