The plasma membrane calcium ATPase 4 does not influence parasite levels but partially promotes experimental cerebral malaria during murine blood stage malaria.

BACKGROUND: Recent genome wide analysis studies have identified a strong association between single nucleotide variations within the human ATP2B4 gene and susceptibility to severe malaria. The ATP2B4 gene encodes the plasma membrane calcium ATPase 4 (PMCA4), which is responsible for controlling the physiological level of intracellular calcium in many cell types, including red blood cells (RBCs). It is, therefore, postulated that genetic differences in the activity or expression level of PMCA4 alters intracellular Ca2+ levels and affects RBC hydration, modulating the invasion and growth of the Plasmodium parasite within its target host cell. METHODS: In this study the course of three different Plasmodium spp. infections were examined in mice with systemic knockout of Pmca4 expression. RESULTS: Ablation of PMCA4 reduced the size of RBCs and their haemoglobin content but did not affect RBC maturation and reticulocyte count. Surprisingly, knockout of PMCA4 did not significantly alter peripheral parasite burdens or the dynamics of blood stage Plasmodium chabaudi infection or reticulocyte-restricted Plasmodium yoelii infection. Interestingly, although ablation of PMCA4 did not affect peripheral parasite levels during Plasmodium berghei infection, it did promote slight protection against experimental cerebral malaria, associated with a minor reduction in antigen-experienced T cell accumulation in the brain. CONCLUSIONS: The finding suggests that PMCA4 may play a minor role in the development of severe malarial complications, but that this appears independent of direct effects on parasite invasion, growth or survival within RBCs.

Temporospatial shifts within commercial laboratory mouse gut microbiota impact experimental reproducibility.

BACKGROUND: Experimental reproducibility in mouse models is impacted by both genetics and environment. The generation of reproducible data is critical for the biomedical enterprise and has become a major concern for the scientific community and funding agencies alike. Among the factors that impact reproducibility in experimental mouse models is the variable composition of the microbiota in mice supplied by different commercial vendors. Less attention has been paid to how the microbiota of mice supplied by a particular vendor might change over time. RESULTS: In the course of conducting a series of experiments in a mouse model of malaria, we observed a profound and lasting change in the severity of malaria in mice infected with Plasmodium yoelii; while for several years mice obtained from a specific production suite of a specific commercial vendor were able to clear the parasites effectively in a relatively short time, mice subsequently shipped from the same unit suffered much more severe disease. Gut microbiota analysis of frozen cecal samples identified a distinct and lasting shift in bacteria populations that coincided with the altered response of the later shipments of mice to infection with malaria parasites. Germ-free mice colonized with cecal microbiota from mice within the same production suite before and after this change followed by Plasmodium infection provided a direct demonstration that the change in gut microbiota profoundly impacted the severity of malaria. Moreover, spatial changes in gut microbiota composition were also shown to alter the acute bacterial burden following Salmonella infection, and tumor burden in a lung tumorigenesis model. CONCLUSION: These changes in gut bacteria may have impacted the experimental reproducibility of diverse research groups and highlight the need for both laboratory animal providers and researchers to collaborate in determining the methods and criteria needed to stabilize the gut microbiota of animal breeding colonies and research cohorts, and to develop a microbiota solution to increase experimental rigor and reproducibility.

Different doses of vitamin C supplementation enhances the Th1 immune response to early Plasmodium yoelii 17XL infection in BALB/c mice.

Vitamin C (ascorbate) is maintained at high levels in most immune cells and can affect many aspects of the immune response. Here, we evaluated the effect of vitamin C supplementation on the immune response to Plasmodium yoelii 17XL (P. yoelii 17XL) infection in BALB/c mice. Two orally administered doses (25 mg/kg/day and 250 mg/kg/day) of vitamin C significantly reduced levels of parasitemia during the early stages of P. yoelii 17XL infection. The numbers of activated Th1 cells and macrophages in the groups receiving vitamin C supplementation were both higher than those in the untreated group. Meanwhile, vitamin C administration reduced the levels of tumor necrosis factor α secreted by splenocytes. Vitamin C also regulated the protective anti-malarial immune response by increasing the number of plasmacytoid dendritic cells, as well as the expression of dendritic cell maturation markers, such as major histocompatibility complex class II and cluster of differentiation 86. In conclusion, the doses of vitamin C (25 mg/kg/day, 250 mg/kg/day) during the early stages of malaria infection may better enhance host protective immunity, but have no dose dependence.

