Targeted autophagy disruption reveals the central role of macrophage iron metabolism in systemic iron homeostasis.

Iron homeostasis depends on both intracellular control through iron-responsive proteins and the systemic level of iron through hepcidin-ferroportin axis. Indeed, the hormone hepcidin downregulates the ferroportin iron exporter to control iron recycling from macrophages and iron uptake from enterocytes. Here, we focused on the role of autophagy in macrophage iron metabolism and systemic iron homeostasis. Mice deficient for autophagy in macrophages (LysM-Atg5-/-) mimicked a primary iron overload phenotype, resulting in high ferroportin expression in both macrophages and enterocytes that correlated with marked parenchymal iron overload. Furthermore, LysM-Atg5-/- mice exhibited increased hematopoietic activity with no sign of anemia but correlating with rather high plasma iron level. Compared with wild-type cells, bone marrow-derived macrophages from LysM-Atg5-/- mice had significantly increased ferroportin expression and decreased iron content, confirming high iron export. In erythrophagocytic macrophages, autophagy regulates hemosiderin storage mechanisms as well as degradation of ferroportin and subsequently its plasma membrane localization and iron export; furthermore, ferroportin colocalization with hepcidin indicates hepcidin autocrine activity. Relatively high hepatic hepcidin expression and decreased hepcidin level in the spleen of LysM-Atg5-/- mice, correlating with low hemosiderin iron storage, as well as in erythrophagocytic Atg5-/- macrophages were evidenced. Therefore, our results highlight the critical role of autophagy in macrophages for iron trafficking and systemic iron homeostasis. We propose that in macrophages, autophagy restricts ferroportin level and iron export, resulting in hepcidin expression with an autocrine-paracrine effect that plays a role in the regulation of ferroportin expression in duodenal enterocytes.

Iron Supplementation Therapy, A Friend and Foe of Mycobacterial Infections?

Iron is an essential element that is required for oxygen transfer, redox, and metabolic activities in mammals and bacteria. Mycobacteria, some of the most prevalent infectious agents in the world, require iron as growth factor. Mycobacterial-infected hosts set up a series of defense mechanisms, including systemic iron restriction and cellular iron distribution, whereas mycobacteria have developed sophisticated strategies to acquire iron from their hosts and to protect themselves from iron's harmful effects. Therefore, it is assumed that host iron and iron-binding proteins, and natural or synthetic chelators would be keys targets to inhibit mycobacterial proliferation and may have a therapeutic potential. Beyond this hypothesis, recent evidence indicates a host protective effect of iron against mycobacterial infections likely through promoting remodeled immune response. In this review, we discuss experimental procedures and clinical observations that highlight the role of the immune response against mycobacteria under various iron availability conditions. In addition, we discuss the clinical relevance of our knowledge regarding host susceptibility to mycobacteria in the context of iron availability and suggest future directions for research on the relationship between host iron and the immune response and the use of iron as a therapeutic agent.

Cell iron status influences macrophage polarization.

Macrophages play crucial roles in innate immune response and in the priming of adaptive immunity, and are characterized by their phenotypic heterogeneity and plasticity. Reprogramming intracellular metabolism in response to microenvironmental signals is required for M1/M2 macrophage polarization and function. Here we assessed the influence of iron on the polarization of the immune response in vivo and in vitro. Iron-enriched diet increased M2 marker Arg1 and Ym1 expression in liver and peritoneal macrophages, while iron deficiency decreased Arg1 expression. Under LPS-induced inflammatory conditions, low iron diet exacerbated the proinflammatory response, while the IL-12/IL-10 balance decreased with iron-rich diet, thus polarizing toward type 2 response. Indeed, in vitro macrophage iron loading reduced the basal percentage of cells expressing M1 co-stimulatory CD86 and MHC-II molecules. Further, iron loading of macrophages prevented the pro-inflammatory response induced by LPS through reduction of NF-κB p65 nuclear translocation with decreased iNOS, IL-1ß, IL-6, IL-12 and TNFα expression. The increase of intracellular iron also reduced LPS-induced hepcidin gene expression and abolished ferroportin down-regulation in macrophages, in line with macrophage polarization. Thus, iron modulates the inflammatory response outcome, as elevated iron levels increased M2 phenotype and negatively regulated M1 proinflammatory LPS-induced response.

An Iron-Rich Diet Decreases the Mycobacterial Burden and Correlates With Hepcidin Upregulation, Lower Levels of Proinflammatory Mediators, and Increased T-Cell Recruitment in a Model of Mycobacterium bovis Bacille Calmette-Guerin Infection.

