Malaria infections: what and how can mice teach us.

JIM-11856; No of Pages 10 Journal of Immunological Methods xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Immunological Methods

Research paper

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Malaria infections: What and how can mice teach us?

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Vanessa Zuzarte-Luis a, Maria M. Mota a,⁎, Ana M. Vigário a,b,⁎⁎

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Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal Unidade de Ciências Médicas, Centro de Competência de Ciências da Vida, Universidade da Madeira, Funchal, Portugal

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Article history: Received 18 February 2014 Received in revised form 24 April 2014 Accepted 1 May 2014 Available online xxxx

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Keywords: Plasmodium Malaria Host Rodent models Pathology Infection

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Malaria imposes a horrific public health burden – hundreds of millions of infections and millions of deaths – on large parts of the world. While this unacceptable health burden and its economic and social impact have made it a focal point of the international development agenda, it became consensual that malaria control or elimination will be difficult to attain prior to gain a better understanding of the complex interactions occurring between its main players: Plasmodium, the causative agent of disease, and its hosts. Practical and ethical limitations exist regarding the ability to carry out research with human subjects or with human samples. In this review, we highlight how rodent models of infection have contributed significantly during the past decades to a better understanding of the basic biology of the parasite, host response and pathogenesis. © 2014 Elsevier B.V. All rights reserved.

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1. Introduction

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Malaria still imposes a significant health and economic burden in large parts of the world, particularly in sub-Saharan Africa and Southeast Asia, where at least 200 million infections and over 600000 deaths are registered annually (WHO, 2013). The disease is caused by protozoan parasites of the genus Plasmodium, which are transmitted by female Anopheline mosquitoes. During a blood meal the infected female mosquitoes deposit Plasmodium sporozoites in the mammalian skin. Within minutes to few hours after inoculation (Sinnis and Zavala, 2012) these highly motile forms enter the circulatory system and reach the liver where they infect hepatocytes establishing the so-called pre-erythrocytic phase of malaria infection. This phase of infection is completely asymptomatic and lasts in humans 5–17 days (the length varies according to Plasmodium species (Coatney et al., 1971)). Each sporozoite

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journal homepage: www.elsevier.com/locate/jim

⁎ Corresponding author. ⁎⁎ Correspondence to: A.M. Vigário, Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal. E-mail addresses: [email protected] (M.M. Mota), [email protected] (A.M. Vigário).

that infects the liver replicates into thousands of new parasites in a process called schizogony. Once parasite replication and cellularization are completed, the newly formed parasites, called merozoites, are released into the bloodstream and infect erythrocytes, initiating the erythrocytic stage of malaria infection (Prudencio et al., 2006). The cycles of parasite multiplication inside erythrocytes are shorter (24, 48 or 72 h, depending on parasite species), and causative of the classic symptoms of the disease (Coatney et al., 1971). When left untreated, the disease can eventually progress to severe syndromes and cause death (Haldar et al., 2007). Human malaria can be caused by five Plasmodium species: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. Of these, P. falciparum and P. vivax are the focus of intense research and targeting strategies due to the high mortality and/or morbidity they cause. P. falciparum is the most virulent species and is responsible for the vast majority of deaths in sub-Saharan Africa, primarily of young children and pregnant women. P. vivax malaria is the most widespread and was previously considered a benign disease but is emerging as a potentially lethal condition outside of Africa (Baird, 2013), as current control measures are successfully reducing P.

http://dx.doi.org/10.1016/j.jim.2014.05.001 0022-1759/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Zuzarte-Luis, V., et al., Malaria infections: What and how can mice teach us?, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.05.001

