Fiona H. Amante

Immune-mediated mechanisms of parasite tissue sequestration during experimental cerebral malaria

Cerebral malaria is a severe complication of malaria. Sequestration of parasitized RBCs in brain microvasculature is associated with disease pathogenesis, but our understanding of this process is incomplete. In this study, we examined parasite tissue sequestration in an experimental model of cerebral malaria (ECM). We show that a rapid increase in parasite biomass is strongly associated with the induction of ECM, mediated by IFN-γ and lymphotoxin α, whereas TNF and IL-10 limit this process. Crucially, we discovered that host CD4+ and CD8+ T cells promote parasite accumulation in vital organs, including the brain. Modulation of CD4+ T cell responses by helminth coinfection amplified CD4+ T cell-mediated parasite sequestration, whereas vaccination could generate CD4+ T cells that reduced parasite biomass and prevented ECM. These findings provide novel insights into immune-mediated mechanisms of ECM pathogenesis and highlight the potential of T cells to both prevent and promote infectious diseases.

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.

Granzyme B Expression by CD8+ T Cells Is Required for the Development of Experimental Cerebral Malaria

Parasite burden predicts disease severity in malaria and risk of death in cerebral malaria patients. In murine experimental cerebral malaria (ECM), parasite burden and CD8+ T cells promote disease by mechanisms that are not fully understood. We found that the majority of brain-recruited CD8+ T cells expressed granzyme B (GzmB). Furthermore, gzmB−/− mice harbored reduced parasite numbers in the brain as a consequence of enhanced antiparasitic CD4+ T cell responses and were protected from ECM. We showed in these ECM-resistant mice that adoptively transferred, Ag-specific CD8+ T cells migrated to the brain, but did not induce ECM until a critical Ag threshold was reached. ECM induction was exquisitely dependent on Ag-specific CD8+ T cell-derived perforin and GzmB, but not IFN-γ. In wild-type mice, full activation of brain-recruited CD8+ T cells also depended on a critical number of parasites in this tissue, which in turn, was sustained by these tissue-recruited cells. Thus, an interdependent relationship between parasite burden and CD8+ T cells dictates the onset of perforin/GzmB-mediated ECM.

Cutting Edge: Conventional Dendritic Cells Are the Critical APC Required for the Induction of Experimental Cerebral Malaria

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection, causing significant morbidity and mortality among young children and nonimmune adults in the developing world. Although previous work on experimental CM has identified T cells as key mediators of pathology, the APCs and subsets therein required to initiate immunopathology remain unknown. In this study, we show that conventional dendritic cells but not plasmacytoid dendritic cells are required for the induction of malaria parasite-specific CD4+ T cell responses and subsequent experimental CM. These data have important implications for the development of malaria vaccines and the therapeutic management of CM.

Prolonged Th1‐like response generated by a Plasmodium yoeli‐specific T cell clone allows complete clearance of infection in reconstituted mice

In the present study, we report the ability of in vitro cultured CD4+ T cells, generated following immunization with dead blood stage P. yoelii parasites, to mediate protection against homologous challenge infection in reconstituted nude mice. P. yoelii‐specific T cell line cells produced IFN‐γ after in vitro stimulation with specific antigen, and were protective when adoptively transferred into athymic nude mice. Following transfer of P. yoelii‐specific T cell lines into nude and SCID mice, elevated levels of nitric oxide (NO) were detected during the first week of infection at a time when parasitaemias were suppressed. However, in vivo blocking of NO production through administration of L‐NMMA, an inhibitor of NO synthase, increased mortality, but did not alter the course of primary parasitaemia in P. yoelii‐specific T cell line‐reconstituted nude mice. In addition, a P. yoelii‐specific CD4+ T cell clone, which produced IFN‐γin vitro, afforded sterile protection via mechanisms other than NO. By ELISA, antibodies were undetectable on all but one day (day 79) post T cell clone transfer and parasite challenge, where very low levels of antibodies were detected, with some evidence of recognition of malaria proteins by Western blot. Collectively, our data suggest that T cell effector functions, independent of NO production and in the absence of high levels of parasite‐specific antibodies, can contribute to sterile immunity to P. yoelii.

CD4+ Natural Regulatory T Cells Prevent Experimental Cerebral Malaria via CTLA-4 When Expanded In Vivo

Studies in malaria patients indicate that higher frequencies of peripheral blood CD4+ Foxp3+ CD25+ regulatory T (Treg) cells correlate with increased blood parasitemia. This observation implies that Treg cells impair pathogen clearance and thus may be detrimental to the host during infection. In C57BL/6 mice infected with Plasmodium berghei ANKA, depletion of Foxp3+ cells did not improve parasite control or disease outcome. In contrast, elevating frequencies of natural Treg cells in vivo using IL-2/anti-IL-2 complexes resulted in complete protection against severe disease. This protection was entirely dependent upon Foxp3+ cells and resulted in lower parasite biomass, impaired antigen-specific CD4+ T and CD8+ T cell responses that would normally promote parasite tissue sequestration in this model, and reduced recruitment of conventional T cells to the brain. Furthermore, Foxp3+ cell-mediated protection was dependent upon CTLA-4 but not IL-10. These data show that T cell-mediated parasite tissue sequestration can be reduced by regulatory T cells in a mouse model of malaria, thereby limiting malaria-induced immune pathology.

