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Recently, there is a paucity of studies focus on the characteristics of myeloid cells which expressed γδTCR. The aim of this study was to observe the properties of γδTCR-expressing myeloid cells in the spleen of C57BL/6 mice infected by P. yoelii nigeriensis NSM. Haematoxylin-eosin (HE) staining was used to observe pathological changes in the spleens from infected mice. The differentially expressed genes (DEGs) between the infection and control groups were analyzed by RNA sequencing (RNA -seq). Flow cytometry (FCM) was used to evaluate the frequency of γδTCR + cells and the characteristics of γδTCR + cells in P. yoelii nigeriensis NSM-infected mice. Obvious infiltration of inflammatory were observed in the spleens from infected C57BL/6 mouse. The proportions of γδTCR + cells and CD11b + γδTCR + cells from infected group were higher than that from normal group. CD11b + γδTCR + cells expressed high levels of activated-mediated genes and inflammatory-mediated genes. The heterogeneous pathway activities among CD11b + γδTCR + cells from normal and infected group were characterized. The oxidative phosphorylation, respiratory electron transport chain and leukocyte activation involved in immune response pathways were up-regulated, while the alpha-beta T cell activation and myeloid leukocyte migration pathways were down-regulated in infected mice. Importantly, Ly6c2 was higher expressed in CD11b + γδTCR + cells than Ly6g. Consistent with it, flow cytometry results revealed that a subset of Ly6C + cells was higher than Ly6G + cells in the spleen. Taken together, our data suggest the existence of a population of γδTCR-expressing myeloid cells and they might be multifunctional cells, which play a role in couse of Plasmodium infection., Competing Interests: Competing interests The authors declare that they have no competing interests., (Copyright © 2022. Published by Elsevier B.V.)

Increased levels of several human ubiquitin ligases, including ring finger protein 123 (RNF123), in red blood cells with Plasmodium falciparum infection, have been reported. RNF123 is an E3 ubiquitin ligase that is highly expressed in erythroid cells. However, the function of the RNF123 gene and the relationship between the RNF123 gene and malarial parasite has not been clarified in vivo. In this study, we generated RNF123-deficient mice using the CRISPR/Cas9 system, and analyzed malaria susceptibility and erythrocyte morphology. The levels of parasitemia 5 days post-infection and mortality 21 days post-infection with the lethal type of rodent malaria (Plasmodium yoelii 17XL) in RNF123-deficient mice was significantly lower than that in wild-type mice. In contrast, red blood cell morphology in RNF123-deficient mice was almost normal. These results suggest that erythrocytic RNF123 plays a role in susceptibility to rodent malaria infection, but does not play a role in erythrocyte morphology., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 Ca 2+ 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.

Background: Malaria has high morbidity and mortality rates in some parts of tropical and subtropical countries. Besides respiratory and metabolic function, lung plays a role in immune system. γδT cells have multiple functions in producing cytokines and chemokines, regulating the immune response by interacting with other cells. It remains unclear about the role of γδT cells in the lung of mice infected by malaria parasites., Methods: Flow cytometry (FCM) was used to evaluate the frequency of γδT cells and the effects of γδT cells on the phenotype and function of B and T cells in Plasmodium yoelii-infected wild-type (WT) or γδTCR knockout (γδT KO) mice. Haematoxylin-eosin (HE) staining was used to observe the pathological changes in the lungs., Results: The percentage and absolute number of γδT cells in the lung increased after Plasmodium infection (p < 0.01). More γδT cells were expressing CD80, CD11b, or PD-1 post-infection (p < 0.05), while less γδT cells were expressing CD34, CD62L, and CD127 post-infection (p < 0.05). The percentages of IL-4 + , IL-5 + , IL-6 + , IL-21 + , IL-1α + , and IL-17 + γδT cells were increased (p < 0.05), but the percentage of IFN-γ-expressing γδT cells decreased (p < 0.05) post-infection. The pathological changes in the lungs of the infected γδT KO mice were not obvious compared with the infected WT mice. The proportion of CD3 + cells and absolute numbers of CD3 + cells, CD3 + CD4 + cells, CD3 + CD8 + cells decreased in γδT KO infected mice (p < 0.05). γδT KO infected mice exhibited no significant difference in the surface molecular expression of T cells compared with the WT infected mice (p > 0.05). While, the percentage of IFN-γ-expressing CD3 + and CD3 + CD8 + cells increased in γδT KO infected mice (p < 0.05). There was no significant difference in the absolute numbers of the total, CD69 + , ICOS + , and CD80 + B cells between the WT infected and γδT KO infected mice (p > 0.05)., Conclusions: The content, phenotype, and function of γδT cells in the lung of C57BL/6 mice were changed after Plasmodium infection. γδT cells contribute to T cell immune response in the progress of Plasmodium infection.

Early type I interferon is essential for antagonizing against malaria infection, which remains a significant global infectious disease. After Plasmodium yoelii YM infection, the activation of MAVS-, STING- and inflammasome-IRF3-mediated pathway could trigger the Socs1 expression to inhibit the TLR7-MyD88-IRF7-induced type I interferon production. However, the dynamic regulatory mechanisms of type I interferon response to YM infection and delicate cross-regulation of these signalling are far from clear. In current study, we established a mathematical model to systematically demonstrate that the MAVS-, STING- and inflammasome-mediated signalling pathways play distinct roles in regulating type I interferon response after YM infection; and the YM dose could significantly affect the difference of resistance to YM infection among MAVS, STING and inflammasome deficiency. Collectively, our study systematically elucidated the precise regulatory mechanisms of type I interferon signalling after YM infection and advanced the research on therapy of plasmodium infection by incorporating multiple signalling pathways at diverse time., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

