Murine Malaria Infection Research Articles

Monitoring Infected Reticulocytes During Murine Malaria Infection

Abstract Malaria infection is a major cause of mortality worldwide. According to the 2022 World Health Organization Malaria Report, malaria caused approximately 247 million infections and 619,000 deaths in 2021, with more than 470,000 children dying of malaria. The gold-standard for measuring parasitemia is counting infected red blood cells (RBCs) by eye using Giemsa staining whole blood smears. This method is time consuming and is subject to error by both who is counting, the number of cells counted, and which frames are selected for counting. Automating the process of measuring malaria infection by flow cytometry decreases both the time required to obtain accurate parasitemia counts and the possible error on behalf of the researcher. Hundreds of thousands of cells can be counted by flow cytometry in a shorter period of time. Animal models of malaria infection are widespread and well-characterized. The rodent malaria strain P. berghei has shown preferential infection of reticulocytes over mature RBCs. Here we outline a method of measuring P. berghei blood stage infection in reticulocytes and mature RBCs and comparing the results to Giemsa stained whole blood smears. Using commercially available P. berghei ANKA expressing green-fluorescent protein (GFP), we infected wild type mice with 1x106 parasitized RBCs by intraperitoneal injection. Whole blood samples were collected daily starting day 3 post infection through day 7 by tail vein collection. Random and representative fields of Giemsa stained peripheral smears were selected to measure the percentage of parasitemia in mature RBCs and reticulocytes for a total cell count of approximately 1000. For flow cytometry, whole blood was incubated in 500uL of 1X SYBR Green I Nucleic Acid Gel Stain in PBS at 37°C for 20 minutes in the dark, washed with PBS, and stained with anti-CD71 to label reticulocytes. During the early stages of infection, the reticulocyte percentage of infected animals is not significant from uninfected animals, approximately 1.86%. As parasitemia increases over time, although the percentage of total reticulocytes decreases, the percentage of infected reticulocytes increases. On day 3 through 7 for one experimental replicate, day 3 demonstrated 1.86% total reticulocytes, and of these 2.56% reticulocytes were infected; day 4 had 0.66% total reticulocytes, and of these 32.92% were infected; day 5 showed 0.18% reticulocytes with 71.81% infected reticulocytes; on day 6 there were 0.04% reticulocytes with 75.44% infected reticulocytes; and day 7 had 0.01% reticulocytes with 69.56% infected reticulocytes. There was a strong correlation (R2 = 0.9147) when comparing flow cytometry to measure parasitemia with counting parasitemia by peripheral smear. This flow cytometry panel is an excellent tool to assess malarial infection of reticulocytes compared to mature RBCs and can be applied to other malarial species while decreasing bias, error and time needed to conduct the experiment.

The deubiquitinating enzyme OTUD7b protects dendritic cells from TNF-induced apoptosis by stabilizing the E3 ligase TRAF2

The cytokine tumor necrosis factor (TNF) critically regulates the intertwined cell death and pro-inflammatory signaling pathways of dendritic cells (DCs) via ubiquitin modification of central effector molecules, but the intrinsic molecular switches deciding on either pathway are incompletely defined. Here, we uncover that the ovarian tumor deubiquitinating enzyme 7b (OTUD7b) prevents TNF-induced apoptosis of DCs in infection, resulting in efficient priming of pathogen-specific CD8+ T cells. Mechanistically, OTUD7b stabilizes the E3 ligase TNF-receptor-associated factor 2 (TRAF2) in human and murine DCs by counteracting its K48-ubiquitination and proteasomal degradation. TRAF2 in turn facilitates K63-linked polyubiquitination of RIPK1, which mediates activation of NF-κB and MAP kinases, IL-12 production, and expression of anti-apoptotic cFLIP and Bcl-xL. We show that mice with DC-specific OTUD7b-deficiency displayed DC apoptosis and a failure to induce CD8+ T cell-mediated brain pathology, experimental cerebral malaria, in a murine malaria infection model. Together, our data identify the deubiquitinating enzyme OTUD7b as a central molecular switch deciding on survival of human and murine DCs and provides a rationale to manipulate DC responses by targeting their ubiquitin network downstream of the TNF receptor pathway.