Type I interferons suppress CD4+ T-cell-dependent parasite control during blood-stage Plasmodium infection

During blood‐stage Plasmodium infection, large‐scale invasion of RBCs often occurs before the generation of cellular immune responses. In Plasmodium berghei ANKA (PbA)‐infected C57BL/6 mice, CD4+ T cells controlled parasite numbers poorly, instead providing early help to pathogenic CD8+ T cells. Expression analysis revealed that the transcriptional signature of CD4+ T cells from PbA‐infected mice was dominated by type I IFN (IFN‐I) and IFN‐γ‐signalling pathway‐related genes. A role for IFN‐I during blood‐stage Plasmodium infection had yet to be established. Here, we observed IFN‐α protein production in the spleen of PbA‐infected C57BL/6 mice over the first 2 days of infection. Mice deficient in IFN‐I signalling had reduced parasite burdens, and displayed none of the fatal neurological symptoms associated with PbA infection. IFN‐I substantially inhibited CD4+ T‐bet+ T‐cell‐derived IFN‐γ production, and prevented this emerging Th1 response from controlling parasites. Experiments using BM chimeric mice revealed that IFN‐I signalled predominantly via radio‐sensitive, haematopoietic cells, but did not suppress CD4+ T cells via direct signalling to this cell type. Finally, we found that IFN‐I suppressed IFN‐γ production, and hampered efficient control of parasitaemia in mice infected with non‐lethal Plasmodium chabaudi. Thus, we have elucidated a novel regulatory pathway in primary blood‐stage Plasmodium infection that suppresses CD4+ T‐cell‐mediated parasite control.

Activation of Invariant NKT Cells Exacerbates Experimental Visceral Leishmaniasis

We report that natural killer T (NKT) cells play only a minor physiological role in protection from Leishmania donovani infection in C57BL/6 mice. Furthermore, attempts at therapeutic activation of invariant NKT (iNKT) cells with α-galactosylceramide (α-GalCer) during L. donovani infection exacerbated, rather than ameliorated, experimental visceral leishmaniasis. The inability of α-GalCer to promote anti-parasitic immunity did not result from inefficient antigen presentation caused by infection because α-GalCer–loaded bone marrow–derived dendritic cells were also unable to improve disease resolution. The immune-dampening affect of α-GalCer correlated with a bias towards increased IL-4 production by iNKT cells following α-GalCer stimulation in infected mice compared to naïve controls. However, studies in IL-4–deficient mice, and IL-4 neutralisation in cytokine-sufficient mice revealed that α-GalCer–induced IL-4 production during infection had only a minor role in impaired parasite control. Analysis of liver cell composition following α-GalCer stimulation during an established L. donovani infection revealed important differences, predominantly a decrease in IFNγ+ CD8+ T cells, compared with control-treated mice. Our data clearly illustrate the double-edged sword of NKT cell–based therapy, showing that in some circumstances, such as when sub-clinical or chronic infections exist, iNKT cell activation can have adverse outcomes.

Type I IFN signaling in CD8– DCs impairs Th1-dependent malaria immunity

Many pathogens, including viruses, bacteria, and protozoan parasites, suppress cellular immune responses through activation of type I IFN signaling. Recent evidence suggests that immune suppression and susceptibility to the malaria parasite, Plasmodium, is mediated by type I IFN; however, it is unclear how type I IFN suppresses immunity to blood-stage Plasmodium parasites. During experimental severe malaria, CD4+ Th cell responses are suppressed, and conventional DC (cDC) function is curtailed through unknown mechanisms. Here, we tested the hypothesis that type I IFN signaling directly impairs cDC function during Plasmodium infection in mice. Using cDC-specific IFNAR1-deficient mice, and mixed BM chimeras, we found that type I IFN signaling directly affects cDC function, limiting the ability of cDCs to prime IFN-γ-producing Th1 cells. Although type I IFN signaling modulated all subsets of splenic cDCs, CD8- cDCs were especially susceptible, exhibiting reduced phagocytic and Th1-promoting properties in response to type I IFNs. Additionally, rapid and systemic IFN-α production in response to Plasmodium infection required type I IFN signaling in cDCs themselves, revealing their contribution to a feed-forward cytokine-signaling loop. Together, these data suggest abrogation of type I IFN signaling in CD8- splenic cDCs as an approach for enhancing Th1 responses against Plasmodium and other type I IFN-inducing pathogens.

High Parasite Burdens Cause Liver Damage in Mice following Plasmodium berghei ANKA Infection Independently of CD8+ T Cell-Mediated Immune Pathology

ABSTRACT Infection of C57BL/6 mice with Plasmodium berghei ANKA induces a fatal neurological disease commonly referred to as experimental cerebral malaria. The onset of neurological symptoms and mortality depend on pathogenic CD8+ T cells and elevated parasite burdens in the brain. Here we provide clear evidence of liver damage in this model, which precedes and is independent of the onset of neurological symptoms. Large numbers of parasite-specific CD8+ T cells accumulated in the liver following P. berghei ANKA infection. However, systemic depletion of these cells at various times during infection, while preventing neurological symptoms, failed to protect against liver damage or ameliorate it once established. In contrast, rapid, drug-mediated removal of parasites prevented hepatic injury if administered early and quickly resolved liver damage if administered after the onset of clinical symptoms. These data indicate that CD8+ T cell-mediated immune pathology occurs in the brain but not the liver, while parasite-dependent pathology occurs in both organs during P. berghei ANKA infection. Therefore, we show that P. berghei ANKA infection of C57BL/6 mice is a multiorgan disease driven by the accumulation of parasites, which is also characterized by organ-specific CD8+ T cell-mediated pathology.