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

Parasite specific CD8⁺ T cells are comparably activated within the brains of mice infected with <i>Pb</i> ANKA and <i>Pb</i> NK65 parasites.

<p>C57BL/6 mice were infected with 10⁴<i>Pb</i> ANKA or <i>Pb</i> NK65 pRBCs. <b>(A)</b> Representative flow cytometric plots and calculated percentages showing frequencies of CD8⁺ T cells (gated on live leukocytes) within the brains of uninfected and infected (day 7 p.i.) mice (n = 5). <b>(B)</b> Absolute numbers of CD8⁺ T cells within the brains of uninfected and infected (day 7 p.i.) mice (n = 19–22). <b>(C)</b> Percentage of CD8⁺ T cells expressing high levels of the surrogate antigen specificity marker, CD11a (n = 5). <b>(D)</b> Activation phenotype of CD8⁺CD11a^high T cells from uninfected and infected (day 7 p.i.) mice (n = 5). Results are representative of two independent experiments <b>(A, C and D)</b> or four combined experiments <b>(B)</b>. Bars represent mean number ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (one-way ANOVA with Tukey's multiple comparisons test).</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

Parasite specific OT-I CD8⁺ T cells that directly cause ECM are perivascular and are highly arrested in the brain.

<p>C57BL/6 (n = 9) and P14 (n = 10) mice were infected with 10⁶ SIINFEKL-expressing <i>Pb</i>-TG pRBCs. Prior to infection, 10⁶ naïve DsRed-expressing OT-I CD8⁺ T cells were adoptively transferred into CFP⁺ P14 host mice (n = 6). Development of ECM (grey area) was monitored by assessing <b>(A)</b> peripheral parasitaemia ± SD and <b>(B)</b> survival. <b>(C)</b> Maximum intensity projections from an intravital two-photon microscopy movie showing accumulation of DsRed⁺CD8⁺ OT-I T cells (orange) within the brain of a P14 recipient on day 6 p.i. infection. Endothelial cells (blue) were visualized by CFP expression. <b>(D)</b> Proportion ± SD of CD8⁺ OT-I T cells located perivascularly on day 6 p.i. (n = 8). <b>(E)</b> Quantification of average perivascular T cell speeds, arrest coefficient and confinement ratio from individual three-dimensional T cell tracks. Results are pooled from two experiments.</p

Hypothetical model for the CD8⁺ T cell-dependent development of ECM.

<p>(1) <i>Pb</i> ANKA infection leads to the upregulation of adhesion molecules and cross-presentation of parasite antigen by MHC-I on brain microvascular endothelial cells [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.ref085" target="_blank">85</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.ref087" target="_blank">87</a>]. (2) This promotes transient interaction of <i>Pb</i> ANKA-pRBCs and rolling of activated CD8⁺ T cells on the luminal aspect of the brain microvessel endothelial cells. Beginning one day prior to signs of ECM, parasite-specific CD8⁺ T cells are recruited to the perivascular space, either via direct diapedesis across the endothelium, or migration via the highly permissive choroid plexus. In the perivascular space, parasite-specific CD8⁺ T cells form immune synapses with (3) parasite Ag-expressing APCs and (4) the basolateral membrane of cross-presenting endothelial cells. Perivascular APCs may acquire parasite antigen as a result of either transport of material across the vessel wall preceding generalized breakdown of the barrier or subsequent to breach of cerebral vascular integrity. (5) The interaction between CD8⁺ T cells and the basolateral membrane of endothelial cells leads to release of cytotoxic perforin and granzyme molecules in the perivascular space that down regulate intercellular tight junction proteins, damaging the vascular integrity and causing vasogenic edema. (6) Through an undefined mechanism, perivascular CD8⁺ T cells also communicate across the glia limitans to induce astrocyte and microglial activation. Activation of these cells further amplifies cerebral inflammation and dysfunction and provides a source of VEGF that concomitantly induces vascular leakage and resistance of endothelial cells to apoptosis. In non-ECM malarial infections, parasites do not accumulate in the brain, brain endothelial cells do not phagocytose or cross-present malaria antigen and perivascular CD8⁺ T cells fail to recognize their cognate antigen, restricting their pathogenic activity and preventing ECM development.</p

pRBCs make transient adhesive contact with endothelial cells and are deposited within the perivascular space of the meninges of mice with ECM.

