Laurent Rénia

On the Pathogenic Role of Brain-Sequestered αβ CD8 + T Cells in Experimental Cerebral Malaria

Cerebral malaria (CM) develops in a small proportion of persons infected with Plasmodium falciparum and accounts for a substantial proportion of the mortality due to this parasite. The actual pathogenic mechanisms are still poorly understood, and in humans investigations of experimental CM are unethical. Using an established Plasmodium berghei-mouse CM model, we have investigated the role of host immune cells at the pathological site, the brain. We report in this study the detailed quantification and characterization of cells, which migrated and sequestered to the brain of mice with CM. We demonstrated that CD8+ αβ T cells, which sequester in the brain at the time when neurological symptoms appear, were responsible for CM mortality. These observations suggest a mechanism which unifies disparate observations in humans.

Protective T Cell Immunity against Malaria Liver Stage after Vaccination with Live Sporozoites under Chloroquine Treatment

In this study we present the first systematic analysis of the immunity induced by normal Plasmodium yoelii sporozoites in mice. Immunization with sporozoites, which was conducted under chloroquine treatment to minimize the influence of blood stage parasites, induced a strong protection against a subsequent sporozoite and, to a lesser extent, against infected RBC challenges. The protection induced by this immunization protocol proved to be very effective. Induction of this protective immunity depended on the presence of liver stage parasites, as primaquine treatment concurrent with sporozoite immunization abrogated protection. Protection was not found to be mediated by the Abs elicited against pre-erythrocytic and blood stage parasites, as demonstrated by inhibition assays of sporozoite penetration or development in vitro and in vivo assays of sporozoite infectivity or blood stage parasite development. CD4+ and CD8+ T cells were, however, responsible for the protection through the induction of IFN-γ and NO.

Dual Role of CCR2 in the Constitution and the Resolution of Liver Fibrosis in Mice

Inflammation has been shown to induce the progression of fibrosis in response to liver injury. Among inflammatory cells, macrophages and lymphocytes play major roles in both the constitution and resolution of liver fibrosis. The chemokine receptor CCR2 is involved in the recruitment of monocytes to injury sites, and it is known to be induced during the progression of fibrosis in humans. However, its specific role during this process has not yet been unveiled. We first demonstrated that, compared with wild-type mice, CCR2 knockout animals presented a delay in liver injury after acute CCl(4) injection, accompanied by a reduction in infiltrating macrophage populations. We then induced fibrosis using repeated injections of CCl(4) and observed a significantly lower level of fibrotic scars at the peak of fibrosis in mutant animals compared with control mice. This diminished fibrosis was associated with a reduction in F4/80(+)CD11b(+) and CD11c(+) populations at the sites of injury. Subsequent analysis of the kinetics of the resolution of fibrosis showed that fibrosis rapidly regressed in wild-type, but not in CCR2(-/-) mice. The persistence of hepatic injury in mutant animals was correlated with sustained tissue inhibitor of metalloproteinase-1 mRNA expression levels and a reduction in matrix metalloproteinase-2 and matrix metalloproteinase-13 expression levels. In conclusion, these findings underline the role of the CCR2 signaling pathway in both the constitution and resolution of liver fibrotic scars.

Invasion of host cells by malaria parasites: a tale of two protein families

Malaria parasites are obligate intracellular parasites whose invasive stages select and invade the unique host cell in which they can develop with exquisite specificity and efficacy. Most studies aimed at elucidating the molecules and the mechanisms implicated in the selection and invasion processes have been conducted on the merozoite, the stage that invades erythrocytes to perpetuate the pathological cycles of parasite multiplication in the blood. Bioinformatic analysis has helped identify the members of two parasite protein families, the reticulocyte‐binding protein homologues (RBL) and erythrocyte binding like (EBL), in recently sequenced genomes of different Plasmodium species. In this article we review data from classical studies and gene disruption experiments that are helping to illuminate the role of these proteins in the selection‐invasion processes. The manner in which subsets of proteins from each of the families act in concert suggests a model to explain the ability of the parasites to use alternate pathways of invasion. Future perspectives and implications are discussed.

Control of pathogenic CD8+ T cell migration to the brain by IFN-γ during experimental cerebral malaria

Previous studies have shown that IFN‐γ is essential for the pathogenesis of cerebral malaria (CM) induced by Plasmodium berghei ANKA (PbA) in mice. However, the exact role of IFN‐γ in the pathway (s) leading to CM has not yet been described. Here, we used 129P2Sv/ev mice which develop CM between 7 and 14 days post‐infection with PbA. In this strain, both CD4+ and CD8+ T cells were involved in the effector phase of CM. When 129P2Sv/ev mice deficient in the IFN‐γ receptor α chain (IFN‐γR1) were infected with PbA, CM did not occur. Migration of leucocytes to the brain at the time of CM was observed in wild type (WT) but not in deficient mice. However, in the latter, there was an accumulation of T cells in the lungs. Analysis of chemokines and their receptors in WT and in deficient mice revealed a complex, organ‐specific pattern of expression. Up‐regulation of RANTES/CCL5, IP‐10/CCL3 and CCR2 was associated with leucocyte migration to the brain and increased expression of MCP‐1/CCL2, IP‐10/CCL3 and CCR5 with leucocyte migration to the lung. This shows that IFN‐γ controls trafficking of pathogenic T cells in the brain, thus providing an explanation for the organ‐specific pathology induced by PbA infection.

