The deterministic and multifactorial nature of ECM.

<p><b>(A)</b> The magnitude of the various pathogenic features within all ten brain regions, plotted separately for individual mice with late stage ECM (n = 4–8). <b>(B)</b> Generalised linear model analyses assessing the contribution of the pathogenic features, in isolation and in combination, in promoting vascular leakage within the brain during late-stage ECM. Each data point represents the mean of the group for each region during late-stage ECM (N = 4–8).</p

Model for ECM-associated vascular leakage.

<p><b>(A)</b> 1) Arrested Schizont ruptures in the cerebral capillary releasing merozoites. 2) pRBC-dependent alterations in cerebral capillary blood flow facilitate EC phagocytosis of merozoites and digestive vacuoles. 3) ECs cross-present parasite antigen on MHC class I molecules to CD8⁺ T-cells. 4) CD8⁺ T-cells degranulate and release cytotoxic perforin and granzyme molecules). 5) The cytolytic molecules induce apoptosis signalling within the target EC. <b>(B)</b> 1) Target EC induces anti-apoptosis signalling pathways to counteract cytolysis via 2) the secretion of growth factors. Growth factors act in an autocrine fashion on the target EC and 3) paracrine fashion on neighbouring ECs. This results in: 4a) the upregulation of caveolae within affected ECs; and 4b) alterations to intercellular EC tight junctions. 5) The enhanced permeability of the cerebrovasculature leads to enlargement of the perivascular spaces and formation of oedematous parenchymal tissue, which precipitate fatal increases in intracranial pressure.</p

Incidence of intracerebral haemorrhage is greater during ECM infection compared to non-ECM infection.

<p>C57BL/6 mice were infected with 1x10⁴ <i>Pb</i> ANKA GFP (n = 8) or 1x10⁴ <i>Pb</i> NK65 GFP (n = 5) pRBCs. Mice were culled at d7 p.i. when <i>Pb</i> ANKA infected mice exhibited signs of late-stage ECM. Brains were removed from transcardially perfused mice and the nature and incidence of haemorrhage examined histologically via H&E staining. <b>(A)</b> Representative H&E images from the olfactory bulbs, cortex, midbrain and cerebellar cortex showing the relative severity and frequency of intracerebral haemorrhage in <i>Pb</i> ANKA and <i>Pb</i> NK65 infected mice. <b>(B)</b> Quantitation of haemorrhage within the different brain regions of <i>Pb</i> ANKA and <i>Pb</i> NK65 infected mice. The type and location of haemorrhage in the brains of mice experiencing ECM including: <b>(C)</b> extensive, amorphous haemorrhage from a large vessel in the olfactory bulbs; <b>(D)</b> extravasation of erythrocytes into the perivascular space of an occluded cortical microvessel; <b>(E)</b> several petechial haemorrhages in the cortex; <b>(F)</b> multiple haemorrhages at the denouements of a branching vessel; <b>(G)</b> thrombosed vessel with perivascular cuff; <b>(H)</b> thrombosed vessel with extravascular erythrocytes in the rarefied perivascular space and adjoining parenchyma; <b>(I)</b> large haemorrhage in the cerebellar grey matter (spanning both granular and Purkinje layers); <b>(J)</b> small haemorrhage in the external capsule white matter. Dots represent mean of individual brains, with red lines mean of total brains. Scale bars: A, I and J 75μm; C-F 50μm; G and H 25μm. *p ≤ 0.05 (unpaired t-test, no assumption made for consistent SD).</p

Vascular leakage is associated with dysregulation of transcellular and paracellular transport mechanisms during ECM.

<p>C57BL/6 mice were infected with 1x10⁴ <i>Pb</i> ANKA GFP (n = 3) or 1x10⁴ <i>Pb</i> NK65 GFP (n = 3) pRBCs. Mice were culled at d7 p.i when <i>Pb</i> ANKA infected mice exhibited signs of late-stage ECM. Brains were removed from transcardially perfused mice and processed for TEM <b>(A)</b> Electron micrographs of representative vessels from <i>Pb</i> ANKA and <i>Pb</i> NK65 infected mice. Note the thickened endothelium and narrowed lumen consistent with vasospasm, cytoplasmic extensions and extensive caveolae seen during <i>Pb</i> ANKA infection. <b>(i and ii)</b> Digitally magnified panels showing endothelial cytoplasmic extensions and extensive caveolae (▲) specifically during <i>Pb</i> ANKA infection. The potential mechanisms of microvessel leakage during <i>Pb</i> ANKA infection include: <b>(B)</b> transendothelial channel (▲); <b>(C)</b> vacuolar cleft (▲) associated with an endothelial cell tight junction, numerous caveolae adjacent; <b>(D)</b> cleft (▲) in an atypical endothelial cell tight junction; <b>(E)</b> cleft within microvascular tight junction (▲) adjacent to fluid accumulation within the perivascular space (◊); <b>(F)</b> caveolae (▲) proximal to astrocyte end-feet swelling (◊), extravasated RBCs (*) can also be visualised. Scale bars: A, E & F 1μm; B-D 2μm.</p

ECM is associated with demyelination and axonal dysfunction.