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The study of human malaria involves a myriad of methods such as epidemiological analysis, population genetics, clinical studies of patients, both in field research studies and in hospital settings, as well as analyses of post-mortem biopsies. However, limitations exist regarding the ability to carry out research with human subjects or with human samples. For example, access to post-mortem tissues is hindered due to religious and cultural objection to autopsy and the lack of proper control subjects for most studies such as samples from infected patients that do not develop the pathology or die are some of the reasons that make research difficult. Moreover, the data obtained from post-mortem studies only represents the end stage of a long process, and the analysis of the sequence of events leading to pathology through monitoring the internal organs and environment is very limited; e.g. the study of the liver during the first phase of infection or the brain during cerebral malaria. Despite their limitation and controversy on replicability of human disease, mouse models of malaria infection have been used for decades and have contributed significantly to a better understanding of the basic biology of the parasite, host response and pathogenesis. Several rodent-infectious Plasmodium parasites are available, Plasmodium berghei, Plasmodium yoelii, Plasmodium chabaudi and Plasmodium vinckei, each including several strains, which lead to distinct courses and outcomes of infection, depending on the host-mouse strain combination (see Box 1), raising questions about which, if any, of the mouse models can be extrapolated to understand human disease or diseases. Concerns exist about the translational utility of animal models in pathogenesis, immunity, vaccine development and drug discovery due to the heterogeneity observed with different parasite and mouse combinations (White et al., 2010; Craig et al., 2012). However, one can argue that the range of disease manifestations in the different mouse models should be considered as a reflection of the diversity

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2. The importance of addressing malaria infection experimentally

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Box 1 Blood stage – Animal models to study severe malaria

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3. How to address malaria infection experimentally

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In addition to studying disease mechanisms, an advantage of using rodent models is the ease of maintaining the entire life cycle of the parasite in controlled and optimized laboratory conditions. The establishment and maintenance of laboratory Anopheles stephensi (as well as Anopheles gambiae) vector colonies and the development of transgenic parasite lines have allowed the dissection of processes occurring during transmission from the mosquito vector to the mammalian host, as well as studies of transmission from the mammalian host to the mosquito vector. Controlled infections can be initiated directly by mosquito bite or, alternatively, by intra-dermal or intra-venous injection of sporozoites. The infection can then be analyzed in the liver or be allowed to progress into the blood, and disease outcome can be monitored. It is also possible to bypass the skin and liver stages of infection by directly injecting parasitized red blood cells (pRBCs) intra-peritoneally or intravenously. The careful choice of transgenic parasite line determines the possibilities of analysis; e.g. the use of fluorescent parasites allows monitoring the infection and the parasite's interaction with host cells in vivo and in real-time (Gomes-Santos et al., 2012). When using chemiluminescent parasites, infection can be analyzed longitudinally over time (within the same infected animal) in a non-invasive (or minimally invasive) manner throughout liver stage into blood stage infection, where bioluminescence is correlated with the level of liver infection and with blood parasitemia (Ploemen et al., 2009; Zuzarte-Luis et al., 2014). Additionally, chemiluminescent parasites have also facilitated the study of infected erythrocyte sequestration (Franke-Fayard et al., 2006). Moreover, the combination of transgenic parasites (lacking or overexpressing parasite or exogenous molecules) with genetically engineered mice lacking key molecules (e.g. immune mediators or their receptors) has proved useful in deciphering the host response to parasite infection throughout the latter's life cycle.

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of the human disease rather than a limitation (Langhorne et al., 2011). Undoubtedly, the availability of inbred/ congenic/transgenic animals and the ability to manipulate and control different aspects of the host, including the immune system, make the mouse model a precious tool. Still, mice are not humans and the Plasmodium spp. that infect rodents are distinct from the ones that infect humans. As such, results arising from studies using rodent models should be interpreted with caution (White et al., 2010; Craig et al., 2012; Langhorne et al., 2011). Aware of the importance and simultaneously the limitation of the mouse models, there has been a constant search for mouse models that better reflect the different field situations (see Boxs 1 and 2).

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falciparum transmission (Cotter et al., 2013). Additionally, P. vivax is capable of forming cryptic forms called hyponozoites during the pre-erythrocytic stage that cause relapses months and even years after blood stage parasite clearance, contributing to the complexity of understanding and treating P. vivax malaria (Shanks and White, 2013; Kondrashin et al., 2014). Despite major advances in the development and implementation of novel intervention strategies, the scientific community is still limited by substantial gaps in understanding the biology of Plasmodium and its complex interaction with the human host (A research agenda for malaria eradication: basic science and enabling technologies, 2011). Further studies addressing fundamental host–parasite interactions, as well as patho-physiological features of infection are necessary, but such studies are difficult to perform in humans.