CD4 + T cell functional inhibition (exhaustion) is a hallmark of malaria and correlates with impaired parasite control and infection chronicity. However, the mechanisms of CD4 + T cell exhaustion are still poorly understood. In this study, we show that Ag-experienced ( Ag-exp ) CD4 + T cell exhaustion during Plasmodium yoelii nonlethal infection occurs alongside the reduction in mammalian target of rapamycin (mTOR) activity and restriction in CD4 + T cell glycolytic capacity. We demonstrate that the loss of glycolytic metabolism and mTOR activity within the exhausted Ag-exp CD4 + T cell population during infection coincides with reduction in T-bet expression. T-bet was found to directly bind to and control the transcription of various mTOR and metabolism-related genes within effector CD4 + T cells. Consistent with this, Ag-exp Th1 cells exhibited significantly higher and sustained mTOR activity than effector T-bet- (non-Th1) Ag-exp T cells throughout the course of malaria. We identified mTOR to be redundant for sustaining T-bet expression in activated Th1 cells, whereas mTOR was necessary but not sufficient for maintaining IFN-γ production by Th1 cells. Immunotherapy targeting PD-1, CTLA-4, and IL-27 blocked CD4 + T cell exhaustion during malaria infection and was associated with elevated T-bet expression and a concomitant increased CD4 + T cell glycolytic metabolism. Collectively, our data suggest that mTOR activity is linked to T-bet in Ag-exp CD4 + T cells but that reduction in mTOR activity may not directly underpin Ag-exp Th1 cell loss and exhaustion during malaria infection. These data have implications for therapeutic reactivation of exhausted CD4 + T cells during malaria infection and other chronic conditions., (Copyright © 2020 The Authors.)

Infection by malaria parasites triggers dynamic immune responses leading to diverse symptoms and pathologies; however, the molecular mechanisms responsible for these reactions are largely unknown. We performed Trans-species Expression Quantitative Trait Locus analysis to identify a large number of host genes that respond to malaria parasite infections. Here we functionally characterize one of the host genes called receptor transporter protein 4 (RTP4) in responses to malaria parasite and virus infections. RTP4 is induced by type I IFN (IFN-I) and binds to the TANK-binding kinase (TBK1) complex where it negatively regulates TBK1 signaling by interfering with expression and phosphorylation of both TBK1 and IFN regulatory factor 3. Rtp4 -/- mice were generated and infected with malaria parasite Plasmodiun berghei ANKA. Significantly higher levels of IFN-I response in microglia, lower parasitemia, fewer neurologic symptoms, and better survival rates were observed in Rtp4 -/- than in wild-type mice. Similarly, RTP4 deficiency significantly reduced West Nile virus titers in the brain, but not in the heart and the spleen, of infected mice, suggesting a specific role for RTP4 in brain infection and pathology. This study reveals functions of RTP4 in IFN-I response and a potential target for therapy in diseases with neuropathology., Competing Interests: The authors declare no competing interest.

Malaria infection induces complex and diverse immune responses. To elucidate the mechanisms underlying host-parasite interaction, we performed a genetic screen during early (24 h) Plasmodium yoelii infection in mice and identified a large number of interacting host and parasite genes/loci after transspecies expression quantitative trait locus (Ts-eQTL) analysis. We next investigated a host E3 ubiquitin ligase gene ( March1 ) that was clustered with interferon (IFN)-stimulated genes (ISGs) based on the similarity of the genome-wide pattern of logarithm of the odds (LOD) scores (GPLS). March1 inhibits MAVS/STING/TRIF-induced type I IFN (IFN-I) signaling in vitro and in vivo. However, in malaria-infected hosts, deficiency of March1 reduces IFN-I production by activating inhibitors such as SOCS1, USP18, and TRIM24 and by altering immune cell populations. March1 deficiency increases CD86 + DC (dendritic cell) populations and levels of IFN-γ and interleukin 10 (IL-10) at day 4 post infection, leading to improved host survival. T cell depletion reduces IFN-γ level and reverse the protective effects of March1 deficiency, which can also be achieved by antibody neutralization of IFN-γ. This study reveals functions of MARCH1 (membrane-associated ring-CH-type finger 1) in innate immune responses and provides potential avenues for activating antimalaria immunity and enhancing vaccine efficacy., Competing Interests: The authors declare no competing interest.

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.

Only a small fraction of the antigens expressed by malaria parasites have been evaluated as vaccine candidates. A successful malaria subunit vaccine will likely require multiple antigenic targets to achieve broad protection with high protective efficacy. Here we describe protective efficacy of a novel antigen, Plasmodium yoelii (Py) E140 (PyE140), evaluated against P. yoelii challenge of mice. Vaccines targeting PyE140 reproducibly induced up to 100% sterile protection in both inbred and outbred murine challenge models. Although PyE140 immunization induced high frequency and multifunctional CD8+ T cell responses, as well as CD4+ T cell responses, protection was mediated by PyE140 antibodies acting against blood stage parasites. Protection in mice was long-lasting with up to 100% sterile protection at twelve weeks post-immunization and durable high titer anti-PyE140 antibodies. The E140 antigen is expressed in all Plasmodium species, is highly conserved in both P. falciparum lab-adapted strains and endemic circulating parasites, and is thus a promising lead vaccine candidate for future evaluation against human malaria parasite species., Competing Interests: The authors have read the journal’s policy and have the following conflicts: ECS, KJL, and JCA are named inventors on a patent application US PCT/US18/25510 covering methods and compositions for vaccinating against malaria. JCA is a named inventor on US patent number 10, 195, 260, US Patent Application No. 16/230,512, and related foreign applications covering the PfE140 antigen. NR, KO, JW, and JCA are/were employed by CAMRIS International, which is a commercial company that owns the rights to the PfE140 antigen under patent number 10, 195, 260. This does not alter our adherence to PLOS ONE policies on sharing data and materials. ERPi Inc., Meso Scale Diagnostics, Sanaria, and General Dynamics Information Technology Inc. are current affiliations for some authors who previously contributed to the study. Neither ERPi Inc., Meso Scale Diagnostics, Sanaria, nor General Dynamics Information Technology Inc. made any contribution to the study.

