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Research Article

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

Felipe H. Santiago-Tirado, Michael D. Onken, John A. Cooper, Robyn S. Klein, Tamara L. Doering
Arturo Casadevall, Editor
Felipe H. Santiago-Tirado
a Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA
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Michael D. Onken
b Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA
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John A. Cooper
b Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA
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Robyn S. Klein
c Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA
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Tamara L. Doering
a Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA
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Arturo Casadevall
Johns Hopkins Bloomberg School of Public Health
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DOI: 10.1128/mBio.02183-16
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  • FIG 1 
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    FIG 1 

    Isolation of loaded macrophages and crossing of the BBB by C. neoformans. (A) Final phase of the sorting strategy used to isolate phagocytes that were healthy (negative for SYTOX dye) and devoid of external fungi (negative for CFW); the complete strategy is shown in Fig. S1. The boxes corresponding to the gates are labeled as follows: gate 1, healthy phagocytes with only internal fungi; gate 2, healthy phagocytes that have external fungi (with or without internal fungi); gate 3, dead or damaged phagocytes. Results are representative of results of 69 independent analyses. (B) Cells collected from the gates shown in panel A (all shown at the same scale; bar = 10 µm). Images are representative of results of 4 independent sorting studies where cells were examined microscopically. (C) Diagram of the BBB model used in this study. (D) Transit of free S. cerevisiae (S. cer) or C. neoformans (C. neo) in the absence or presence of serum; means and standard deviations (SD) of results are shown for one of two similar studies. (E) Mean and SD values over time for transit (bars, left axis) and TEER (points, right axis), for various starting inocula of opsonized C. neoformans. Stars represent TEER values at 0 h. Results from one of two similar studies are shown. (F) Time-dependent transmigration of free fungi, a 1:1 mix of free fungi and empty THPs, and fungus-loaded THPs (1 to 1.49 fungi/host cell). Means and standard errors of the means (SEM) are shown for results of one of seven similar independent experiments. Values plotted for all time course transit assays are cumulative values.

  • FIG 2 
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    FIG 2 

    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.

  • FIG 3 
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    FIG 3 

    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]).

  • FIG 4 
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    FIG 4 

    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).

  • FIG 5 
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    FIG 5 

    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.

  • FIG 6 
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    FIG 6 

    Mechanisms of brain infection by C. neoformans. (A) Depicted are wild-type fungi (red ovals) crossing the BBB either free (1) or within Trojan horse phagocytes (2). Mutant fungi (orange ovals) cannot cross alone (3) but can use Trojan horse transit as an alternative route. See text for details on transendothelial pore formation for Trojan horse transit. (B) Loaded phagocytes potentially contribute to brain invasion by pathways that do not involve true Trojan horse transit (2). Phagocytes can bring the fungus to the CNS internally and then exit the phagocyte by nonlytic exocytosis and cross the BBB as free yeast by transcytosis (1). Finally, we observed two instances of direct cell-to-cell transfer of fungal cells from phagocytes to endothelial cells (3), supporting the hypothesis of a “taxi” mechanism, where loaded phagocytes deliver fungal cells directly into brain endothelial cells. Pink, blood vessel lumen; pale green, brain parenchyma; blue cells, brain endothelial cells; green cells, infected phagocytes; shaded gray rectangle, the extracellular matrix that forms the BBB basal membrane.

Supplemental Material

  • Figures
  • FIG S1 

    Optimization of THP-1 loading and sorting strategy for isolation of singly loaded phagocytes. (A) Fungi were incubated with THPs at various MOIs for quantitation of the number of internal fungi/host cell by confocal microscopy. (B) The same conditions were assessed to determine phagocytic and adherence indices (number of cell-associated fungi/100 host cells). (C to F) Sorting strategy, applied to optimized uptake reactions, to exclude free fungi (near origin) and dead or damaged host cells (at left) (C); to select single particles (D); to exclude unassociated host cells (left, stained only with DFFDA) and fungi (lower right, stained only with mCherry) (E); and to collect the desired population (region 1) of cells that were differentiated from CFW+ cells bearing adherent external fungi (region 2) and SYTOX+ damaged cells (region 3) (F). Download FIG S1, TIF file, 1.2 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • MOVIE S1 

    A Z-stack of confocal slices 5 µm apart across the permeable membrane of a model BBB. Loaded macrophages were added to the top of an hCMEC monolayer and incubated for 1 h prior to washing and fixation performed as described in Materials and Methods. The first half of the movie shows xyz views, while the second half shows the same images rendered as a three-dimensional (3D) object with rotation. Download MOVIE S1, MOV file, 5 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S2 

