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In JoVE (1)

Other Publications (20)

Articles by Diana S. Hansen in JoVE

 JoVE Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

1The Walter and Eliza Hall Institute of Medical Research


JoVE 50112

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Other articles by Diana S. Hansen on PubMed

Regulation of Murine Cerebral Malaria Pathogenesis by CD1d-restricted NKT Cells and the Natural Killer Complex

NKT cells are specialized cells coexpressing NK and T cell receptors. Upon activation they rapidly produce high levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) and are therefore postulated to influence T(H)1/T(H)2 immune responses. The precise role of the CD1/NKT cell pathway in immune response to infection remains unclear. We show here that CD1d-restricted NKT cells from distinct genetic backgrounds differentially influence T(H)1/T(H)2 polarization, proinflammatory cytokine levels, pathogenesis, and fatality in the P. berghei ANKA/rodent model of cerebral malaria. The functional properties of CD1d-restricted NKT cells vary according to expression of loci of the natural killer complex (NKC) located on mouse chromosome 6, which is shown here to be a significant genetic determinant of murine malarial fatalities.

The Lack of Suppressor of Cytokine Signalling-1 (SOCS1) Protects Mice from the Development of Cerebral Malaria Caused by Plasmodium Berghei ANKA

Cerebral malaria is a severe complication of infection with Plasmodium berghei ANKA involving the Th1 cytokines TNF-alpha and IFN-gamma. Suppressor of cytokine signalling-1 (SOCS1) is an important component in the regulatory cascade controlling inflammatory responses and signalling through IFN-gamma. Contrary to the expectation that SOCS1-deficient mice, in which IFN-gamma responses are uncontrolled and which are more sensitive to IFN-gamma, may show heightened susceptibility, mice lacking SOCS1 were protected from cerebral malaria. Unlike the controls and despite similar parasitaemia, infected SOCS1 null mice showed no inflammation or haemorrhaging in the brains. Mice lacking SOCS1 exhibited decreased splenic cellularity and a reduced ratio of CD4 : CD8 lymphocytes, which were maintained during infection. However, the ratio of IFN-gamma to IL-4 mRNA expression during infection was similar in SOCS1 -/- and control mice suggesting that a dramatic shift in the ratio of Th1 : Th2 responses does not account for the resistance to disease. Resistance conferred by the lack of SOCS1 is specific since the related SOCS2, also implicated in Th1-mediated responses, did not seem to be involved in the development of disease. Understanding the mechanism by which SOCS1 deficiency protects mice from cerebral malaria may allow the manipulation of its activity and alleviate pathology.

CD1d-restricted NKT Cells Contribute to Malarial Splenomegaly and Enhance Parasite-specific Antibody Responses

CD1d-restricted NKT cells are a novel T cell lineage with unusual features. They co-express some NK cell receptors and recognize glycolipid antigens through an invariant T cell receptor (TCR) in the context of CD1d molecules. Upon activation through the TCR, NKT cells produce large amounts of IFN-gamma and IL-4. It has been proposed that rapid cytokine output by activated NKT cells may induce bystander activation of other lymphoid lineages. The impact of CD1d-restricted NKT cell activation in the induction of B cell-mediated immune responses to infection is still unclear. We show here that CD1-restricted NKT cells contribute to malarial splenomegaly associated with expansion of the splenic B cell pool and enhance parasite-specific antibody formation in response to Plasmodium berghei infection. The increased B cell-mediated response correlates with the ability of NKT cells to promote Th2 immune responses. Additionally, antibody responses against the glycosylphosphatidylinositol (GPI)-anchored protein merozoite surface protein 1 (MSP-1) were found to be significantly lower in CD1(-/-) mice compared to wild-type animals. P. berghei-infected MHC class II (MHCII)(-/-) mice also generated antibodies against MSP-1, suggesting that antibody production against GPI-anchored antigens in response to malaria infection can arise from both MHCII-dependent and independent pathways.