Detection of host pathways universally inhibited after Plasmodium yoelii infection for immune intervention.

Malaria is a disease with diverse symptoms depending on host immune status and pathogenicity of Plasmodium parasites. The continuous parasite growth within a host suggests mechanisms of immune evasion by the parasite and/or immune inhibition in response to infection. To identify pathways commonly inhibited after malaria infection, we infected C57BL/6 mice with four Plasmodium yoelii strains causing different disease phenotypes and 24 progeny of a genetic cross. mRNAs from mouse spleens day 1 and/or day 4 post infection (p.i.) were hybridized to a mouse microarray to identify activated or inhibited pathways, upstream regulators, and host genes playing an important role in malaria infection. Strong interferon responses were observed after infection with the N67 strain, whereas initial inhibition and later activation of hematopoietic pathways were found after infection with 17XNL parasite, showing unique responses to individual parasite strains. Inhibitions of pathways such as Th1 activation, dendritic cell (DC) maturation, and NFAT immune regulation were observed in mice infected with all the parasite strains day 4 p.i., suggesting universally inhibited immune pathways. As a proof of principle, treatment of N67-infected mice with antibodies against T cell receptors OX40 or CD28 to activate the inhibited pathways enhanced host survival. Controlled activation of these pathways may provide important strategies for better disease management and for developing an effective vaccine.

α-Tocopheryl succinate-suppressed development of cerebral malaria in mice.

α-Tocopheryl succinate (α-TOS), a derivative of vitamin E, is synthesized by esterification of α-tocopherol. It has been reported that α-TOS inhibits the mitochondrial complex II resulting in generation of reactive oxygen species, which triggers selective apoptosis in a large number of cancer cells, while it appears largely non-toxic towards normal cells. Plasmodium parasites are well known to have high sensitivity to oxidative stress. Thus, α-TOS is suspected to impact Plasmodium parasites by oxidative stress. In this study, to ascertain whether α-TOS is an appropriate candidate for an anti-malarial drug, C57BL/6J mice were infected with P. yoelii 17XL and P. berghei ANKA, a lethal strain of rodent malaria and experimental cerebral malaria (ECM), and treated with several concentrations of α-TOS by intraperitoneal administration on 1, 3, 5, and 7 days post infection (dpi). In addition, the permeability of the blood brain barrier (BBB) was examined by Evans blue staining in ECM on 7 dpi. As a result of α-TOS treatment, parasitemia was decreased and survival rate was significantly increased in mice infected with both parasites. Furthermore, the intensity of Evans blue staining on brains taken from α-TOS-treated mice was weaker than that of untreated mice. This means that α-TOS might inhibit the breakdown of BBB and progress of cerebral malaria. These findings indicate that vitamin E derivatives like α-TOS might be a potential candidate for treatment drugs against malaria.

Plasmodium yoelii infection inhibits murine leukaemia WEHI-3 cell proliferation in vivo by promoting immune responses.

BACKGROUND: Leukaemia is a malignant leukocyte disorder with a high fatality rate, and current treatments for this disease are unsatisfactory. Therefore, new therapeutic strategies for leukaemia must be developed. Malaria parasite infection has been shown to be effective at combating certain neoplasms in animal experiments. This study is to demonstrate the anti-leukaemia activity of malaria parasite Plasmodium yoelii (P. yoelii) infection,. METHODS: In this study, the proportion of CD3, CD19, CD11b and Mac-3 cells was analysed by flow cytometry; the levels of IFN-γ and TNF-α in individual serum samples were measured by enzyme-linked immunosorbent assay, and the phagocytic activity of macrophages and natural killer (NK) cell activity were measured by flow cytometry. RESULTS: We found that P. yoelii infection significantly attenuated the growth of WEHI-3 cells in mice. In addition, tumor cell infiltration into the murine liver and spleen was markedly reduced. We also demonstrated that malaria parasite infection elicited anti-leukaemia activity by promoting immune responses, including increasing the surface markers of T cells (CD3) and B cells (CD19); decreasing the surface markers of monocytes (CD11b) and macrophages (Mac-3); inducing the secretion of IFN-γ and TNF-α; and increasing NK cell and macrophage activity. CONCLUSIONS: Malaria parasite infection significantly decreases the number of myeloblasts and inhibits neoplasm proliferation in mice. In addition, malaria parasite infection inhibits murine leukaemia by promoting immune responses.