Background: Recent evidence indicates a robust competition between the host and mycobacteria for iron acquisition during mycobacterial infection. Variable effects of iron supplementation on the susceptibility to mycobacterial infection have been reported. In this study, we revisited the effects of an experimental iron-enriched diet on Mycobacterium bovis bacille Calmette-Guerin (BCG) infection. Methods: Mice fed a standard diet or a diet moderately enriched with iron were infected with M. bovis BCG expressing green fluorescent protein. Colony-forming unit numbers, host myeloid cell counts, cell recruitment, cytokine production, and iron gene expression were determined at different stages of infection. Bone marrow-derived macrophages incubated with or without iron were also used to measure bacterial uptake, levels of inflammation markers, and iron gene expression. Results: In vivo analysis of BCG-infected mice revealed that moderate iron supplementation reduced inflammation, as measured by decreased proinflammatory cytokine levels and neutrophil recruitment and enhanced T-cell recruitment in granulomas, and decreased the bacterial load. Enhanced bacterial clearance in the liver correlated with upregulation of the gene encoding hepcidin, which is known to have antimicrobial proprieties, and with sequestration of iron in tissues. In cultured macrophages, iron supplementation induced reactive oxygen species and reduced uptake and intracellular growth of BCG. Conclusion: Moderate iron diet supplementation diminished inflammation and growth of M. bovis BCG via enhanced reactive oxygen species production, immune cell activation, and local hepcidin expression.

Inflammation-induced up-regulation of hepcidin and down-regulation of ferroportin transcription are dependent on macrophage polarization.

Iron is essential in all organisms. In mammals systemic iron homeostasis relies on hepcidin, a peptide hormone with defensin properties, and its target, the cell iron exporter ferroportin. Hepcidin and ferroportin transcription are both upregulated by high iron levels, but are inversely regulated upon inflammation, leading to hypoferremia. Thus, host iron genes regulation may affect the innate immune responses against infectious microorganisms. Since macrophages, which are crucial innate immune cells, express both hepcidin and ferroportin, we explored in these cells their transcriptional regulation upon inflammation which is not completely understood. Macrophages represent an heterogenous population of immune cells resulting from cytokine and pathogen sensing, indeed macrophages polarized especially into pro-inflammatory M1 and regulatory/anti-inflammatory M2 phenotypes. We found that hepcidin mRNA upregulation depends on M1 polarization and ferroportin mRNA downregulation depends on M2 subtype polarization. All TLR agonists, except TLR2 agonist, polarize to pro-inflammatory macrophages and upregulate hepcidin mRNA expression. Cell pretreatment with IFNγ or inhibitor of PI3K, p38-MAPK and NF-κB pathway involved in M1 polarization prior TLR4 activation, enhanced hepcidin upregulation. Conversely, ferroportin mRNA downregulation upon inflammation was strongly increased by macrophage polarization through TLR2- and 4-PI3K-dependent pathways, or through IL-1ß and TNFα priming prior to LPS activation.

Inflammasome, IL-1 and inflammation in ozone-induced lung injury.

Exposure to ambient ozone causes airway hyperreactivity and lung inflammation, which represent an important health concern in humans. Recent clinical and experimental studies contributed to the understanding of the mechanisms of epithelial injury, inflammation and airway hyperreactivity, which is reviewed here. The present data suggest that ozone induced oxidative stress causes inflammasome activation with the release of IL-1, other cytokines and proteases driving lung inflammation leading to the destruction of alveolar epithelia with emphysema and respiratory failure. Insights in the pathogenic pathway may allow to identify novel biomarkers of ozone-induced lung disease and therapeutic targets.

Ecosystem screening approach for pathogen-associated microorganisms affecting host disease.

The microbial community in which a pathogen evolves is fundamental to disease outcome. Species interacting with a pathogen on the host surface shape the distribution, density, and genetic diversity of the inoculum, but the role of these species is rarely determined. The screening method developed here can be used to characterize pathogen-associated species affecting disease. This strategy involves three steps: (i) constitution of the microbial community, using the pathogen as a trap; (ii) community selection, using extracts from the pathogen as the sole nutrient source; and (iii) molecular identification and the screening of isolates focusing on their effects on the growth of the pathogen in vitro and host disease. This approach was applied to a soilborne plant pathogen, Phytophthora parasitica, structured in a biofilm, for screening the microbial community from the rhizosphere of Nicotiana tabacum (the host). Two of the characterized eukaryotes interfered with the oomycete cycle and may affect the host disease. A Vorticella species acted through a mutualistic interaction with P. parasitica, disseminating pathogenic material by leaving the biofilm. A Phoma species established an amensal interaction with P. parasitica, strongly suppressing disease by inhibiting P. parasitica germination. This screening method is appropriate for all nonobligate pathogens. It allows the definition of microbial species as promoters or suppressors of a disease for a given biotope. It should also help to identify important microbial relationships for ecology and evolution of pathogens.