<p>C57BL/6 mice were infected with 10⁴<i>Pb</i> ANKA-GFP (n = 3) or <i>Pb</i> NK65-GFP (n = 3) pRBCs. Representative images demonstrating the <b>(A)</b> presence and <b>(B)</b> absence of cytoadherent GFP⁺ pRBCs (green) within the cortical vasculature (red) of brains taken on d7 p.i. from transcardially perfused mice infected with <i>Pb</i> ANKA or <i>Pb</i> NK65 respectively. Cell nuclei are shown in blue. <b>(C)</b> Quantification of cytoadherent GFP⁺ pRBCs within brains taken from transcardially perfused mice infected with <i>Pb</i> ANKA or <i>Pb</i> NK65. CFP and DsRed mice showing blue and orange endothelial cells, respectively, were infected with 10⁶<i>Pb</i> ANKA-GFP pRBCs and monitored for symptoms of ECM. Transcranial two-photon microscopy of the meninges was performed on days 5 (n = 2), 6 (n = 7) and 7 p.i. (n = 3). Circulatory blood flow was visualized by intravenous injection of Evans blue (red) prior to two-photon imaging. <b>(D)</b> Example of rare GFP⁺ pRBCs (green) in contact with the luminal side of endothelial cells (orange, left panel or blue, right panel) of DsRed and CFP mice with ECM. <b>(E)</b> Quantification of adherent intraluminal pRBCs in cortical pial microvessels on days 5, 6 and 7 p.i. <b>(F)</b> Orthogonal (left) and maximum intensity projection (right) examples of GFP⁺ pRBCs (green) located within the perivascular space surrounding the pial microvessels (orange) of a DsRed mouse with ECM. <b>(G)</b> Quantification of pRBCs located within the perivascular space surrounding the pial microvessels on days 5, 6 and 7 p.i. with <i>Pb</i> ANKA. Endothelial cells are identified by expression of CFP (blue) or DsRed (orange) <b>(H)</b> Proportion of pRBCs found either adhering to the luminal vessel wall or located within the perivascular space on day 6 p.i (n = 4). Bars represent mean number ± SD. Scale bars: 5 μm. **p<0.01 (unpaired t test with Welch’s correction).</p

Widespread apoptosis or loss of vasculature does not occur in the brains of mice with ECM.

