Complex genetic control of susceptibility to malaria: positional cloning of the Char9 locus

Mouse strains AcB55 and AcB61 are resistant to malaria by virtue of a mutation in erythrocyte pyruvate kinase (PklrI90N). Linkage analysis in [AcB55 × A/J] F2 mice detected a second locus (Char9; logarithm of odds = 4.74) that regulates the blood-stage replication of Plasmodium chabaudi AS independently of Pklr. We characterized the 77 genes of the Char9 locus for tissue-specific expression, strain-specific alterations in gene expression, and polymorphic variants that are possibly associated with differential susceptibility. We identified Vnn1/Vnn3 as the likely candidates responsible for Char9. Vnn3/Vnn1 map within a conserved haplotype block and show expression levels that are strictly cis-regulated by this haplotype. The absence of Vnn messenger RNA expression and lack of pantetheinase protein activity in tissues are associated with susceptibility to malaria and are linked to a complex rearrangement in the Vnn3 promoter region. The A/J strain also carries a unique nonsense mutation that leads to a truncated protein. Vanin genes code for a pantetheinase involved in the production of cysteamine, a key regulator of host responses to inflammatory stimuli. Administration of cystamine in vivo partially corrects susceptibility to malaria in A/J mice, as measured by reduced blood parasitemia and decreased mortality. These studies suggest that pantetheinase is critical for the host response to malaria

Pyruvate kinase deficiency confers susceptibility to Salmonella typhimurium infection in mice

The mouse response to acute Salmonella typhimurium infection is complex, and it is under the influence of several genes, as well as environmental factors. In a previous study, we identified two novel Salmonella susceptibility loci, Ity4 and Ity5, in a (AcB61 × 129S6)F2 cross. The peak logarithm of odds score associated with Ity4 maps to the region of the liver and red blood cell (RBC)–specific pyruvate kinase (Pklr) gene, which was previously shown to be mutated in AcB61. During Plasmodium chabaudi infection, the Pklr mutation protects the mice against this parasite, as indicated by improved survival and lower peak parasitemia. Given that RBC defects have previously been associated with resistance to malaria and susceptibility to Salmonella, we hypothesized that Pklr is the gene underlying Ity4 and that it confers susceptibility to acute S. typhimurium infection in mice. Using a fine mapping approach combined with complementation studies, comparative studies, and functional analysis, we show that Pklr is the gene underlying Ity4 and that it confers susceptibility to acute S. typhimurium infection in mice through its effect on the RBC turnover and iron metabolism

C5 deficiency and C5a or C5aR blockade protects against cerebral malaria

Experimental infection of mice with Plasmodium berghei ANKA (PbA) provides a powerful model to define genetic determinants that regulate the development of cerebral malaria (CM). Based on the hypothesis that excessive activation of the complement system may confer susceptibility to CM, we investigated the role of C5/C5a in the development of CM. We show a spectrum of susceptibility to PbA in a panel of inbred mice; all CM-susceptible mice examined were found to be C5 sufficient, whereas all C5-deficient strains were resistant to CM. Transfer of the C5-defective allele from an A/J (CM resistant) onto a C57BL/6 (CM-susceptible) genetic background in a congenic strain conferred increased resistance to CM; conversely, transfer of the C5-sufficient allele from the C57BL/6 onto the A/J background recapitulated the CM-susceptible phenotype. The role of C5 was further explored in B10.D2 mice, which are identical for all loci other than C5. C5-deficient B10.D2 mice were protected from CM, whereas C5-sufficient B10.D2 mice were susceptible. Antibody blockade of C5a or C5a receptor (C5aR) rescued susceptible mice from CM. In vitro studies showed that C5a-potentiated cytokine secretion induced by the malaria product P. falciparum glycosylphosphatidylinositol and C5aR blockade abrogated these amplified responses. These data provide evidence implicating C5/C5a in the pathogenesis of CM

Cytomegalovirus generates long-lived antigen-specific NK cells with diminished bystander activation to heterologous infection