Malaria sporozoite penetration: a new approach by double staining

To determine, whether a sporozoite is outside the hepatocyte membrane or internalized, a double staining test was carried out using, successively, antibody labeled with peroxidase and fluorescein. This test permits the quantification of sporozoite entry and outline sporozoite-hepatocyte interactions.

Sterile Protection against Malaria Is Independent of Immune Responses to the Circumsporozoite Protein

Background Research aimed at developing vaccines against infectious diseases generally seeks to induce robust immune responses to immunodominant antigens. This approach has led to a number of efficient bacterial and viral vaccines, but it has yet to do so for parasitic pathogens. For malaria, a disease of global importance due to infection by Plasmodium protozoa, immunization with radiation-attenuated sporozoites uniquely leads to long lasting sterile immunity against infection. The circumsporozoite protein (CSP), an important component of the sporozoites surface, remains the leading candidate antigen for vaccines targeting the parasites pre-erythrocytic stages. Difficulties in developing CSP-based vaccines that reproduce the levels of protection afforded by radiation-attenuated sporozoites have led us to question the role of CSP in the acquisition of sterile immunity. We have used a parasite transgenic for the CSP because it allowed us to test whether a major immunodominant Plasmodium antigen is indeed needed for the induction of sterile protective immunity against infection. Methodology/Main Findings We employed a P. berghei parasite line that expresses a heterologous CSP from P. falciparum in order to assess the role of the CSP in the protection conferred by vaccination with radiation-attenuated P. berghei parasites. Our data demonstrated that sterile immunity could be obtained despite the absence of immune responses specific to the CSP expressed by the parasite used for challenge. Conclusions We conclude that other pre-erythrocytic parasite antigens, possibly hitherto uncharacterised, can be targeted to induce sterile immunity against malaria. From a broader perspective, our results raise the question as to whether immunodominant parasite antigens should be the favoured targets for vaccine development.

Vaccination with Live Plasmodium yoelii Blood Stage Parasites under Chloroquine Cover Induces Cross-Stage Immunity against Malaria Liver Stage

Immunity to malaria has long been thought to be stage-specific. In this study we show that immunization of BALB/c mice with live erythrocytes infected with nonlethal strains of Plasmodium yoelii under curative chloroquine cover conferred protection not only against challenge by blood stage parasites but also against sporozoite challenge. This cross-stage protection was dose-dependent and long lasting. CD4+ and CD8+ T cells inhibited malaria liver but not blood stage. Their effect was mediated partially by IFN-γ, and was completely dependent of NO. Abs against both pre-erythrocytic and blood parasites were elicited and were essential for protection against blood stage and liver stage parasites. Our results suggest that Ags shared by liver and blood stage parasites can be the foundation for a malaria vaccine that would provide effective protection against both pre-erythrocytic and erythrocytic asexual parasites found in the mammalian host.

Chagasic patients develop a type 1 immune response to Trypanosoma cruzi trans-sialidase.

Infective forms of Trypanosoma cruzi, the parasite that causes Chagas’ disease, express on their surface an enzyme denominated trans‐sialidase (TS). The present study was designed to evaluate the naturally acquired immune responses to a bacterial recombinant protein representing the catalytic domain of TS in chronically infected chagasic individuals. The cellular immune response was measured by in‐vitro T‐cell proliferation and by interferon (INF)‐g, interleukin (IL)‐4 and IL‐10 production in response to a whole‐parasite homogenate and the recombinant protein. The peripheral blood mononuclear cells of 78·6% of 28 chagasic patients responded to the recombinant protein as estimated by T‐cell proliferation. With respect to cytokine production, 88% of the cells of the chagasic individuals produced IFN‐g on stimulation with the recombinant protein. In contrast, IL‐4 or IL‐10 were minimally produced in response to TS. The cellular immune response was specific because most healthy individuals never exposed to T. cruzi failed to react with this recombinant protein. The plasma of 71·4% or 100% of chagasic patients had IgG antibodies as determined by ELISA or by the presence of TS inhibitory antibodies, respectively. We conclude that the catalytic domain of TS is recognized by IFN‐g producing type 1 cells and antibodies in a large proportion of patients infected with T. cruzi.

Iron‐ and inflammation‐induced hepcidin gene expression in mice is not mediated by Kupffer cells in vivo

Hepcidin, a recently discovered iron regulatory peptide, is believed to inhibit the release of iron from absorptive enterocytes and macrophages. Liver hepcidin synthesis is induced in vivo by iron stores and inflammation. The molecular basis of the regulation of hepcidin gene expression by these effectors in hepatocytes is currently unknown, although there is strong evidence that indirect mechanisms are involved. The aims of this study were to gain insight into these mechanisms and to determine to what extent other liver cell types are responsible for transducing the signal by which hepcidin expression is regulated in mouse hepatocytes. For this, we depleted Kupffer cells by injection of liposome‐encapsulated clodronate and then studied iron‐ and inflammation‐induced hepcidin gene expression. In addition, we directly evaluated the role of the inflammatory cytokine interleukin 6 (IL‐6) by using IL‐6–deficient mice. Our results show that iron is able to induce hepcidin gene expression independently of Kupffer cells in the liver and circulating IL‐6. In contrast, we show that hepcidin gene induction by inflammation is also independent of Kupffer cells, but involves, at least partly, IL‐6. In conclusion, these results show that two independent regulatory pathways control hepcidin gene expression and suggest that hepatocytes play a key role in the regulation of hepcidin gene expression by sensing iron and inflammatory signals. (HEPATOLOGY 2005.)