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5. In the liver – replicating at full speed

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Sporozoites that successfully invade blood vessels rapidly home to the liver where they arrest in the liver sinusoids through specific interactions between parasite surface proteins and host molecules. These molecular interactions have been extensively characterized in the 1990s using biochemical and molecular approaches (Ejigiri and Sinnis, 2009), but the details of liver invasion were still unclear. Early observations of sporozoites going in and out of cells (Vanderberg et al., 1990) inspired the discovery that sporozoites have the ability to traverse cells prior to establishing infection in the liver (Mota et al., 2001), which in turn strongly impacted the view on how parasites reach the liver from the mosquito bite site (Ishino et al., 2004; Amino et al., 2008; Tavares et al., 2013). Imaging techniques, both in vitro (using cell lines) and in vivo (using rodent models), were pivotal to these discoveries (Mota et al., 2001; Frevert et al., 2005). Once inside hepatocytes, sporozoites undergo a remarkable process of transformation and intense replication that lasts 5–17 days in humans and ~2 days in rodents. The complex interactions that occur between the host cell and Plasmodium parasites during this phase of infection only recently have begun to be elucidated. The application of high-throughput technologies, namely genomics, proteomics or lipidomics to the study of isolated infected hepatocytes was and still is, fundamental to the understanding of how the host cell responds to the presence of a developing and highly replicative parasite and which host and parasite pathways are engaged during the successful establishment of infection (Tarun et al., 2008; Albuquerque et al., 2009). Several relevant and novel questions regarding host-Plasmodium interactions in the liver have emerged from the analysis of such complex data sets (Epiphanio et al., 2008; Vaughan et al., 2009). The similarly unbiased analysis of whole-liver samples has further contributed to the understanding of the responses at the organ/organism level (Portugal et al., 2011; Liehl et al., 2014). In fact, such a recent analysis using rodent models of infection enabled us to demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver (Liehl et al., 2014), a stage that until now was thought to be undetected by the host (Liehl and Mota, 2012). The use of genetic mouse models in combination with classical immunology techniques, such as flow cytometry for the ex vivo analysis of liver immune cell populations, immunohistochemistry analysis for positional information, and molecular techniques (such as PCR or Western

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Until recently, details regarding the initiation of malaria infection when an infected female mosquito injects sporozoitecontaining saliva as it probes the dermis for blood, remained inscrutable. Questions such as “where are sporozoites deposited?” or “how long do sporozoites stay at the injection site?” remained unsolved until recently. The small number of sporozoites injected and their highly motile behavior hindered the study of the dermal phase of the infection. The notion that sporozoites are deposited in the skin, rather than directly into the blood circulation, was first suggested in the 1930s upon histological analysis of skin tissue of volunteers exposed to P. vivax-infected mosquitoes (Boyd and S.F., 1939). Nevertheless, it was the use of rodent models that allowed the experimental confirmation of such results. Experiments of excision of the bitten tissue (Sidjanski and Vanderberg, 1997) and interruption of mosquito feeding (Matsuoka et al., 2002; Ponnudurai et al., 1991) resulted in a delayed onset of blood parasitemia, indirectly suggesting that sporozoites are indeed deposited in the skin. The direct observation of this phenomenon was possible due to advances in molecular biology and imaging technologies, once again using rodent models of infection. Using intravital fluorescence microscopy and genetically engineered parasites expressing fluorescent tags, researchers directly observed sporozoites being injected in the host skin when the mosquito ejects saliva (Vanderberg and Frevert, 2004). Similarly, the study of the dynamics of sporozoites in the skin has significantly evolved with the advances in technologies that enabled the direct observation, in real-time, of the interactions between parasites and their hosts. Upon injection, and contrary to the initial assumption, sporozoites may spend several hours at the inoculation site. The first experimental evidence of this fact came from skin transplantation experiments in monkeys (Lloyd and Sommerville, 1949). Later use of rodent models, in which sporozoite load determination in excised tissue was performed through histological analysis or quantitative molecular methods (PCR), further supported the concept that the transit to the bloodstream can take several hours (reviewed in Sinnis and Zavala, 2012). Nevertheless, insights on sporozoite numbers, characteristics of movement and positional information could only be gathered using high-resolution and high-speed microscopy. The combination of in vivo imaging of rodent models with genetically engineered P. berghei parasites enabled the determination of sporozoite numbers and parasite release rate through