Regulation of Immunity and Pathogenesis in Infectious Diseases by CD1d-restricted NKT Cells

CD1d-restricted NKT cells are emerging as an unusual lymphoid lineage with important immunoregulatory properties. To date, much of our understanding of the biology of the CD1/NKT system comes from studies that utilise non-natural glycolipid ligands. Recent evidence suggests that NKT cells play an important role in the response to pathogens, manifesting a range of functions including cytotoxicity, help for antibody formation and regulation of Th1/Th2 differentiation. Infectious disease models provide appropriate physiological and pathophysiological systems to explore the biological roles of this lineage in immunity and disease. Novel insights are emerging from infection models, particularly with respect to the nature of ligands recognised by the T cell receptor of NKT cells, and to the role of diverse non-T cell receptor NK activation and inhibitory receptors in regulation of the lineage. Such insights have the potential to add considerably to our understanding of the CD1/NKT cell system and to the immunology and pathogenesis of infectious diseases.

Transcriptional Profiling Reveals Suppressed Erythropoiesis, Up-regulated Glycolysis, and Interferon-associated Responses in Murine Malaria

The primary pathophysiological events contributing to fatal malaria are the cerebral syndrome, anemia, and lactic acidosis. The molecular basis of each event has been unclear. In the present study, microarray analysis of murine transcriptional responses during the development of severe disease revealed temporal, organ-specific, and pathway-specific patterns. More than 400 genes in the brain and 600 genes in the spleen displayed transcriptional changes. Dominant patterns revealed strongly suppressed erythropoiesis, starting early during infection, and highly up-regulated transcription of genes that control host glycolysis, including lactate dehydrogenase. The latter presents a mechanism that may contribute to metabolic acidosis. No evidence for hypoxia-mediated regulation of these events was observed. Interferon-regulated gene transcripts dominated the inflammatory response to cytokines. These results demonstrate previously unknown transcriptional changes in the host that may underlie the development of malarial syndromes, such as anemia and metabolic dysregulation, and increase the utility of murine models in investigation of basic malarial pathogenesis.

The Natural Killer Complex Regulates Severe Malarial Pathogenesis and Influences Acquired Immune Responses to Plasmodium Berghei ANKA

The natural killer complex (NKC) is a genetic region of highly linked genes encoding several receptors involved in the control of NK cell function. The NKC is highly polymorphic, and allelic variability of various NKC loci has been demonstrated in inbred mice. Making use of BALB.B6-Cmv1r congenic mice, in which the NKC from disease-susceptible C57BL/6 mice has been introduced into the disease-resistant BALB/c background, we show here that during murine malaria infection, the NKC regulates a range of pathophysiological syndromes such as cerebral malaria, pulmonary edema, and severe anemia, which contribute to morbidity and mortality in human malaria. Parasitemia levels were not affected by the NKC genotype, indicating that control of malarial fatalities by the NKC cells does not operate through effects on parasite growth rate. Parasite-specific antibody responses and the proinflammatory gene transcription profile, as well as the TH1/TH2 balance, also appeared to be influenced by NKC genotype, providing evidence that this region, known to control innate immune responses via NK and/or NK T-cell activation, can also significantly regulate acquired immunity to infection. To date, NKC-encoded innate system receptors have been shown mainly to regulate viral infections. Our data provide evidence for critical NKC involvement in the broad immunological responses to a protozoan parasite.