Parasite-Host Dynamics throughout Antimalarial Drug Development Stages Complicate the Translation of Parasite Clearance.

Ensuring continued success against malaria depends on a pipeline of new antimalarials. Antimalarial drug development utilizes preclinical murine and experimental human malaria infection studies to evaluate drug efficacy. A sequential approach is typically adapted, with results from each stage informing the design of the next stage of development. The validity of this approach depends on confidence that results from murine malarial studies predict the outcome of clinical trials in humans. Parasite clearance rates following treatment are key parameters of drug efficacy. To investigate the validity of forward predictions, we developed a suite of mathematical models to capture parasite growth and drug clearance along the drug development pathway and estimated parasite clearance rates. When comparing the three infection experiments, we identified different relationships of parasite clearance with dose and different maximum parasite clearance rates. In Plasmodium berghei-NMRI mouse infections, we estimated a maximum parasite clearance rate of 0.2 (1/h); in Plasmodium falciparum-SCID mouse infections, 0.05 (1/h); and in human volunteer infection studies with P. falciparum, we found a maximum parasite clearance rate of 0.12 (1/h) and 0.18 (1/h) after treatment with OZ439 and MMV048, respectively. Sensitivity analysis revealed that host-parasite driven processes account for up to 25% of variance in parasite clearance for medium-high doses of antimalarials. Although there are limitations in translating parasite clearance rates across these experiments, they provide insight into characterizing key parameters of drug action and dose response and assist in decision-making regarding dosage for further drug development.

A Conserved TCRβ Signature Dominates a Highly Polyclonal T-Cell Expansion During the Acute Phase of a Murine Malaria Infection.

CD4+ αβ T-cells are key mediators of the immune response to a first Plasmodium infection, undergoing extensive activation and splenic expansion during the acute phase of an infection. However, the clonality and clonal composition of this expansion has not previously been described. Using a comparative infection model, we sequenced the splenic CD4+ T-cell receptor repertoires generated over the time-course of a Plasmodium chabaudi infection. We show through repeat replicate experiments, single-cell RNA-seq, and analyses of independent RNA-seq data, that following a first infection - within a highly polyclonal expansion - T-effector repertoires are consistently dominated by TRBV3 gene usage. Clustering by sequence similarity, we find the same dominant clonal signature is expanded across replicates in the acute phase of an infection, revealing a conserved pathogen-specific T-cell response that is consistently a hallmark of a first infection, but not expanded upon re-challenge. Determining the host or parasite factors driving this conserved response may uncover novel immune targets for malaria therapeutic purposes.

Biosynthesized silver nanoparticles protect against hepatic injury induced by murine blood-stage malaria infection

Biosynthesized nanoparticles proposed to have antiplasmodial activities have attracted increasing attention for malaria that considered being one of the foremost hazardous diseases. In this study, Indigofera oblongifolia leaf extracts were used for the synthesis of silver nanoparticles (AgNPs), which were characterized utilizing transmission electron microscopy. We investigated the antiplasmodial and hepatoprotective effects of AgNPs against Plasmodium chabaudi-induced infection in mice. Treatment of the infected mice with 50mg/kg AgNPs for seven days caused a significant decrease in parasitemia and reduced the histopatholoical changes in the liver, as indicated by Ishak's histology index. Further, the AgNPs alleviated the oxidative damage in the liver infected with P. chabaudi. This was evidenced by the changed levels of malondialdehyde, nitric oxide, and glutathione, as well as increased catalase activity after treatment with AgNPs. In addition, levels of the liver enzymes alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase were increased after treatment. Moreover, the findings showed the efficiency of AgNPs in improving the infected mice's erythrocyte counts and hemoglobin content. Generally, our results reported that AgNPs possess antiplasmodial and hepatoprotective properties.