Identifying host regulators and inhibitors of liver stage malaria infection using kinase activity profiles.

Plasmodium parasites have extensive needs from their host hepatocytes during the obligate liver stage of infection, yet there remains sparse knowledge of specific host regulators. Here we assess 34 host-targeted kinase inhibitors for their capacity to eliminate Plasmodium yoelii-infected hepatocytes. Using pre-existing activity profiles of each inhibitor, we generate a predictive computational model that identifies host kinases, which facilitate Plasmodium yoelii liver stage infection. We predict 47 kinases, including novel and previously described kinases that impact infection. The impact of a subset of kinases is experimentally validated, including Receptor Tyrosine Kinases, members of the MAP Kinase cascade, and WEE1. Our approach also predicts host-targeted kinase inhibitors of infection, including compounds already used in humans. Three of these compounds, VX-680, Roscovitine and Sunitinib, each eliminate >85% of infection. Our approach is well-suited to uncover key host determinants of infection in difficult model systems, including field-isolated parasites and/or emerging pathogens.

Mechanism of splenic cell death and host mortality in a Plasmodium yoelii malaria model.

Malaria is a fatal disease that displays a spectrum of symptoms and severity, which are determined by complex host-parasite interactions. It has been difficult to study the effects of parasite strains on disease severity in human infections, but the mechanisms leading to specific disease phenotypes can be investigated using strains of rodent malaria parasites that cause different disease symptoms in inbred mice. Using a unique mouse malaria model, here we investigated the mechanisms of splenic cell death and their relationship to control of parasitemia and host mortality. C57BL/6 mice infected with Plasmodium yoelii nigeriensis N67C display high levels of pro-inflammatory cytokines and chemokines (IL-6, IFN-γ, TNF-α, CXCL1, and CCL2) and extensive splenic damage with dramatic reduction of splenic cell populations. These disease phenotypes were rescued in RAG2-/-, IFN-γ-/-, or T cell depleted mice, suggesting IFN-γ and T cell mediated disease mechanisms. Additionally, apoptosis was one of the major pathways involved in splenic cell death, which coincides with the peaks of pro-inflammatory cytokines. Our results demonstrate the critical roles of T cells and IFN-γ in mediating splenic cell apoptosis, parasitemia control, and host lethality and thus may provide important insights for preventing/reducing morbidity associated with severe malaria in humans.

The rodent malaria liver stage survives in the rapamycin-induced autophagosome of infected Hepa1-6 cells.

It has been reported that non-selective autophagy of infected hepatocytes could facilitate the development of malaria in the liver stage, but the fate of parasites following selective autophagy of infected hepatocytes is still not very clear. Here, we confirmed that sporozoite infection can induce a selective autophagy-like process targeting EEFs (exo-erythrocytic forms) in Hepa1-6. Rapamycin treatment greatly enhanced this process in EEFs and non-selective autophagy of infected Hepa1-6 cells and enhanced the development of the malaria liver stage in vivo. Although rapamycin promoted the fusion of autophagosomes containing the malaria parasite with lysosomes, some parasites inside the autophagosome survived and replicated normally. Further study showed that the maturation of affected autolysosomes was greatly inhibited. Therefore, in addition to the previously described positive role of rapamycin-induced nonselective autophagy of hepatocytes, we provide evidence that the survival of EEFs in the autophagosome of the infected hepatocytes also contributes to rapamycin-enhanced development of the malaria liver stage, possibly due to the suppression of autolysosome maturation by EEFs. These data suggest that the inhibition of autolysosome maturation might be a novel escape strategy used by the malaria liver stage.

An Atypical Splenic B Cell Progenitor Population Supports Antibody Production during Plasmodium Infection in Mice.

