Disruption of blood-brain barrier (BBB) function is a key feature of cerebral malaria. Increased barrier permeability occurs due to disassembly of tight and adherens junctions between endothelial cells, yet the mechanisms governing junction disassembly and vascular permeability during cerebral malaria remain poorly characterized. We found that EphA2 is a principal receptor tyrosine kinase mediating BBB breakdown during Plasmodium infection. Upregulated on brain microvascular endothelial cells in response to inflammatory cytokines, EphA2 is required for the loss of junction proteins on mouse and human brain microvascular endothelial cells. Furthermore, EphA2 is necessary for CD8+ T cell brain infiltration and subsequent BBB breakdown in a mouse model of cerebral malaria. Blocking EphA2 protects against BBB breakdown highlighting EphA2 as a potential therapeutic target for cerebral malaria.

Malaria is a disease caused by transmission of the mosquito-borne Plasmodium parasite that remains a severe global public health issue. Advancements in parasite control measures such as prevention, treatment, and surveillance have reduced the incidence of malaria worldwide. However, current reports indicate that progress towards reducing global malaria cases and deaths in recent years has stalled. Therefore, it is imperative that we continue to explore new therapeutic avenues that can synergize with existing treatment methods. In particular, there is currently no adjunctive treatment available for cerebral malaria which is a serious complication of Plasmodium infection characterized by blood-brain barrier breakdown. Here, we have identified that a receptor EphA2 is required for the breakdown of the blood-brain barrier during Plasmodium infection in mice. We found that expression of this receptor is critical for inducing brain inflammation, recruiting immune cells to the brain, and disruption brain endothelial cell junctions. Inhibiting activation of this receptor using two different treatment approaches also prevented blood-brain barrier breakdown in mice. Thus, along with identifying a new molecule critical for cerebral malaria in mice we also provide a basis for exploring this receptor as a novel therapeutic target in human cerebral malaria in the future.

Funding: This work was funded by grants from the National Institute of Neurological Disorders and Stroke (R21NS085382 and R01NS097819), the Emory Egleston Children’s Research Fund, and Royal Society to TJL. TKD was supported by training grants T32AI007610, T32AI106699, and individual fellowship F31NS098736. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of this manuscript.

Erythropoietin-producing hepatocellular (Eph) receptors constitute the largest family of receptor tyrosine kinases in humans and are ubiquitously expressed in nearly all tissues, including the brain[ 20 ] in both mice and humans. There are nine different functional EphA receptors in the mouse and human genome (EphA1-EphA9) that have the ability to interact with five membrane-bound Eph receptor interacting (ephrin) ligands (ephrin-A1-ephrin-A5) with varying affinities[ 21 ]. The unique expression patterns of EphA receptors and ephrin-A ligands in different tissues and cell types allows for functional specificity, and EphA-ephrin-A binding between cells canonically leads to events such as cellular migration, adhesion, and changes in cellular morphology[ 22 ]. As the interaction between EphA receptors and membrane-bound ephrin-A ligands is of high-affinity, this initial binding event will often lead to strong adhesion between the two cells involved. This can progress to either extended adhesion or repulsion and separation of the two cell surfaces once signaling pathways are propagated depending on the context[ 21 ]. As a prime example of these multifunctional receptors, one particular EphA family member, EphA2, can be utilized by CD8+ T cells for chemotaxis[ 23 ] and adhesion[ 24 ]. Additionally, EphA2 has also been previously shown to be instrumental in the disassembly of both tight and adherens junction protein complexes on endothelial cells reducing cell-cell contact[ 25 , 26 ]. Given that EphA receptors play a role in regulating both brain endothelial junction formation and immune cell migration and adhesion, processes highly relevant to the development of CM, here we have investigated the role of EphA receptors in malaria-associated BBB breakdown. We found that EphA2 is upregulated on both human and mouse primary brain microvascular endothelial cells in response to tumor necrosis factor family cytokines. In mice, EphA2 is upregulated by LT-α, a cytokine required for BBB breakdown in PbA infection[ 10 , 27 ]. EphA2 deficient mice exhibit significantly improved survival in comparison to EphA2 sufficient mice, likely as a result of reduced CD8+ T cell brain infiltration and inflammation along with maintenance of brain microvascular endothelial cell junctions. Collectively, this results in enhanced BBB integrity. Interestingly, brain EphA2 upregulation is a unique feature of infection with the ECM-causing PbA strain and does not occur upon infection with strains that do not cause ECM. This suggests EphA2 upregulation on brain microvascular endothelial cells is critical for Plasmodium-associated cerebral pathology. Blocking the interaction between EphA2 and its cognate ephrin-A ligands increases the integrity of the BBB during ECM which demonstrates a rationale for exploring EphA2 antagonism as a novel therapeutic strategy for maintaining BBB integrity during CM.