SNP and haplotype analysis reveals new HFE variants associated with iron overload trait.

Hereditary hemochromatosis is a common-recessive-autosomal disease characterized by progressive iron overload, and its prevalence correlates with c.845G>A (p. C282Y) mutation of the HFE gene. Two other variants c.187C>G and c.193A>T are associated with a mild iron overload phenotype. The correlation studies have revealed incompletely penetrance of the HFE mutations, as well as the lack of mutation on some chromosomes from patients. We screened for SNPs before examining allele and haplotype association with elevated iron parameters. We confirmed that the c.845G>A mutation is in complete linkage disequilibrium with a unique haplotype, whereas two haplotypes proved to account for 79.8 and 20.2% of the c.187G chromosomes whose only difference was the g.4694C>G variation. A greater prevalence of the g.4694G allele among patients' chromosomes, compared to controls, was observed. In addition, among non-mutant chromosomes the analyses revealed a risk haplotype and a protective haplotype, and the g.4694G and the c.1007-47A alleles were associated with a higher risk of elevated iron parameters. We determined that the g.4694C allele was located within a putative hypoxia-response element, protein binding was evidenced and was reduced with the g.4694C>G change. In addition, IVS4 was not spliced as well in the c.1007-47A allele compared to the c.1007-47G allele.

Iron metabolism in macrophages from HFE hemochromatosis patients.

HFE-linked hereditary hemochromatosis is a common form of iron-overload disease in European populations. We studied the role of HFE in macrophage iron metabolism. Patients under venesection treatment had higher EPO levels and drastically reduced levels of transferrin receptor (TfRC and TfR2) mRNA, and also decreased levels of HAMP mRNA in macrophages cultured in autologous serum. Macrophages from C282Y/C282Y patients cultured either in autologous serum or in FBS with or without iron supplementation, had elevated CYBRD1 (cytochrome b reductase 1), SLC40A1 (ferroportin) and FTL (ferritin L) mRNA levels. Those incubated with holo-Tf also showed lower levels of TfRC and TfR2 mRNA. Iron flux from C282Y/C282Y macrophages incubated with a low concentration of non-transferrin-bound iron (NTBI) was similar to that from wild-type macrophages, but incubation with holo-Tf or high NTBI did not trigger a continuous increase in the cytosolic calcein-chelatable iron pool in C282Y/C282Y macrophages conversely to wild-type cells. All culture conditions revealed a high level of intracellular ferritin in C282Y/C282Y macrophages compared to wild-type cells. These results suggest that the non-functional C282Y form of HFE may alter the balance between cytosolic calcein-chelatable iron and sequestered iron, thereby disrupting the iron uptake and release equilibrium in cells involved in iron storage.

Iron responses in hepatic, intestinal and macrophage/monocyte cell lines under different culture conditions.

Iron homeostasis is mainly controlled by the liver-produced hepcidin peptide, which induces the degradation of the ferroportin iron exporter and thus regulates serum iron level. Hepcidin transcription is clearly up-regulated by the pro-inflammatory cytokine IL-6 and down-regulated, in the case of iron depletion, at least via HIF transcription factors. In addition, in vivo iron overload up-regulates hepcidin, but this cannot be reproduced in cell culture or isolated hepatocytes. Here, we investigated the steady state mRNA levels of a series of genes involved in iron metabolism in hepatic HepG2, intestinal Caco-2, and monocyte/macrophage THP-1 cell lines under different iron and culture conditions. Our results showed that iron-saturated transferrin up-regulated hepcidin mRNA synthesis from HepG2 via cross-talk with macrophages or enterocyte cytokine-producing cells, whereas non-transferrin-bound iron down-regulated hepcidin, likely due to missing TfR-iron-transferrin uptake.

Impact of HFE genetic testing on clinical presentation of hereditary hemochromatosis: new epidemiological data.