Severe Malarial Anemia of Low Parasite Burden in Rodent Models Results from Accelerated Clearance of Uninfected Erythrocytes

Severe malarial anemia (SMA) is the most frequent life-threatening complication of malaria and may contribute to the majority of malarial deaths worldwide. To explore the mechanisms of pathogenesis, we developed a novel murine model of SMA in which parasitemias peaked around 1.0% of circulating red blood cells (RBCs) and yet hemoglobin levels fell to 47% to 56% of baseline. The severity of anemia was independent of the level of peak or cumulative parasitemia, but was linked kinetically to the duration of patent infection. In vivo biotinylation analysis of the circulating blood compartment revealed that anemia arose from accelerated RBC turnover. Labeled RBCs were reduced to 1% of circulating cells by 8 days after labeling, indicating that the entire blood compartment had been turned over in approximately one week. The survival rate of freshly transfused RBCs was also markedly reduced in SMA animals, but was not altered when RBCs from SMA donors were transferred into naive recipients, suggesting few functional modifications to target RBCs. Anemia was significantly alleviated by depletion of either phagocytic cells or CD4+ T lymphocytes. This study demonstrates that immunologic mechanisms may contribute to SMA by promoting the accelerated turnover of uninfected RBCs.

CD4+ CD25+ Regulatory T Cells Suppress CD4+ T-cell Function and Inhibit the Development of Plasmodium Berghei-specific TH1 Responses Involved in Cerebral Malaria Pathogenesis

The infection of mice with Plasmodium berghei ANKA constitutes the best available mouse model for human Plasmodium falciparum-mediated cerebral malaria, a devastating neurological syndrome that kills nearly 2.5 million people every year. Experimental data suggest that cerebral disease results from the sequestration of parasitized erythrocytes within brain blood vessels, which is exacerbated by host proinflammatory responses mediated by cytokines and effector cells including T lymphocytes. Here, T cell responses to P. berghei ANKA were analyzed in cerebral malaria-resistant and -susceptible mouse strains. CD4+ T-cell proliferation and interleukin-2 (IL-2) production in response to parasite-specific and polyclonal stimuli were strongly inhibited in cerebral malaria-resistant mice. In vitro and in vivo depletion of CD4+ CD25+ regulatory T (T(reg)) cells significantly reversed the inhibition of CD4+ T-cell proliferation and IL-2 production, indicating that this cell population contributes to the suppression of T-cell function during malaria. Moreover, in vivo depletion of T(reg) cells prevented the development of parasite-specific TH1 cells involved in the induction of cerebral malaria during a secondary parasitic challenge, demonstrating a regulatory role for this cell population in the control of pathogenic responses leading to fatal disease.

NK Cells Stimulate Recruitment of CXCR3+ T Cells to the Brain During Plasmodium Berghei-mediated Cerebral Malaria

NK cells are cytotoxic lymphocytes that also secrete regulatory cytokines and can therefore influence adaptive immune responses. NK cell function is largely controlled by genes present in a genomic region named the NK complex. It has been shown that the NK complex is a genetic determinant of murine cerebral malaria pathogenesis mediated by Plasmodium berghei ANKA. In this study, we show that NK cells are required for cerebral malaria disease induction and the control of parasitemia. NK cells were found infiltrating brains of cerebral malaria-affected mice. NK cell depletion resulted in inhibition of T cell recruitment to the brain of P. berghei-infected animals. NK cell-depleted mice displayed down-regulation of CXCR3 expression and a significant reduction of T cells migrating in response to IFN-gamma-inducible protein 10, indicating that this chemokine pathway plays an essential role in leukocyte trafficking leading to cerebral disease and fatalities.

Gammadelta-T Cells Expressing NK Receptors Predominate over NK Cells and Conventional T Cells in the Innate IFN-gamma Response to Plasmodium Falciparum Malaria

