Current approach to hemochromatosis

Iron overload diseases of genetic origin are an ever changing world, due to major advances in genetics and molecular biology. Five major categories are now established: HFE-related or type1 hemochromatosis, frequently found in Caucasians, and four rarer diseases which are type 2 (A and B) hemochromatosis (juvenile hemochromatosis), type 3 hemochromatosis (transferrin receptor 2 hemochromatosis), type 4 (A and B) hemochromatosis (ferroportin disease), and a(hypo)ceruloplasminemia. Increased duodenal iron absorption and enhanced macrophagic iron recycling, both due to an impairment of hepcidin synthesis, account for the development of cellular excess in types 1, 2, 3, and 4B hemochromatosis whereas decreased cellular iron egress is involved in the main form of type 4A) hemochromatosis and in aceruloplasminemia. Non-transferrin bound iron plays an important role in cellular iron excess and damage. The combination of magnetic resonance imaging (for diagnosing visceral iron overload) and of genetic testing has drastically reduced the need for liver biopsy. Phlebotomies remain an essential therapeutic tool but the improved understanding of the intimate mechanisms underlying these diseases paves the road for innovative therapeutic approaches.     

Toll‐like receptors expressed on embryonic macrophages couple inflammatory signals to iron metabolism during early ontogenesis

Mammalian TLRs in adult animals serve indispensable functions in establishing innate and adaptive immunity and contributing to the homeostasis of surrounding tissues. However, the expression and function of TLRs during mammalian embryonic development has not been studied so far. Here, we show that CD45⁺ CD11b⁺ F4/80⁺ macrophages from 10.5‐day embryo (E10.5) co‐express TLRs and CD14. These macrophages, which have the capability to engulf both apoptotic cells and bacteria, secrete a broad spectrum of proinflammatory cytokines and chemokines upon TLR stimulation, demonstrating that their TLRs are functional. Comparative microarray analysis revealed an additional set of genes that were significantly upregulated in E10.5 TLR2⁺ CD11b⁺ macrophages. This analysis, together with our genetic, microscopic, and biochemical evidence, showed that embryonic phagocytes express protein machinery that is essential for the recycling of cellular iron and that this expression can be regulated by TLR engagement in a MyD88‐dependent manner, leading to typical inflammatory M1 responses. These results characterize the utility of TLRs as suitable markers for early embryonic phagocytes as well as molecular triggers of cellular responses, the latter being demonstrated by the involvement of TLRs in an inflammation‐mediated regulation of embryonic homeostasis via iron metabolism.     

Deciphering the molecular basis of ferroportin resistance to hepcidin: Structure/function analysis of rare SLC40A1 missense mutations found in suspected hemochromatosis type 4 patients

Genetic medicine applied to the study of hemochromatosis has identified the systemic loop controlling iron homeostasis, centered on hepcidin-ferroportin interaction. Current challenges are to dissect the molecular pathways underlying liver hepcidin synthesis in response to circulatory iron, HFE, TFR2, HJV, TMPRSS6 and BMP6 functions, and to define the major structural elements of hepcidin-ferroportin interaction. We built a first 3D model of human ferroportin structure, using the crystal structure of EmrD, a bacterial drug efflux transporter of the Major Facilitator Superfamily, as template. The model enabled study of disease-associated mutations, and guided mutagenesis experiments to determine the role of conserved residues in protein stability and iron transport. Results revealed novel amino acids that are critical for the iron export function and the hepcidin-mediated inhibition mechanism: for example, tryptophan 42, localized in the extracellular end of the ferroportin pore and involved in both biological functions. Here, we propose a strategy that is not limited to structure analysis, but integrates information from different sources, including human disease-associated mutations and functional in vitro assays. The first major hypothesis of this PhD thesis is that ferroportin resistance to hepcidin relies on different molecular mechanisms that are critical for ferroportin endocytosis, and include at least three fundamental steps: (i) hepcidin binding to ferroportin, (ii) structural reorganization of the N- and C-ter ferroportin lobes, and (iii) ferroportin ubiquitination.     

缺氧缺血对O-2A祖细胞膜铁转运蛋白1表达的影响

目的 观察膜铁转运蛋白1（FPN1）在O-2A祖细胞缺氧缺血损伤后的表达变化,探讨其在O-2A祖细胞缺氧缺血损伤中的作用。方法 体
外培养O-2A祖细胞,以特异性抗体A2B5鉴定,观察FPN1在O-2A祖细胞上的定位表达;以糖氧剥夺（OGD）法建立缺氧缺血细胞模型,CCK8法检测
细胞存活率;应用免疫荧光染色法、实时荧光定量PCR法和Western blotting法观察FPN1在细胞缺氧缺血后的表达变化。结果 FPN1定位表达于
O-2A祖细胞的细胞膜、细胞质和突起;OGD3h、6h、12h及24h细胞存活率呈时间依赖性降低（P＜0.05）;OGD12h内细胞FPN1免疫荧光强度进行
性减弱;与OGD0h相比,FPN1 mRNA和蛋白水平在OGD3h表达下调,OGD6h进一步降低,OGD12h最低,差异具有统计学意义（P＜0.05）。结论 O-2A
祖细胞缺氧缺血损伤后FPN1表达明显下调,细胞存活率显著降低,提示FPN1可能参与O-2A祖细胞的缺氧缺血损伤过程。     

Optimizing the diagnosis and the treatment of iron overload diseases

A number of human disorders are related to chronic iron overload, either of genetic or acquired origin. The multi-organ damage produced by iron excess leads, in adults and in children, to severe clinical consequences, affecting both quality of life and life expectancy. The diagnosis is increasingly based on a non-invasive strategy, resorting to clinical, biological and imaging data. The treatment rests on either venesection or chelation therapy, depending on the etiology. Major advances in the fields of molecular biology, pharmacology, and biotechnology pave the road for key improvements in the diagnostic and therapeutic management of the patients.     