Activation of receptor tyrosine kinases has been previously shown to play a role in endothelial junction disruption[ 18 ] and barrier integrity during ECM which can be maintained by global inhibition of the receptor tyrosine kinase family[ 17 ]. However, therapeutic potential of this observation is limited by the simultaneous inhibition of receptor tyrosine kinases that are also involved in mounting an effective immune response[ 19 ] which could detrimentally affect control of Plasmodium infection. Identification of the major receptor tyrosine kinases necessary for junction disruption during CM is required to capitalize on strategies to specifically target receptor tyrosine kinases for therapeutic benefit.

Cerebral malaria (CM) is a severe manifestation of infection with the Plasmodium falciparum (Pf) parasite and has a 20% fatality rate[ 1 ]. Presenting as a plethora of neurological symptoms that lead to coma, pediatric CM is a complex disease that has been shown to involve alterations to, and breakdown of, the blood-brain barrier (BBB)[ 2 – 5 ]. This is thought to result from vascular activation in response to sequestration of Plasmodium-infected red blood cells (pRBCs) on the endothelium via adhesion molecules that include endothelial protein C receptor (EPCR)[ 6 ] and intercellular adhesion molecule 1 (ICAM-1)[ 7 , 8 ]. Infection of mice with Plasmodium berghei ANKA (PbA) has been used to demonstrate the importance of inflammatory cytokines such as interferon-γ (IFN-γ)[ 9 ] and tumor necrosis factor-β, also known as lymphotoxin-α (LT-α)[ 10 ], in the development of experimental cerebral malaria (ECM), a disease that shares several key features with human CM[ 11 , 12 ]. Inflammation in ECM is T cell-mediated with CD8+ T cells playing a critical role in breakdown of the BBB[ 13 – 16 ]. However, apoptosis of brain endothelial cells does not appear to be sufficient to cause significant disruption of the barrier[ 15 , 17 ]. The molecular mechanisms underlying BBB breakdown during Plasmodium infection are poorly understood, but the disruption of endothelial junctions is thought to be instrumental in this pathophysiological process.

Results

EphA2 is upregulated in human and mouse primary brain endothelial cells in response to inflammatory cytokines EphA2 expression on endothelial cells has previously been associated with impairment of junction formation[26]. Since the majority of pRBCs[15] and CD8+ T cells[30] are known to adhere to the brain microvascular endothelium on the luminal surface of blood vessels during ECM, we sought to determine the impact of PbA infection specifically on endothelial-expressed EphA2. We confirmed that EphA2 is upregulated at the protein level in brains of PbA-infected wild-type mice and colocalizes primarily with the brain vasculature (S2 Fig). To determine if the pRBCs or inflammatory cytokines were responsible for this observed upregulation of EphA2 on brain endothelial cells, we isolated both human primary brain microvascular endothelial cells (HBMECs) and mouse primary brain microvascular endothelial cells (MBMECs) as confirmed by staining for the endothelial-specific markers von Willebrand factor (VWF) (Fig 3A, left) and CD31 (Fig 3A, right) and the formation of cell-cell contacts by transmission electron microscopy (Fig 3B). We cultured the cell monolayers with their respective parasites (Pf pRBCs with HBMECs and PbA pRBCs with MBMECs) along with the inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and LT-α which are known to be produced in the brain during Plasmodium infections. EphA2 expression was significantly increased in HBMECs pulsed with TNF-α (Fig 3C). The addition of Plasmodium falciparum-infected red blood cells (Pf pRBC) had no synergistic effect which supports previous studies showing upregulation of EphA2 in human microvascular endothelial cells and monocytes by TNF-α[31, 32]. On the other hand, MBMECs upregulated EphA2 primarily in response to LT-α (Fig 3D and 3E). This result is biologically significant because production of TNF-β/LT-α by non-hematopoietic cells in PbA infection is required for ECM development[10]. The PbA-associated upregulation