Splenic Innate B1 B Cell Plasmablasts Produce Sustained Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-3 Cytokines during Murine Malaria Infections.

The physiopathology of malaria, one of the most deadly human parasitic diseases worldwide, is complex, as it is a systemic disease involving multiple parasitic stages and hosts and leads to the activation of numerous immune cells and release of inflammatory mediators. While some cytokines increased in the blood of patients infected with Plasmodium falciparum have been extensively studied, others, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), have not received much attention. GM-CSF and IL-3 belong to the β common (βc/CD131) chain family of cytokines, which exhibit pleiotropic functions, including the regulation of myeloid cell growth, differentiation, and activation. GM-CSF can be secreted by multiple cell types, whereas IL-3 is mostly restricted to T cells, yet innate response activator (IRA) B cells, a subset of innate B1 B cells, also produce significant amounts of these cytokines during bacterial sepsis via Toll-like receptor 4 (TLR4)/MyD88 sensing of lipopolysaccharides. Herein, using murine models of malaria, we report a sustained production of GM-CSF and IL-3 from IgM+ and IgM-/IgG+ CD138+ Blimp-1+ innate B1b B cell plasmablasts. IgM+ B1b B cells include IRA-like and non-IRA B cells and express higher levels of both cytokines than do their IgG+ counterparts. Interestingly, as infection progresses, the relative proportion of IgM+ B1 B cells decreases while that of IgG+ plasmablasts increases, correlating with potential isotype switching of GM-CSF- and IL-3-producing IgM+ B1 B cells. GM-CSF/IL-3+ B1 B cells originate in the spleen of infected mice and are partially dependent on type I and type II interferon signaling to produce both cytokines. These data reveal that GM-CSF and IL-3 are produced during malaria infections, initially from IgM+ and then from IgG+ B1b B cell plasmablasts, which may represent important emergency cellular sources of these cytokines. These results further highlight the phenotypic heterogeneity of innate B1 B cell subsets and of their possible fates in a relevant murine model of parasitic infection in vivo.

Potential of Vitamin E Deficiency, Induced by Inhibition of α-Tocopherol Efflux, in Murine Malaria Infection.

Although epidemiological and experimental studies have suggested beneficial effects of vitamin E deficiency on malaria infection, it has not been clinically applicable for the treatment of malaria owing to the significant content of vitamin E in our daily food. However, since α-tocopherol transfer protein (α-TTP) has been shown to be a determinant of vitamin E level in circulation, manipulation of α-tocopherol levels by α-TTP inhibition was considered as a potential therapeutic strategy for malaria. Knockout studies in mice indicated that inhibition of α-TTP confers resistance against malaria infections in murines, accompanied by oxidative stress-induced DNA damage in the parasite, arising from vitamin E deficiency. Combination therapy with chloroquine and α-TTP inhibition significantly improved the survival rates in murines with malaria. Thus, clinical application of α-tocopherol deficiency could be possible, provided that α-tocopherol concentration in circulation is reduced. Probucol, a recently found drug, induced α-tocopherol deficiency in circulation and was effective against murine malaria. Currently, treatment of malaria relies on the artemisinin-based combination therapy (ACT); however, when mice infected with malarial parasites were treated with probucol and dihydroartemisinin, the beneficial effect of ACT was pronounced. Protective effects of vitamin E deficiency might be extended to manage other parasites in future.

TCRβ Combinatorial Immunoreceptor Expression by Neutrophils Correlates with Parasite Burden and Enhanced Phagocytosis during a Plasmodium berghei ANKA Malaria Infection.