Rapid production of interferon-gamma (IFN-gamma) in response to malaria by the innate immune system may determine resistance to infection, or inflammatory disease. However, conflicting reports exist regarding the identity of IFN-gamma-producing cells that rapidly respond to Plasmodium falciparum. To clarify this area, we undertook detailed phenotyping of IFN-gamma-producing cells across a panel of naive human donors following 24-h exposure to live schizont-infected red blood cells (iRBC). Here, we show that NK cells comprise only a small proportion of IFN-gamma-responding cells and that IFN-gamma production is unaffected by NK cell depletion. Instead, gammadelta-T cells represent the predominant source of innate IFN-gamma, with the majority of responding gammadelta-T cells expressing NK receptors. Malaria-responsive gammadelta-T cells more frequently expressed NKG2A compared to non-responding gammadelta-T cells, while non-responding gammadelta-T cells more frequently expressed CD158a/KIR2DL1. Unlike long-term gammadelta-T cell responses to iRBC, alphabeta-T cell help was not required for innate gammadelta-T cell responses. Diversity was observed among donors in total IFN-gamma output. This was positively associated with CD94 expression on IFN-gamma(+) NK-like gammadelta-T cells. Applied to longitudinal cohort studies in endemic regions, similar comparative phenotyping should allow assessment of the contribution of diverse cell populations and regulatory receptors to risk of infection and disease.

The Role of Leukocytes Bearing Natural Killer Complex Receptors and Killer Immunoglobulin-like Receptors in the Immunology of Malaria

The biology of Natural Killer (NK) cells and other NK Receptor (NKR)(+) leukocytes has largely been elucidated in viral or cancer systems, and involvement in other diseases or infectious states is less clearly defined. Recently, however, clear evidence has emerged for a role in malaria. NK cells and NKR(+) leukocytes significantly control susceptibility and resistance to both malaria infection and severe disease syndromes in murine models, in dependence upon receptors encoded within the Natural Killer Complex (NKC). Plasmodium falciparum can rapidly activate human NKR(+) gammadelta T cells and NK cells in vitro, and these responses are controlled partly by NKR loci encoded within the human syntenic NKC and Killer Immunoglobulin-like Receptor (KIR) genomic regions. Neither erythrocytes nor malaria parasites express HLA or MHC Class I-like homologues, or obvious stress-type ligands, suggesting the possibility of novel NKR recognition mechanisms. Parasite-derived ligands such as P. falciparum Erythrocyte Membrane Protein-1 (PfEMP-1) and glycosylphosphatidylinositol (GPI) regulate some of these diverse responses. Population-based immunogenetic analyses should allow the identification of NKC and KIR loci controlling innate and adaptive immune responses to malaria and associated with altered risk of infection and disease.

Putative IKDCs Are Functionally and Developmentally Similar to Natural Killer Cells, but Not to Dendritic Cells

Interferon-producing killer dendritic cells (IKDCs) have been described as possessing the lytic potential of NK cells and the antigen-presenting capacity of dendritic cells (DCs). In this study, we examine the lytic function and antigen-presenting capacity of mouse spleen IKDCs, including those found in DC preparations. IKDCs efficiently killed NK cell targets, without requiring additional activation stimuli. However, in our hands, when exposed to protein antigen or to MHC class II peptide, IKDCs induced little or no T cell proliferation relative to conventional DCs or plasmacytoid DCs, either before or after activation with CpG, or in several disease models. Certain developmental features indicated that IKDCs resembled NK cells more than DCs. IKDCs, like NK cells, did not express the transcription factor PU.1 and were absent from recombinase activating gene-2-null, common gamma-chain-null (Rag2(-/-)Il2rg(-/-)) mice. When cultured with IL-15 and -18, IKDCs proliferated extensively, like NK cells. Under these conditions, a proportion of expanded IKDCs and NK cells expressed high levels of surface MHC class II. However, even such MHC class II(+) IKDCs and NK cells induced poor T cell proliferative responses compared with DCs. Thus, IKDCs resemble NK cells functionally, and neither cell type could be induced to be effective antigen-presenting cells.