During a blood meal, female Anopheles mosquitoes are potentially exposed to diverse microbes in addition to the malaria parasite, Plasmodium. Human and animal African trypanosomiases are frequently co-endemic with malaria in Africa. It is not known whether exposure of Anopheles to trypanosomes influences their fitness or ability to transmit Plasmodium. Using cell and molecular biology approaches, we found that Trypanosoma brucei brucei parasites survive for at least 48h after infectious blood meal in the midgut of the major malaria vector, Anopheles coluzzii before being cleared. This transient survival of trypanosomes in the midgut is correlated with a dysbiosis, an alteration in the abundance of the enteric bacterial flora in Anopheles coluzzii. Using a developmental biology approach, we found that the presence of live trypanosomes in mosquito midguts also reduces their reproductive fitness, as it impairs the viability of laid eggs by affecting their hatching. Furthermore, we found that Anopheles exposure to trypanosomes enhances their vector competence for Plasmodium, as it increases their infection prevalence. A transcriptomic analysis revealed that expression of only two Anopheles immune genes are modulated during trypanosome exposure and that the increased susceptibility to Plasmodium was microbiome-dependent, while the reproductive fitness cost was dependent only on the presence of live trypanosomes but was microbiome independent. Taken together, these results demonstrate multiple effects upon Anopheles vector competence for Plasmodium caused by eukaryotic microbes interacting with the host and its microbiome, which may in turn have implications for malaria control strategies in co-endemic areas., Competing Interests: The authors have declared that no competing interests exist.

Erythrocyte-binding-like (EBL) proteins are known to play an important role in malaria parasite invasion of red blood cells (RBCs); however, any roles of EBL proteins in regulating host immune responses remain unknown. Here, we show that Plasmodium yoelii EBL (PyEBL) can shape disease severity by modulating the surface structure of infected RBCs (iRBCs) and host immune responses. We identified an amino acid substitution (a change of C to Y at position 741 [C741Y]) in the protein trafficking domain of PyEBL between isogenic P. yoellii nigeriensis strain N67 and N67C parasites that produce different disease phenotypes in C57BL/6 mice. Exchanges of the C741Y alleles altered parasite growth and host survival accordingly. The C741Y substitution also changed protein processing and trafficking in merozoites and in the cytoplasm of iRBCs, reduced PyEBL binding to band 3, increased phosphatidylserine (PS) surface exposure, and elevated the osmotic fragility of iRBCs, but it did not affect invasion of RBCs in vitro The modified iRBC surface triggered PS-CD36-mediated phagocytosis of iRBCs, host type I interferon (IFN-I) signaling, and T cell differentiation, leading to improved host survival. This study reveals a previously unknown role of PyEBL in regulating host-pathogen interaction and innate immune responses, which may be explored for developing disease control strategies. IMPORTANCE Malaria is a deadly parasitic disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild symptoms, or fatal, depending on a delicate balance of host immune responses. Malaria parasites enter host red blood cells (RBCs) through interactions between parasite ligands and host receptors, such as erythrocyte-binding-like (EBL) proteins and host Duffy antigen receptor for chemokines (DARC). Plasmodium yoelii EBL (PyEBL) is known to play a role in parasite invasion of RBCs. Here, we show that PyEBL also affects disease severity through modulation of host immune responses, particularly type I interferon (IFN-I) signaling. This discovery assigns a new function to PyEBL and provides a mechanism for developing disease control strategies.

Immunization with attenuated whole Plasmodium sporozoites constitutes a promising vaccination strategy. Compared to replication-deficient parasites, immunization with replication-competent parasites confers better protection and also induces a type I IFN (IFN-1) response, but whether this IFN-1 response has beneficial or adverse effects on vaccine-induced adaptive immunity is not known. Here, we show that IFN-1 signaling-deficient mice immunized with replication-competent sporozoites exhibit superior protection against infection. This correlates with superior CD8 T cell memory including reduced expression of the exhaustion markers PD-1 and LAG-3 on these cells and increased numbers of memory CD8 T cells in the liver. Moreover, the adoptive transfer of memory CD8 T cells from the livers of previously immunized IFN-1 signaling-deficient mice confers greater protection against liver stage parasites. However, the detrimental role of IFN-1 signaling is not CD8 T cell intrinsic. Together, our data demonstrate that liver stage-engendered IFN-1 signaling impairs hepatic CD8 T cell memory via a CD8 T cell-extrinsic mechanism.

CRISPR/Cas9 is a powerful genome editing method that has greatly facilitated functional studies in many eukaryotic organisms including malaria parasites. Due to the lack of genes encoding enzymes necessary for the non-homologous end joining DNA repair pathway, genetic manipulation of malaria parasite genomes is generally accomplished through homologous recombination requiring the presence of DNA templates. Recently, an alternative double-strand break repair pathway, microhomology-mediated end joining, was found in the Plasmodium falciparum parasite. Taking advantage of the MMEJ pathway, we developed a MMEJ-based CRISPR/Cas9 (mCRISPR) strategy to efficiently generate multiple mutant parasites simultaneously in genes with repetitive sequences. As a proof of principle, we successfully produced various size mutants in the central repeat region of the Plasmodium yoelii circumsporozoite surface protein without the use of template DNA. Monitoring mixed parasite populations and individual parasites with different sizes of CSP-CRR showed that the CSP-CRR plays a role in the development of mosquito stages, with severe developmental defects in parasites with large deletions in the repeat region. However, the majority of the csp mutant parasite clones grew similarly to the wild type P. yoelii 17XL parasite in mice. This study develops a useful technique to efficiently generate mutant parasites with deletions or insertions, and shows that the CSP-CRR plays a role in parasite development in mosquito., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The enzyme acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and is thereby involved in several cellular processes such as differentiation, proliferation, and apoptosis in different cell types. However, the function of ASM in T cells is still not well characterized. Here, we used T cell-specific ASM overexpressing mice (t-ASM/CD4cre) to clarify the impact of cell-intrinsic ASM activity on T cell function in vitro and in vivo . We showed that t-ASM/CD4cre mice exhibit decreased frequencies of Foxp3 + T regulatory cells (Tregs) within the spleen. Enforced T cell-specific ASM expression resulted in less efficient induction of Tregs and promoted differentiation of CD4 + CD25 - naïve T cells into IFN-γ producing Th1 cells in vitro . Further analysis revealed that ASM-overexpressing T cells from t-ASM/CD4cre mice show elevated T cell receptor (TCR) signaling activity accompanied with increased proliferation upon stimulation in vitro . Plasmodium yoelii infection of t-ASM/CD4cre mice resulted in enhanced T cell activation and was associated with reduced parasitemia in comparison to infected control mice. Hence, our results provide evidence that ASM activity modulates T cell function in vitro and in vivo .

