Cerebral malaria claims more than 1 million lives per year. We report that heme oxygenase-1 (HO-1, encoded by Hmox1) prevents the development of experimental cerebral malaria (ECM). BALB/c mice infected with Plasmodium berghei ANKA upregulated HO-1 expression and activity and did not develop ECM. Deletion of Hmox1 and inhibition of HO activity increased ECM incidence to 83% and 78%, respectively. HO-1 upregulation was lower in infected C57BL/6 compared to BALB/c mice, and all infected C57BL/6 mice developed ECM (100% incidence). Pharmacological induction of HO-1 and exposure to the end-product of HO-1 activity, carbon monoxide (CO), reduced ECM incidence in C57BL/6 mice to 10% and 0%, respectively. Whereas neither HO-1 nor CO affected parasitemia, both prevented blood-brain barrier (BBB) disruption, brain microvasculature congestion and neuroinflammation, including CD8(+) T-cell brain sequestration. These effects were mediated by the binding of CO to hemoglobin, preventing hemoglobin oxidation and the generation of free heme, a molecule that triggers ECM pathogenesis.

Intracellular bacteria and parasites typically invade host cells through the formation of an internalization vacuole around the invading pathogen. Plasmodium sporozoites, the infective stage of the malaria parasite transmitted by mosquitoes, have an alternative mechanism to enter cells. We observed breaching of the plasma membrane of the host cell followed by rapid repair. This mode of entry did not result in the formation of a vacuole around the sporozoite, and was followed by exit of the parasite from the host cell. Sporozoites traversed the cytosol of several cells before invading a hepatocyte by formation of a parasitophorous vacuole, in which they developed into the next infective stage. Sporozoite migration through several cells in the mammalian host appears to be essential for the completion of the life cycle.

Plasmodium sporozoites are deposited in the skin of their vertebrate hosts through the bite of an infected female Anopheles mosquito. Most of these parasites find a blood vessel and travel in the peripheral blood circulation until they reach the liver sinusoids. Once there, the sporozoites cross the sinusoidal wall and migrate through several hepatocytes before they infect a final hepatocyte, with the formation of a parasitophorous vacuole, in which the intrahepatic form of the parasite grows and multiplies. During this period, each sporozoite generates thousands of merozoites. As the development of Plasmodium sporozoites inside hepatocytes is an obligatory step before the onset of disease, understanding the parasite's requirements during this period is crucial for the development of any form of early intervention. This Review summarizes our current knowledge on this stage of the Plasmodium life cycle.

Sequestration of malaria-parasite-infected erythrocytes in the microvasculature of organs is thought to be a significant cause of pathology. Cerebral malaria (CM) is a major complication of Plasmodium falciparum infections, and PfEMP1-mediated sequestration of infected red blood cells has been considered to be the major feature leading to CM-related pathology. We report a system for the real-time in vivo imaging of sequestration using transgenic luciferase-expressing parasites of the rodent malaria parasite Plasmodium berghei. These studies revealed that: (i) as expected, lung tissue is a major site, but, unexpectedly, adipose tissue contributes significantly to sequestration, and (ii) the class II scavenger-receptor CD36 to which PfEMP1 can bind is also the major receptor for P. berghei sequestration, indicating a role for alternative parasite ligands, because orthologues of PfEMP1 are absent from rodent malaria parasites, and, importantly, (iii) cerebral complications still develop in the absence of CD36-mediated sequestration, dissociating parasite sequestration from CM-associated pathology. Realtime in vivo imaging of parasitic processes may be used to evaluate the molecular basis of pathology and develop strategies to prevent pathology.imaging ͉ Plasmodium ͉ P. berghei ͉ luciferase ͉ real-time in vivo imaging I nfected red blood cells (irbc) of many species of malaria parasites adhere to the endothelial cells of the microvasculature of numerous deep tissues (1, 2). Termed sequestration, this characteristic may facilitate parasite multiplication, avoiding removal of the irbc by the spleen (3, 4). In some parasite-host combinations, the process of sequestration is associated with pathogenesis, for example, Plasmodium falciparum in humans (1, 2, 5) and Plasmodium berghei in certain mouse strains (6, 7). Cerebral malaria (CM) is a major complication of P. falciparum infections, and the sequestration of irbc has been considered to be the major feature leading to CM-related pathology. Sequestration may lead to vascular obstruction, local endothelial cell activation, and the release of proinflammatory cytokines, resulting in damage to adjacent tissues (2, 7, 8). In P. falciparum, the class II scavenger receptor CD36 is a major endothelial receptor. CD36 is involved in not only the adherence of irbc (1, 9, 10) through specific domains of the surface variant antigen PfEMP-1 but also in the modulation of innate and adaptive immune responses (11,12). To date, most investigations of the dynamics of irbc-receptor interactions rely on in vitro observations with cultured cells and immobilized receptors (2). Despite the increase in knowledge of the molecules involved in the binding of irbc to endothelial cells, the specific interactions that lead to pathology have yet to be established. Infection with P. berghei in laboratory rodents is a well established model for the investigation of associations among CM, proinflammatory cytokines, and endothelial receptors involved in the sequestration of irbc, leukocytes, and platelet...

Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a novel pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-α/β receptor–dependent manner. This signaling pathway is critical for immune cell–mediated host resistance to liver-stage Plasmodium infection, which can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN.

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.

Immunization with Plasmodium sporozoites that have been attenuated by ␥-irradiation or specific genetic modification can induce protective immunity against subsequent malaria infection. The mechanism of protection is only known for radiation-attenuated sporozoites, involving cell-mediated and humoral immune responses invoked by infected hepatocytes cells that contain longlived, partially developed parasites. Here we analyzed sporozoites of Plasmodium berghei that are deficient in P36p (p36p ؊ ), a member of the P48͞45 family of surface proteins. P36p plays no role in the ability of sporozoites to infect and traverse hepatocytes, but p36p ؊ sporozoites abort during development within the hepatocyte. Immunization with p36p ؊ sporozoites results in a protective immunity against subsequent challenge with infectious wild-type sporozoites, another example of a specifically genetically attenuated sporozoite (GAS) conferring protective immunity. Comparison of biological characteristics of p36p ؊ sporozoites with radiation-attenuated sporozoites demonstrates that liver cells infected with p36p ؊ sporozoites disappear rapidly as a result of apoptosis of host cells that may potentiate the immune response. Such knowledge of the biological characteristics of GAS and their evoked immune responses are essential for further investigation of the utility of an optimized GAS-based malaria vaccine.apoptosis ͉ attenuated vaccines ͉ host-pathogen interaction ͉ Plasmodium

In regions of high malaria transmission, mosquitoes repeatedly transmit liver-tropic Plasmodium sporozoites into individuals who already have blood-stage parasitaemia1. This manifests itself in older semi-immune children by concurrent carriage of different parasite genotypes at low asymptomatic parasitaemias2. Superinfection presents an increased risk of hyperparasitaemia and death in less immune individuals, but counter-intuitively is not frequently observed in the young3,4. Here, we show in a rodent model, that ongoing blood-stage infections, above a minimum threshold, impair the growth of subsequently inoculated sporozoites such that they become growth arrested in liver hepatocytes and fail to develop into blood-stage parasites. Inhibition of the liver-stage can be mediated by the host iron regulatory hormone hepcidin5, the synthesis of which is stimulated by blood-stage parasites in a density-dependent manner. We model this phenomenon and show how density-dependent protection against liver-stage malaria can shape the epidemiological patterns of age-related risk and complexity of malaria infections seen in young children. The interaction between these two Plasmodium stages and host iron metabolism has relevance for the global efforts to reduce malaria transmission and for nutritional programmes of iron supplementation in malaria-endemic regions.