<p>C57BL/6 mice were infected with 1x10⁴ <i>Pb</i> ANKA GFP (n = 7) or 1x10⁴ <i>Pb</i> NK65 GFP (n = 5) pRBCs. Mice were culled at d7 p.i. when <i>Pb</i> ANKA infected mice exhibited signs of late-stage ECM. Brains were removed from transcardially perfused mice and processed for immunofluorescent and histological examination. <b>(A)</b> Representative images show presence and absence, respectively, of β-APP (green) accumulation within white matter tracts (Pons) during <i>Pb</i> ANKA and <i>Pb</i> NK65 infection. Cell nuclei are blue and auto-fluorescent (AF) RBCs red. <b>(B)</b> Patterns of β-APP staining seen during ECM. β-APP accumulation adjacent to: <b>(C)</b> erythrocyte congested capillaries, DAPI labelled erythrocytes are likely pRBCs (Δ); <b>(D)</b> leukocyte-packed vessel (denoted by dashed line), and haemorrhage. <b>(E)</b> Disruption of neuronal architecture during <i>Pb</i> ANKA and <i>Pb</i> NK65 infection in the olfactory bulbs. NeuN+ neurons (green), auto-fluorescent (AF) RBCs red and cell nuclei blue. <b>(F)</b> Extensive white matter disruption visualised by H&E staining during <i>Pb</i> ANKA, but not <i>Pb</i> NK65, infection (dotted black lines demarcate boundaries of the white matter (external capsule)). Myelin pallor, fragmentation and/or gross demyelination seen adjacent to <b>(G)</b> erythrocyte-congested capillaries (Δ), <b>(H)</b> leukocyte packed vessel and haemorrhage. Scale bars: A, D, E & F 75μm; B, C & G 25μm and H 50μm.</p

A single, pRBC is sufficient to occlude a capillary in the brains of mice infected with <i>Pb</i> ANKA.

<p>C57BL/6 mice were infected with 1x10⁴ Pb ANKA GFP pRBCs and euthanised upon developing ECM (d7 p.i.) Transcardially perfused brains were dissected out and processed for TEM <b>(Figs A-C)</b> (n = 3), smeared for cytological examination <b>(Figs D-M)</b> (n = 5) or processed for histological investigation <b>(Figs N-O)</b> (n = 8). Electron micrographs of: <b>(A)</b> a sequestered pRBC in the cross-section of a capillary, <b>(i)</b> with the adjacent digitally magnified panel highlighting an electron-dense spot (↑) on the infected erythrocyte surface, proximal to the endothelium. <b>(B)</b> An uninfected, highly deformed RBC squeezing through a capillary. <b>(C)</b> An uninfected RBC trapped behind a sequestered pRBC in a capillary longitudinal-section. Brain smears stained via Quik-Diff depicting: a <b>(D)</b> mid to late-stage trophozoite (↑) and a <b>(E)</b> late-stage schizont (↑) sequestered within capillaries. Capillaries with <b>(F)</b> 1, <b>(G)</b> 2 and <b>(H)</b> 4 uninfected RBCs (▲) trapped behind a single, mature pRBC (↑). H&E stained brain smears demonstrating: <b>(I)</b> uninfected erythrocytes (▲), trapped behind cytoadherent pigmented pRBCs (↑), deformed by the narrow capillary lumen. <b>(J)</b> A pigmented, mid-stage trophozoite (↑) sequestered within a capillary entraps two uninfected RBCs (▲). <b>(K)</b> A sequestered schizont (↑) occludes a capillary, with a “tail” of uninfected RBCs (▲) and immature pRBCs (★) behind it. <b>(L)</b> A larger calibre vessel, congested primarily by macrophages dense with parasite material. Extra-erythrocytic parasite (↑) is associated with macrophage, rather than endothelium. <b>(M)</b> Extravascular macrophage enriched with parasite material. 30μm thick, conventional histological sections stained for H&E demonstrate extensive erythrocytic accumulation (in which pRBCs are a minority) in both <b>(N)</b> cortical and <b>(O)</b> cerebellar grey matter, also shown <b>(ii, iii)</b> at higher magnification in adjacent panels.. Scale bars: A-C 1μm; D-M 5μm; N and O 50μm.</p

Survival, parasitemia and weight loss during the course of blood stage <i>Pb</i> ANKA and <i>Pb</i> NK65 infection.

<p>C57BL/6 mice were infected with 1x10⁴ <i>Pb</i> ANKA GFP (n = 13) or 1x10⁴ <i>Pb</i> NK65 GFP (n = 12) pRBCs. <b>(A)</b> Survival was monitored daily during the window period (days 6–12 p.i.) for ECM, and <b>(B)</b> peripheral parasitemia ±SEM and <b>(C)</b> weight loss ±SD were monitored every other day during the course of infection.</p

T cells exhibit equivalent perivascular compartmentalisation but distinct behaviours during <i>Pb</i> ANKA and <i>Pb</i> NK65 infections.