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., (Copyright © 2019. Published by Elsevier B.V.)

Malaria is a devastating disease resulting in significant morbidity and mortality, especially in the developing world. Previously, we showed that the gut microbiome modulates severity of malaria in mice, though the exact mechanism was unknown. One well-studied mechanism by which the intestinal microbiota exerts an effect on host health is by synthesis of short-chain fatty acids (SCFAs). SCFAs have pleiotropic effects on the host, including modulating the immune system and altering susceptibility to pathogens. The objective of the current work was to explore if gut microbiota-mediated resistance and susceptibility to malaria in mice is through differential production of SCFAs. Of the eight detected SCFAs, only propionic acid (C3) was different between two groups of resistant and two groups of susceptible mice, with higher levels in feces of susceptible mice compared to resistant mice. Nevertheless, subsequent analysis revealed no robust correlation between malaria severity and levels of fecal propionic acid. In spite of the broad effect of SCFAs on host physiology, including host immunity, this study shows that gut microbiota-mediated modulation of malaria severity in mice is independent of fecal SCFA levels. Additionally, our data indicates that intestinal SCFAs do not function as biomarkers for prediction of malaria disease severity., Competing Interests: The authors have declared that no competing interests exist.

Experimental models of malaria have shown that infection with specific Plasmodium species in certain mouse strains can transiently modulate gut microbiota and cause intestinal shortening, indicating a disruption of gut homeostasis. Importantly, changes in gut homeostasis have not been characterized in the context of mild versus severe malaria. We show that severe Plasmodium infection in mice disrupts homeostasis along the gut-liver axis in multiple ways compared to mild infection. High parasite burden results in a larger influx of immune cells in the lamina propria and mice with high parasitemia display specific metabolomic profiles in the ceca and plasma during infection compared to mice with mild parasitemia. Liver damage was also more pronounced and longer lasting during severe infection, with concomitant changes in bile acids in the gut. Finally, severe Plasmodium infection changes the functional capacity of the microbiota, enhancing bacterial motility and amino acid metabolism in mice with high parasite burden compared to a mild infection. Taken together, Plasmodium infections have diverse effects on host gut homeostasis relative to the severity of infection that may contribute to enteric bacteremia that is associated with malaria.

Plasmodium malaria parasites multiply within erythrocytes and possess a repertoire of proteins whose function is to recognize and invade these vertebrate host cells. One such protein involved in erythrocyte invasion is the micronemal protein, Erythrocyte Binding-Like (EBL), which has been studied as a potential target of vaccine development in Plasmodium vivax (PvDBP) and Plasmodium falciparum (EBA-175). In the rodent malaria parasite model Plasmodium yoelii, specific substitutions in the EBL regions responsible for intracellular trafficking (17XL parasite line) or receptor recognition (17X1.1pp. parasite line), paradoxically increase invasion ability and virulence rather than abolish EBL function. Attempts to disrupt the ebl gene locus in the 17XL and 17XNL lines were unsuccessful, suggesting EBL essentiality. To understand the mechanisms behind these potentially conflicting outcomes, we generated 17XL-based transfectants in which ebl expression is suppressed with anhydrotetracycline (ATc) and investigated merozoite behavior during erythrocyte invasion. In the absence of ATc, EBL was secreted to the merozoite surface, whereas following ATc administration parasitemia was negligible in vivo. Merozoites lacking EBL were unable to invade erythrocytes in vitro, indicating that EBL has a critical role for erythrocyte invasion. Quantitative time-lapse imaging revealed that with ATc administration a significant number of merozoites were detached from the erythrocyte after the erythrocyte deformation event and no echinocytosis was observed, indicating that EBL is required for merozoites to establish an irreversible connection with erythrocytes during invasion., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Malaria transmission-blocking vaccines aim to inhibit the development of malaria parasites in mosquitoes by inducing antibodies targeting surface proteins of sexual stage parasites. We have recently identified PyMiGS, a protein specifically expressed in the osmiophilic body of male gametocytes of Plasmodium yoelii (Py). PyMiGS is translocated to the surface of microgametes, and potent transmission-blocking activity was observed in mosquitoes fed on mice passively immunized with antibodies against PyMiGS. Here we demonstrate using a direct feeding assay that recombinant PyMiGS successfully induces anti-PyMiGS antibodies in mice and that the antibodies block parasite development in mosquitoes. We also show using the membrane-feeding assay that rabbit anti-PyMiGS antibody inhibits parasite development in mosquitoes in a dose-dependent manner without complement involvement. To investigate the mode of action of anti-PyMiGS antibodies against parasite development, we observed exflagellation after mixing Py gametocytes with activation medium containing anti-PyMiGS or anti-GST control antibodies. Whereas most microgametes were released from activated male gametocytes in the control group, a significantly reduced number of microgametes were released in the anti-PyMiGS group, with most of the microgametes left attached to the activated male gametocytes. Moreover, anti-PyMiGS antibodies shortened the duration of the active movement of microgametes after the onset of exflagellation. Taken together, these findings suggest that anti-PyMiGS antibodies bind to the microgamete surface immediately after exflagellation, thereby reducing microgamete motility and inhibiting microgamete release from the activated male gametocytes. These results strongly suggest that PyMiGS orthologues in Plasmodium falciparum and Plasmodium vivax can be promising TBV candidates., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The inflammasome plays a critical role in inflammation and immune responses against pathogens. However, whether or how inflammasome activation regulates type I interferon (IFN-I) signaling in the context of malaria infection remain unknown. Here we show mice deficient in inflammasome sensors AIM2, NLRP3 or adaptor Caspase-1 produce high levels of IFN-I cytokines and are resistant to lethal Plasmodium yoelii YM infection. Inactivation of inflammasome signaling reduces interleukin (IL)-1β production, but increases IFN-I production. Mechanistically, we show inflammsome activation enhances IL-1β-mediated MyD88-TRAF3-IRF3 signaling and SOCS1 upregulation. However, SOCS1 inhibits MyD88-IRF7-mediated-IFN-I signaling and cytokine production in plasmacytoid dendritic cells. By contrast, ablation of inflammsome components reduces SOCS1 induction, and relieves its inhibition on MyD88-IRF7-dependent-IFN-I signaling, leading to high levels of IFN-α/β production and host survival. Our study identifies a previously unrecognized role of inflammasome activation in the negative regulation of IFN-I signaling pathways and provides potential targets for developing effective malaria vaccines.