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the mosquito proboscis (Amino et al., 2006) and evidenced the randomness of movement of sporozoites in the dermis until contacting dermal blood vessels (Frischknecht, 2007). Finally, this technology allowed the quantification of the proportion of blood vessels (Amino et al., 2006, reviewed in Sinnis and Zavala, 2012; Menard et al., 2013). Sporozoites that remain at the inoculum site are likely destroyed by the innate immune cells contributing to the initiation of the immune response. Surgical excision of local lymph nodes or pharmacological inhibition of T-cell egress from these organs demonstrated, in a vaccination rodent model, that sporozoites that reach lymphatic circulation and the draining lymph nodes (15–20%) are critical for priming CD8+ T cell response against liver stage probably by being captured and their antigens presented by dendritic cells (reviewed in Sinnis and Zavala, 2012).

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Further technological progress such as the development of high-throughput Omics technologies employed to study Plasmodium infection at the DNA, RNA, protein, and metabolite levels has prompted further advances in our understanding of both host as well as parasite biology (Tarun et al., 2008; Albuquerque et al., 2009; Olszewski et al., 2009). Equally important was the recent development of clinical diagnostic techniques for small animals, including non-invasive imaging techniques such as computer tomography (CT) and magnetic resonance imaging (MRI), as well as monitoring systems for cardio-vascular and respiratory function (Penet et al., 2005; Martins et al., 2013). Such advances are crucial for the detailed characterization of malaria pathology in animal models and help determine the degree of similitude with the human pathology.

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The possibility of maintaining P. falciparum blood stage cycles in vitro using human RBCs has contributed to the fact that the majority of studies attempting to establish the parasite and RBC determinants of infection have been performed in vitro without

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the need of animal models (Bei and Duraisingh, 2012; Cowman et al., 2012). Similarly, while the recent development of humanized mouse models for blood stage of P. falciparum has allowed to answer several questions related with in vivo multiplication of the human parasite P. falciparum and contributed to drug development (discussed elsewhere in this issue), the majority of these studies have been performed using P. falciparum in vitro cultures. Nevertheless, animal models have been critical in dissecting key processes and players of the immune response mounted during the blood stage of infection. In particular, the use of genetically engineered mice lacking different molecules of the immune system, in combination with depleting antibodies, unveiled some of the complex immune mechanisms and immuno-pathogenesis of Plasmodium infection (Schofield, 2007). These studies contributed to establish potential biomarkers to predict risks of malaria related mortality (such as IP-10 or CXCL10). The influence of genetic polymorphisms for several of these molecules has also been studied on susceptibility/resistance to P. falciparum malaria (reviewed in Driss et al., 2011). If left untreated the infection may eventually progress to severe disease. In the case of some Plasmodium strains, pRBCs have the ability to cytoadhere to the endothelium of different organs, which has been associated with the development of severe pathology in human malaria. Hence, a detailed analysis of the organs during disease progression is of major importance. Similarly to the studies of liver stage, approaches from classical histopathology, high-throughput Omics techniques and in vivo imaging (for whole-organ or specific cell population analysis), provided additional contributions to understanding pathogenesis at the tissue level. Histological and ultra-structural analysis of brains from animals developing experimental cerebral malaria (ECM) evidenced the sequestration of leukocytes (Polder et al., 1992; Hearn et al., 2000; Vigario et al., 2007) but also platelets (Combes et al., 2004) and some infected erythrocytes (Hearn et al., 2000). It was later demonstrated, using flow cytometry analysis of isolated brain leucocytes, that although very low in number, CD8 T cells are the key leukocytes and the terminal effector cells associated with ECM (Belnoue et al., 2002; Nitcheu et al., 2003; Haque et al., 2011a). This important technical advance contributed to the recognition of the importance of these cells in the development of the pathology, an aspect that remained unnoticed until recently due to their low frequency in the brain. Still, they have occasionally been detected by microscopy on both mouse models (Belnoue et al., 2002) as well as on the brain of Malawian children who died of CM (Dorovini-Zis et al., 2011). A similar strategy of isolating and analyzing immune cells from an entire organ has also been used to study lung and placental pathology in mouse models in order to dissect the players on the immune response occurring in these organs (Hee et al., 2011; Van den Steen et al., 2010). Despite the advances in the understanding of immunopathology, these approaches to study ECM have failed to detect sequestration of pRBCs within the brain vasculature, a hallmark of severe P. falciparum malaria. The recent availability of transgenic chemiluminescent parasite lines allowed the in vivo real-time imaging of pRBCs, bringing valuable information on the dynamic of parasite biomass/sequestration in different organs, including placenta of pregnant mice, brain, lungs and its association with pathogenesis (Franke-Fayard et al., 2006; Franke-Fayard et al.,