BACKGROUND: Hereditary hemochromatosis (HH) is a common inherited disorder of iron metabolism in Northern European populations. The discovery of a candidate gene in 1996 (HFE), and of its main mutation (C282Y), has radically altered the way to diagnose this disease. The aim of this study was to assess the impact of the HFE gene discovery on the clinical presentation and epidemiology of HH. METHODS: We studied our cohort of 415 patients homozygous for the C282Y allele and included in a phlebotomy program in a blood centre in western Brittany, France. RESULTS: In this cohort, 56.9% of the patients were male and 21.9% began their phlebotomy program before the implementation of the genetic test. A significant decrease in the sex ratio was noticed following implementation of this DNA test, from 3.79 to 1.03 (p < 10(-5)), meaning that the proportion of diagnosed females relatives to males greatly increased. The profile of HH patients at diagnosis changed after the DNA test became available. Serum ferritin and iron values were lower and there was a reduced frequency of clinical signs displayed at diagnosis, particularly skin pigmentation (20.1 vs. 40.4%, OR = 0.37, p < 0.001) and hepatomegaly (11.0 vs. 22.7%, OR = 0.42, p = 0.006). In contrast, fatigue became a more common symptom at diagnosis (68.0 vs. 51.2%, OR = 2.03, p = 0.004). CONCLUSION: This study highlights the importance of the HFE gene discovery, which has simplified the diagnosis of HH and modified its clinical presentation and epidemiology. This study precisely measures these changes. Enhanced diagnosis of HFE-related HH at an early stage and implementation of phlebotomy treatment are anticipated to maintain normal life expectancy for these patients.

A 6-year survey of HFE gene test for hemochromatosis diagnosis.

PURPOSE: A 6-year survey of HFE gene test was conducted to evaluate its helpfulness for hereditary hemochromatosis diagnosis. METHODS: We analyzed C282Y, H63D, and S65C mutations on 3525 individuals. RESULTS: The test produced 89.7% and 30% of positive results for individuals clinically diagnosed hemochromatosis before HFE gene-test availability and those prospectively tested because of elevated serum iron parameter and/or family history, respectively; among them there were 90.4% and 48.7% of C282Y homozygotes. CONCLUSIONS: The HFE gene test confirmed a genetic defect that may lead to iron loading in individuals when iron parameter values, especially for the C282Y/C282Y, were still low as well as for genotypes usually associated with low expressivity and penetrance (C282Y/H63D, H63D/H63D). This gene-test should allow a biochemical follow-up of patients carrying a disease-related genotype.

Transcriptional regulation of the human HFE gene indicates high liver expression and erythropoiesis coregulation.

The human HFE gene is clearly involved in hereditary hemochromatosis, a common autosomal recessive genetic disorder of iron homeostasis. However, the precise role of the HFE protein is still undefined. Here, to obtain new insight, we analyzed the transcriptional regulation of HFE gene and defined the functional organization of the HFE promoter. Both in vitro transcription and reporter gene assay in transient transfection evidenced a high liver expression of the HFE mRNA. The 5' end analysis of mRNA showed two major initiation sites localized at -265 and -195 directed by TATA-like sequences and a window of initiation within the -120 to -10 GC-rich region upstream of the first coding nucleotide. Positive cis-regulating elements were characterized within the -1057/-8 region, and a negative one extending upstream (-1485/-1057) was identified. DNase I footprinting analysis and gel shift assay revealed several protein binding sites, and subsequent functional analysis evidenced transactivation of HFE by liver-enriched C/EBPalpha, erythropoietic-specific GATA-1, and ubiquitous Sp1 transcription factors. These data bring some evidence of a role of HFE in the liver and a coregulation with erythropoiesis as other genes involved in iron homeostasis.

The recently identified type 2A juvenile haemochromatosis gene (HJV), a second candidate modifier of the C282Y homozygous phenotype.

The most common form of hereditary haemochromatosis is an adult-onset condition usually associated with the HFE C282Y/C282Y genotype. The phenotypic expression of this genotype is heterogeneous and depends on a complex interplay of genetic and non-genetic factors. The aim of the present study was to determine if mutations in the recently identified HJV gene were associated with more severe iron overload phenotypes in C282Y homozygous patients. From a cohort of 310 C282Y homozygous patients, we found nine (six males and three females) with an additional HJV missense mutation in the heterozygous state (S105L, E302K, N372D, R335Q or the previously described L101P and G320V). The iron indices of eight patients appeared to be more severe than those observed in C282Y homozygous patients of identical sex and similar age ranges. The mean serum ferritin concentration of the six males with an HJV mutation was significantly higher than that of C282Y homozygous males without an additional mutation [2350.3 (sigma=1429.9) versus 1227.2 (sigma=1130.1) microg/l; P=0.0233, Student's t-test]. We have recently reported that mutations in the gene that encodes hepcidin (HAMP) could explain one part of the C282Y/C282Y-related phenotypic heterogeneity by accentuating the iron burden. Our new data reveal that mutations in the HJV gene could be associated with a similar effect. Taken together, these results emphasize that a search for modifier genes could enable us to more precisely distinguish those C282Y homozygous patients with a higher risk to develop a severe iron overload and, consequently, clinical complications.

