Faculty Mentor: Tracey Lamb (Pathology, University of Utah)

Did you know that the World Health Organization in 2012 reported that of the global population, those found in 109 different countries carried a 50% risk of malaria? Not only that, but the most vulnerable population under this concerning statistic happen to be young children. This data is highlighted in the following paper, Cerebral malaria – Mysteries at the Blood Brain Barrier, where authors L. Rénia, Wu Howland S., et. al. present the main argument is to prove that the blood brain barrier plays an essential role in the complications brought on by the pathogenesis of cerebral malaria, a severe form of malaria.

According to the 2023 World Malaria report, the World Health Organization (WHO) African Region hosted approximately 94% of malaria cases, as well as 95% of malaria-induced deaths in 2022. As mentioned previously, young children are particularly at risk for severe cases and fatalities. In 2022, children under the age of 5 years made up 78% of deaths in the WHO African Region. To place the severity of this staggering majority into perspective, the second most affected region, WHO South-East Asia Region, only made up 10% of malaria cases and 3% of deaths in the same year. With global efforts, WHO has recorded that 44 countries and 1 territory have been certified as malaria-free. Countries in the African Region make up roughly 11% of those who have successfully eradicated malaria. One reason why African countries struggle to eradicate malaria may be due to the climate. Increasing temperatures and environmental rainfall and humidity may worsen efforts due to facilitating the idea environment for mosquitoes.

Malaria is a vector-caused disease. Vector-borne diseases require transmittance via a vector. This can be an insect, rodent, or other animal that carries a particular pathogen. There are six Plasmodium species that can cause malaria; however, P. falciparum is the prevalent species. Infection occurs through the bite of infected Anopheles mosquitoes (the vector), who get infected through ingesting blood of another infected individual. As such, places where malaria run rampart are usually humid and can facilitate an advantageous environment for mosquitoes.

When an infected mosquito bites a human or other animal, they introduce an infantile and motile spore form of P. falciparum, called a sporozoite, into the host’s system. In the liver, the sporozoite matures into the schizont intermediate form. When the parasite develops further, the parasite in its merozoite form, ruptures from hepatocytes and flow into the bloodstream. In the bloodstream, the infection is allowed to spread through the invasion on red blood cells where merozoites reproduce and infect more red blood cells.

Cerebral malaria (CM) is a complication of Plasmodium infection that entails severe and often fatal swelling of the brain. CM is often characterized by coma and increased intracranial pressure [Laurent, et. al, 2012]. L. Rénia et.al. describe that “In more recent studies, it was shown that during [experimental cerebral malaria], brain edema resulting from leakage and accumulation of fluid into the parenchymal extracellular space was observed… and was associated with enlarged perivascular spaces” (Rénia, 2013).

It seems as though the most logical solution to this issue would be a preventative inoculation to create widespread immunity. However, “[d]espite intense efforts, a universally effective anti-malaria vaccine is not yet available. There are multiple pathogen-specific barriers to effective vaccine development” [Postels, et. al., 2013]. The parasite that causes malaria is considered quite genetically and developmentally complex. With over 5000+ genes and a complicated life cycle that varies between vector (mosquito) and host (human), comprehension of its methods of gene regulation proves to be a bit difficult. Furthermore, additional studies show that quinine and artemisinin compounds have been used to treat severe cases of malaria, however prevailing drug-resistant strains of Plasmodium rendered the treatment ineffective [Nishanth, 2019]. P. falciparum, P. vivax, and P. malariae are three strains that have been confirmed to be drug-resistant. P. falciparum in particular is resistant to a majority of developed antimalarials, primarily chloroquine. Even those that receive treatment face a mortality rate of 20%. In 2010, 86% of roughly 655,000 people who died of CM, were children under the age of 5 [Postels et al., 2012]. Many of those who do survive faced long-lasting effects such as neurologic sequelae, speech impairment, and epilepsy [Idro et al., 2010]. While a vaccine may not be feasible at the current moment, there is a particular aspect of CM that can be highlighted upon, as it pertains to future therapies.