Hematopoietic stem and progenitor cells (HSPCs) function to replenish the immune cell repertoire under steady-state conditions and in response to inflammation due to infection or stress. Whereas the bone marrow serves as the primary niche for hematopoiesis, extramedullary mobilization and differentiation of HSPCs occur in the spleen during acute Plasmodium infection, a critical step in the host immune response. In this study, we identified an atypical HSPC population in the spleen of C57BL/6 mice, with a lineage(-)Sca-1(+)c-Kit(-) (LSK(-)) phenotype that proliferates in response to infection with nonlethal Plasmodium yoelii 17X. Infection-derived LSK(-) cells upon transfer into naive congenic mice were found to differentiate predominantly into mature follicular B cells. However, when transferred into infection-matched hosts, infection-derived LSK(-) cells gave rise to B cells capable of entering into a germinal center reaction, and they developed into memory B cells and Ab-secreting cells that were capable of producing parasite-specific Abs. Differentiation of LSK(-) cells into B cells in vitro was enhanced in the presence of parasitized RBC lysate, suggesting that LSK(-) cells expand and differentiate in direct response to the parasite. However, the ability of LSK(-) cells to differentiate into B cells was not dependent on MyD88, as myd88(-/-) LSK(-) cell expansion and differentiation remained unaffected after Plasmodium infection. Collectively, these data identify a population of atypical lymphoid progenitors that differentiate into B lymphocytes in the spleen and are capable of contributing to the ongoing humoral immune response against Plasmodium infection.

Co-administration of α-GalCer analog and TLR4 agonist induces robust CD8(+) T-cell responses to PyCS protein and WT-1 antigen and activates memory-like effector NKT cells.

In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8(+) T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8(+) T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii, and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immunization regimens were able to induce higher levels of CD8(+) T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44(+)CD62L(-)NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation.

Dendritic cells subsets mediated immune response during Plasmodium berghei ANKA and Plasmodium yoelii infection.

The roles of dendritic cells (DCs) in mediating immunity against Plasmodium infection have been extensively investigated, but immune response during pathogenesis of malaria is still poorly understood. In the present study, we compared the splenic DCs phenotype and function during P. berghei ANKA (PbA) or P. yoelii (P. yoelii) infection in Swiss mice. We observed that PbA-infected mice developed more myeloid and mature DCs capable of secreting IL-12, while P. yoelii-infected mice had more plasmacytoid and immature DCs secreting higher levels of IL-10. Expression of FoxP3, IL-17, TGF-ß and IL-6 were also different between these two infections. Thus, these results suggest that the phenotypic and functional subsets of splenic DCs are key factors for regulating immune responses to PbA and P. yoelii infections.

Cytotoxic activities of CD8⁺ T cells collaborate with macrophages to protect against blood-stage murine malaria.

The protective immunity afforded by CD8(+) T cells against blood-stage malaria remains controversial because no MHC class I molecules are displayed on parasite-infected human erythrocytes. We recently reported that rodent malaria parasites infect erythroblasts that express major histocompatibility complex (MHC) class I antigens, which are recognized by CD8(+) T cells. In this study, we demonstrate that the cytotoxic activity of CD8(+) T cells contributes to the protection of mice against blood-stage malaria in a Fas ligand (FasL)-dependent manner. Erythroblasts infected with malarial parasites express the death receptor Fas. CD8(+) T cells induce the externalization of phosphatidylserine (PS) on the infected erythroblasts in a cell-to-cell contact-dependent manner. PS enhances the engulfment of the infected erythroid cells by phagocytes. As a PS receptor, T-cell immunoglobulin-domain and mucin-domain-containing molecule 4 (Tim-4) contributes to the phagocytosis of malaria-parasite-infected cells. Our findings provide insight into the molecular mechanisms underlying the protective immunity exerted by CD8(+) T cells in collaboration with phagocytes.

CD47 regulates the phagocytic clearance and replication of the Plasmodium yoelii malaria parasite.