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blot), has been critical for the characterization of the immune populations and molecular pathways involved in the immune response elicited by the immunization with radiation-attenuated Plasmodium spp. sporozoites or other protocols known to induce sterile protective immunity against parasite challenge (reviewed in Doolan and Martinez-Alier, 2006). Complete sterile protection against liver stage, first demonstrated following immunization with radiation-attenuated sporozoites in mice (Nussenzweig et al., 1967), and latter in non-human primates (Gwadz et al., 1979) and humans (Clyde, 1975), can also be achieved in mice with genetically-attenuated sporozoites (Mueller et al., 2005a, 2005b; van Dijk et al., 2005) or with sporozoites in combination with chemoprophylaxis in mice and humans (Belnoue et al., 2004; Roestenberg et al., 2009). After an intense period of liver schizogny, the newly formed merozoites are released into the blood stream, where they initiate the cycles of erythrocyte infection. The use of time-lapse intravital, confocal and electron microscopy allowed the visualization of the mechanism by which hepatic merozoites reach blood circulation, escaping the immune system (Sturm et al., 2006; Baer et al., 2007). At the end of the liver stage, merozoites are packed into merosomes, covered by host cell membrane, and released into the bloodstream (Sturm et al., 2006). Once in circulation, merosomes can reach the lung capillaries where they rupture releasing the erythrocyte-infectious merozoites (Baer et al., 2007). The timing of appearance/detection of infected erythrocytes in circulation can be used as an indirect measure of viable parasite load in the previous stage (the liver). The accurate and fast detection of the first generation of infected erythrocytes is therefore important to evaluate interventions targeting the liver stage. This can be done by analysis of blood smears, by qRT-PCR or by a luciferase assay using transgenic chemiluminescent parasite lines (Zuzarte-Luis et al., 2014). Altogether, the use of rodent models has been absolutely critical to the current understanding of Plasmodium liver stage, recently recognized as the ideal target for the development of novel anti-malarial strategies, such as drug development or vaccines (Derbyshire et al., 2011; Duffy et al., 2012; Rodrigues et al., 2012). Still, not all aspects of human Plasmodium spp. biology can be modeled using rodent malaria. The recent engineering of chimeric humanized mouse models to study P. falciparum and P. vivax infections constitutes a great evolution in the study of the human parasites, enabling the study of parasite biology in vivo, but also the evaluation of specific anti-malarial interventions in a more physiological setting (Vaughan et al., 2012). The technology is still evolving and while it is already possible to investigate separately P. falciparum liver and blood-stage development in vivo, the critical step of merozoite release from hepatocytes and transition to RBC invasion still awaits further elucidation. Please refer to Box 2 for further details on the current models to study liver stage infection and for a summary of humanized models to study pre-erythrocytic stage malaria infection.

