Beyond the well-defined role of the Eph receptor tyrosine kinases in developmental processes, cell motility, cell trafficking/adhesion and cancer, nothing is known about their involvement in liver pathologies. During blood-stage rodent malaria infection we have found that EphB2 transcripts and proteins were upregulated in the liver, a result likely driven by elevated surface expression on immune cells including macrophages. This was significant for malaria pathogenesis because EphB2−/− mice were protected from malaria-induced liver fibrosis despite having a similar liver parasite burden compared with littermate control mice. This protection was correlated with a defect in the inflammatory potential of hepatocytes from EphB2−/− mice resulting in a reduction in adhesion molecules, chemokines/chemokines receptors RNA levels and infiltration of leukocytes including macrophages/Kupffer cells which mediate liver fibrosis during rodent malaria infections. These observations are recapitulated in the well-established carbon tetrachloride (CCL4) model of liver fibrosis in which EphB2−/− CCL4-treated mice showed a significant reduction of liver fibrosis compared to CCL4-treated littermate mice. Depletion of macrophages by clodronate-liposome abrogates liver EphB2 mRNA and proteins up-regulation and fibrosis in malaria-infected mice. Conclusion: During rodent malaria, EphB2 expression promotes malaria-associated liver fibrosis. To our knowledge, our data is the first to reveal the implication of the EphB family of receptor tyrosine kinases in liver fibrosis or in the pathogenesis of malaria infection.

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.

Beyond the well-defined role of Eph receptor tyrosine kinases in biological processes, cell migration, adhesion, nothing is known about their implication in liver pathologies. During blood-stage rodent malaria infection, EphB2 mRNA and proteins are upregulated in the liver, a result likely driven by elevated surface expression on macrophages. This is significant for malaria pathogenesis because EphB2-/- mice are protected from malaria-induced liver fibrosis despite having a similar liver parasite burden compared with littermate mice. This protection is correlated with a defect in inflammatory potential of hepatocytes from EphB2-/- mice resulting in a reduction in adhesion molecules, chemokine/chemokine receptors RNA levels and infiltration of leukocytes including Kupffer cells which mediate liver fibrosis during malaria. These observations are recapitulated in the well-established carbon tetrachloride (CCL4) model of liver fibrosis in which EphB2-/- CCL4-treated mice showed a significant reduction of liver fibrosis compared to CCL4-treated littermate mice. EphB2 is predominantly expressed by Kupffer cells and depletion of macrophages abrogates liver EphB2 mRNA and protein increase in malaria infected mice, as well as fibrosis. Altogether these results reinforce the critical role played by EphB2 in promoting liver inflammation and fibrosis. To our knowledge, this work is the first to reveal the potential profibrotic nature of EphB receptor tyrosine kinases in liver injury.

Cerebral malaria (CM) is a leading cause of death from Plasmodium falciparum infection, yet the mechanisms of disease pathology are poorly understood. While Eph receptors have been linked to several neurological disorders, their roles in malaria remain largely unknown. Experimental cerebral malaria (ECM) is a lethal condition resulting from Plasmodium berghei ANKA (PbANKA) infection of mice on a C57BL/6J background that recapitulates key features of human CM including blood-brain barrier (BBB) breakdown. Here we show that PbANKA-infected EphA2 −/− mice, but not EphA2 +/+ mice, have an intact BBB at ECM onset. CD8+ T cells mediate ECM development, and EphA2 −/− mice exhibit less accumulation of CD8+ T cells in the brain despite normal splenic expansion providing a mechanism by which EphA2 deficiency may preserve BBB integrity. Upregulation of EphA2 mRNA in brains of PbANKA-infected C57BL/6J mice is a feature of ECM absent in non-lethal Plasmodium infections such as PbNK65 where CD8+ T cells are present in the brain but do not disrupt the BBB. Unlike EphA2 +/+ mice, PbANKA-infected EphA2 −/− mice are protected from death even with peripheral and organ-sequestered parasite levels similar to EphA2 +/+ mice suggesting BBB integrity, not parasite burden, is responsible for improved survival in EphA2 −/− mice. Therapeutically targeting EphA2 by blocking EphA2 activation or interaction with membrane-bound ephrin-A ligands on T cells leads to increased survival of PbANKA-infected C57BL/6J mice. We also observe a parasite-dependent upregulation of both ephrin-A mRNA in CD3+ human PBMCs and EphA2 mRNA in human brain endothelial cells. Taken together, our data suggests a novel and crucial role for EphA2 in ECM with potential translation to human CM.

Cerebral malaria is a severe complication of infection with the Plasmodium falciparum parasite, yet the etiology of the disease is not fully understood. In the well-established Plasmodium berghei ANKA (PbANKA) mouse model of experimental cerebral malaria (ECM), sequestration of parasitized red blood cells (pRBCs) and CD8+ T cells in the brain microvasculature is required for ECM development. However, our understanding of the receptors involved in CD8+ T cell adherence to the brain endothelium during ECM is currently incomplete. We hypothesize that EphA2, a member of the Eph family of receptor tyrosine kinases, is involved in the sequestration of CD8+ T cells in the brain during ECM through its interactions with membrane-bound ephrin-A ligands on CD8+ T cells. Using the PbANKA model of ECM, we show that EphA2 is upregulated in the brains of mice six days post-infection at the onset of cerebral symptoms in a lymphotoxin-α dependent manner. Ephrin-A ligands are also upregulated on splenic T cells of mice infected with PbANKA three days post-infection prior to potential trafficking to the brain. Importantly, EphA2-/- mice show significant resistance to death from ECM compared to EphA2+/+ mice. Similar numbers of pRBCs accumulate in the brains of EphA2-/- and EphA2+/+ mice, but EphA2-/- mice have less CD8+ T cells in the brain six days post-infection correlating with improved survival. Our data demonstrates a novel potential role for the EphA2 receptor in the pathogenesis of ECM.