<p><b>(A-C and K)</b> 10⁶ DsRed⁺CD8⁺ OT-I T cells were adoptively transferred into CFP⁺ P14 host mice, which were subsequently infected with 10⁶ SIINFEKL-expressing <i>Pb</i>-TG pRBCs (n = 4) or left uninfected (n = 5). Mice were intravenously injected with CellEvent Caspase-3/7 Detection Reagent when displaying signs of ECM (day 6 p.i). Brains were subsequently isolated and processed for histological examination. <b>(A)</b> Representative snapshot of an apoptotic cell (green) associated with the cortical vasculature (blue) of the brain taken from a mouse with ECM. <b>(B)</b> Quantification ± SD of apoptotic cells stained with CellEvent per mm³ in brains from infected and uninfected control mice. <b>(C)</b> Representative snapshot showing an apoptotic cell (green) in contact with a parasite-specific CD8⁺ OT-I T cell (red). Endothelial cells (blue) were visualized by CFP expression. <b>(D-J)</b> C57BL/6 mice were infected with 10⁴<i>Pb</i> ANKA pRBCs or left uninfected. Brains were removed and processed for histological examination when <i>Pb</i> ANKA mice developed signs of ECM (day 7 p.i.). Representative images demonstrating the <b>(D)</b> absence and <b>(E)</b> rarity of apoptotic cells (green) within the cortex of brains from uninfected and ECM affected mice respectively. Cell nuclei are shown in blue. <b>(F)</b> Quantification ± SD of apoptotic cells stained for activated caspase 3 per mm² in brains from infected and uninfected control mice. <b>(G-H)</b> Co-staining of activated caspase 3 (green) and CD31 (red) demonstrating an <b>(G)</b> apoptotic endothelial cell and an <b>(H)</b> apoptotic leukocyte within the cortical vasculature of brains taken from ECM affected mice. Cell nuclei are shown in blue. Quantification ± SD of total <b>(I)</b> number and <b>(J)</b> area per mm² of cortical blood vessels in the brain from uninfected and ECM affected mice. <b>(K)</b> Representative tile scan showing the localization of CellEvent⁺ apoptotic cells (green/asterisk) in relation to vascular leakage (red) within the brain of an ECM affected (day 6 p.i.) CFP⁺ P14 mouse. Endothelial cells (blue) were identified by CFP. Scale bars: (A,C) 25 μm, (D,E) 50 μm, (G,H) 25 μm, (L) 1 mm. Results are pooled from mice infected in one experiment. Significance determined by unpaired t test with Welch’s correction.</p

ECM with associated late stage vascular leakage.

<p>C57BL/6 (n = 14) mice were intravenously infected with 10⁶<i>Pb</i> ANKA-pRBCs. <b>(A)</b> Survival and <b>(B)</b> peripheral parasitemia ± SD were monitored daily during development of ECM (grey area). (n = 14, pooled from 2 experiments). <b>(C)</b> Representative example of Evans blue leakage in the brain of an uninfected mouse and a mouse with ECM (day 6 p.i). <b>(D)</b> Quantification by spectrophotometry of Evans blue leakage in the brains of infected mice on days 4–7 p.i. Dashed line indicates baseline Evans blue signal (no leakage) from uninfected brains. (n = 23, pooled from 4 experiments).</p

The intracerebral CX₃CR1^+/GFP cellular response is comparable during <i>Pb</i> ANKA and <i>Pb</i> NK65 infections.

<p>CX₃CR1^+/GFP mice were infected with 10⁴<i>Pb</i> ANKA or <i>Pb</i> NK65 pRBCs. Brains from uninfected and infected (day 7 p.i.) mice were analyzed by flow cytometry. <b>(A)</b> Representative plots and calculated percentages of CX₃CR1^+/GFP cells (gating on live leukocytes) within the brains of uninfected and infected (day 7 p.i.) mice (n = 6). <b>(B)</b> Representative plots showing the subdivision of GFP⁺ cells into: R1—CD45^intCD11b^hi microglia; R2—CD45^hiCD11b^hi meningeal, perivascular macrophages and inflammatory monocytes; R3—CD45^hiCD11b^int leukocytes. <b>(C)</b> Calculated percentages of R1, R2 and R3 sub-gated populations within the brains of uninfected and infected (day 7 p.i.) mice (gating on live GFP⁺ leukocytes) (n = 15–17). <b>(D)</b> Representative dot plots showing the expression of phenotypic markers CD11c and Ly6C on the gated R1, R2 and R3 GFP⁺ populations from brains of uninfected and infected (day 7 p.i.) mice. <b>(E)</b> Representative histograms and calculated geometric means of CD40, CD80 and MHC I expression on the gated R1, R2 and R3 GFP⁺ populations from brains of uninfected and infected (day 7 p.i.) mice (n = 6). Results are representative of at least two independent experiments <b>(A, B, D and E)</b> or are pooled from three combined experiments <b>(C)</b>. Bars represent mean number ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (one-way ANOVA with Tukey's multiple comparisons test).</p