Suppression of hepcidin expression and iron overload mediate Salmonella susceptibility in ankyrin 1 ENU-induced mutant.

International audienceSalmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1(Ity16/Ity16) mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1(Ity16/Ity16) mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1(Ity16/Ity16), the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1(+/Ity16) mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1(Ity16/Ity16) and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1(+/Ity16) mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1(+/Ity16) heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants

The Erotic and the Vulgar: Visual Culture and Organized Labor's Critique of U.S. Hegemony in Occupied Japan

This essay engages the colonial legacy of postwar Japan by arguing that the political cartoons produced as part of the postwar Japanese labor movement’s critique of U.S. cultural hegemony illustrate how gendered discourses underpinned, and sometimes undermined, the ideologies formally represented by visual artists and the organizations that funded them. A significant component of organized labor’s propaganda rested on a corpus of visual media that depicted women as icons of Japanese national culture. Japan’s most militant labor unions were propagating anti-imperialist discourses that invoked an engendered/endangered nation that accentuated the importance of union roles for men by subordinating, then eliminating, union roles for women

X-linked myotubular myopathy is associated with epigenetic alterations and is ameliorated by HDAC inhibition

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention

Transcription and Translation Products of the Cytolysin Gene psm-mec on the Mobile Genetic Element SCCmec Regulate Staphylococcus aureus Virulence

The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus

Fine-mapping and phenotypic analysis of the Ity3 Salmonella susceptibility locus identify a complex genetic structure.

Experimental animal models of Salmonella infections have been widely used to identify genes important in the host immune response to infection. Using an F2 cross between the classical inbred strain C57BL/6J and the wild derived strain MOLF/Ei, we have previously identified Ity3 (Immunity to Typhimurium locus 3) as a locus contributing to the early susceptibility of MOLF/Ei mice to infection with Salmonella Typhimurium. We have also established a congenic strain (B6.MOLF-Ity/Ity3) with the MOLF/Ei Ity3 donor segment on a C57BL/6J background. The current study was designed to fine map and characterize functionally the Ity3 locus. We generated 12 recombinant sub-congenic strains that were characterized for susceptibility to infection, bacterial load in target organs, cytokine profile and anti-microbial mechanisms. These analyses showed that the impact of the Ity3 locus on survival and bacterial burden was stronger in male mice compared to female mice. Fine mapping of Ity3 indicated that two subloci contribute collectively to the susceptibility of B6.MOLF-Ity/Ity3 congenic mice to Salmonella infection. The Ity3.1 sublocus controls NADPH oxidase activity and is characterized by decreased ROS production, reduced inflammatory cytokine response and increased bacterial burden, thereby supporting a role for Ncf2 (neutrophil cytosolic factor 2 a subunit of NADPH oxidase) as the gene underlying this sublocus. The Ity3.2 sub-locus is characterized by a hyperresponsive inflammatory cytokine phenotype after exposure to Salmonella. Overall, this research provides support to the combined action of hormonal influences and complex genetic factors within the Ity3 locus in the innate immune response to Salmonella infection in wild-derived MOLF/Ei mice

Genetic dissection of the Ity3 locus identifies a role for Ncf2 co-expression modules and suggests Selp as a candidate gene underlying the Ity3.2 locus.

Typhoid fever and salmonellosis, which are caused by Salmonella Typhi and Typhimurium respectively, are responsible for significant morbidity and mortality in both developed and developing countries. We model typhoid fever using mice infected with Salmonella Typhimurium, which results in a systemic disease, whereby the outcome of infection is variable in different inbred strains of mice. This model recapitulates several clinical aspects of the human disease and allows the study of the host response to Salmonella Typhimurium infection in vivo. Previous work in our laboratory has identified three loci (Ity, Ity2 and Ity3) in the wild-derived MOLF/Ei mice influencing survival after infection with Salmonella Typhimurium. Fine mapping of the Ity3 locus indicated that two sub-loci contribute collectively to the susceptibility of B6.MOLF-Ity/Ity3 congenic mice to Salmonella infection. In the current paper, we provided further evidence supporting a role for Ncf2 (neutrophil cytosolic factor 2 a subunit of NADPH oxidase) as the gene underlying the Ity3.1 sublocus. Gene expression profiling indicated that the Ity3.1 sub-locus defined a global gene expression signature with networks articulated around Ncf2. Furthermore, based on differential expression and complementation analysis using Selp (Selectin-P) knock-out mice, Selp was identified as a strong candidate gene for the Ity3.2 sub-locus

IL-6 production by explanted splenocytes and BMDM from <i>Ity</i>, <i>Ity3, Ity3.RecN</i> and <i>Ity3.RecG</i> after stimulation with PAMPs or infection with <i>Salmonella</i> Typhimurium.