Plasmodium Falciparum Erythrocyte Membrane Protein-1 Specifically Suppresses Early Production of Host Interferon-gamma

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a variable antigen expressed by P. falciparum, the malarial parasite. PfEMP-1, present on the surface of infected host erythrocytes, mediates erythrocyte binding to vascular endothelium, enabling the parasite to avoid splenic clearance. In addition, PfEMP-1 is proposed to regulate host immune responses via interactions with the CD36 receptor on antigen-presenting cells. We investigated the immunoregulatory function of PfEMP-1 by comparing host cell responses to erythrocytes infected with either wild-type parasites or transgenic parasites lacking PfEMP-1. We showed that PfEMP-1 suppresses the production of the cytokine interferon-gamma by human peripheral blood mononuclear cells early after exposure to P. falciparum. Suppression of this rapid proinflammatory response was CD36 independent and specific to interferon-gamma production by gammadelta-T, NK, and alphabeta-T cells. These data demonstrate a parasite strategy for downregulating the proinflammatory interferon-gamma response and further establish transgenic parasites lacking PfEMP-1 as powerful tools for elucidating PfEMP-1 functions.

CXCR3 Determines Strain Susceptibility to Murine Cerebral Malaria by Mediating T Lymphocyte Migration Toward IFN-gamma-induced Chemokines

Cerebral malaria (CM) results from the binding of infected erythrocytes and leukocytes to brain endothelia. The precise mechanisms underlying lymphocyte recruitment and activation in CM remain unclear. Therefore, the expression of various chemokines was quantified in brains of mice infected with Plasmodium berghei ANKA (PbA). Several chemokines attracting monocytes and activated T-lymphocytes were expressed at high levels. Their expression was almost completely abrogated in IFN-gamma ligand and receptor KO mice, indicating that IFN-gamma is an essential chemokine inducer in vivo. Surprisingly, the expression levels of chemokines, IFN-gamma and also adhesion molecules in the brain were not lower in CM-resistant Balb/c and DBA/2 mice compared to CM-sensitive C57BL/6 and DBA/1 mice, although T lymphocyte sequestration in the brain was significantly less in CM-resistant than in CM-sensitive mice. This difference correlated with a higher up-regulation of the CXC chemokine receptor (CXCR)-3 on splenic T cells and a higher chemotactic response to IFN-gamma-inducible protein-10 (IP-10) in C57BL/6 compared to Balb/c mice. In conclusion, parasite-induced IFN-gamma in the brain results in high local expression levels of specific chemokines for monocytes and lymphocytes. The strain-dependent susceptibility to develop CM is more related to the expression of CXCR3 in circulating leukocytes than to the chemokine expression levels in the brain.

Blood-stage Plasmodium Infection Induces CD8+ T Lymphocytes to Parasite-expressed Antigens, Largely Regulated by CD8alpha+ Dendritic Cells

Although CD8(+) T cells do not contribute to protection against the blood stage of Plasmodium infection, there is mounting evidence that they are principal mediators of murine experimental cerebral malaria (ECM). At present, there is no direct evidence that the CD8(+) T cells mediating ECM are parasite-specific or, for that matter, whether parasite-specific CD8(+) T cells are generated in response to blood-stage infection. To resolve this and to define the cellular requirements for such priming, we generated transgenic P. berghei parasites expressing model T cell epitopes. This approach was necessary as MHC class I-restricted antigens to blood-stage infection have not been defined. Here, we show that blood-stage infection leads to parasite-specific CD8(+) and CD4(+) T cell responses. Furthermore, we show that P. berghei-expressed antigens are cross-presented by the CD8alpha(+) subset of dendritic cells (DC), and that this induces pathogen-specific cytotoxic T lymphocytes (CTL) capable of lysing cells presenting antigens expressed by blood-stage parasites. Finally, using three different experimental approaches, we provide evidence that CTL specific for parasite-expressed antigens contribute to ECM.