Recent studies have demonstrated that a subpopulation of neutrophils express the TCRαβ combinatorial immunoreceptor in humans and mice. Here, we report that a Plasmodium berghei ANKA murine malaria infection induces expansion of TCRβ expressing CD11b+ Ly6G+ neutrophils in the spleen during the early phase of infection. Measurement of TCRβ transcript and protein levels of neutrophils in wild-type versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of nonspecific antibody staining or passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells. Remarkably, on day 3 postinfection, we observed a highly significant correlation between the proportion of neutrophils that express TCRβ and peripheral blood parasite burden. In addition, TCRβ+ neutrophils phagocytose parasitized erythrocytes with 4-fold greater efficiency than TCRβ- neutrophils. Together these results signify that TCR expression by the neutrophil plays an important role in the regulation of parasite burden by enhancing the phagocytic capacity of the neutrophil.

Role of Activins in Hepcidin Regulation during Malaria.

ABSTRACTEpidemiological observations have linked increased host iron with malaria susceptibility, and perturbed iron handling has been hypothesized to contribute to the potentially life-threatening anemia that may accompany blood-stage malaria infection. To improve our understanding of these relationships, we examined the pathways involved in regulation of the master controller of iron metabolism, the hormone hepcidin, in malaria infection. We show that hepcidin upregulation in Plasmodium berghei murine malaria infection was accompanied by changes in expression of bone morphogenetic protein (BMP)/sons of mothers against decapentaplegic (SMAD) pathway target genes, a key pathway involved in hepcidin regulation. We therefore investigated known agonists of the BMP/SMAD pathway and found that Bmp gene expression was not increased in infection. In contrast, activin B, which can signal through the BMP/SMAD pathway and has been associated with increased hepcidin during inflammation, was upregulated in the livers of Plasmodium berghei-infected mice; hepatic activin B was also upregulated at peak parasitemia during infection with Plasmodium chabaudi. Concentrations of the closely related protein activin A increased in parallel with hepcidin in serum from malaria-naive volunteers infected in controlled human malaria infection (CHMI) clinical trials. However, antibody-mediated neutralization of activin activity during murine malaria infection did not affect hepcidin expression, suggesting that these proteins do not stimulate hepcidin upregulation directly. In conclusion, we present evidence that the BMP/SMAD signaling pathway is perturbed in malaria infection but that activins, although raised in malaria infection, may not have a critical role in hepcidin upregulation in this setting.

Oxidative stress and genes regulation of cerebral malaria upon Zizyphus spina-christi treatment in a murine model.

The development and spread of multidrug-resistant strains of malarial parasites have led to an overwhelming increase in the resistance to current antimalarial drugs. The urgent need for alternative antimalarial drugs has directed some of the current studies toward folkloric medicine approaches. Interestingly, the Zizyphus spina Cristi leaf extract (ZLE) has been found to exhibit antiplasmodial activity. This study evaluated the protective effect of ZLE against Plasmodium berghei-induced cerebral tissue injuries in mice. Male C57Bl/6 mice received an injection of P.berghei-infected red blood cells. Mice were divided into three groups (control, infected, and ZLE-treated), and were subjected to histological, biochemical, and molecular analyses. Murine malaria infections induced significant weight loss; however, upon ZLE treatment, the weight of mice was markedly restored. Additionally, infected mice showed brain histopathological changes and induction of oxidative damage. Significantly, ZLE treatment restored the levels of oxidative markers and antioxidant enzyme to the normal ranges. The mRNA expression of several genes in the brain of mice including Cacnb4, Adam23, Glrb, Vdac3, and Cabp1 was significantly upregulated during P.berghei infection. In contrast, ZLE markedly reduced the mRNA expression of these genes. To conclude, the results indicate that ZLE could play an important role in reducing the destructive effect of P.berghei-induced cerebral malaria owing to its antiplasmodial and antioxidant activities.

A quantitative brain map of experimental cerebral malaria pathology.