of EphA2 mRNA appeared specific to the brain tissue as transcript levels remained essentially unchanged in liver and lung tissues (S3A and S3B Fig). Furthermore, Plasmodium-reactive CD8+ T cell accumulation in pulmonary tissue was identical in the presence or absence of EphA2 (S3C and S3D Fig). This brain-specific EphA2 upregulation likely results from the fact that LT-α is only upregulated in the brain during ECM with little to no increase in LT-α mRNA levels in the liver, lung, or spleen at day 6 post-infection with PbA (Fig 3F). In agreement with these observations, EphA2 transcript was not upregulated in the brains of LT-α deficient and tumor necrosis factor receptor 2 (TNFR2) deficient mice infected with PbA in contrast with wild-type mice (Fig 3G). Additionally, treatment of PbA-infected C57BL/6J mice with an anti-TNFR2 blocking antibody resulted in significantly reduced mRNA levels of both LT-α and EphA2 in the brain in comparison to isotype control treated mice (Fig 3H). These data provide further support for the role of LT-α in inducing EphA2 upregulation in the brains of mice at the onset of ECM. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. EphA2 is upregulated in human and mouse primary brain endothelial cells in response to inflammatory cytokines. Representative data showing von Willebrand factor (VWF) immunofluorescence staining (cell nuclei stained with DAPI-blue) (A, left), CD31 flow cytometry staining (gray histogram: isotype control, blue histogram: anti-CD31) (A, right), and transmission electron microscopy (B) of cultured MBMECs isolated from C57BL/6J mice. White arrows in (B) indicate endothelial cell contact points. Scale bars represent 25μm (A) and 0.5μm (B). (C-D) EphA2 transcription in human (C) and mouse (D) BMECs incubated for 24 hours with naïve RBC lysates (nRBC), P. falciparum 3D7-infected RBC lysates (Pf pRBC), PbA-infected RBC lysates (PbA pRBC), or no RBC lysate (∅ RBC) plus human (C) or mouse (D) LT-α, TNF-α, or media (M) (n = 3–6 endothelial preparations/group). Values are normalized to untreated cells. (E) Immunofluorescence images and fluorescence quantification of EphA2 (red) on MBMECs unstimulated or stimulated with PbA-infected RBC lysates (PbA pRBC) in the presence of absence of LT-α for 24 hours. Cell nuclei stained with DAPI (blue). Scale bars represent 25μm. Images representative of two endothelial preparations. (F) LT-α transcription relative to naïve mice (dashed line) in whole brains, livers, lungs, and spleens of C57BL/6J mice (n = 8-9/group) at day 6 post-infection with PbA. (G) EphA2 transcription relative to naïve mice (dashed line) in brains of LT-α-/- (n = 7) and TNFR2-/- (n = 3) mice at day 6 post-infection with PbA compared to wild-type C57BL/6J mice (n = 6–11). (H) LT-α and EphA2 transcription relative to naïve mice (dashed line) in brains of isotype control (n = 9) and anti-TNFR2 (n = 8) treated mice at day 6 post-infection with PbA. Bars in all graphs represent the mean ± SEM. Statistical analyses: Kruskal-Wallis and Dunn’s multiple comparisons tests (C, D, E, F) and Mann-Whitney test (G, H). Only statistically significant (p<0.05) values are shown. Figures represent combined data from 1 (G-right panel), 2 (E, F, G-left panel, H), or 3 (C, D) independent experiments. https://doi.org/10.1371/journal.ppat.1008261.g003

EphA2 expression in the brain is a hallmark of pathogenesis in ECM CD8+ T cell accumulation in the brain is necessary[51] but not sufficient[28] for the development of ECM. Previous studies have attributed CD8+ T cell participation in ECM in the brain microvasculature as dependent on MHC-I cross-presentation of malaria peptides, a phenomenon driven by IFN-γ. In addition to pMHC-I/TCR interactions, we hypothesized that a signal provided by EphA2 ligation is required to mediate BBB disruption. In support of this hypothesis, upregulation of EphA2 was not observed in brains of C57BL/6J mice infected with PbNK65 or another Plasmodium strain, Plasmodium chabaudi AS (PcAS), neither of which causes ECM (Fig 5A, left). Significant upregulation only occurs in the brains of mice infected with the ECM-causing strain PbA (Fig 1A). We