Susan Gauthier

Birc1e is the gene within the Lgn1 locus associated with resistance to Legionella pneumophila

In inbred mouse strains, permissiveness to intracellular replication of Legionella pneumophila is controlled by a single locus (Lgn1), which maps to a region within distal Chromosome 13 that contains multiple copies of the gene baculoviral IAP repeat–containing 1 (Birc1, also called Naip; refs. 1–3). Genomic BAC clones from the critical interval were transferred into transgenic mice to functionally complement the Lgn1-associated susceptibility of A/J mice to L. pneumophila. Here we report that two independent BAC clones that rescue susceptibility have an overlapping region of 56 kb in which the entire Lgn1 transcript must lie. The only known full-length transcript coded in this region is Birc1e (also called Naip5).

Genetic interaction between members of the Vangl family causes neural tube defects in mice

Neural tube defects (NTDs) are very frequent congenital abnormalities in humans. Recently, we have documented independent association of Vangl1 and Vangl2 gene mutations with NTDs. In the Looptail mouse, homozygosity (but not heterozygosity) for loss-of-function alleles at Vangl2 causes the severe NTD craniorachischisis, whereas heterozygosity for mutant variants of VANGL1 is associated with NTDs in a human cohort of sporadic and familial cases. To understand the role of Vangl1 in normal development, we created a mouse mutant with an inactivating mutation at Vangl1 (Vangl1gt). Vangl1 shows a dynamic pattern of expression in the developing neural tube and notochord at the time of neural tube closure. Vangl1gt/+ heterozygotes and Vangl1gt/gt homozygotes are viable and fertile, although Vangl1gt/gt display subtle alterations in polarity of inner hair cells of the cochlea. Remarkably, and as opposed to healthy Vangl1gt/+ and Vangl2lp/+ heterozygotes, Vangl1gt/+;Vangl2lp/+ double heterozygotes show profound developmental defects that include severe craniorachischisis, inner ear defects (disorganization of the stereociliary bundles of hair cells of the organ of Corti), and cardiac abnormality (aberrant right subclavian artery). These results show that genetic interaction between Vangl1 and Vangl2 genes causes neural tube defects and raise the possibility that interaction between individual Vangl genes and other genetic loci and/or environmental factors may additionally contribute to the etiology of NTDs.

Cell-specific and inducible Nramp1 gene expression in mouse macrophages in vitro and in vivo.

Mutations in the Nrampl gene abolish natural resistance to infections with many unrelated intracellular parasites in vivo. Global cDNA amplification was used to analyze Nrampl mRNA expression in bone marrow‐derived cell colonies corresponding to either undifferentiated progenitors or to mature lymphoid, erythroid, and myeloid lineages. Nramp1 mRNA was detected in mature myeloid colonies expressing molecular markers for either the monocyte/macrophage or granulocytic lineages. Having established constitutive expression of Nramp1 in phagocytic cells, the parameters of inducible Nramp1 expression by cytokines and lipopolysaccharide (LPS) were studied in the mouse macrophage cell line RAW 264.7. LPS caused upregulation of Nramp1 expression in a time‐ and dose‐dependent fashion. This induction required de novo protein synthesis and was abrogated by treatment with cycloheximide. Treatment with interferon‐γ (IFN‐γ) also caused a modest but reproducible twofold induction of Nramp1 mRNA expression. In addition, maximum Nramp1 mRNA induction in RAW 264.7 cells was observed after pretreatment with IFN‐γ followed by LPS exposure. In vivo, Nramp1 mRNA expression could be upregulated in macrophage populations by intraperitoneal injection of either LPS or thioglycollate. Together these results indicate that Nramp1 is expressed in professional phagocytes of the myeloid lineage and can be further upregulated during macrophage activation in response to infectious, inflammatory, or cytokine stimuli. Finally, the patterns of constitutive and inducible expression of Nramp1 have been compared to those of the inducible nitric oxide synthase (iNOS) gene in the same cells. J. Leukoc. Biol. 62: 277–286; 1997.

A mutation in the Icsbp1 gene causes susceptibility to infection and a chronic myeloid leukemia–like syndrome in BXH-2 mice