The murine model of experimental cerebral malaria (ECM) has been utilised extensively in recent years to study the pathogenesis of human cerebral malaria (HCM). However, it has been proposed that the aetiologies of ECM and HCM are distinct, and, consequently, no useful mechanistic insights into the pathogenesis of HCM can be obtained from studying the ECM model. Therefore, in order to determine the similarities and differences in the pathology of ECM and HCM, we have performed the first spatial and quantitative histopathological assessment of the ECM syndrome. We demonstrate that the accumulation of parasitised red blood cells (pRBCs) in brain capillaries is a specific feature of ECM that is not observed during mild murine malaria infections. Critically, we show that individual pRBCs appear to occlude murine brain capillaries during ECM. As pRBC-mediated congestion of brain microvessels is a hallmark of HCM, this suggests that the impact of parasite accumulation on cerebral blood flow may ultimately be similar in mice and humans during ECM and HCM, respectively. Additionally, we demonstrate that cerebrovascular CD8+ T-cells appear to co-localise with accumulated pRBCs, an event that corresponds with development of widespread vascular leakage. As in HCM, we show that vascular leakage is not dependent on extensive vascular destruction. Instead, we show that vascular leakage is associated with alterations in transcellular and paracellular transport mechanisms. Finally, as in HCM, we observed axonal injury and demyelination in ECM adjacent to diverse vasculopathies. Collectively, our data therefore shows that, despite very different presentation, and apparently distinct mechanisms, of parasite accumulation, there appear to be a number of comparable features of cerebral pathology in mice and in humans during ECM and HCM, respectively. Thus, when used appropriately, the ECM model may be useful for studying specific pathological features of HCM.

Merozoite-Protein Loaded Liposomes Protect against Challenge in Two Murine Models of Plasmodium Infection.

Liposomes loaded with GPI-anchored proteins induce a strong and effective response against protozoan pathogens when immunized in mice. On the basis of results using Plasmodium falciparum merozoite proteins loaded on liposomes, we tested whether a similar approach would be protective in the P. yoelii XL and P. berghei NK65 models of lethal murine malaria infections. We also analyzed the enhancing capacity of additional adjuvants. As expected, merozoite protein loaded liposomes provided strong humoral responses (IgG titers of 105 against MSP119) in BALB/c and C57BL/6 host backgrounds and showed total protection in lethal challenges (100% survival) with P. yoelii, while protection against lethal challenge with P. berghei NK was at least partial (11%-20% in C57BL/6 mice and no protection in BALB/c). Thus, immunization with proteoliposomes is not only highly immunogenic but also provides a protective response in widely used murine models of malaria.

Plasmodium berghei bio-burden correlates with parasite lactate dehydrogenase: application to murine Plasmodium diagnostics.

BackgroundThe spectrum of techniques to detect malaria parasites in whole blood is limited to measuring parasites in circulation. One approach that is currently used to enumerate total parasite bio-burden involves the use of bio-luminescent parasites. As an alternative approach, this study describes the use of a commercial ELISA human parasite lactate dehydrogenase (pLDH) detection kit to estimate total parasite bio-burden in murine malaria models.MethodsThe cross reactivity of pLDH in a commercial human malaria pLDH diagnostic kit was established in different components of blood for different murine malaria models. The use of pLDH as a measure of parasite bio-burden was evaluated by examining pLDH in relation to peripheral blood parasitaemia as determined by microscopy and calculating total parasite bio-burden using a bio-luminescent Plasmodium berghei ANKA luciferase parasite.ResultsThe pLDH antigen was detected in all four murine Plasmodium species and in all components of Plasmodium-infected blood. A significant correlation (r = 0.6922, P value <0.0001) was observed between total parasite bio-burden, measured as log average radiance, and concentration of pLDH units.ConclusionsThis high throughput assay is a suitable measure of total parasite bio-burden in murine malaria infections. Unlike existing methods, it permits the estimation of both circulating and sequestered parasites, allowing a more accurate assessment of parasite bio-burden.

Effect of anti-hyperlipidemia drugs on the alpha-tocopherol concentration and their potential for murine malaria infection.