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.

Early plasmablast induction is a hallmark of Plasmodium infection and is thought to contribute to the control of acute parasite burden. Although long understood to be a T-cell dependent phenomenon, regulation of early plasmablast differentiation, however, is poorly understood. Here, we identify a population of CD4 + T cells that express the innate NK cell marker NK1.1 as an important source of T cell help for early plasmablast and parasite-specific Ab production. Interestingly, NK1.1 + CD4 + T cells arise from conventional, naive NK1.1 - CD4 + T cells, and their generation is independent of CD1d but critically reliant on MHC-II. CD4 + T cells that express NK1.1 early after activation produce IFN-γ and IL-21, and express the follicular helper T (Tfh) cell markers ICOS, PD-1 and CXCR5 more frequently than NK1.1 - CD4 + T cells. Further analysis of this population revealed that NK1.1 + Tfh-like cells were more regularly complexed with plasmablasts than NK1.1 - Tfh-like cells. Ultimately, depletion of NK1.1 + cells impaired class-switched parasite-specific antibody production during early Plasmodium yoelii infection. Together, these data suggest that expression of NK1.1 defines a population of rapidly expanding effector CD4 + T cells that specifically promote plasmablast induction during Plasmodium infection and represent a subset of T cells whose modulation could promote effective vaccine design.

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.

Tissue-resident memory CD8 + T (Trm) cells in the liver are critical for long-term protection against pre-erythrocytic Plasmodium infection. Such protection can usually be induced with three to five doses of i.v. administered radiation-attenuated sporozoites (RAS). To simplify and accelerate vaccination, we tested a DNA vaccine designed to induce potent T cell responses against the SYVPSAEQI epitope of Plasmodium yoelii circumsporozoite protein. In a heterologous "prime-and-trap" regimen, priming using gene gun-administered DNA and boosting with one dose of RAS attracted expanding Ag-specific CD8 + T cell populations to the liver, where they became Trm cells. Vaccinated in this manner, BALB/c mice were completely protected against challenge, an outcome not reliably achieved following one dose of RAS or following DNA-only vaccination. This study demonstrates that the combination of CD8 + T cell priming by DNA and boosting with liver-homing RAS enhances formation of a completely protective liver Trm cell response and suggests novel approaches for enhancing T cell-based pre-erythrocytic malaria vaccines., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Background: Plasmodium 18S rRNA is a biomarker used to monitor blood-stage infections in malaria clinical trials. Plasmodium sporozoites also express this biomarker, and there is conflicting evidence about how long sporozoite-derived 18S rRNA persists in peripheral blood. If present in blood for an extended timeframe, sporozoite-derived 18S rRNA could complicate use as a blood-stage biomarker., Methods: Blood samples from Plasmodium yoelii infected mice were tested for Plasmodium 18S rRNA and their coding genes (rDNA) using sensitive quantitative reverse transcription PCR and quantitative PCR assays, respectively. Blood and tissues from Plasmodium falciparum sporozoite (PfSPZ)-infected rhesus macaques were similarly tested., Results: In mice, when P. yoelii sporozoite inoculation and blood collection were performed at the same site (tail vein), low level rDNA positivity persisted for 2 days post-infection. Compared to intact parasites with high rRNA-to-rDNA ratios, this low level positivity was accompanied by no increase in rRNA-to-rDNA, indicating detection of residual, non-viable parasite rDNA. When P. yoelii sporozoites were administered via the retro-orbital vein and blood sampled by cardiac puncture, neither P. yoelii 18S rRNA nor rDNA were detected 24 h post-infection. Similarly, there was no P. falciparum 18S rRNA detected in blood of rhesus macaques 3 days after intravenous injection with extremely high doses of PfSPZ. Plasmodium 18S rRNA in the rhesus livers increased by approximately 101-fold from 3 to 6 days post infection, indicating liver-stage proliferation., Conclusions: Beyond the first few hours after injection, sporozoite-derived Plasmodium 18S rRNA was not detected in peripheral blood. Diagnostics based on 18S rRNA are unlikely to be confounded by sporozoite inocula in human clinical trials.

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.

Background: Cerebral malaria (CM) is a fatal complication of Plasmodium infection, mostly affecting children under the age of five in the sub-Saharan African region. CM pathogenesis remains incompletely understood, although sequestered infected red blood cells, inflammatory cells aggregating in the cerebral blood vessels, and the microvesicles (MV) that they release in the circulation, have been implicated. Plasma MV numbers increase in CM patients and in the murine model, where blocking their release, genetically or pharmacologically, protects against brain pathology, suggesting a role of MV in CM neuropathogenesis. In this work, the microRNA (miRNA) cargo of MV is defined for the first time during experimental CM with the overarching hypothesis that this characterization could help understand CM pathogenesis., Results: The change in abundance of miRNA was studied following infection of CBA mice with Plasmodium berghei ANKA strain (causing experimental CM), and Plasmodium yoelii, which causes severe malaria without cerebral complications, termed non-CM (NCM). miRNA expression was analyzed using microarrays to compare MV from healthy (NI) and CM mice, yielding several miRNA of interest. The differential expression profiles of these selected miRNA (miR-146a, miR-150, miR-193b, miR-205, miR-215, miR-467a, and miR-486) were analyzed in mouse MV, MV-free plasma, and brain tissue by quantitative reverse transcription PCR (RT-qPCR). Two miRNA-miR-146a and miR-193b-were confirmed as differentially abundant in MV from CM mice, compared with NCM and NI mice. These miRNA have been shown to play various roles in inflammation, and their dysregulation during CM may be critical for triggering the neurological syndrome via regulation of their potential downstream targets., Conclusions: These data suggest that, in the mouse model at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.

Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete-specific circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) promoter (S4/CelTOS CTRP ), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOS CTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild-type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility., (© 2017 John Wiley & Sons Ltd.)

Malaria parasites have a complex life cycle with multiple developmental stages in mosquito and vertebrate hosts, and different developmental stages express unique sets of genes. Unexpectedly, many transcription factors (TFs) commonly found in eukaryotic organisms are absent in malaria parasites; instead, a family of genes encoding proteins similar to the plant Apetala2 (ApiAP2) transcription factors is expanded in the parasites. Several malaria ApiAP2 genes have been shown to play a critical role in parasite development; however, the functions of the majority of the ApiAP2 genes remain to be elucidated. In particular, no study on the Plasmodium yoelii ApiAP2 (PyApiAP2) gene family has been reported so far. This study systematically investigated the functional roles of PyApiAP2 genes in parasite development. Twenty-four of the 26 PyApiAP2 genes were selected for disruption, and 12 were successfully knocked out using the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) method. The effects of gene knockout (KO) on parasite development in mouse and mosquito stages were evaluated. Ten of 12 successfully disrupted genes, including two genes that have not been functionally characterized in any Plasmodium species previously, were shown to be critical for P. yoelii development of sexual and mosquito stages. Additionally, seven of the genes were labeled for protein expression analysis, revealing important information supporting their functions. This study represents the first systematic functional characterization of the P. yoelii ApiAP2 gene family and discovers important insights on the roles of the ApiAP2 genes in parasite development. IMPORTANCE Malaria is a parasitic disease that infects hundreds of millions of people, leading to an estimated 0.35 million deaths in 2015. A better understanding of the mechanism of gene expression regulation during parasite development may provide important clues for disease control and prevention. In this study, systematic gene disruption experiments were performed to study the functional roles of members of the Plasmodium yoelii ApiAP2 (PyApiAP2) gene family in parasite development. Genes that are critical for the development of male and female gametocytes, oocysts, and sporozoites were characterized. The protein expression profiles for seven of the PyApiAP2 gene products were also analyzed, revealing important information on their functions. This study provides expression and functional information for many PyApiAP2 genes, which can be explored for disease management.

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.

Transmission of the malaria parasite occurs in an unpredictable moment, when a mosquito takes a blood meal. Plasmodium has therefore evolved strategies to prepare for transmission, including translationally repressing and protecting mRNAs needed to establish the infection. However, mechanisms underlying these critical controls are not well understood, including whether Plasmodium changes its translationally repressive complexes and mRNA targets in different stages. Efforts to understand this have been stymied by severe technical limitations due to substantial mosquito contamination of samples. Here using P. yoelii, for the first time we provide a proteomic comparison of a protein complex across asexual blood, sexual and sporozoite stages, along with a transcriptomic comparison of the mRNAs that are affected in these stages. We find that the Apicomplexan-specific ALBA4 RNA-binding protein acts to regulate development of the parasite's transmission stages, and that ALBA4 associates with both stage-specific and stage-independent partners to produce opposing mRNA fates. These efforts expand our understanding and ability to interrogate both sexual and sporozoite transmission stages and the molecular preparations they evolved to perpetuate their infectious cycle., (© 2017 John Wiley & Sons Ltd.)

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.

Background: The transmission of drug-resistant parasites by the mosquito may be influenced by the altered biological fitness of drug-resistant parasites and different immune reactions or metabolic change in the mosquito. At this point, little is known about the variations in mosquito immunity and metabolism when mosquitoes are infected with drug-resistant parasites. To understand the differential gene expression in Anopheles following infection with drug-resistant Plasmodium, we conducted a genome-wide transcriptomic profiling analysis of Anopheles stephensi following feeding on mice with drug-resistant or drug-sensitive P. yoelii, observed changes in gene expression profiles and identified transcripts affected by the drug-resistant parasite., Results: To study the impact of drug-resistant Plasmodium infections on An. stephensi gene transcription, we analyzed the three major transition stages of Plasmodium infections: at 24 h and 13 and 19 days after blood-feeding. Six cDNA libraries (R-As24h, R-As13d, R-As19d,S-As24h, S-As13dand S-As19d) were constructed, and RNA sequencing was subsequently performed. In total, approximately 50.1 million raw reads, 47.9 million clean reads and 7.18G clean bases were obtained. Following differentially expressed gene (DEG) analysis, GO enrichment analysis of DEGs, and functional classification by KEGG, we showed that the variations in gene expression in An. stephensi infected by the drug-resistant P. yoelii NSM occurred mainly at 13 days after blood meal during sporozoite migration through the hemolymph. The differentially expressed genes included those functioning in some important immune reaction and iron metabolism pathways, such as pattern recognition receptors, regulators of the JNK pathway, components of the phagosome pathway, regulators of the melanization response, activators of complement reactions, insulin signaling cascade members, oxidative stress and detoxification proteins., Conclusions: Our study shows that drug-resistant P. yoelii NSM has an impact on the transcript abundance levels of An.stephensi mostly at 13 days after blood meal during sporozoite migration through the hemolymph and that most differentially expressed genes were downregulated. Our results highlight the need for a better understanding of selective pressures from these differentially expressed genes of the drug-resistant Plasmodium in the mosquito and the different transmission patterns of drug-resistant Plasmodium by Anopheles.

Severe malaria and associated high parasite burdens occur more frequently in humans lacking robust adaptive immunity to Plasmodium falciparum Nevertheless, the host may partly control blood-stage parasite numbers while adaptive immunity is gradually established. Parasite control has typically been attributed to enhanced removal of parasites by the host, although in vivo quantification of this phenomenon remains challenging. We used a unique in vivo approach to determine the fate of a single cohort of semisynchronous, Plasmodium berghei ANKA- or Plasmodium yoelii 17XNL-parasitized red blood cells (pRBCs) after transfusion into naive or acutely infected mice. As previously shown, acutely infected mice, with ongoing splenic and systemic inflammatory responses, controlled parasite population growth more effectively than naive controls. Surprisingly, however, this was not associated with accelerated removal of pRBCs from circulation. Instead, transfused pRBCs remained in circulation longer in acutely infected mice. Flow cytometric assessment and mathematical modeling of intraerythrocytic parasite development revealed an unexpected and substantial slowing of parasite maturation in acutely infected mice, extending the life cycle from 24 h to 40 h. Importantly, impaired parasite maturation was the major contributor to control of parasite growth in acutely infected mice. Moreover, by performing the same experiments in rag1 -/- mice, which lack T and B cells and mount weak inflammatory responses, we revealed that impaired parasite maturation is largely dependent upon the host response to infection. Thus, impairment of parasite maturation represents a host-mediated, immune system-dependent mechanism for limiting parasite population growth during the early stages of an acute blood-stage Plasmodium infection., Competing Interests: The authors declare no conflict of interest.