Natural killer (NK) cells play a key role in the host response to cytomegalovirus (CMV) and can mediate an enhanced response to secondary challenge with CMV. We assessed the ability of mouse CMV (MCMV)–induced memory Ly49H(+) NK cells to respond to challenges with influenza, an acute viral infection localized to the lung, and Listeria monocytogenes, a systemic bacterial infection. MCMV-memory NK cells did not display enhanced activation or proliferation after infection with influenza or Listeria, as compared with naive Ly49H(+) or Ly49H(−) NK cells. Memory NK cells also showed impaired activation compared with naive cells when challenged with a mutant MCMV lacking m157, highlighting their antigen-specific response. Ex vivo, MCMV-memory NK cells displayed reduced phosphorylation of STAT4 and STAT1 in response to stimulation by IL-12 and type I interferon (IFN), respectively, and IFN-γ production was reduced in response to IL-12 + IL-18 compared with naive NK cells. However, costimulation of MCMV-memory NK cells with IL-12 and m157 antigen rescues their impaired response compared with cytokines alone. These findings reveal that MCMV-primed memory NK cells are diminished in their response to cytokine-driven bystander responses to heterologous infections as they become specialized and antigen-specific for the control of MCMV upon rechallenge

CD36 deficiency attenuates experimental mycobacterial infection

<p>Abstract</p> <p>Background</p> <p>Members of the CD36 scavenger receptor family have been implicated as sensors of microbial products that mediate phagocytosis and inflammation in response to a broad range of pathogens. We investigated the role of CD36 in host response to mycobacterial infection.</p> <p>Methods</p> <p>Experimental <it>Mycobacterium bovis </it>Bacillus Calmette-Guérin (BCG) infection in <it>Cd36^+/+</it>and <it>Cd36^-/-</it>mice, and <it>in vitro </it>co-cultivation of <it>M. tuberculosis</it>, BCG and <it>M. marinum </it>with <it>Cd36^+/+</it>and <it>Cd36^-/-</it>murine macrophages.</p> <p>Results</p> <p>Using an <it>in vivo </it>model of BCG infection in <it>Cd36^+/+</it>and <it>Cd36^-/-</it>mice, we found that mycobacterial burden in liver and spleen is reduced (83% lower peak splenic colony forming units, p < 0.001), as well as the density of granulomas, and circulating tumor necrosis factor (TNF) levels in <it>Cd36^-/-</it>animals. Intracellular growth of all three mycobacterial species was reduced in <it>Cd36^-/-</it>relative to wild type <it>Cd36^+/+</it>macrophages <it>in vitro</it>. This difference was not attributable to alterations in mycobacterial uptake, macrophage viability, rate of macrophage apoptosis, production of reactive oxygen and/or nitrogen species, TNF or interleukin-10. Using an <it>in vitro </it>model designed to recapitulate cellular events implicated in mycobacterial infection and dissemination <it>in vivo </it>(i.e., phagocytosis of apoptotic macrophages containing mycobacteria), we demonstrated reduced recovery of viable mycobacteria within <it>Cd36^-/-</it>macrophages.</p> <p>Conclusions</p> <p>Together, these data indicate that CD36 deficiency confers resistance to mycobacterial infection. This observation is best explained by reduced intracellular survival of mycobacteria in the <it>Cd36^-/-</it>macrophage and a role for CD36 in the cellular events involved in granuloma formation that promote early bacterial expansion and dissemination.</p

Natural killer cells: walking three paths down memory lane

Immunological memory has traditionally been regarded as a unique feature of the adaptive immune response, mediated in an antigen-specific manner by T and B lymphocytes. All other hematopoietic cells, including natural killer (NK) cells, are classified as innate immune cells, which have been considered short-lived but can respond rapidly against pathogens in a manner not thought to be driven by antigen. Interestingly, NK cells have recently been shown to survive long term after antigen exposure and subsequently mediate antigen-specific recall responses. In this review, we address the similarities between, and the controversies surrounding, three major viewpoints of NK memory that have arisen from these recent studies: (i) mouse cytomegalovirus (MCMV)-induced memory; (ii) cytokine-induced memory; and (iii) liver-restricted memory cells

The genetic basis of malaria susceptibility: uncovering novel host factors in a mouse model of blood-stage infection