<p>hCD2-DsRed C57BL/6 mice were infected with 10⁴<i>Pb</i> ANKA or <i>Pb</i> NK65 pRBCs or left uninfected. Transcranial two-photon microscopy of the meninges was performed on days 5 p.i. (<i>Pb</i> ANKA), and 7 p.i. (<i>Pb</i> ANKA and <i>Pb</i> NK65). <b>(A)</b> Maximum intensity projections from intravital two-photon microscopy movies (283x283x30 μm) showing Qtracker non-targeted quantum dot-labeled blood vessels (cyan) and DsRed T cells (red) within the meninges of infected mice on day 7 p.i. with <i>Pb</i> ANKA and <i>Pb</i> NK65 and in uninfected mice. <b>(B)</b> Mean number ± SD of hCD2-DsRed T cells (including luminal and perivascular) in imaged tissue sites in uninfected control mice and on day 5 (<i>Pb</i> ANKA, n = 2) and 7 p.i. (<i>Pb</i> ANKA, n = 7; and <i>Pb</i> NK65, n = 5). <b>(C)</b> Mean proportion ± SD of hCD2-DsRed T cells located perivascularly in the meninges of <i>Pb</i> ANKA and <i>Pb</i> NK65 infected (day 7 p.i.) mice. <b>(D)</b> Mean distance ± SD (μm) of perivascularly located T cells from abluminal vessel wall in mice infected with <i>Pb</i> ANKA and <i>Pb</i> NK65 (day 7 p.i.). <b>(E)</b> Representative cropped tile scanned images showing heterogeneity of T cell clusters around pial vessels within the brains of mice infected with <i>Pb</i> ANKA and <i>Pb</i> NK65 (day 7 p.i.). <b>(F)</b> Quantification of average perivascular T cell speeds, arrest coefficient (proportion of time points when instantaneous velocity is <2 μm/min) and confinement ratio (track displacement/track length) from individual three-dimensional T cell tracks. Each point represents an individual DsRed T cell. Results are pooled two experiments, n = 4 for both groups. <b>(G)</b> Graphical illustrations summarizing the XY movement of individual perivascularly located T cells from normalized starting positions in the brains of mice on day 7 p.i. with <i>Pb</i> ANKA and <i>Pb</i> NK65 parasites. Scale bars: (A) 30 μm, (C) 150 μm. *p ≤ 0.05, ****p<0.0001 (Student’s unpaired t test or one-way ANOVA with Tukey's multiple comparisons test).</p

Depletion of systemically and perivascularly located phagocytic cells from day 5 p.i. does not prevent ECM.

<p><b>(A)</b> C57BL/6 mice were infected with 10⁴<i>Pb</i> ANKA pRBCs. On day 5 p.i. mice were injected i.p. with 300 μL clodronate liposomes with (n = 22) or without (n = 6) 8 μL clodronate liposomes i.c.v., or left untreated (n = 9). Mice were monitored daily for development of ECM (grey area). Results are pooled from five experiments. <b>(B)</b> C57BL/6 (n = 11) and CX₃CR1-iDTR mice (n = 5) were infected with 10⁶<i>Pb</i>ANKA pRBCs and treated with tamoxifen and diphtheria toxin as described in the methods to systemically deplete CX₃CR1⁺ cells in the latter group. Mantel-Cox test showed no statistically significant differences in survival. <b>(C)</b> Representative coronal cryosection of brains showing depletion of Iba1⁺microglia (green), a CX₃CR1⁺ population, in CX₃CR1-iDTR mice (right), compared to parallel C57BL/6 control (left), with ECM. Scale bars: (C) 50 μm.</p

Perivascular T cells form long-lasting interactions with CX₃CR1^+/GFP in the brains of mice infected with <i>Pb</i> ANKA.

<p>hCD2-DsRed X CX₃CR1^GFP/GFP dual reporter mice were infected with 10⁴<i>Pb</i> GFP ANKA (n = 5 from three experiments). <b>(A)</b> Maximum intensity projections from intravital two-photon microscopy movies (283x283x30 μm) showing interaction of perivascular T cells (red) with CX₃CR1^+/GFP cells (green) in brains of mice on day 7 p.i. Blood vessels (cyan) were visualized by i.v. injection of Qtracker non-targeted quantum dots. GFP⁺ pRBCs (green) can also be seen within the lumen of the vessels. White arrows highlight selected perivascular T cells forming stable interactions with CX₃CR1^+/GFP cells. <b>(B)</b> Selected cropped frames from a time-lapse movie showing a perivascular T cell [number one in <b>(A)</b> in contact with a CX₃CR1^+/GFP cell over a 17-minute period. <b>(C)</b> Three dimensional section showing the same T cell and CX₃CR1^+/GFP cell interacting in the XY, XZ and YZ planes. Scale bars: (A) 30 μm, (B) 5 μm, (C) 10 μm. (D) Mean duration ± SD of individual hCD2-DsRed T cell contacts with CX₃CR1^+/GFP cells in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.s021" target="_blank">S8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.s022" target="_blank">S9</a> Videos</b>. (E) Mean number ± SD of CX₃CR1^+/GFP cells contacted by individual perivascular hCD2-DsRed T cells in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.s021" target="_blank">S8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.s022" target="_blank">S9</a> Videos</b>, over a 17-minute period.</p