Several Plasmodium species exhibit a strong age-based preference for the red blood cells (RBC) they infect, which in turn is a major determinant of disease severity and pathogenesis. The molecular basis underlying this age constraint on the use of RBC and its influence on parasite burden is poorly understood. CD47 is a marker of self on most cells, including RBC, which, in conjunction with signal regulatory protein alpha (expressed on macrophages), prevents the clearance of cells by the immune system. In this report, we have investigated the role of CD47 on the growth and survival of nonlethal Plasmodium yoelii 17XNL (PyNL) malaria in C57BL/6 mice. By using a quantitative biotin-labeling procedure and a GFP-expressing parasite, we demonstrate that PyNL parasites preferentially infect high levels of CD47 (CD47(hi))-expressing young RBC. Importantly, C57BL/6 CD47(-/-) mice were highly resistant to PyNL infection and developed a 9.3-fold lower peak parasitemia than their wild-type (WT) counterparts. The enhanced resistance to malaria observed in CD47(-/-) mice was associated with a higher percentage of splenic F4/80(+) cells, and these cells had a higher percentage of phagocytized parasitized RBC than infected WT mice during the acute phase of infection, when parasitemia was rapidly rising. Furthermore, injection of CD47-neutralizing antibody caused a significant reduction in parasite burden in WT C57BL/6 mice. Together, these results strongly suggest that CD47(hi) young RBC may provide a shield to the malaria parasite from clearance by the phagocytic cells, which may be an immune escape mechanism used by Plasmodium parasites that preferentially infect young RBC.

Immunization of mice with Plasmodium TCTP delays establishment of Plasmodium infection.

Translationally controlled tumour protein (TCTP) may play an important role in the establishment or maintenance of parasitemia in a malarial infection. In this study, the potential of TCTP as a malaria vaccine was investigated in two trials. In the initial vaccine trial, Plasmodium falciparum TCTP (PfTCTP) was expressed in Saccharomyces cerevisiae and used to immunize BALB/c mice. Following challenge with Plasmodium yoelii YM, parasitemia was significantly reduced during the early stages of infection. In the second vaccine trial, the TCTP from P. yoelii and P. berghei was expressed in Escherichia coli and used in several mouse malaria models. A significant reduction in parasitemia in the early stages of infection was observed in BALB/c mice challenged with P. yoelii YM. A significantly reduced parasitemia at each day leading up to a delayed and reduced peak parasitemia was also observed in BALB/c mice challenged with the nonlethal Plasmodium chabaudi (P.c.) chabaudi AS. These results suggest that TCTP has an important role for parasite establishment and may be important for pathogenesis.

Susceptibility to Plasmodium yoelii preerythrocytic infection in BALB/c substrains is determined at the point of hepatocyte invasion.

After transmission by Anopheles mosquitoes, Plasmodium sporozoites travel to the liver, infect hepatocytes, and rapidly develop as intrahepatocytic liver stages (LS). Rodent models of malaria exhibit large differences in the magnitude of liver infection, both between parasite species and between strains of mice. This has been mainly attributed to differences in innate immune responses and parasite infectivity. Here, we report that BALB/cByJ mice are more susceptible to Plasmodium yoelii preerythrocytic infection than BALB/cJ mice. This difference occurs at the level of early hepatocyte infection, but expression levels of reported host factors that are involved in infection do not correlate with susceptibility. Interestingly, BALB/cByJ hepatocytes are more frequently polyploid; thus, their susceptibility converges on the previously observed preference of sporozoites to infect polyploid hepatocytes. Gene expression analysis demonstrates hepatocyte-specific differences in mRNA abundance for numerous genes between BALB/cByJ and BALB/cJ mice, some of which encode hepatocyte surface molecules. These data suggest that a yet-unknown receptor for sporozoite infection, present at elevated levels on BALB/cByJ hepatocytes and also polyploid hepatocytes, might facilitate Plasmodium liver infection.

Co-infection restrains Litomosoides sigmodontis filarial load and plasmodial P. yoelii but not P. chabaudi parasitaemia in mice.