This thesis examines the genetic factors controlling host response to malaria. Recombinant congenic strains AcB55 and AcB61 were identified as uniquely resistant to malaria despite a susceptible genetic background. These mice display splenomegaly, anemia, reticulocytosis and extra-medullary erythropoiesis. Genome wide mapping led to the identification of a mutation in pyruvate kinase (PklrI90N) that is associated with increased survival and decreased peak parasite levels. We found a significant malaria-protective effect with a second Pklr mutation (PklrG338D) on a distinct genetic background (CBA/N), thus validating our initial findings. The mechanism of protection was linked to increased erythrocyte uptake and compensatory erythropoiesis. Subsequently, we showed that PK-deficiency in humans was protective against P.falciparum replication in vitro. A second genetic locus in AcB55, Char9, was identified and localized to a 14Mb C57Bl/6-derived congenic segment. The Vnn1/Vnn3 genes map within the minimal interval and encode a pantetheinase enzyme; they show cis-regulated gene expression A/J-like alleles associated with an absence of mRNA. In addition, no enzymatic activity was detected in A/J tissues. Administration of cysteamine, the product of pantetheinase, to susceptible A/J mice resulted in increased survival and decreased parasite levels. The inhibition of parasite replication by cysteamine suggests a potential for this compound to be used as a novel anti-malarial monotherapy or in combination with current therapeutics. Finally, a third recombinant congenic strain, AcB62, shows rapid parasite replication with an early peak of infection, despite the presence of the PklrI90N mutation. AcB62 mice do display PK-deficiency associated anemia, erythropoiesis, tissue iron overload, and concomitant increases in iron-regulated proteins. We identified a novel locus controlling peak parasite levels in an [AcB6Cette thèse examine les facteurs génétiques contrôlant la réponse de l'hôte au paludisme. Les souches recombinantes congéniques (RCS) AcB55 et AcB61 ont été identifiées comme présentant une résistance unique au paludisme malgré un fond génétique de susceptibilité. Ces souris développent une splénomégalie, de l'anémie, de la réticulose et une érythropoïèse extra médullaire. Un génome scan a conduit à l'identification d'une mutation de la pyruvate kinase (PklrI90N), laquelle est associée à une survie accrue et à une diminution des niveaux de pic de parasitémie. Un effet protecteur significatif contre le paludisme, du à une seconde mutation de la pyruvate kinase (PklrG338D) sur un fond génétique différent (CBA/N), a également été mis en évidence, validant ainsi nos premiers résultats. Le mécanisme de protection est associé à une augmentation de la phagocytose érythrocytaire et de l'érythropoïèse compensatoire. Nous avons également montré, chez l'humain, qu'un déficit en pyruvate kinase protégeait contre la réplication de P. falciparum in vitro. Un second locus, Char9, a été identifié sur la RCS AcB55 et a été localisé sur un segment congénique de 14Mb dérivé de C57Bl/6. Les gènes Vnn1/Vnn3 sont situés à l'intérieur de l'intervalle minimum et codent pour une enzyme pantéthéinase. Leur expression est régulée en cis et les allèles de type A/J ne codent pour aucun ARNm. Par ailleurs, aucune activité enzymatique n'a été détectée dans les tissus A/J. L'administration de cystéamine (un produit de la pantéthéinase) à des souris susceptibles A/J est responsable d'une survie accrue et d'une diminution des niveaux de parasitémie. L'inhibition de la réplication du parasite par la cystéamine suggère que ce composé pourrait potentiellement être utilisé comme nouvel antipaludéen, en monothérapie ou en combinaison avec des drogues actu

Cytomegalovirus generates long-lived antigen-specific NK cells with diminished bystander activation to heterologous infection.

Natural killer (NK) cells play a key role in the host response to cytomegalovirus (CMV) and can mediate an enhanced response to secondary challenge with CMV. We assessed the ability of mouse CMV (MCMV)-induced memory Ly49H(+) NK cells to respond to challenges with influenza, an acute viral infection localized to the lung, and Listeria monocytogenes, a systemic bacterial infection. MCMV-memory NK cells did not display enhanced activation or proliferation after infection with influenza or Listeria, as compared with naive Ly49H(+) or Ly49H(-) NK cells. Memory NK cells also showed impaired activation compared with naive cells when challenged with a mutant MCMV lacking m157, highlighting their antigen-specific response. Ex vivo, MCMV-memory NK cells displayed reduced phosphorylation of STAT4 and STAT1 in response to stimulation by IL-12 and type I interferon (IFN), respectively, and IFN-γ production was reduced in response to IL-12 + IL-18 compared with naive NK cells. However, costimulation of MCMV-memory NK cells with IL-12 and m157 antigen rescues their impaired response compared with cytokines alone. These findings reveal that MCMV-primed memory NK cells are diminished in their response to cytokine-driven bystander responses to heterologous infections as they become specialized and antigen-specific for the control of MCMV upon rechallenge