CM is the result of the disruption of the blood brain barrier (BBB), which is a selectively semipermeable structure made up of the brain endothelial cells with imbedded tight and adherens junctions that limit the passage of unwanted solutes from the lumen of the blood vessels into the brain parenchyma. As put in the 2021 paper, Profiling of Blood-Brain Barrier Disruption in Mouse

Intracerebral Hemorrhage Models […], “As a dynamic structure, the BBB separates peripheral circulation from the brain” (Jia P, et. al., 2021). This is a vital role. The Central Nervous System (CNS) carry particular environmental requirements that differ from outside circulation and help maintain stability for the brain. As such, a semipermeable BBB carries the job of a gatekeeper, ensuring homeostasis for both environments. The endothelial cells receive signals from other cell types such as astrocytes, pericytes, and microglia which enhance barrier function. This cellular communication is facilitated by chemical messengers such as growth factors and cytokines. Fatal cases of CM are predominantly the result of intracranial hemorrhage and fluid collection in the brain after the BBB is disrupted (Postels, Douglas G., et al, 2012). The breakdown of the BBB is facilitated by the disruption of tight and adherens junction proteins. The direct cause of the tight and adherens junctions is still not understood. Understanding why this disruption occurs is necessary to lead for developing novel therapies to prevent death among CM patients.

Scientific research on cerebral malaria has vastly improved due to the implantation of a reliable mouse model. But why? There used to not be a commonly agreed upon pathogenesis of human cerebral malaria since studies were limited by the availability of deceased human brain tissue as well as tests that can be safely administered to living patients. In the 2000 paper, Immunopathology of Cerebral Malaria: Morphological Evidence of Parasite Sequestration in Murine Brain Microvasculature, the authors strived to prove an effective mouse model that could be utilized to study the pathogenesis of cerebral malaria. Their work proved to be successful as stated that “… this is the first time that a murine CM model has been defined in which parasitized erythrocytes appear in close contact with the microvascular endothelium of the brain, resembling the pattern seen in humans” (Hearn, et. al., 2000). Infecting C57Bl/6J mice with Plasmodium berghei ANKA (Pb-A) provides an effective model of human cerebral malaria, as infected mice die of brain hemorrhage 6 to 10 days after infection. This is due to CD8+ T-cells, which are necessary for BBB disruption and eventual fatality. This model is referred to as experimental cerebral malaria (ECM) in the literature.

EphA2, a part of the Erythropoietin-producing hepatocellular receptor (Eph) family, plays an integral role in cerebral malaria. EphA-ephrin-A binding can result in adhesion, cellular morphology alterations, and chemotaxis among cells. A previous study expressed that EphA2 is also associated with the reduction of endothelial cell-to-cell connections through the disruption of “…tight and adherens junction protein complexes” [Darling et al., 2020]. Evidence suggests that the pathogenic contribution to death and BBB breakdown by EphA2 originates from EphA2 that is expressed in endothelial cells located within the brain. A recent study found that “[i]n the absence of EphA2, PbA-infected mice maintained an intact BBB…at the onset of ECM in comparison to EphA2 sufficient mice” [Darling et al., 2020]. Furthermore, a global knock out (KO) of EphA2 has been shown to protect against death and BBB disruption in the mouse model of cerebral malaria. For clarification purposes, a global KO constitutes a complete absence of EphA2 from the the entire murine genetic makeup. It was found that an absence of EphA2 correlated with the absence of CD8+ T-cell accumulation in the brain [Darling et al., 2020]. EphA2knockout was also shown to prevent cell to cell junctions’ downregulation in endothelia cells found in brain tissue.

In a model proposed by a 2020 study, disruption of the BBB begins with blood cells infected with PbA attaching to receptors on endothelial cells located in brain tissue. This results in the start of the inflammatory response with cytokines LT-α and TNF-α upregulate EphA2 and ephrin-A1 ligand respectively. Released chemokines are responsible for the CD8+ T cell accumulation, which as previous stated, are essential for interferon-gamma accumulation. These T-cells also bring ephrin-A1 ligand to EphA2 on cerebral endothelial cells, activating said EphA2, a vital step in the pathogenesis of ECM. This results in the start of the NFκB pathway which causes the breakdown of tight and adherens junction proteins. As such, “vascular leakage, brain edema, and the development of other neurological symptoms…” arise [Darling et al., 2020]. From this proposed model, it is heavily implied that the deactivation of EphA2 localized on endothelial cells will prevent the breakdown of cell junctions, thus halting BBB disruption. To test this more directly, I seek to knock out EphA2 specifically in endothelial cells. I will compare these results with the effects of a global KO of EphA2 for similarities.

If the results are similar to those reported in the paper, this supports the model that the protection conferred by EphA2 deletion comes from the inactivation of EphA2 in endothelial cells. If the cell specific KO of EphA2 yields substantially different results from those reported in the paper, then our model is likely incorrect, and EphA2 KO’s protection is mediated by EphA2 inactivation in another cell type or types.

Further research is needed to determine the mechanism of EphA2 as it pertains to its relevance in BBB breakdown. If proven to play a vital part, EphA2 may be the key to prevent BBB disruption in its entirety. Furthermore, by isolating EphA2 in neuronal cells, it could be proven that this mechanism is localized and can be prevented in the affected area, as opposed to the whole body. In order to effectively target EphA2 in endothelial brain cells, a Cre-loxP system can be utilized.