The current preventions of malaria are protection against mosquito bites and taking chemoprophylactic anti-malarial drugs. However, drug therapies are usually associated with adverse events and emergency of drug-resistant malaria parasites. Previous study showed that host plasma alpha-tocopherol deficiency enhanced resistance against malaria infection in mice. Here, we report a new prevention strategy against malaria by using anti-hyperlipidemia drugs, ezetimibe, berberine, cholestyramine, and probucol to modify the host plasma alpha-tocopherol concentration. The drugs were mixed with diet and fed to C57BL/6J mice for 2 weeks. Although all drugs reduced plasma alpha-tocopherol concentration after 2 weeks of feeding, probucol-treated mice showed 90 % reduction and it was the lowest alpha-tocopherol concentration among the four drugs. Ezetimibe, berberine, and combination of ezetimibe and berberine pretreatment for 2 weeks were not effective against infection of Plasmodium yoelii XL17, a lethal strain, for survival and parasitemia in mice. Two-week pretreatment and 1-week treatment after infection of cholestyramine had also no effect on malaria infection. Survival rates of cholestyramine, ezetimibe, and/or berberine treated mice were 0-22 %. However, probucol caused significant decrease in parasitemia and increased in mice survival following 2-week pretreatment and 1-week treatment after infection. All control mice died while all probucol treated mice survived during the course of infection. Thus, probucol which reduced plasma alpha-tocopherol concentration was effective in enhancing the host to resist malaria infection in mice. Our finding indicates that plasma alpha-tocopherol reducing drugs like probucol might be a candidate for beneficial prevention strategy for travelers from malaria-free area.

A Novel Role for Von Willebrand Factor in the Pathogenesis of Experimental Cerebral Malaria

Cerebral malaria (CM) is a major cause of death in young children in sub-Saharan Africa. Although the pathogenesis of this condition remains poorly understood, Plasmodium falciparum infection results in adhesion of infected erythrocytes (IE) to microvascular endothelium, and acute endothelial cell (EC) activation. Recent studies have shown that plasma VWF:Ag and VWF propeptide levels are markedly elevated in children with severe falciparum malaria. Moreover elevated VWF:Ag levels inversely correlate with clinical outcomes. In addition, circulating ultra-large VWF multimers have also been observed in children with severe malaria. Importantly, platelet-decorated UL-VWF strings have been shown to recruit trophozoite-stage falciparum-IE to EC surfaces in shear-based assays. Collectively, these emerging data suggest that VWF may play a novel role in the pathogenesis of CM. To further investigate this hypothesis, we utilized an established murine model of experimental CM which involves wild type (WT) C57BL/6J mice infected with Plasmodium berghei ANKA.In brief, WT C57Bl/6J mice were inoculated with 2x106P. berghei ANKA via I.P. injection. These mice typically developed CM and died within 6-8 days. In keeping with findings in children with falciparum malaria, acute EC activation was also an early and consistent feature in the murine model of CM. Mean plasma VWF:Ag levels were significantly elevated from Day+3 in infected mice compared to controls (Day +3 1.8 fold increase; Day +5 2.5 fold increase; p<0.01). In addition, plasma angiopoietin-2 and osteoprotegerin levels were also both significantly elevated from Day +3 and Day +5 respectively in mice infected with P. berghei. Interestingly, despite the fact that murine plasma ADAMTS13 activity levels were not significantly reduced, pathological ultra-large VWF multimers (UL-VWF) were also observed in murine plasma from Day +3 following P. berghei inoculation. Cumulatively these findings suggest that early marked elevation in VWF:Ag levels, and the appearance of pathological UL-VWF multimers in the plasma, represent hallmarks of both human and murine severe malaria infection.To determine whether VWF plays a direct role in modulating the pathogenesis of CM in vivo, we further investigated P. berghei infection in VWF-/- C57BL/6J mice. Importantly, although there was no difference in blood parasitaemia levels, overall survival was significantly prolonged in VWF-/- mice compared to wild type mice (6 versus 7.25 days; p=0.0106). Moreover, a significant delay in malaria clinical progression in the VWF-/- mice was also observed using a previously validated clinical scoring algorithm for experimental cerebral malaria. (Amante et al, Am J Path 2007). Recent studies have demonstrated that platelets may play a direct role in modulating malaria parasite killing. Consequently, to investigate potential mechanisms through which VWF-/- mice are protected against experimental CM, daily platelet counts were determined in WT and VWF-/- mice following infection. In keeping with observations in human patients infected with falciparum malaria, significant thrombocytopenia was also a consistent feature in the murine model. For example, by Day +4 following inoculation of WT mice mean platelet count had fallen by 71.6 ± 26 %. Interestingly however, significant thrombocytopenia was also observed in VWF-/- mice infected with P. berghei (84.2 ± 9 %). Despite the significant differences in clinical progression and overall survival no significant differences in platelet counts were observed between VWF-/- and WT mice at any time point. These findings suggest that VWF-/-mice are protected against CM through a platelet-independent mechanism.In conclusion, we demonstrate that early and consistent EC activation is a feature of the murine model of experimental CM similar to previous findings of children infected with P. falciparum. In addition, our findings show that marked elevation of VWF:Ag, and the appearance of pathological UL-VWF multimers in the plasma, both represent hallmarks of human and murine severe malaria infection. Finally, we report that VWF-/- mice exhibit significantly prolonged survival against CM, and that this effect is mediated through a platelet-independent mechanism. Given the significant morbidity and mortality associated with CM, these novel data have direct translational significance. DisclosuresNo relevant conflicts of interest to declare.