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mice following intraperitoneal injection of P. berghei ANKAparasitized red blood cells (pRBC). C57BL/6 mice infected with P. berghei ANKA-related parasite isolates, such as P. berghei NK65 or P. berghei K173, do not develop neurological symptoms and, as such, have been used as controls on ECM studies. Similarly, mouse strains shown to be resistant to ECM when injected with P. berghei ANKA, like BALB/c mice, have been used as controls. Placental malaria (PM), a major malaria complication occurring during pregnancy, is estimated to be the cause of up to 200 000 infant deaths per year (WHO, 2013). It is usually associated with fetal growth restriction and/or preterm delivery, stillbirths and maternal anemia. Different parasite/mouse strain combinations have been used to study PM. Early P. berghei ANKA infection of pregnant BALB/c mice generally causes abortion, while infection at around gestational day 13 induces a syndrome that resembles severe PM in women, with clear signs of pathology in the mother as well as the offspring (Neres et al., 2008; Oduola et al., 1982; Hioki et al., 1990). To take advantage of the available C57BL/6 mutant strains, experimental models of C57BL/6 mice infected, either early in gestation with P. chabaudi (Poovassery and Moore, 2006), or at gestational day 13 with different lines of P. berghei (such as PbNK65 or PbK173) (Rodrigues-Duarte et al., 2012) were also developed. Given that in areas where malaria is endemic, women generally develop considerable clinical immunity to malaria before reproductive age, PM has also been studied in mice that had been immunized prior to mating (Megnekou et al., 2009; Marinho et al., 2009; van Zon and Eling, 1980). Malaria-associated acute lung injury (ALI) and its more severe form, malaria-associated acute respiratory distress syndrome (ARDS) often occur in association with other severe forms of malaria infection (reviewed in Taylor et al., 2012). Although, pulmonary pathology has been described in early studies of P. berghei experimental severe/cerebral malaria prior to the development of ECM, only recently was it used as a model with the specific objective of studying ALI/ ARDS (Lovegrove et al., 2008). P. berghei ANKA-infected DBA/ 2 mice (Epiphanio et al., 2010) and either P. berghei K173(Hee et al., 2011) or P. berghei NK65-infected C57BL/6 mice (Van den Steen et al., 2010) were also described as models to investigate the mechanisms leading to lung disease but without any associated cerebral complication. Acute kidney injury (AKI) is a malaria-associated complication observed especially in adults. Although no specific mice model exists, acute kidney injury has been studied in P. berghei infected BALB/c (Elias et al., 2012) or C57BL/6 (Sinniah et al., 1999) mice. Severe malarial anemia (SMA) is a life-threatening complication of malaria, highly prevalent in regions with high malaria transmission. Several mouse/parasite combinations have been used to study this severe complication. However, in the majority of these models severe anemia is associated with hyperparasitemia, which is not a characteristic of human SMA. As such, other models like P. berghei ANKA infection in semiimmune BALB/c mice (Evans et al., 2006) or sequential infection with two different Plasmodium species (Harris et al., 2012) have been described. Liver injury (LI) can happen as consequence of malaria infection. Immune-mediated liver damage was first described on P. berghei NK65 infection of C57BL/6 and BALB/c mice