HAMP as a modifier gene that increases the phenotypic expression of the HFE pC282Y homozygous genotype.

Hereditary hemochromatosis is a genetically heterogeneous disease of iron metabolism. The most common form of the disorder is an adult-onset form that has mainly been associated with the HFE pC282Y/pC282Y genotype. The phenotypic expression of this genotype is very heterogeneous and could be modulated by both environmental factors and modifier genes. The non-HFE hereditary hemochromatosis forms include a juvenile onset form associated with mutations in HAMP. From a cohort of 392 C282Y homozygous patients, we found 5 carriers of an additional HAMP mutation at the heterozygous state (pR59G, pG71D, or pR56X). We found that iron indices of these 5 patients were among the most elevated of the cohort. Moreover, we specified that the HAMP mutations were not detected in 300 control subjects. These results revealed that mutations in HAMP might increase the phenotypic expression of the pC282Y/pC282Y genotype. From a cohort of 31 patients with at least one chromosome lacking an HFE mutation, we further identified 4 males carrying a heterozygous HAMP mutation (pR59G or pG71D). Based on a digenic model of inheritance, these data suggest that the association of heterozygous mutations in the HFE and HAMP genes could lead, at least in some cases, to an adult-onset form of primary iron overload.

Hereditary hemochromatosis: effect of excessive alcohol consumption on disease expression in patients homozygous for the C282Y mutation.

Hereditary hemochromatosis is a common inherited disorder characterized by iron overload. A single mutation (C282Y) in the HFE gene is present in 80-95% of cases in populations of northern European extraction. The disorder presents a large phenotypic heterogeneity, and its expression can be influenced by environmental factors. This 1977-2002 study aimed to identify the influence of alcohol consumption on expression of the disease. The authors retrospectively registered 378 C282Y-homozygous patients treated in a blood center of western Brittany, France. In this cohort, 33 patients reported excessive alcohol consumption (8.7%). Those subjects presented significantly increased iron parameters (serum ferritin: 1745.2 vs. 968.7 microg/liter, p< 0.0001; serum iron: 39.9 vs. 36.0 micromol/liter, p = 0.0040; transferrin saturation: 87.1 vs. 80.1%, p = 0.0071) and elevated liver enzymes (alanine aminotransferase: 66.3 vs. 41.1 IU/liter, p = 0.0003; aspartate aminotransferase: 56.2 vs. 34.9 IU/liter, p = 0.0002). Their risk of skin pigmentation was also higher (odds ratio = 3.4, p = 0.0006). Results remained unchanged after adjustment. This study provides precise quantitative data about the impact of alcohol on expression of hereditary hemochromatosis in C282Y-homozygous patients. Excessive alcohol consumption accentuates disease expression and therefore the risk of cirrhosis and cancer. Consequently, these patients should be encouraged to consume very moderate quantities of alcohol.

Phenotypic expression of the C282Y/Q283P compound heterozygosity in HFE and molecular modeling of the Q283P mutation effect.

In Caucasians, from 4 to 35% of hereditary hemochromatosis (HH) patients carry a least one chromosome without a common assigned HFE mutation (i.e., C282Y, H63D, and S65C). We have undertaken a D-HPLC scanning of the HFE coding region in such patients in order to identify uncommon mutations liable to explain their high transferrin saturation level. Twenty HH patients from Brittany carrying at least one chromosome without an assigned mutation were selected on the basis of a transferrin saturation level with the following threshold: > or = 60% in men and > or = 50% in women, in the absence of other known causes of iron disorders. This strategy allowed us to detect a heterozygous sequence variant in exon 4 of the HFE gene from one individual who was also heterozygous for C282Y. Subsequent DNA sequencing analysis identified an adenine to cytosine transversion at position 848 which changes amino acid 283 from glutamine to proline (Q283P). Family study revealed a clear association between the C282Y/Q283P compound heterozygote genotype and the development of HH. Molecular modeling studies are in favor of a destabilizing effect of the Q283P mutation on the tertiary structure of the HFE protein. This is the first report of a natural protein variant describing the introduction of a proline in a central beta-strand position. Our approach may have practical implications in screening strategies for hereditary hemochromatosis, molecular diagnosis, and HFE structure-function relationships.