found no upregulation of ephrin-A1 ligand in the brains of mice infected with either PbA or PbNK65 (Fig 5A, right) suggesting that this is not a factor required for the development of ECM. PbNK65 infection of C57BL/6J mice led to equivalent levels of Plasmodium transcript (Fig 5B) and accumulation of Plasmodium-reactive CD8+ T cells in the brain at day 6 post-infection to those measured in PbA infection (Fig 5C) consistent with previous reports[15, 28]. CD8+ T cells found in the brains of PbNK65-infected mice expressed similar levels of surface EphA2 (Fig 5D) and ephrin-A1 ligand (Fig 5E) to those that accumulated in brains of PbA-infected C57BL/6J mice at the onset of ECM. However, unlike PbA-induced CD8+ T cell accumulation in the brain which we found was dependent on EphA2 (Fig 1E), Plasmodium-reactive CD8+ T cells were found at equal numbers in the brains of PbNK65-infected EphA2-/- mice compared to EphA2+/+ control mice (Fig 5F). This is likely due to the fact that levels of chemokines responsible for CD8+ T cell recruitment to the brain are present at equivalent levels in the brains of PbNK65-infected EphA2+/+ and EphA2-/- mice (Fig 5G) suggesting that CD8+ T cells are recruited to the brain independently of EphA2 in this non-ECM malaria model. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. EphA2 is not required for trafficking of CD8+ T cells in the brain during PbNK65 infection. (A) Transcription of EphA2 (left) and ephrin-A1 (right) in whole brain lysates of C57BL/6J mice infected with PbA (n = 12), PbNK65 (n = 12) or PcAS (n = 8) relative to naïve mice (dashed line). (B) Quantification of 18S parasite DNA transcript in whole brain lysates of PbA- and PbNK65-infected mice (n = 12-13/group) at day 6 post-infection. (C) Frequency and total number of Plasmodium GAP50-reactive CD8+ T cells in the brains of mice at day 6 post-infection with PbA (n = 14–22), PbNK65 (n = 14–21), or naïve (n = 12). (D) Total number of EphA2+ CD8+ T cells in the brains of mice at day 6 post-infection with PbA (n = 14), PbNK65 (n = 14), or naïve (n = 8). (E) Frequency of ephrin-A+ CD8+ T cells in the brains of mice at day 6 post-infection with PbA (n = 13), PbNK65 (n = 13), or naïve (n = 6). (F) Total number of CD8+T cells and frequency of Plasmodium GAP50-reactive CD8+ T cells in the brains of EphA2-/- and littermate control mice at day 6 post-infection with PbNK65 (n = 9/group) compared to naïve mice (N) (n = 4/group). Naïve and PbNK65-infected groups are significantly different within each genotype for all graphs except EphA2+/+ in left panel. (G) Transcription of inflammatory chemokines in whole brain lysates of EphA2-/- and littermate control mice at day 6 post-infection with PbNK65 (n = 7-10/group) relative to naïve mice (dashed lines). Bars in all graphs represent the mean ± SEM. Statistical analyses: Kruskal-Wallis and Dunn’s multiple comparisons tests (A-left panel, C, D, E, F) and Mann-Whitney test (A-right panel, B, G). Only statistically significant (p<0.05) values are shown unless otherwise noted in the legend. Figures represent combined data from 2 (E, F, G), 3 (A, B, D), or 4 (C) independent experiments. https://doi.org/10.1371/journal.ppat.1008261.g005 There are considerable challenges in obtaining brain sections from children who have died from CM as well as control brain sections precluding confirmation that endothelial-expressed EphA2 is the main correlate of BBB breakdown in CM. However, these data from mouse models of Plasmodium infection suggest that EphA2 expression on endothelial cells, which is mediated by the interactions of sequestered pRBCs with MBMECs, is a critical mediator of ECM pathogenesis. While expression of ephrin-A1 ligand in whole brains (Fig 5A) and CD8+ T cells (Fig 5C) along with soluble ephrin-A1 ligand in the plasma (Fig 4D) are similar in PbA and PbNK65 infections, the differential requirement of EphA2 for CD8+ T cell accumulation in the brain in these two mouse models of malaria suggests that the unique and significant difference in brain EphA2 expression may be a contributing factor to the different neurological damage that occurs in these two Plasmodium models.