IP-10-mediated T Cell Homing Promotes Cerebral Inflammation over Splenic Immunity to Malaria Infection

Plasmodium falciparum malaria causes 660 million clinical cases with over 2 million deaths each year. Acquired host immunity limits the clinical impact of malaria infection and provides protection against parasite replication. Experimental evidence indicates that cell-mediated immune responses also result in detrimental inflammation and contribute to severe disease induction. In both humans and mice, the spleen is a crucial organ involved in blood stage malaria clearance, while organ-specific disease appears to be associated with sequestration of parasitized erythrocytes in vascular beds and subsequent recruitment of inflammatory leukocytes. Using a rodent model of cerebral malaria, we have previously found that the majority of T lymphocytes in intravascular infiltrates of cerebral malaria-affected mice express the chemokine receptor CXCR3. Here we investigated the effect of IP-10 blockade in the development of experimental cerebral malaria and the induction of splenic anti-parasite immunity. We found that specific neutralization of IP-10 over the course of infection and genetic deletion of this chemokine in knockout mice reduces cerebral intravascular inflammation and is sufficient to protect P. berghei ANKA-infected mice from fatality. Furthermore, our results demonstrate that lack of IP-10 during infection significantly reduces peripheral parasitemia. The increased resistance to infection observed in the absence of IP-10-mediated cell trafficking was associated with retention and subsequent expansion of parasite-specific T cells in spleens of infected animals, which appears to be advantageous for the control of parasite burden. Thus, our results demonstrate that modulating homing of cellular immune responses to malaria is critical for reaching a balance between protective immunity and immunopathogenesis.

Murine Cerebral Malaria: the Whole Story

Natural Regulatory T Cells in Malaria: Host or Parasite Allies?

Plasmodium falciparum malaria causes 500 million clinical cases with approximately one million deaths each year. After many years of exposure, individuals living in endemic areas develop a form of clinical immunity to disease known as premunition, which is characterised by low parasite burdens rather than sterilising immunity. The reason why malaria parasites persist under a state of premunition is unknown but it has been suggested that suppression of protective immunity might be a mechanism leading to parasite persistence. Although acquired immunity limits the clinical impact of infection and provides protection against parasite replication, experimental evidence indicates that cell-mediated immune responses also result in detrimental inflammation and contribute to the aetiology of severe disease. Thus, an appropriate regulatory balance between protective immune responses and immune-mediated pathology is required for a favourable outcome of infection. As natural regulatory T (T(reg)) cells are identified as an immunosuppressive lineage able to modulate the magnitude of effector responses, several studies have investigated whether this cell population plays a role in balancing protective immunity and pathogenesis during malaria. The main findings to date are summarised in this review and the implication for the induction of pathogenesis and immunity to malaria is discussed.

NK Cells and Conventional Dendritic Cells Engage in Reciprocal Activation for the Induction of Inflammatory Responses During Plasmodium Berghei ANKA Infection

Cerebral malaria (CM) is the most severe syndrome associated with Plasmodium falciparum infections. Experimental evidence suggests that disease results from the sequestration of parasitized-red blood cells (pRBCs) together with inflammatory leukocytes within brain capillaries. We have previously shown that NK cells stimulate migration of CXCR3(+) T cells to the brain of Plasmodium berghei ANKA-infected mice. Here we investigated whether interactions between NK cells and dendritic cells (DCs) are required for the induction of T cell responses involved in disease. For that, NK cell-depleted and control mice were infected with transgenic parasites expressing model T cell epitopes. T cells from TCR transgenic mice specific for those epitopes were adoptively transferred and proliferation was determined. NK cell depletion significantly reduced CD8(+) but not CD4(+) DC-mediated T cell priming. Lack of NK cells did not compromise CD8(+) T cell responses in IL-12(-/-) mice, suggesting that NK cells stimulate IL-12 output by DCs required for optimal T cell priming. The contribution of DCs to NK cell function was also investigated. DC depletion and genetic deletion of IL-12 dramatically reduced NK cell-mediated IFN-γ responses to malaria. Thus NK cells and DCs engage in reciprocal activation for the induction of inflammatory responses involved in severe malaria.

Advances in Infection and Immunity: from Bench to Bedside

This report summarizes recent advances on host-pathogen interactions, innate and adaptive responses to infection, as well as novel strategies for the control of infectious diseases.

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