Alpha-tocopherol transfer protein gene inhibition enhances the acquired immune response during malaria infection in mice

Immune response to malaria infection is complex and seems to be regulated by innate and adaptive immune response as well as environmental factors such as host genetics and nutritional status. Previously, we have reported that α-tocopherol transfer protein knockout (α-ttp(Δ)) mice, showing low concentrations of α-tocopherol in circulation, infected with Plasmodium berghei NK65 survived significantly longer as compared with the wild-type mice. In addition, Plasmodium yoelii XL-17, a lethal strain, showed non-lethal virulence in α-ttp(Δ) mice. Thus, we hypothesized that the ability of the α-ttp(Δ) mice to control P. yoelli XL-17 proliferation may allow them to build an efficient immune response against murine malaria infection. On 15 days after infection with P. yoelli XL-17, α-ttp(Δ) mice were challenged to infection with P. berghei NK65. Results indicated that α-ttp(Δ) mice infected with P. yoelli XL-17 built a protective immunity against P. berghei NK65 associated to extremely low levels of parasitemia, a controlled inflammatory response, and a robust antibody response. Moreover, the importance of α-tocopherol for parasite proliferation was remarkable. The results suggest that inhibition of α-tocopherol transfer protein activity is effective for the enhancement of acquired immunity in murine malaria infection.

Allicin enhances host pro-inflammatory immune responses and protects against acute murine malaria infection

