Carla Claser

A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development

Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.

CD8+ T cells and IFN-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria.

Background Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood. Methods and Findings Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8+ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4+ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs. Conclusions CD8+ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues.

A Pathogenic Role for CD4+ T Cells during Chikungunya Virus Infection in Mice

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. Injury to the joint is believed to occur because of viral and host immune-mediated effects. However, the exact involvement of the different immune mediators in CHIKV-induced pathogenesis is unknown. In this study, we assessed the roles of T cells in primary CHIKV infection, virus replication and dissemination, and virus persistence, as well as in the mediation of disease severity in adult RAG2−/−, CD4−/−, CD8−/−, and wild-type CHIKV C57BL/6J mice and in wild-type mice depleted of CD4+ or CD8+ T cells after Ab treatment. CHIKV-specific T cells in the spleen and footpad were investigated using IFN-γ ELISPOT. Interestingly, our results indicated that CHIKV-specific CD4+, but not CD8+, T cells are essential for the development of joint swelling without any effect on virus replication and dissemination. Infection in IFN-γ−/− mice demonstrated that pathogenic CD4+ T cells do not mediate inflammation via an IFN-γ–mediated pathway. Taken together, these observations strongly indicate that mechanisms of joint pathology induced by CHIKV in mice resemble those in humans and differ from infections caused by other arthritogenic viruses, such as Ross River virus.

Brain microvessel cross-presentation is a hallmark of experimental cerebral malaria.

Cerebral malaria is a devastating complication of Plasmodium falciparum infection. Its pathogenesis is complex, involving both parasite‐ and immune‐mediated events. CD8+ T cells play an effector role in murine experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA) infection. We have identified a highly immunogenic CD8 epitope in glideosome‐associated protein 50 that is conserved across rodent malaria species. Epitope‐specific CD8+ T cells are induced during PbA infection, migrating to the brain just before neurological signs manifest. They are functional, cytotoxic and can damage the blood–brain barrier in vivo. Such CD8+ T cells are also found in the brain during infection with parasite strains/species that do not induce neuropathology. We demonstrate here that PbA infection causes brain microvessels to cross‐present parasite antigen, while non‐ECM‐causing parasites do not. Further, treatment with fast‐acting anti‐malarial drugs before the onset of ECM reduces parasite load and thus antigen presentation in the brain, preventing ECM death. Thus our data suggest that combined therapies targeting both the parasite and host antigen‐presenting cells may improve the outcome of CM patients.

Plasmodium vivax: restricted tropism and rapid remodeling of CD71-positive reticulocytes

Plasmodium vivax merozoites only invade reticulocytes, a minor though heterogeneous population of red blood cell precursors that can be graded by levels of transferrin receptor (CD71) expression. The development of a protocol that allows sorting reticulocytes into defined developmental stages and a robust ex vivo P vivax invasion assay has made it possible for the first time to investigate the fine-scale invasion preference of P vivax merozoites. Surprisingly, it was the immature reticulocytes (CD71(+)) that are generally restricted to the bone marrow that were preferentially invaded, whereas older reticulocytes (CD71(-)), principally found in the peripheral blood, were rarely invaded. Invasion assays based on the CD71(+) reticulocyte fraction revealed substantial postinvasion modification. Thus, 3 to 6 hours after invasion, the initially biomechanically rigid CD71(+) reticulocytes convert into a highly deformable CD71(-) infected red blood cell devoid of host reticular matter, a process that normally spans 24 hours for uninfected reticulocytes. Concurrent with these changes, clathrin pits disappear by 3 hours postinvasion, replaced by distinctive caveolae nanostructures. These 2 hitherto unsuspected features of P vivax invasion, a narrow preference for immature reticulocytes and a rapid remodeling of the host cell, provide important insights pertinent to the pathobiology of the P vivax infection.

Inflammatory Flt3l is essential to mobilize dendritic cells and for T cell responses during Plasmodium infection

Innate sensing mechanisms trigger a variety of humoral and cellular events that are essential to adaptive immune responses. Here we describe an innate sensing pathway triggered by Plasmodium infection that regulates dendritic cell homeostasis and adaptive immunity through Flt3 ligand (Flt3l) release. Plasmodium-induced Flt3l release in mice requires Toll-like receptor (TLR) activation and type I interferon (IFN) production. We found that type I IFN supports the upregulation of xanthine dehydrogenase, which metabolizes the xanthine accumulating in infected erythrocytes to uric acid. Uric acid crystals trigger mast cells to release soluble Flt3l from a pre-synthesized membrane-associated precursor. During infection, Flt3l preferentially stimulates expansion of the CD8-α+ dendritic cell subset or its BDCA3+ human dendritic cell equivalent and has a substantial impact on the magnitude of T cell activation, mostly in the CD8+ compartment. Our findings highlight a new mechanism that regulates dendritic cell homeostasis and T cell responses to infection.