CRISPR/Cas9 has been successfully adapted for gene editing in malaria parasites including Plasmodium falciparum and Plasmodium yoelii. However, the reported methods were limited to editing one gene at a time. In practice, it is often desired to modify multiple genetic loci in a parasite genome. Here we describe a CRISPR/Cas9 mediated genome editing method that allows successive modification of more than one gene in the genome of P. yoelii using an improved single-vector system (pYCm) we developed previously. Drug resistant genes encoding human dihydrofolate reductase (hDHFR) and a yeast bifunctional protein (yFCU), with cytosine deaminase (CD) and uridyl phosphoribosyl transferase (UPRT) activities in the plasmid, allowed sequential positive (pyrimethamine, Pyr) and negative (5-fluorocytosine, 5FC) selections and generation of transgenic parasites free of the episomal plasmid after genetic modification. Using this system, we were able to efficiently tag a gene of interest (Pyp28) and subsequently disrupted two genes (Pyctrp and Pycdpk3) that are individually critical for ookinete motility. Disruption of the genes either eliminated (Pyctrp) or greatly reduced (Pycdpk3) ookinete forward motility in matrigel in vitro and completely blocked oocyst development in mosquito midgut. The method will greatly facilitate studies of parasite gene function, development, and disease pathogenesis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

A major challenge in disease ecology is to understand how co-infecting parasite species interact. We manipulate in vivo resources and immunity to explain interactions between two rodent malaria parasites, Plasmodium chabaudi and P. yoelii. These species have analogous resource-use strategies to the human parasites Plasmodium falciparum and P. vivax: P. chabaudi and P. falciparum infect red blood cells (RBC) of all ages (RBC generalist); P. yoelii and P. vivax preferentially infect young RBCs (RBC specialist). We find that: (1) recent infection with the RBC generalist facilitates the RBC specialist (P. yoelii density is enhanced ~10 fold). This occurs because the RBC generalist increases availability of the RBC specialist's preferred resource; (2) co-infections with the RBC generalist and RBC specialist are highly virulent; (3) and the presence of an RBC generalist in a host population can increase the prevalence of an RBC specialist. Thus, we show that resources shape how parasite species interact and have epidemiological consequences., (© 2016 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd.)

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., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Background: Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites., Methods: We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25., Results: Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group., Conclusions: This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Macrophage migration inhibitory factor (PMIF) expressed by Plasmodium parasites has been proved to be similar to the mammalian MIF in both structure and biological activity and is a critical upstream regulator in antimalaria innate immunity. In this work, using a genetically modified (MIF-KO) strain of highly lethal rodent Plasmodium yoelii 17XL (Py17XL), we found that PyMIF could increase the secretion of pro-inflammatory factors by eliciting the CD11b(+) Ly6C(+) cells accumulated in the spleen of infected mouse. In addition, the chemotactic effect of PyMIF was demonstrated to associate with cell receptors CXCR2, CXCR4 and the cell surface markers ICAM-1, LFA-4., (© 2016 John Wiley & Sons Ltd.)

Background: More than half of the world's population is at risk of malaria and simultaneously, many malaria-endemic regions are facing dramatic increases in the prevalence of type 2 diabetes. Studies in murine malaria models have examined the impact of malaria infection on type 2 diabetes pathology, it remains unclear how this chronic metabolic disorder impacts the transmission of malaria. In this report, the ability type 2 diabetic rodents infected with malaria to transmit parasites to Anopheles stephensi mosquitoes is quantified., Methods: The infection prevalence and intensity of An. stephensi mosquitoes that fed upon control or type 2 diabetic C57BL/6 db/db mice infected with either lethal Plasmodium berghei NK65 or non-lethal Plasmodium yoelii 17XNL murine malaria strains were determined. Daily parasitaemias were also recorded., Results: A higher percentage of mosquitoes (87.5 vs 61.5 % for P. yoelii and 76.9 vs 50 % for P. berghei) became infected following blood feeding on Plasmodium-infected type 2 diabetic mice compared to mosquitoes that fed on infected control animals, despite no significant differences in circulating gametocyte levels., Conclusions: These results suggest that type 2 diabetic mice infected with malaria are more efficient at infecting mosquitoes, raising the question of whether a similar synergy exists in humans.

The antimalarial activity of heparin, against which there are no resistances known, has not been therapeutically exploited due to its potent anticoagulating activity. Here, we have explored the antiplasmodial capacity of heparin-like sulfated polysaccharides from the sea cucumbers Ludwigothurea grisea and Isostichopus badionotus, from the red alga Botryocladia occidentalis, and from the marine sponge Desmapsamma anchorata. In vitro experiments demonstrated for most compounds significant inhibition of Plasmodium falciparum growth at low-anticoagulant concentrations. This activity was found to operate through inhibition of erythrocyte invasion by Plasmodium, likely mediated by a coating of the parasite similar to that observed for heparin. In vivo four-day suppressive tests showed that several of the sulfated polysaccharides improved the survival of Plasmodium yoelii-infected mice. In one animal treated with I. badionotus fucan parasitemia was reduced from 10.4% to undetectable levels, and Western blot analysis revealed the presence of antibodies against P. yoelii antigens in its plasma. The retarded invasion mediated by sulfated polysaccharides, and the ensuing prolonged exposure of Plasmodium to the immune system, can be explored for the design of new therapeutic approaches against malaria where heparin-related polysaccharides of low anticoagulating activity could play a dual role as drugs and as potentiators of immune responses.