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2005). Ex vivo imaging revealed random parasite accumulation/sequestration in the brain of mice developing ECM (Baptista et al., 2010). The engineering of transgenic parasites expressing luciferase under the control of a schizont-specific promoter allowed the analysis of the organ-specific distribution of these mature forms of the P. berghei parasite (reviewed in Franke-Fayard et al., 2010). The development of imaging technologies has also significantly contributed to the characterization of malaria pathology. The recent availability of high performance computer tomography (CT) and magnetic resonance imaging (MRI) equipment for small animals, allowed in vivo monitoring of neuropathology during ECM (Penet et al., 2005). These and related non-invasive imaging techniques, applied for the first time to the study of ECM in 2005 (Penet et al., 2005), have huge potential since they can provide a large spectrum of information such as localization of structural lesions, edema, hemorrhages, and thrombosis. Importantly, magnetic resonance techniques also revealed differences in the blood flow and metabolic changes in the brains of infected but ECM-resistant mice (Penet et al., 2007). Intravital microscopy (IVM) enables the in vivo analysis of parasite-host interaction at the cellular level, allowing the measure of blood flow, vascular leakage, cellular adhesion to the endothelium, leukocyte recruitment and migration and it has been used mainly for ECM (reviewed in Frevert et al., 2014). Moreover, the use of this technique in combination with the newly available fluorescent parasite lines (Graewe et al., 2009) increases the possibilities of analyses. The use of these imaging technologies can also be of great importance to understand other severe malaria complications, such as placental malaria (de Moraes et al., 2013; Conroy et al., 2013). Blood stage malaria infection has variable clinical outcomes, ranging from mild or uncomplicated malaria, usually non-lethal, to severe or complicated malaria, with a mortality rate of 20–30%. Severe and complicated malaria includes different clinical features with different organs being affected (reviewed in Haldar et al., 2007 and see references in Fig. 1 for individual syndromes Trang et al., 1992; Joshi et al., 1986; Rogerson et al., 2007; Douglas et al., 2012; Perkins et al., 2011; Taylor et al., 2012), which have been modeled in rodents using different parasite/mouse strain combinations. (See Fig. 2.) Cerebral malaria is responsible for most of deaths occurring due to malaria infection especially in children in endemic areas (Haldar et al., 2007). CM can also develop in non-immune adults but with some symptomatological differences (Mishra and Wiese, 2009). Even in children it is clear that CM is not a homogenous syndrome and 3 patterns of histopathological changes (based on presence or absence of brain pRBC sequestration and/or microvascular pathology) have been described in children dying with clinically defined CM (Dorovini-Zis et al., 2011). The reasons for this heterogeneity are unclear but are probably due to host or parasite genetic variations as well as environmental factors. Notably, different parasite/mouse strain combinations also show some heterogeneity on histopathological patterns (reviewed in Brian de Souza et al. 2009). Nevertheless, while different combinations of parasite/mouse strains have been used in the past, most recent studies on experimental cerebral malaria (ECM) have been conducted using C57BL/6

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Fig. 1. https://www.dropbox.com/s/zonlhyxqeo634p6/Fig. 1.tif. Top. Table summarizing the possible severe malaria syndromes developed depending on the rodent host-parasite combinations. Bottom. Pictures representing three different outcomes of P. berghei ANKA infection in different host-mouse strains. Left: Brains illustrating blood-brain barrier disruption (evans blue) and parasite sequestration (luminescence) in infected C57BL/6 mice that developed ECM. Middle: Impaired fetal development in infected pregnant BALB/c females with PM. Right: Histological analysis of lung tissue evidencing hemorrhage and edema in DBA/2 mice that developed ALI/ARDS.

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(Yoshimoto et al., 1998; Adachi et al., 2001), but other models were also described such as P. chabaudi infection in DBA/2 mice (Seixas et al., 2001) and P. berghei ANKA in C57BL/6 mice (Haque et al., 2011b). Often, different disease mechanisms have been described for the same malaria complication based on different animal/ parasite combination models. In spite of being used as an argument against animal models this can also be seen as an advantage. In fact, differences in the mechanism or outcome

of the human disease exist, either related to host differences (age, genetic variability, previous exposure and environment) or parasite variations. Human severe malaria is complex and very likely implicates several of these mechanisms. Undoubtedly the use of models has significantly contributed to dissect the mechanisms of severe pathology. However, their input for therapeutic interventions has been limited possibly because most studies test the intervention strategies during a controlled infection and prior or at early stage of symptoms onset,


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disregarding the fact that patients only present to the hospital in an advanced stage of disease. The liver forms of Plasmodium parasites were first identified in 1948 (Cox, 2010). Still, our understanding of the biology of Plasmodium hepatic stages remains limited. Despite being asymptomatic, the hepatic stage is an obligatory phase of Plasmodium development. During this phase parasite numbers increase by four orders of magnitude (Prudencio et al., 2006) but the load of parasites in the liver is significantly lower than in blood stage. This feature makes the liver stage an attractive target for the development of malaria prophylaxis strategies. Humans ultimately constitute the ideal system to study infection by Plasmodium parasites. However, given the difficulties in obtaining human liver samples, animal models remain the closest surrogates available to researchers. In fact, the vast majority of our knowledge has emerged by using P. bergheiand P. yoelii-infected mice. However, not all aspects of P. falciparum biology can be modeled using rodent malaria, for example, the vaccine candidate LSA-1 has no ortholog in rodent malaria species (Frech and Chen, 2011). Moreover, the human parasites P. ovale and P. vivax, are able to generate cryptic forms called hypnozoites, never identified in rodent parasites. These dormant forms may remain in the liver for long periods and cause disease relapses when re-activated. Therefore, intervention during this stage of infection is essential to achieve complete parasite elimination. As such, an understanding the biology of this enigmatic form of the parasite and the development of effective hypnozoiticides are crucial for the goal of malaria eradication.

