Crossroad between Inflammation, Iron and Lipids in Atherogenesis

Atherosclerosis (ATH) is recognized as a chronic inflammatory condition and it is the leading cause of cardiovascular disease. The process of atherogenesis is characterized by the accumulation and oxidation of LDL (oxLDL) in the vessel wall and subsequent infiltration and activation of immune cells, particularly monocytes in an earlier stage and, later on, lymphocytes. The infiltrated monocytes differentiate into macrophages which then could differentiate into foam cells as a consequence of oxLDL uptake [1]. The recruitment of immune cells to the site of ATH lesion contributes to a local pro-inflammatory state that will promote the development of the atheroma plaque and progression of the disease. However, the exact mechanisms involved in this process are not fully understood. One hypothesis is the contribution of oxidative stress mediated by metals such as iron [2]. Previous authors have shown high iron content in foam cells and also accumulation of hemoglobin and ferritin in the areas rich in foam cells [3]. Herein, we investigate a possible mechanism for cellular iron accumulation by testing the effect of pro-inflammatory as well as pro-atherogenic stimuli in the expression of proteins involved in iron efflux in macrophages.This work was supported by National Institute of Health Doutor Ricardo Jorge, I.P, INSERM (Institut National de la Santé et de la Recherche Médicale), CNRS (Centre National de la Recherche Scientifique), ANR (Agence Nationale de la Recherche, France; ANR- 08- GENO-000) , Fundação para a Ciência e Tecnologia (Grant SFRH/BD/48671/2008) and BioFIG (Center for Biodiversity, Functional and Integrative Genomics)

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

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

Les rafts , radeaux lipidiques membranaires, participent à la régulation du transporteur de fer, la ferroportine, dans les macrophages

Le fer est un élément vital mais en excès il peut devenir toxique pour l organisme. Une régulation fine de son adsorption (au niveau du duodénum) et de son recyclage (au niveau des macrophages tissulaires) est donc indispensable. Cette régulation implique l hepcidine, un peptide produit majoritairement par les hépatocytes, et qui induit l endocytose et la dégradation du seul exportateur de fer connu chez les mammifères, la ferroportine. Nous avons étudié, dans les macrophages, la localisation subcellulaire de la ferroportine, sa régulation et nous avons recherché des partenaires protéiques fonctionnels ou régulateurs potentiels de cette protéine. Nous avons montré que ce transporteur est localisé dans des radeaux lipidiques (rafts) à la surface des macrophages. De plus, la désorganisation des rafts induit une diminution de la dégradation de la ferroportine par l hepcidine. Le transport de fer au niveau cellulaire semble impliquer une ferroxidase, la céruloplasmine. Nos macrophages en culture expriment une céruloplasmine cytosolique et une céruloplasmine membranaire liée à la membrane par une ancre GPI (glycosylphosphatidylinositol). Après un traitement au fer, la ferroportine et les deux formes de céruloplasmine sont surexprimées avec une céruloplasmine membranaire localisée dans des rafts de même densité que ceux de la ferroportine. Ensemble, nos observations soulignent l importance fonctionnelle des rafts dans le recyclage du fer macrophagique. Une approche protéomique des fractions rafts contenant la ferroportine nous suggèrent déjà certains acteurs moléculaires impliqués dans l activité et la régulation de la ferroportine macrophagique.ORSAY-PARIS 11-BU Sciences (914712101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Ferroportin is transcriptionally upregulated by oxldl through Nrf2 and is counteracted by LPS/IFNγ in murine macrophages

This work was supported by National Institute of Health Dr Ricardo Jorge, I.P, INSERM (Institut National de la Santé et de la Recherche Médicale), CNRS (Centre National de la Recherche Scientifique), ANR (Agence Nationale de la Recherche, France; ANR- 08-GENO-000) , Fundação para a Ciência e Tecnologia (Grant SFRH/BD/48671/2008) and BioFIG (Center for Biodiversity, Functional and Integrative Genomics). The authors report no conflit of interests