    TEM of hCMECs grown on PA pads. (A) Low-magnification views of PA fibers and the hCMEC monolayer (top row; scale bars = 10 µm) and higher-magnification examples of cellular junctions (bottom row; scale bars = 0.5 µm for the first two images and 2 µm for the last). Arrows indicate the cell junctions. (B) Loaded primary monocytes interacting with hCMECs; scale bars = 2 µm. (C) Free C. neoformans within an endothelial cell, highlighting host cell distortion and cytoskeletal elements surrounding the fungus-containing vacuole. Scale bar = 2 µm. Download FIG S2, TIF file, 4.9 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • MOVIE S2 

    Live-cell recording of loaded hPBMs interacting with and traversing the hCMEC monolayer. Arrows indicate the start of membrane activity prior to transmigration. Download MOVIE S2, MOV file, 13.2 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • MOVIE S3 

    Live-cell recording of loaded hPBM interacting with and traversing the hCMEC monolayer. Arrows indicate the start of membrane activity prior to transmigration. Download MOVIE S3, MOV file, 8.5 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • MOVIE S4 

    Examples of fungal escape from phagocytes. (Left) A loaded macrophage (arrow) that lyses and allows fungal escape. (Right) Nonlytic escape of fungal cells from a loaded macrophage; arrows indicate the positions of impending fungal extrusion. Download MOVIE S4, MOV file, 11.5 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • MOVIE S5 

    Live-cell recording showing cell-to-cell transmission of fungi between a loaded macrophage and an endothelial cell. Yellow arrows indicate the transferred fungi, while magenta arrows indicate extracellular fungal cells as a reference. Download MOVIE S5, MOV file, 6.3 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • MOVIE S6 

    Live-cell recording of a free fungal cell moving across the field that is captured and engulfed by an hCMEC. It later buds intracellular, exits nonlytically, and then continues replicating extracellularly. Download MOVIE S6, MOV file, 9.5 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • MOVIE S7 

    Live-cell recording of a loaded macrophage interacting with the hCMEC monolayer, where the presence of fungi apparently prevents complete transmigration during the duration of the movie. Yellow arrows indicate the start of membrane activity prior to transmigration; the red arrow points to the fungal cell that remains above the monolayer; and the white arrow indicates the direction of the futile movement of the host cell, which seems to be stuck. Download MOVIE S7, MOV file, 8.7 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S3 

    Isolation of S. cerevisiae-loaded THP-1 cells and the effect of phagocyte loading on transmigration. (A) Flow plots and gating strategy for S. cerevisiae loading (essentially the same as that used for C. neoformans in the experiment whose results are shown Fig. S1). Gate 1, loaded macrophages with no external fungi; gate 2, phagocytes with adherent CFW+ fungi; gate 3, damaged/dead SYTOX+ phagocytes. (B) Transmigration of empty THPs or THPs containing either S. cerevisiae (S. cer) or C. neoformans (C. neo), assayed by fluorescence. In these studies, model BBBs were generated in 96-well plates with Corning FluoroBlok multiwell inserts, which block light transmission for wavelengths between 400 and 700 nm. This allows DFFDA fluorescence of particles that cross the barrier to be directly measured from below with the FITC filter set of a fluorescence plate reader (Cytation3 system). The data plotted represent the increase in fluorescence compared to control wells without THPs. *, P < 0.002 (by Tukey’s multiple-comparison test compared to either loaded population at that time point). (C) Transmigration assays prepared as described for panel B were performed by direct counting. At the desired time points, the inserts were dipped sequentially into HBSS+, fixative (4% formaldehyde–PBS), and HBSS+. A Cytation3 system was then used with the manufacturer’s software to image the bottom of the inserts and count the DFFDA-stained transmigrated THPs. *, P < 0.0001 (by Tukey’s multiple-comparison test compared to either loaded population at that time point). Download FIG S3, TIF file, 1.3 MB.

    Copyright © 2017 Santiago-Tirado et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

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Trojan Horse Transit Contributes to Blood-Brain Barrier Crossing of a Eukaryotic Pathogen
Felipe H. Santiago-Tirado, Michael D. Onken, John A. Cooper, Robyn S. Klein, Tamara L. Doering
mBio Jan 2017, 8 (1) e02183-16; DOI: 10.1128/mBio.02183-16

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Trojan Horse Transit Contributes to Blood-Brain Barrier Crossing of a Eukaryotic Pathogen
Felipe H. Santiago-Tirado, Michael D. Onken, John A. Cooper, Robyn S. Klein, Tamara L. Doering
mBio Jan 2017, 8 (1) e02183-16; DOI: 10.1128/mBio.02183-16
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KEYWORDS

blood-brain barrier
Cryptococcus neoformans
immune evasion
phagocytes

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