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Fig. 2. https://www.dropbox.com/s/tqm7suxoniniwxh/Fig. 2.tif. Table summarizing the possibility of studies when using wild-type mice models infected with rodent Plasmodium parasites (left) versus liver humanized mice models infected with human Plasmodium parasites (right).

The development of a small-animal model capable of efficiently supporting human liver-stage development in vivo became an important challenge. The proof-of-concept that P. falciparum infected hepatocytes could develop in a small animal model was demonstrated with the transplantation of human hepatocytes into immunosuppressed severe combined immunodeficiency (SCID) mice (Sacci et al., 1992). To promote human hepatocyte expansion by giving them a competitive growth advantage over the endogenous murine hepatocytes, genetic approaches were employed. SCID mice homozygous for the urokinase type plasminogen activator transgene (SCID/Alb-uPA), known to cause murine liver injury, were engrafted with human hepatocytes (Mercer et al., 2001; Meuleman et al., 2005). These humanized liver-chimeric mice are able to support complete development of P. falciparum liver stage (Morosan et al., 2006; Sacci et al., 2006), culminating with the release of merozoites capable of invading hRBCs ex-vivo (Sacci et al., 2006), and were shown to be useful to study the phenotype of P. falciparum gene knockouts (VanBuskirk et al., 2009; Mikolajczak et al., 2011). Nevertheless, this model cannot be used to study the parasite transition from the liver stage to the blood within the mouse. When SCID/Alb-uPA mice are depleted in NK cells and macrophages, the level of human hepatocytes chimerism and consequently of P. falciparum liver-stage development improves (Morosan et al., 2006). Nevertheless, the human liver chimeric SCID/Alb-uPA mouse model presents some drawbacks, mainly due to the severe liver injury induced by the uPA transgene expression and to mice hypofertility (Vaughan et al., 2012). These weaknesses, and the


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The authors gratefully acknowledge Sabrina Epiphanio and Cláudio Marinho for providing pictures used in this review, and Iset Vera, Ana Pamplona and Miguel Prudêncio for critical reading of the manuscript. V.Z.-L. is funded by FCT fellowship (SFRH/BPD/81953/2011). We apologize to all authors whose work was not cited due to constraints on the number of references.

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need for high-quality adult human hepatocytes for transplantation, make this model extremely costly and time-consuming, which drastically limits the number of experimental mice and consequently the strength of the conclusions. To overcome the above-mentioned weakness of the SCID/Alb-uPA liver injury model, an alternative mouse model of human hepatocyte engraftment was developed (Azuma et al., 2007; Bissig et al., 2007). In this new model, the liver injury is caused by the ablations of fumarylacetoacetate hydrolase (FAH) gene and mice are rescued from death by providing them 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). FAH−/− mice can be crossed with extremely immunocompromised mice, without B, T and NK cells (due to disruption of the Rag2 and the IL2Rγ genes). These FAH−/−Rag2−/− IL2Rγnull (FRG) mice can then be efficiently engrafted with human adult hepatocytes (Bissig et al., 2010). These mice breed efficiently, do not show liver injury, and have a long lifespan. Moreover, human hepatocytes from a donor FRG mouse can be transplanted into recipient mice, reducing the need of adult human hepatocytes (Azuma et al., 2007). There is no doubt that liver-humanized mice are an important tool to understand particular aspects of the human hepatic infection such the biology of the human parasite, its sensitivity to new drugs, and may be critical for future studies of Plasmodium hypnozoite forms. Importantly, not all aspects of liver infection can be addressed with these models, in particular the immune response to the parasite, either in primary infection or in vaccine studies. Moreover, the high cost of these models also hampers their regular use, thus rodent models of infection together with novel models of in vitro infection developed recently for P. falciparum and P. vivax infection (March et al., 2013; Ng et al., 2013) will always be critical to improve our still scarce knowledge of the liver stage of infection.

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