Infection with multiple parasite species is clearly the norm rather than the exception, in animals as well as in humans. Filarial nematodes and Plasmodium spp. are important parasites in human public health and they are often co-endemic. Interactions between these parasites are complex. The mechanisms underlying the modulation of both the course of malaria and the outcome of filarial infection are poorly understood. Despite increasing activity in recent years, studies comparing co- and mono-infections are very much in their infancy and results are contradictory at first sight. In this study we performed controlled and simultaneous co-infections of BALB/c mice with Litomosoides sigmodontis filaria and with Plasmodium spp. (Plasmodium yoelii 17 XNL or Plasmodium chabaudi 864VD). An analysis of pathological lesions in the kidneys and lungs and a parasitological study were conducted at different times of infection. Whatever the plasmodial species, the filarial recovery rate was strongly decreased. The peak of parasitaemia in the plasmodial infection was decreased in the course of P. yoelii infection but not in that of P. chabaudi. Regarding pathological lesions, L. sigmodontis can reverse lesions in the kidneys due to the presence of both Plasmodium species but does not modify the course of pulmonary lesions. The filarial infection induces granulomas in the lungs.

Strain-specific innate immune signaling pathways determine malaria parasitemia dynamics and host mortality.

Malaria infection triggers vigorous host immune responses; however, the parasite ligands, host receptors, and the signaling pathways responsible for these reactions remain unknown or controversial. Malaria parasites primarily reside within RBCs, thereby hiding themselves from direct contact and recognition by host immune cells. Host responses to malaria infection are very different from those elicited by bacterial and viral infections and the host receptors recognizing parasite ligands have been elusive. Here we investigated mouse genome-wide transcriptional responses to infections with two strains of Plasmodium yoelii (N67 and N67C) and discovered differences in innate response pathways corresponding to strain-specific disease phenotypes. Using in vitro RNAi-based gene knockdown and KO mice, we demonstrated that a strong type I IFN (IFN-I) response triggered by RNA polymerase III and melanoma differentiation-associated protein 5, not Toll-like receptors (TLRs), binding of parasite DNA/RNA contributed to a decline of parasitemia in N67-infected mice. We showed that conventional dendritic cells were the major sources of early IFN-I, and that surface expression of phosphatidylserine on infected RBCs might promote their phagocytic uptake, leading to the release of parasite ligands and the IFN-I response in N67 infection. In contrast, an elevated inflammatory response mediated by CD14/TLR and p38 signaling played a role in disease severity and early host death in N67C-infected mice. In addition to identifying cytosolic DNA/RNA sensors and signaling pathways previously unrecognized in malaria infection, our study demonstrates the importance of parasite genetic backgrounds in malaria pathology and provides important information for studying human malaria pathogenesis.

Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Elevated levels of the Plasmodium yoelii homologue of macrophage migration inhibitory factor attenuate blood-stage malaria.

The excessive production of proinflammatory cytokines plays a significant role in the pathogenesis of severe malaria. Mammalian macrophage migration inhibitory factor (MIF) (mMIF) is an immune mediator that promotes a sustained proinflammatory response by inhibiting the glucocorticoid-mediated downregulation of inflammation. In addition, Plasmodium parasites also encode a homologue of mammalian MIF that is expressed in asexual-stage parasites. We used the Plasmodium yoelii murine model to study the potential role of parasite-encoded MIF in the pathogenesis of malaria. Antibodies raised against purified, non-epitope-tagged P. yoelii MIF (PyMIF) were used to localize expression in trophozoite- and schizont-stage parasites and demonstrate extracellular release. In vitro, recombinant PyMIF was shown to actively induce the chemotaxis of macrophages but did not induce or enhance tumor necrosis factor alpha (TNF-α) production from peritoneal macrophages. To examine the role of parasite-derived PyMIF in vivo, two transgenic parasite lines that constitutively overexpress PyMIF were generated, one in a nonlethal P. yoelii 17X background [Py17X-MIF(+)] and the other in a lethal P. yoelii 17XL background [Py17XL-MIF(+)]. Challenge studies with transgenic parasites in mice showed that the increased expression of PyMIF resulted in a reduction in disease severity. Mice infected with Py17X-MIF(+) developed lower peak parasitemia levels than controls, while malaria-associated anemia was unaltered. Infection with Py17XL-MIF(+) resulted in a prolonged course of infection and a reduction in the overall mortality rate. Combined, the data indicate that parasite-derived MIF does not contribute significantly to immunopathology but, through its chemotactic ability toward macrophages, may attenuate disease and prolong infection of highly virulent parasite isolates.