Cutting Edge: Clec9A+ Dendritic Cells Mediate the Development of Experimental Cerebral Malaria

Plasmodium infections trigger strong innate and acquired immune responses, which can lead to severe complications, including the most feared and often fatal cerebral malaria (CM). To begin to dissect the roles of different dendritic cell (DC) subsets in Plasmodium-induced pathology, we have generated a transgenic strain, Clec9A-diphtheria toxin receptor that allows us to ablate in vivo Clec9A+ DCs. Specifically, we have analyzed the in vivo contribution of this DC subset in an experimental CM model using Plasmodium berghei, and we provide strong evidence that the absence of this DC subset resulted in complete resistance to experimental CM. This was accompanied with dramatic reduction of brain CD8+ T cells, and those few cerebral CD8+ T cells present had a less activated phenotype, unlike their wildtype counterparts that expressed IFN-γ and especially granzyme B. This almost complete absence of local cellular responses was also associated with reduced parasite load in the brain.

The CTLA-4 and PD-1/PD-L1 Inhibitory Pathways Independently Regulate Host Resistance to Plasmodium-induced Acute Immune Pathology.

The balance between pro-inflammatory and regulatory immune responses in determining optimal T cell activation is vital for the successful resolution of microbial infections. This balance is maintained in part by the negative regulators of T cell activation, CTLA-4 and PD-1/PD-L, which dampen effector responses during chronic infections. However, their role in acute infections, such as malaria, remains less clear. In this study, we determined the contribution of CTLA-4 and PD-1/PD-L to the regulation of T cell responses during Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM) in susceptible (C57BL/6) and resistant (BALB/c) mice. We found that the expression of CTLA-4 and PD-1 on T cells correlates with the extent of pro-inflammatory responses induced during PbA infection, being higher in C57BL/6 than in BALB/c mice. Thus, ECM develops despite high levels of expression of these inhibitory receptors. However, antibody-mediated blockade of either the CTLA-4 or PD-1/PD-L1, but not the PD-1/PD-L2, pathways during PbA-infection in ECM-resistant BALB/c mice resulted in higher levels of T cell activation, enhanced IFN-γ production, increased intravascular arrest of both parasitised erythrocytes and CD8+ T cells to the brain, and augmented incidence of ECM. Thus, in ECM-resistant BALB/c mice, CTLA-4 and PD-1/PD-L1 represent essential, independent and non-redundant pathways for maintaining T cell homeostasis during a virulent malaria infection. Moreover, neutralisation of IFN-γ or depletion of CD8+ T cells during PbA infection was shown to reverse the pathologic effects of regulatory pathway blockade, highlighting that the aetiology of ECM in the BALB/c mice is similar to that in C57BL/6 mice. In summary, our results underscore the differential and complex regulation that governs immune responses to malaria parasites.

Cerebral malaria: mysteries at the blood-brain barrier.

Cerebral malaria is the most severe pathology caused by the malaria parasite, Plasmodium falciparum. The pathogenic mechanisms leading to cerebral malaria are still poorly defined as studies have been hampered by limited accessibility to human tissues. Nevertheless, histopathology of post-mortem human tissues and mouse models of cerebral malaria have indicated involvement of the blood-brain barrier in cerebral malaria. In contrast to viruses and bacteria, malaria parasites do not infiltrate and infect the brain parenchyma. Instead, rupture of the blood-brain barrier occurs and may lead to hemorrhages resulting in neurological alterations. Here, we review the most recent findings from human studies and mouse models on the interactions of malaria parasites and the blood-brain barrier, shedding light on the pathogenesis of cerebral malaria, which may provide directions for possible interventions.

Pathogenic CD8+ T cells in experimental cerebral malaria

Cerebral malaria (CM) is one the major complications occurring during malaria infection. The mechanisms leading to this syndrome are still not completely understood. Although it is clear that parasite sequestration is the key initiation factor, the downstream pathological processes are still highly debated. The experimental cerebral malaria (ECM) model, in which susceptible mice are infected with Plasmodium berghei ANKA, has led to the identification of CD8+ T cells as the major mediator of ECM death. In this review, we discuss the recent advances and future developments in the understanding of the role of CD8+ T cells in CM.