<p>A) IL-6 production by explanted splenocytes after stimulation with flagellin (Flg), lipoteichoic acid (LTA), lipopolysaccharide (LPS), zymosan (Zym), CpG-DNA (CpG), and PolyIC. The culture medium (Med) was used as negative control. <i>Ity</i> (n = 4), <i>Ity3</i> (n = 4), <i>Ity3.RecN</i> (n = 4) and <i>Ity3.RecG</i> (n = 4). Significant differences in IL-6 production are shown with respect to the reference <i>Ity</i> strain. Splenocytes from <i>Ity3.RecG</i> strain were hypo-responsive to stimulation with <i>Tlr4</i> and <i>Tlr5</i> ligands as shown by significant lower levels of IL-6 after stimulation with LPS and flagellin, respectively, as compared to the <i>Ity</i> strain. No major differences were seen in IL-6 production in <i>Ity3</i> and <i>Ity3.RecN</i> strains implying that these strains do not have a defect in TLR-dependent activation of IL-6 production. The data shown are representative of two experiments with n = 4 per experiment B) IL-6 production measured in the supernatant of BMDM derived from <i>Ity</i> (n = 4), <i>Ity3</i> (n = 4), <i>Ity3.RecN</i> (n = 4) and <i>Ity3.RecG</i> (n = 4) male mice. The data shown are representative of three experiments with n = 4 per experiment. BMDM were infected with <i>Salmonella</i> Typhimurium. After a period of 45 min, RPMI 1640 containing 100 µg/ml gentamycin was added for a period of 1 h (T0). The medium was subsequently replaced by RPMI 1640 containing 10 µg/ml gentamycin and IL-6 was measured after 2 (T2), 4 (T4) and 6 (T6) hrs of incubation. Significant differences in IL-6 production are shown with respect to the reference <i>Ity</i> strain. The data were analysed by two-way ANOVA followed by multiple-comparisons testing. *corresponds to a P-value of ≤0.05, **corresponds to a P-value of ≤0.01, ***corresponds to P-value ≤0.001, while ****implies a P≤0.0001.</p

Serum cytokine levels in <i>Ity</i>, <i>Ity3, Ity3.RecN</i> and <i>Ity3.RecG</i> male mice during infection with <i>Salmonella</i> Typhimurium.

<p>TNF (A), IL-12 (B), IL-6 (C) and IFN-γ (D) were measured in the serum of congenic <i>Ity</i> (n = 4) and <i>Ity3</i> (n = 4) and sub-congenic <i>Ity3.RecN</i> (n = 4) and <i>Ity3.RecG</i> (n = 4) mice before infection and at day 3 and day 5 post infection. The data shown are representative of two experiments with n = 4 per experiment. All levels were compared to the resistant strain <i>Ity</i> at each time point. No major differences were observed in the expression of TNF across the four strains (A). The data were analysed by two-way ANOVA followed by Dunnett’s multiple-comparisons testing. *corresponds to a P-value of ≤0.05, **corresponds to a P-value of ≤0.01, ***corresponds to P-value ≤0.001, while ****implies a P≤0.0001.</p

Fine mapping of the <i>Ity3</i> interval using 12 sub-congenic strains.

a<p>Position (Mb) based on Ensembl Build 37;</p>b<p>B6 corresponds to the C57BL/6J allele;</p>c<p>M corresponds to the MOLF/Ei allele;</p>d<p>n represents the number of mice tested;</p>e<p>the sub-congenic strains nomenclature is shortened showing only the letter attributed to each strain.</p>f<p>The survival was classified, as either <i>Ity</i>-like (Ity) or <i>Ity3</i>-like (Ity3) or intermediate (INT).</p

Bacterial load in the spleen and liver of male congenic and subcongenic strains at day 3 and day 5 post-infection with <i>Salmonella</i> Typhimurium.

<p>The bacterial burden in the spleen (A, B) and liver (C, D), is shown at day 3 (A, C) and day 5 (B, D). Significant differences in log CFU of congenic and subcongenic strains are shown with respect to the reference <i>Ity</i> strain. At day 3-post infection, there was a significant higher bacterial burden in the liver of the <i>Ity3</i> and <i>Ity3.RecG</i> strains as compared to the <i>Ity</i> strain. At day 5 post infection, the bacterial loads in both the liver and spleen were significantly higher in <i>Ity3.RecG</i> and <i>Ity3</i> males. The data were analysed by one-way ANOVA followed by Dunnett’s multiple-comparisons testing. *corresponds to a P-value of ≤0.05, **corresponds to a P-value of ≤0.01, ***corresponds to P-value ≤0.001, while ****implies a P≤0.0001.</p