BXH-2 mice develop a fatal myeloid leukemia by a two-step mutagenic process. First, a BXH-2–specific recessive mutation causes a myeloproliferative syndrome. Second, retroviral insertions alter oncogenes or tumor suppressors, resulting in clonal expansion of leukemic cells. We have identified a recessive locus on chromosome 8 (Myls) that is responsible for myeloproliferation in BXH-2. This Myls interval has been narrowed down to 2 Mb and found to contain several positional candidates, including the interferon consensus sequence–binding protein 1 gene (Icsbp, also known as interferon regulatory factor 8 [IRF8]). We show that BXH-2 mice carry a mutation (915 C to T) resulting in an arginine-to-cysteine substitution at position 294 within the predicted IRF association domain of the protein. Although expression of Icsbp1 mRNA transcripts is normal in BXH-2 splenocytes, these cells are unable to produce interleukin 12 and interferon-γ in response to activating stimuli, confirming that R294C behaves as a loss-of-function mutation. Myeloproliferation in BXH-2 mice is concomitant to increased susceptibility to Mycobacterium bovis (BCG) despite the presence of resistance alleles at the Nramp1 locus. These results suggest a two-step model for chronic myeloid leukemia in BXH-2, in which inactivation of Icsbp1 predisposes to myeloproliferation and immunodeficiency. This event is required for retroviral replication, and subsequent insertional mutagenesis that causes leukemia in BXH-2 mice.

Dysregulated inflammatory response to Candida albicans in a C5-deficient mouse strain.

ABSTRACT Experimental infection of inbred mouse strains with Candida albicans provides a good model system to identify host genetic determinants that regulate onset of, response to, and ultimate outcome of disseminated candidiasis. The A/J mouse strain is exquisitely sensitive to infection with C. albicans, while the C57BL/6J strain is relatively resistant, as measured by survival following intravenous injection of Candida blastospores. This differential susceptibility is caused by an A/J-specific loss-of-function mutation in the C5 component of the complement pathway. C5 plays several critical roles in host response to infection, including target lysis and phagocyte recruitment. Therefore, to determine which of its functions were required for host resistance to candidiasis, a detailed comparative analysis of pathophysiology and host response to acute C. albicans infection was conducted in A/J and C57BL/6J mice. C5-sufficient C57BL/6J mice were found to succumb late in infection due to severe kidney pathology, typified by fungal replication and robust neutrophil-based inflammatory response associated with extensive tissue damage. In contrast, A/J mice were moribund within 24 h postinfection but displayed little if any kidney damage despite an inability to mobilize granulocytes and a high fungal load in the kidney. Rather, C5 deficiency in A/J mice was associated with higher levels of circulating cytokines tumor necrosis factor alpha, interleukin-6, monocyte chemotactic protein 1 (MCP-1), MCP-5, and eotaxin in response to C. albicans. Transfer of the C5-defective allele from A/J onto a C57BL/6J genetic background in recombinant congenic strain BcA17 recapitulated the phenotypic aspects of the susceptibility of A/J mice to C. albicans, confirming the causative role of C5 deficiency in the dysregulated cytokine response.

Icsbp1/IRF-8 Is Required for Innate and Adaptive Immune Responses against Intracellular Pathogens

The chronic myeloid leukemia syndrome of the BXH-2 mouse strain (Mus musculus) is caused by a recessive mutation (R294C) in the transcriptional regulator Icsbp1/IRF-8. In trans activation assays using an IL-12p40 gene reporter construct introduced in RAW 264.7 mouse macrophages, we show that the Icsbp1C294 isoform behaves as a partial loss-of-function. The Icsbp1C294 hypomorph allele appears to have a threshold effect on IL-12 production, with pleiotropic consequences on resistance to different types of infections in vivo. Despite the presence of a resistance Nramp1G169 allele, BXH-2 mice (Icsbp1C294) show impaired control of Mycobacterium bovis (bacille Calmette-Guérin) multiplication both early and late during infection, with uncontrolled replication linked to inability to form granulomas in infected liver and spleen. Studies in informative (BXH-2 × BALB/cJ)F2 mice show that homozygosity for Icsbp1C294 causes susceptibility to Salmonella enterica serovar Typhimurium to a level comparable to that seen for mice lacking functional Nramp1 or TLR4. Finally, impaired Icsbp1C294 function is associated with the following: 1) increased replication of the Plasmodium chabaudi AS malarial parasite during the first burst of blood parasitemia, and 2) recurring waves of high blood parasitemia late during infection. These results show that Icsbp1 is required for orchestrating early innate responses and also long-term immune protection against unrelated intracellular pathogens.

Cysteamine, the natural metabolite of pantetheinase, shows specific activity against Plasmodium

In mice, loss of pantetheinase activity causes susceptibility to infection with Plasmodium chabaudi AS. Treatment of mice with the pantetheinase metabolite cysteamine reduces blood-stage replication of P. chabaudi and significantly increases survival. Similarly, a short exposure of Plasmodium to cysteamine ex vivo is sufficient to suppress parasite infectivity in vivo. This effect of cysteamine is specific and not observed with a related thiol (dimercaptosuccinic acid) or with the pantethine precursor of cysteamine. Also, cysteamine does not protect against infection with the parasite Trypanosoma cruzi or the fungal pathogen Candida albicans, suggesting cysteamine acts directly against the parasite and does not modulate host inflammatory response. Cysteamine exposure also blocks replication of P. falciparum in vitro; moreover, these treated parasites show higher levels of intact hemoglobin. This study highlights the in vivo action of cysteamine against Plasmodium and provides further evidence for the involvement of pantetheinase in host response to this infection.

