Cerebral Malaria: Immune Cell and Parasite Interactions
Ute Frevert, D.V.M., Ph.D.
New York University School of Medicine, New York, NY
David Mahoney Neuroimaging Program
June 2007, for 3 years
Imaging Cerebral Malaria in New Animal Model May Reveal Immune Cell-Parasite Interactions
Researchers will develop a mouse model that produces similar disease processes and clinical symptoms as those that occur in human cerebral malaria, to facilitate imaging studies of the interactions between parasite and immune cells of the host.
Cerebral malaria causes the death of several million children worldwide each year, yet researchers do not know why only some infected children develop it or how it produces damage and death. Capillaries in affected children become occluded by sequestered red blood cells infected by the malaria parasite and by immune cells, producing unresponsiveness and coma leading to death. Good cerebral malaria animal models are currently lacking, but researchers have found that mice infected with lethal P. yoelii develop tissue damage and clinical symptoms that are basically identical to those of human cerebral malaria.
They now will further develop this model and use various intravital microscopic techniques to image the sequestration of red blood cells infected with fluorescent parasites and immune cells within brain capillaries in real time. Simultaneously, they will undertake light and electron microscopic examination of mouse brain to identify alterations produced by immune responses, such as changes in the production of immune cytokines, infiltration of inflammatory immune cells, and changes in the permeability of the blood-brain barrier. They will seek to understand the causal and temporal relationship between sequestration of infected red blood cells and clinical symptoms; whether parasite numbers or parasite virulence is key; and the roles of immune cytokines in the disease process.
Significance: Development of an effective mouse model to study cerebral malaria through imaging and tissue research may advance understanding of the roles of immune cells in this often fatal brain infection, and could contribute to development of effective preventive vaccines or therapies.
Cerebral Malaria: Immune Cell and Parasite Interactions
Cerebral malaria (CM), the most lethal manifestation of Plasmodium falciparum, is caused by sequestration of infected red blood cells in the brain. Best evidence indicates that the pathogenesis of this deadliest of the four human malaria species is driven by vascular occlusion and excessive immune responses to infected cells. Because human brain infections cannot be observed directly and there are many differences between human and existing animal model responses, we aim to establish a new model using infection of mice with Plasmodium yoelii. Intravital imaging of fluorescent parasites and immune cells in naïve and immune hosts is expected to provide a better understanding of the effect of severe malaria infections on the microvasculature of the brain and the individual steps leading to the manifestation of CM. Immunohistochemistry or immunofluorescence analyses conducted in parallel aim to complement and expand the intravital data by defining micro-environmental changes such as infection-induced modulation of cytokine secretion, alterations of the expression pattern of adhesion molecules, attraction of inflammatory and immune cells, and endothelial damage leading to breakdown of the blood-brain barrier. Our goal is to generate a model that allows recent and anticipated improvements in clinical therapeutic immuno-modulation to be exploited for control or blockage of the frequently fatal crisis presented by human CM.
Ute Frevert, D.V.M., Ph.D.
Dr. Ute Frevert studied veterinary medicine at the Freie Universität Berlin, Berlin, Germany. She received her D.V.M. in 1982 and her Ph.D. in 1983. She was a postdoctoral fellow in the Department of Veterinary Biochemistry, Freie Universität Berlin, where she became Assistant Professor in 1988. In 1991, she completed her Habilitation. From 1991 to 1992, she was a Visiting Scholar at the Department of Pathology, NYU School of Medicine. In 1992, she was appointed Assistant Professor of the Department of Medical and Molecular Parasitology and since 2004, Dr. Frevert has been Associate Professor in the same Department.
Her laboratory focuses on cellular and molecular interactions between malaria parasites and the liver of the infected host. After injection into the skin by an infected mosquito, sporozoites enter dermal capillaries, use the bloodstream to travel to the liver, and develop inside hepatocytes to thousands of merozoites. To reach hepatocytes, sporozoites must traverse the layer of sinusoidal cells, which is composed of endothelia and Kupffer cells, the resident macrophages of the liver. Malaria sporozoites use their major surface proteins, the circumsporozoite protein (CSP) and the thrombospondin-related adhesive protein (TRAP) to interact with distinct cell type-specific surface proteoglycans expressed on hepatocytes, Kupffer cells, and stellate cells, but not from liver endothelia. According to the current model, the parasites are arrested in the sinusoid by binding to stellate cell-derived extracellular matrix proteoglycans, which are thought to protrude from the space of Disse across the sinusoidal fenestration into the sinusoidal lumen. Intravital studies documented that arrested sporozoites initially glide along the sinusoidal cell layer until they eventually encounter a Kupffer cell, which they use to traverse the sinusoidal cell barrier. Using two different Kupffer cell-deficient mouse models, Dr. Frevert’s group showed that these hepatic macrophages are obligatory for sporozoite infection of the liver. They also found that malaria sporozoites invade Kupffer cells actively by vacuole formation, avoid destruction by blocking lysosomal fusion of the vacuole, and safely pass exit these phagocytes towards the space of Disse. After migrating through several hepatocytes, the parasites eventually settle down in a final one for differentiation to merozoites.
Most recently, Dr. Frevert’s lab discovered another evasion mechanism of Plasmodium sporozoites: inhibition of the respiratory burst in Kupffer cells. To do this, the parasite CSP engages a multifunctional high-affinity scavenger receptor, the low density lipoprotein receptor-related protein (LRP), and Kupffer cell surface proteoglycans. This interaction induces an intracellular signaling cascade that prevents the assembly of the NADPH oxidase, the enzyme responsible for the production of reactive oxygen intermediates. It is thought that Kupffer cell deactivation allows sporozoites to enter the liver, develop inside hepatocytes in the immediate neighborhood to large schizonts, and differentiate to merozoites. Unpublished data indicate that sporozoites also modify the cytokine secretion pattern in Kupffer cells, suggesting that parasite survival in the liver is aided by the generation of an overall anti-inflammatory milieu.
Another focus of Dr. Frevert’s lab is on the release of Plasmodium merozoites into the blood. This parasite stage is sensitive to phagocytosis and has to avoid the gauntlet of Kupffer cells on the way out of the liver. They showed that mature liver schizonts bulge out into the sinusoidal lumen and release extrusomes, large bags containing hundreds of merozoites enclosed in membrane derived from the infected hepatocyte and therefore unrecognizable by the innate immune system of the host. This mechanism allows the parasites to safely exit the liver and infect erythrocytes at extrahepatic sites.
A new focus will be on the pathogenesis of cerebral malaria, a frequently fatal complication in particular young children. By imaging fluorescent Plasmodium parasites in a mouse model, Dr. Frevert aims to understand the mode of sequestration of malaria-infected erythrocytes and immune cells of the host in brain capillaries.
Overall, Dr. Frevert’s lab is interested in understanding the molecular basis of the various evasion mechanisms Plasmodium has acquired in the course of evolution that enable this deadly parasite to survive in the vertebrate host.