EphA2 deficiency leads to a reduced neuroinflammatory response to PbA To determine why CD8+ T cells did not accumulate in the brains of PbA-infected EphA2-/- mice despite their abundance in the bloodstream, we examined if there was a defect in the inflammatory response in EphA2-/- mice compared to EphA2+/+ littermate control mice. Transcription of inflammatory cytokines associated with PbA pathogenesis was significantly reduced in brains of EphA2-/- mice compared to littermate control mice (Fig 6A). In contrast to what was observed in the brains of PbNK65-infected EphA2-/- mice (Fig 5G), PbA-infected EphA2-/- mice exhibited a significant reduction in the mRNA levels of key chemokines responsible for CD8+ T cell recruitment to the brain during ECM (Fig 6B). This suggests that Plasmodium-reactive CD8+ T cells do not accumulate in the brain microvasculature of EphA2-/- mice because the chemokine signals required for their recruitment to the brain are not present at sufficient levels. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. Brain inflammatory response is reduced in PbA-infected EphA2-/- mice. Transcription of inflammatory cytokines (A) and chemokines (B) in whole brain lysates of EphA2-/- and littermate control mice at day 6 post-infection with PbA (n = 11-13/group) relative to naïve mice (dashed lines). (C) Western blots and densitometry quantification of MBMECs derived from EphA2-/- or littermate control mice and incubated with media (M), naïve red blood cell lysate (nRBC), or PbA-infected red blood cell lysate (PbA) for 30 minutes. Blots are representative of 3 endothelial preparations. The horizontal dashed line indicates two separate Western blots. (D) TNF-α secreted from identical culture conditions as described in C after 24 hours incubation (n = 4 endothelial preparations). (E) Mouse primary brain endothelial cells derived from EphA2-/- or littermate control mice were incubated with naïve red blood cell (nRBC) or PbA-infected red blood cell lysate (PbA) for 24 hours and the fold change in the transcription of chemokines relative to unstimulated controls is shown (n = 2 endothelial cultures/group). Chemokines secreted from identical culture conditions are shown in (F) (n = 4–6 endothelial preparations/group). Bars in all graphs represent the mean ± SEM. Only statistically significant (p<0.05) values are shown. Statistical analyses: Mann-Whitney test (A-B) and Wilcoxon matched-pairs test (D-F). Only statistically significant (p<0.05) values are shown. Figures represent combined data from 2 (E), 3 (A-C), 4 (D, F-middle and right panels), or 6 (F-left panel) independent experiments. https://doi.org/10.1371/journal.ppat.1008261.g006 Plasmodium-infected RBCs are able to induce a potent inflammatory response in endothelial cells in vitro[52]. This includes inducing the production of C-X-C motif chemokine 10 (CXCL10/IP-10)[53, 54], C-C motif chemokine ligand 2 (CCL2/MCP1)[55], and C-C motif chemokine ligand 5 (CCL5/RANTES)[56] which are essential for drawing leukocytes into brain capillaries where pRBCs have adhered to the endothelium. Since EphA2 has been implicated in activation of the NF-κB pathway[57], a critical signaling cascade that mediates inflammation, we investigated if EphA2-/- derived MBMECs had reduced NF-κB signaling and inflammatory responses. Stimulation of EphA2+/+ MBMECs with PbA pRBCs induced phosphorylation of NFκB p65 and IKKα/β, but this effect was abolished in the absence of EphA2 (Fig 6C). The reduced phosphorylation of these members of the NF-κB signaling pathway was associated with significantly reduced TNF-α secretion from EphA2-/- MBMECs (Fig 6D) as well as a clear reduction in transcript levels (Fig 6E) and secreted protein (Fig 6F) of CXCL10, CCL2 and RANTES in MBMECs stimulated with PbA pRBCs. Together, the reduced cytokine and chemokine production from brain endothelial cells in the absence of EphA2 explain the lack of CD8+ T cells found in the brains of EphA2-/- mice during PbA infection and the resulting improvement in survival.