Macrophage iron metabolism profile in proatherogenic conditions

Atherosclerosis is currently considered as a chronic inflammatory disease, in which the subendothelial accumulation of macrophages and their differentiation in lipid−laden foam cells through the uptake of oxidized LDL (oxLDL) constitute key events in atherogenesis [1]. In such process, iron could constitute a modifiable factor [2] and characterization of the iron metabolism profile of the different macrophages phenotypes present in atherosclerotic lesions may provide insight on their potential protective or proatherogenic role. A new macrophage phenotype (Mox) driven by exposure to oxidized phospholipids was recently described in murine models of atherosclerosis [3]. Herein, we investigated the effect of oxLDL as well as proinflammatory stimuli on macrophage polarization and iron metabolism profile.This work was supported by Instituto Nacional de Saúde Doutor Ricardo Jorge; INSERM (Institut National de la Santé et de la Recherche Médicale); CNRS (Centre National de la Recherche Scientifique); ANR (Agence Nationale de la Recherche; ANR- 08-GENO-000); Fundação para a Ciência e Tecnologia (Grant SFRH/BD/48671/2008); BioISI (Biosystems & Integrative Sciences Institute)

The effect of pro-inflammatory conditions on iron homeostasis and atherogenesis

This work was supported by National Institute of Health Dr Ricardo Jorge, I.P, INSERM (Institut National de la Santé et de la Recherche Médicale), CNRS (Centre National de la Recherche Scientifique), ANR (Agence Nationale de la Recherche, France; ANR- 08-GENO-000) , Fundação para a Ciência e Tecnologia (Grant SFRH/BD/48671/2008) and BioFIG (B Center for Biodiversity, Functional and Integrative Genomics).Atherosclerosis (ATH) is recognized as a chronic inflammatory condition and it is the leading cause of cardiovascular disease. Atherogenesis is characterized by the accumulation and oxidation of LDL (oxLDL) in the vessel wall and subsequent infiltration, activation of lymphocytes and monocytes, these later ones differentiating into macrophages and subsequently into foam cells. The recruitment of immune cells to the site of lesion contributes to a local pro-inflammatory state that will promote the development of the atheroma plaque and progression of the disease. However, the exact mechanisms involved in this process are not fully understood. One hypothesis is the contribution of oxidative stress mediated by metals such as iron. Previous authors have shown high iron content in foam cells and also accumulation of hemoglobin and ferritin in the areas rich in foam cells. Herein, we investigate a possible pathway for cellular iron accumulation by testing the effect of pro-inflammatory as well as pro-atherogenic stimuli in the expression of proteins involved in iron efflux from macrophages. Mouse bone marrow-derived macrophages (BMDM) were treated with LPS, iron or/and oxLDL. The expression of ferroportin (Fpn, iron exporter), beta-amyloid precursor protein (APP, ferroxidase), ceruloplasmin (Cp, ferroxidase) and hemoxygenase-1 (HO-1, heme catabolism) were analyzed by western blot of subcellular fractions (cytosol, membrane and lipid raft fractions). Oil Red O staining was used to follow foam cell differentiation by oxLDL treatment. APP and HO-1 were shown to be upregulated by both iron and LPS, being recruited to lipid rafts enriched fractions in BMDM. Fpn, present also in lipid rafts, was upregulated by iron and downregulated by LPS confirming our previous observations. Such modulation of proteins involved in iron efflux by inflammatory stimuli could also contribute to disruption of iron metabolism in plaque macrophages. In addition, foam cell differentiation of BMDM by oxLDL was accelerated in the presence of iron. These observations suggest that iron in plaque could be a pro-atherogenic factor. Moreover, effect of oxLDL expression and localization of iron-related proteins on lipid raft microdomains may constitute an important pathway for iron efflux disruption in the plaque environment and is under investigation

Erythrocytes: Central Actors in Multiple Scenes of Atherosclerosis

International audienceThe development and progression of atherosclerosis (ATH) involves lipid accumulation, oxidative stress and both vascular and blood cell dysfunction. Erythrocytes, the main circulating cells in the body, exert determinant roles in the gas transport between tissues. Erythrocytes have long been considered as simple bystanders in cardiovascular diseases, including ATH. This review highlights recent knowledge concerning the role of erythrocytes being more than just passive gas carriers, as potent contributors to atherosclerotic plaque progression. Erythrocyte physiology and ATH pathology is first described. Then, a specific chapter delineates the numerous links between erythrocytes and atherogenesis. In particular, we discuss the impact of extravasated erythrocytes in plaque iron homeostasis with potential pathological consequences. Hyperglycaemia is recognised as a significant aggravating contributor to the development of ATH. Then, a special focus is made on glycoxidative modifications of erythrocytes and their role in ATH. This chapter includes recent data proposing glycoxidised erythrocytes as putative contributors to enhanced atherothrombosis in diabetic patients