In the current study, we examined the effects of depletion of phagocytes on the progression of Plasmodium yoelii 17XNL infection in mice. Strikingly, the depletion of phagocytic cells, including macrophages, with clodronate in the acute phase of infection significantly reduced peripheral parasitemia but increased mortality. Moribund mice displayed severe pathological damage, including coagulative necrosis in liver and thrombi in the glomeruli, fibrin deposition, and tubular necrosis in kidney. The severity of infection was coincident with the increased sequestration of parasitized erythrocytes, the systematic upregulation of inflammation and coagulation, and the disruption of endothelial integrity in the liver and kidney. Aspirin was administered to the mice to minimize the risk of excessive activation of the coagulation response and fibrin deposition in the renal tissue. Interestingly, treatment with aspirin reduced the parasite burden and pathological lesions in the renal tissue and improved survival of phagocyte-depleted mice. Our data imply that the depletion of phagocytic cells, including macrophages, in the acute phase of infection increases the severity of malarial infection, typified by multiorgan failure and high mortality., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Childhood malaria is a risk factor for disseminated infections with non-typhoidal Salmonella (NTS) in sub-Saharan Africa. While hemolytic anemia and an altered cytokine environment have been implicated in increased susceptibility to NTS, it is not known whether malaria affects resistance to intestinal colonization with NTS. To address this question, we utilized a murine model of co-infection. Infection of mice with Plasmodium yoelii elicited infiltration of inflammatory macrophages and T cells into the intestinal mucosa and increased expression of inflammatory cytokines. These mucosal responses were also observed in germ-free mice, showing that they are independent of the resident microbiota. Remarkably, P. yoelii infection reduced colonization resistance of mice against S. enterica serotype Typhimurium. Further, 16S rRNA sequence analysis of the intestinal microbiota revealed marked changes in the community structure. Shifts in the microbiota increased susceptibility to intestinal colonization by S. Typhimurium, as demonstrated by microbiota reconstitution of germ-free mice. These results show that P. yoelii infection, via alterations to the microbial community in the intestine, decreases resistance to intestinal colonization with NTS. Further they raise the possibility that decreased colonization resistance may synergize with effects of malaria on systemic immunity to increase susceptibility to disseminated NTS infections.

The emergence of malaria pathogens having resistance against antimalarials implies the necessity for the development of new drugs. Recently, we have demonstrated a resistance against malaria infection of α-tocopherol transfer protein knockout mice showing undetectable plasma levels of α-tocopherol, a lipid-soluble antioxidant. However, dietary restriction induced α-tocopherol deficiency is difficult to be applied as a clinical antimalarial therapy. Here, we report on a new strategy to potentially treat malaria by using probucol, a drug that can reduce the plasma α-tocopherol concentration. Probucol pre-treatment for 2 weeks and treatment throughout the infection rescued from death of mice infected with Plasmodium yoelii XL-17 or P. berghei ANKA. In addition, survival was extended when the treatment started immediately after parasite inoculation. The ratio of lipid peroxidation products to parent lipids increased in plasma after 2 weeks treatment of probucol. This indicates that the protective effect of probucol might be mediated by the oxidative stressful environment induced by α-tocopherol deficiency. Probucol in combination with dihydroartemisin suppressed the proliferation of P. yoelii XL-17. These results indicated that probucol might be a candidate for a drug against malaria infection by inducing α-tocopherol deficiency without dietary α-tocopherol restriction.

Rodent malaria is a useful model for evaluating the efficacy of malaria vaccine candidates; however, labor-intensive microscopic parasite counting hampers the use of an in vivo parasite challenge in high-throughput screening. The measurement of malaria parasite lactate dehydrogenase (pLDH) activity, which is commonly used in the in vitro growth inhibition assay of Plasmodium falciparum, may be the cheapest and simplest alternative to microscopic parasite counting. However, the pLDH assay has not been applied in the in vivo rodent malaria model. Here, we showed that the pLDH assay is reliable and accurately determines parasitemia in the rodent malaria model. pLDH activity measured using a chromogenic substrate reflects the parasite number in the blood; it allows fast and easy assessment using a conventional microplate reader. To validate this approach, we synthesized recombinant PyMSP1-19 protein (rPyMSP1-19) using a wheat germ cell-free protein synthesis system and immunized mice with rPyMSP1-19. The antisera showed specific reactivity on the surface of the Plasmodium yoelii merozoite and immunized mice were protected against a lethal P. yoelii 17 XL challenge. The pLDH assay quickly and easily demonstrated a significant reduction of the parasite numbers in the immunized mice. Accordingly, the pLDH assay proved to be an efficient alternative to rodent malaria parasite counting, and may therefore accelerate in vivo vaccine candidate screening., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: The life cycle of Plasmodium is complex, requiring invasion of two different hosts, humans and mosquitoes. In humans, initiation of an effective Th1 response during early infection is critical for the control of parasite multiplication. In mosquitoes, inhibition of the development of sexual-stage parasites interrupts the parasite transmission. In this study, we aim to investigate whether dietary supplementation of L-arginine (L-Arg) in mice affects Plasmodium yoelii 17XL (Py17XL) transmission in mosquitoes., Methods: BALB/c mice were orally administered with 1.5 mg/g L-Arg daily for 7 days and infected with Py17XL. The mRNA levels of inducible nitric oxide synthase (iNOS) and arginase 1 in spleen cells were determined by real-time RT-PCR. The amount of nitric oxide (NO) released by spleen cells in vitro was determined by the Griess method. The effect of L-Arg supplementation on subsequent development of P. yoelii gametocytes was evaluated by an in vitro ookinete culture assay and mosquito feeding assay., Results: Pretreatment of mice with L-Arg significantly increased the transcript level of iNOS in spleen cells and the amount of NO synthesized. Dietary L-Arg supplementation also significantly reduced the number of zygotes and ookinetes formed during in vitro culture and the number of oocysts formed on mosquito midguts after blood feeding., Conclusions: L-Arg enhances host immunity against blood-stage parasites as well as suppressing subsequent parasite development in mosquitoes. L-Arg as an inexpensive and safe supplement may be used as a novel adjunct treatment against malarial 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-β 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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)