EphA2 contributes to destabilization of tight and adherens junctions Since EphA2 was found to be involved in CD8+ T cell retention in the brain during ECM along with BBB breakdown, we next examined the mechanism by which EphA2 could be contributing to BBB destabilization. In comparison to EphA2-/- mice, EphA2+/+ littermate control mice infected with PbA had significantly reduced transcription of several tight junction proteins (Fig 7A) but not the adherens junction protein vascular endothelial cadherin (VE-cadherin). This suggests that the maintenance of an intact BBB in EphA2-/- mice was the result of preserved tight junction protein expression. A link between EphA2 activation and dysregulation of adherens and tight junctions in the BBB has been previously shown in other homeostatic and disease contexts[25, 58, 59]. Additional immunofluorescence analyses of MBMECs isolated from EphA2-/- and EphA2+/+ mice and stimulated with ephrin-A1 ligand along with the inflammatory cytokine LT-α revealed significantly reduced expression of both adherens junction (Fig 7B) and tight junction (Fig 7C) proteins in EphA2 sufficient endothelial cells upon stimulation. On the contrary, expression of these junction proteins was fully maintained in stimulated EphA2-/- MBMECs. Using a transwell system we found that in the absence of EphA2, baseline MBMEC barrier integrity appeared more robust than in the presence of EphA2 (Fig 7D, left) presumably as a result of enhanced adherens and tight junction protein expression. Activation of EphA2 on MBMECs with ephrin-A1 ligand resulted in the disruption of endothelial barrier integrity (Fig 7D, right) with a trend towards decreased permeability in EphA2-/- MBMEC cultures. The permeability observed in EphA2-/- cultures stimulated with ephrin-A1 ligand could be due to the expression of several other EphA receptors in the brains of EphA2-/- mice (Fig 1F) that have the potential to bind ephrin-A1 ligand with lower affinity. A similar phenomenon was also observed in HBMECs stimulated with human TNF-α and ephrin-A1 ligand which showed a significant reduction of VE-cadherin expression upon stimulation with both TNF-α and ephrin-A1 ligand (Fig 7E). It is possible that activation of EphA2 upon ephrin-A1 binding leads to either internalization and recycling of junction proteins or EphA2-induced ADAM- and MMP-mediated shedding of junction proteins (Fig 4D and S5A and S5B Fig). However, plasma levels of the adherens junction proteins VE-cadherin and epithelial cadherin (E-cadherin) are equivalent or lower in PbA-infected mice compared to naïve mice (S5C Fig). These findings favor the hypothesis that destabilization of junctions occurs through protein internalization either as a direct or indirect (e.g. via suppression of RhoA[25]) result of EphA2 signaling. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 7. Endothelial cell barrier integrity is enhanced in the absence of EphA2. (A) Transcription levels of adherens and tight junction proteins in whole brains of EphA2-/- and EphA2+/+ mice at day 6 post-infection with PbA (n = 8-10/group) relative to naïve mice (dashed line). (B-C) Immunofluorescence images and fluorescence quantification of mouse primary brain endothelial cells derived from EphA2-/- or littermate control mice showing expression of adherens junction protein vascular endothelial cadherin (VE-cadherin, red) (B) and tight junction protein zonula occludens-2 (ZO-2, red) (C) after stimulation for 24 hours with recombinant mouse LT-α, recombinant mouse ephrin-A1-Fc, or IgG-Fc as a negative control. Cell nuclei are stained with DAPI (blue). Scale bars represent 25μm. (D) Baseline MBMEC transendothelial electrical resistance (TEER, left) and relative permeability (right) of MBMECs from EphA2-/- and EphA2+/+ mice. On right, transwell cultures were incubated for 2 hours with naïve RBC lysate (nRBC), PbA-infected RBC lysate (PbA pRBC), or mouse ephrin-A1-Fc ligand. Permeability is relative to IgG-stimulated EphA2+/+ endothelial cultures (n = 3 endothelial preparations/group). (E) Immunofluorescence images and fluorescence quantification of human primary brain endothelial cells showing expression of VE-cadherin (red) after stimulation for 24 hours with recombinant human TNF-α, recombinant human ephrin-A1-Fc, or IgG-Fc as a negative control. Cell nuclei are stained with DAPI (blue). Scale bars represent 10μm. (F-G) Brain permeability in C57BL/6J mice either orally gavaged with 100μL Nilotinib or vehicle control (100 mg/kg/day; n = 8-10/group) (F) or injected intraperitoneally with 200μL recombinant EphA2-Fc or vehicle control on days 4–6 post-infection (13.3 μg/mouse/day; n = 4-12/group) (G). Mice were injected intravenously with 200μL of 1% Evan’s Blue at day 6 post-infection with PbA. OD values are normalized to naïve mice from each respective treatment group. Bars in all graphs represent the mean ± SEM. Statistical analyses: Mann-Whitney test (A, D—left, F, G) and Kruskal-Wallis and Dunn’s multiple comparisons tests (B, C, E). Only statistically significant (p<0.05) values are shown. All figures are representative of 2 (A, B, C, E-G) or 3 (D) independent experiments. https://doi.org/10.1371/journal.ppat.1008261.g007