BackgroundDuring malaria infection, multiple pro-inflammatory mediators including IFN-γ, TNF and nitric oxide (NO) play a crucial role in the protection against the parasites. Modulation of host immunity is an important strategy to improve the outcome of malaria infection. Allicin is the major biologically active component of garlic and shows anti-microbial activity. Allicin is also active against protozoan parasites including Plasmodium, which is thought to be mediated by inhibiting cysteine proteases. In this study, the immunomodulatory activities of allicin were assessed during acute malaria infection using a rodent malaria model Plasmodium yoelii 17XL.MethodsTo determine whether allicin modulates host immune responses against malaria infection, mice were treated with allicin after infection with P. yoelii 17XL. Mortality was checked daily and parasitaemia was determined every other day. Pro-inflammatory mediators and IL-4 were quantified by ELISA, while NO level was determined by the Griess method. The populations of dendritic cells (DCs), macrophages, CD4+ T and regulatory T cells (Treg) were assessed by FACS.ResultsAllicin reduced parasitaemia and prolonged survival of the host in a dose-dependent manner. This effect is at least partially due to improved host immune responses. Results showed that allicin treatment enhanced the production of pro-inflammatory mediators such as IFN-γ, TNF, IL-12p70 and NO. The absolute numbers of CD4+ T cells, DCs and macrophages were significantly higher in allicin-treated mice. In addition, allicin promoted the maturation of CD11c+ DCs, whereas it did not cause major changes in IL-4 and the level of anti-inflammatory cytokine IL-10.ConclusionsAllicin could partially protect host against P. yoelii 17XL through enhancement of the host innate and adaptive immune responses.

Induction of ssDNA-binding autoantibody secreting B cell immunity during murine malaria infection is a critical part of the protective immune responses

Although it has been hypothesized that autoimmune-like phenomena may play a critical role in the protective immune responses to both human and animal malaria, there are still no evidence-based data to support this view. In this study we demonstrate that the majority of anti-single stranded (ss) DNA autoantibody secreting B cells were confined to B220+CD21+CD23− cells and that these cells expanded significantly in the spleen of C57BL/6 mice infected with Plasmodium yoelii 17X non-lethal (PyNL). To determine the role of ssDNA-binding autoantibody secreting B cell responses in murine malaria, we conjugated generation 6 (poly) amidoamine dendrimer nanoparticles with ssDNA to deplete ssDNA-binding autoreactive B cells in vivo. Our data revealed that 55.5% of mice died after DNA-coated nanoparticle-mediated in vivo depletion of ssDNA-specific autoreactive B cells and subsequent challenge using PyNL. Adoptive transfer of B cells with ssDNA specificity to mice, followed by PyNL infection, caused a later appearance and inhibition of parasitemia. The possible mechanism by which the ssDNA-binding autoantibody secreting B cells is involved in the protection against murine malaria has also been demonstrated.

N-acetylglucosaminyltransferase V-deficiency increases susceptibility to murine malaria

It is considered that several glycoproteins on erythrocytes in mammalian species are involved in malaria parasite infection. To elucidate the role of N-glycans on malaria parasite infection, we induced experimental murine malaria infection (using Plasmodium berghei ANKA) in mice deficient in N-acetylglucosaminyltransferase V (Mgat5), which is one of the enzymes involved in β1,6-GlcNAc N-glycan biosynthesis. After infection, Mgat5-/- mice showed severe body weight loss and parasitemia compared with wild-type mice. The Mgat5-/- mice, but not wild-type mice, also showed severe pathology accompanied by marked infiltration of plasma cells into the lungs and liver. These results suggest that β1,6-GlcNAc N-glycans on/in host erythrocytes may interfere with invasion of the parasites and progression to severe malaria.

Expression of erythropoietic cytokines in α-tocopherol transfer protein knockout mice with murine malaria infection

Malaria infection leads to anemia in humans which generally occurs during the chronic phase of the infection. The role that erythropoietic molecules play for anemia during malaria at low parasitemia levels is still controversial due to the lack of suitable animal models which might mimic this condition. In this regard, α-tocopherol transfer protein knockout mice, with undetectable levels of vitamin E in circulation, were possibly used as a model to investigate the role that erythropoietic molecules such as erythropoietin (EPO), erythropoietin receptor (EPOR), and macrophage migration inhibitory factor (MIF) play on the outcome of anemia during uncomplicated malaria infection at low parasitemias. The results indicate that the degree of parasitemia unlikely plays any important effect on mRNA expression of EPO and EPOR in different organs. Moreover, even though EPO and EPOR productions are impaired in the kidney and bone marrow, respectively, other organs such as the liver and spleen intend to compensate production of these cytokines to prevent anemia in the infected animals.