Past research has demonstrated that CD4+ and CD8+ T-cells are an important source of interferon-gamma in the brain in the cerebral malaria mouse model. Interferon-gamma is a cytokine that plays a role in the immune response. Astrocytes and microglia, both of which are important for brain immune signaling and blood brain barrier maintenance, had yet to be investigated to see if they make a significant contribution to interferon-gamma production in ECM. Last spring, I conducted a study utilizing flow cytometry, single-cell RNA sequencing, and in vitro assays, to show that microglia and astrocytes do not increase production of interferon-gamma protein or transcript in the brains of mice with ECM, suggesting that they are not important sources of interferon-gamma in ECM. In contrast, CD8+ T-cells were an important source of interferongamma in the brain, which allows for BBB disruption. Diving deeper, it is hypothesized that the cause of BBB disruption is due to endothelial junction damage.

I proposed that an endothelial-cell specific knockout of EphA2 would be completed on C57Bl/6J mice infected with Plasmodium berghei ANKA (Pb-A) in order to observe if the absence of this gene of interest could prevent the disruption of the blood brain barrier. This notion is supported through the research presented in the literature review. By restricting EphA2 knockout to endothelial cells, we can observe if EphA2’s activity in endothelial cells is the source of its contribution to pathogenesis of ECM. To procure a model necessary to carry out this experiment, a special mouse with a Cre-LoxP system needed to be bred. While my main outcome has remained constant, the introduction of the Cre-LoxP system has presented novel.

In order to conduct this study, I began with a survival experiment to compare the effect of endothelial-specific Epha2 KO mice on death in ECM with the wild type and global KO mice. I strived to repeat the experiment but perform an Evans blue assay in order to observe BBB breakdown and brain edema in endothelial-specific Epha2 KO, wild type and global KO mice. I also envisioned performing a brain luciferase assay looking at schizont brain sequestration in Endo specific Epha2 KO, WT and global KO mice in ECM using an engineered strain of PbA that expresses luciferase. qPCR would allow me to investigate how to maximize the harvest of cerebral endothelial cells for future analysis. My work would contribute by determining whether the protection conferred by global knockout of EphA2 is replicated in the endothelial cell-specific knockout of EphA2, which would further validate that Epha2 function in endothelial cells is necessary for ECM pathogenesis.

Unfortunately, my survival experiment did not produce statistically significant results. I ran into issues with my control not behaving as it should and as a result, went through a long period of troubleshooting. I have since then, changed directions with my final thesis project. Instead, I will be looking at how Beta-Catenin is localized in the brain of healthy versus infected mice, as well as looking into its regulation as it is impacted by ECM pathogenesis.

Bibliography

Darling, Thayer K et al. “EphA2 contributes to disruption of the blood-brain barrier in cerebral malaria.” PLoS pathogens vol. 16,1 e1008261. 30 Jan. 2020, doi:10.1371/journal.ppat.1008261

Darling, Thayer K, and Tracey J Lamb. “Emerging Roles for Eph Receptors and Ephrin Ligands in Immunity.” Frontiers in immunology vol. 10 1473. 4 Jul. 2019, doi:10.3389/fimmu.2019.01473

Hearn J, Rayment N, Landon DN, Katz DR, de Souza JB. Immunopathology of Cerebral Malaria: Morphological Evidence of Parasite Sequestration in Murine Brain Microvasculature. Infect Immun 2000; 68:5364-76; PMID:10948166; http://dx.doi.org/10. 1128/IAI.68.9.5364-5376.2000

Kim H, Kim M, Im SK, Fang S. Mouse Cre-LoxP system: general principles to determine tissuespecific roles of target genes. Lab Anim Res. 2018 Dec;34(4):147-159. doi: 10.5625/lar.2018.34.4.147. Epub 2018 Dec 31. PMID: 30671100; PMCID: PMC6333611.

Postels, Douglas G., et al. “Pediatric Cerebral Malaria: A Scourge of Africa Pediatric Cerebral Malaria: A Scourge of Africa.” Future Neurology, vol. 8, no. 1, 2013, pp. 67-85. ProQuest, https://www.proquest.com/scholarly-journals/pediatric-cerebral-malariascourge-africa/docview/1285096201/se-2?accountid=15172, doi:https://doi.org/10.2217/fnl.12.84.

Rénia, Laurent et al. “Cerebral malaria: mysteries at the blood-brain barrier.” Virulence vol. 3,2 (2012): 193-201. doi:10.4161/viru.19013