Rescue of the neural tube defect of loop-tail mice by a BAC clone containing the Ltap gene

The mouse mutant loop-tail (Lp) is an accepted model for the study of neural tube defects (NTDs) in humans. Whereas Lp/+ heterozygotes show a mild tail defect (looped), homozygous Lp/Lp embryos show a very severe form of NTD, with a completely open neural tube from the hindbrain region to the caudal portion of the spinal cord (craniorachischisis). We have recently identified a positional candidate for Lp on chromosome 1, designated as Ltap. Here, we have used an in vivo complementation approach in transgenic mice to attempt to correct the looped-tail phenotype with a bacterial artificial chromosome clone (BAC280A23) that harbors a full-length copy of the Ltap gene. Genotype:phenotype correlations in Lp/+ heterozygotes carrying BAC280A23 show that this clone can rescue the looped-tail phenotype in two independent founder lines (P < 0.05 and P < 0.0001). Importantly, BAC280A23 is also observed to rescue the lethal NTD of Lp/Lp homozygotes, because several viable transgenic Lp/Lp mice could be identified and appeared normal (P < 0.05). Results from these gain-of-function transgenic animals strongly suggest that the positional candidate Ltap present in this BAC is indeed the gene that is defective in loop-tail.

Identification of a novel cerebral malaria susceptibility locus (Berr5) on mouse chromosome 19.

Cerebral malaria (CM) is an acute, generally lethal condition characterized by high fever, seizures and coma. The genetic component to CM can be investigated in mouse models that vary in degree of susceptibility to infection with Plasmodium berghei ANKA. Using survival time to measure susceptibility in an informative F2 cross (n=257), we identified linkage to chromosome 19 (Berr5 (Berghei resistance locus 5), LOD=4.69) controlling, in part, the differential response between resistant BALB/c and susceptible C57BL/6 progenitors. BALB/c alleles convey increased survival through the cerebral phase of infection but have no quantitative effect on parasitemia during the later, anemic phase. The Berr5 locus colocalizes with three other immune loci, including Trl-4 (tuberculosis resistance), Tsiq2 (T-cell secretion of IL-4) and Eae19 (experimental allergic encephalitis 19), suggesting the possibility of a common genetic effect underlying these phenotypes. Potential positional candidates include the family of Ifit1–3 (interferon-inducible protein with tetratricopeptide repeats 1–3) and Fas.

Cysteamine broadly improves the anti-plasmodial activity of artemisinins against murine blood stage and cerebral malaria

BackgroundThe potential emergence and spread of resistance to artemisinins in the Plasmodium falciparum malaria parasite constitutes a major global health threat. Hence, improving the efficacy of artemisinins and of artemisinin-based combination therapy (ACT) represents a major short-term goal in the global fight against malaria. Mice defective in the enzyme pantetheinase (Vnn3) show increased susceptibility to blood-stage malaria (increased parasitaemia, reduced survival), and supplementation of Vnn3 mutants with the reaction product of pantetheinase, cysteamine, corrects in part the malaria-susceptibility phenotype of the mutants. Cysteamine (Cys) is a small, naturally occurring amino-thiol that has very low toxicity in vivo and is approved for clinical use in the life-long treatment of the kidney disorder nephropathic cystinosis.MethodsThe ability of Cys to improve the anti-plasmodial activity of different clinically used artemisinins was tested. The effect of different CYS/ART combinations on malarial phenotypes (parasite blood-stage replication, overall and survival from lethal infection) was assessed in a series of in vivo experiments using Plasmodium strains that induce either blood-stage (Plasmodium chabaudi AS) or cerebral disease (Plasmodium berghei ANKA). This was also evaluated in an ex vivo experimental protocol that directly assesses the effect of such drug combinations on the viability of Plasmodium parasites, as measured by the ability of tested parasites to induce a productive infection in vivo in otherwise naïve animals.ResultsCys is found to potentiate the anti-plasmodial activity of artesunate, artemether, and arteether, towards the blood-stage malaria parasite P. chabaudi AS. Ex vivo experiments, indicate that potentiation of the anti-plasmodial activity of artemisinins by Cys is direct and does not require the presence of host factors. In addition, potentiation occurs at sub-optimal concentrations of artemisinins and Cys that on their own have little or no effect on parasite growth. Cys also dramatically enhances the efficacy and protective effect of artemisinins against cerebral malaria induced by infection with the P. berghei ANKA parasite.ConclusionThese findings indicate that inclusion of Cys in current formulations of ACT, or its use as adjunct therapy could improve the anti-plasmodial activity of artemisinin, decrease mortality in cerebral malaria patients, and prevent or delay the development and spread of artemisinin resistance.