Hepcidin Regulation in Wild-Type and Hfe Knockout Mice in Response to Alcohol Consumption: Evidence for an Alcohol-Induced Hypoxic Response

Background /Aims:  Expression of Hamp1 , the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice ( Hfe ^−/− ). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe ^−/− mice.
Methods:  Hfe ^−/− and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1α) was measured by western blot.
Results:  Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe ^−/− mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1α protein levels were elevated in alcohol-fed wild-type animals compared with controls.
Conclusion:  Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.     

Pathophysiology and classification of iron overload diseases; update 2018

Iron overload pathophysiology has benefited from significant advances in the knowledge of iron metabolism and in molecular genetics. As a consequence, iron overload nosology has been revisited. The hematologist may be confronted to a number of iron overload syndromes, from genetic or acquired origin. Hemochromatoses, mostly but not exclusively related to the HFE gene, correspond to systemic iron overload of genetic origin in which iron excess is the consequence of hepcidin deficiency, hepcidin being the hormone regulating negatively plasma iron. Iron excess develops following hypersideremia and the formation of non-transferrin-bound iron, which targets preferentially parenchymal cells (hepatocytes). The ferroportin disease has a totally different iron overload mechanism consisting of defective egress of cellular iron into the plasma, iron deposition taking place mostly within the macrophages (spleen). Hereditary aceruloplasminemia is peculiar since systemic iron overload involves the brain. Two main types of acquired iron overload can be seen by the hematologist, one related to dyserythropoiesis (involving hypohepcidinemia ), the other related to multiple transfusions (thalassemias, myelodysplasia, hematopoietic stem cell transplantation). Congenital sideroblastic anemias, either monosyndromic (anemia) or polysyndromic (anemia plus extra-hematological syndromes), develop both compartimental iron excess within the erythroblast mitochondria, and systemic iron overload (through dyserythropoiesis and/or transfusions).     

Iron homeostasis and toxicity in retinal degeneration

Iron is essential for many metabolic processes but can also cause damage. As a potent generator of hydroxyl radical, the most reactive of the free radicals, iron can cause considerable oxidative stress. Since iron is absorbed through diet but not excreted except through menstruation, total body iron levels buildup with age. Macular iron levels increase with age, in both men and women. This iron has the potential to contribute to retinal degeneration.

Here we present an overview of the evidence suggesting that iron may contribute to retinal degenerations. Intraocular iron foreign bodies cause retinal degeneration. Retinal iron buildup resulting from hereditary iron homeostasis disorders aceruloplasminemia, Friedreich's ataxia, and panthothenate kinase-associated neurodegeneration cause retinal degeneration. Mice with targeted mutation of the iron exporter ceruloplasmin have age-dependent retinal iron overload and a resulting retinal degeneration with features of age-related macular degeneration (AMD). Post mortem retinas from patients with AMD have more iron and the iron carrier transferrin than age-matched controls.

Over the past 10 years much has been learned about the intricate network of proteins involved in iron handling. Many of these, including transferrin, transferrin receptor, divalent metal transporter-1, ferritin, ferroportin, ceruloplasmin, hephaestin, iron-regulatory protein, and histocompatibility leukocyte antigen class I-like protein involved in iron homeostasis (HFE) have been found in the retina. Some of these proteins have been found in the cornea and lens as well. Levels of the iron carrier transferrin are high in the aqueous and vitreous humors. The functions of these proteins in other tissues, combined with studies on cultured ocular tissues, genetically engineered mice, and eye exams on patients with hereditary iron diseases provide clues regarding their ocular functions.

Iron may play a role in a broad range of ocular diseases, including glaucoma, cataract, AMD, and conditions causing intraocular hemorrhage. While iron deficiency must be prevented, the therapeutic potential of limiting iron-induced ocular oxidative damage is high. Systemic, local, or topical iron chelation with an expanding repertoire of drugs has clinical potential.     

Alcoholic liver injury： Pathological features and models—Role of alcohol in the regulation of iron metabolism

Patients with alcoholic liver disease frequently exhibit increased body iron stores, as reflected by elevated serum iron indices (transferrin saturation, ferritin) and hepatic iron concentration. Even mild to moderate alcohol consumption has been shown to increase the prevalence of iron overload. Moreover, increased hepatic iron content is associated with greater mortality from alcoholic cirrhosis, suggesting a pathogenic role for iron in alcoholic liver disease. Alcohol increases the severity of disease in patients with genetic hemochromatosis, an iron overload disorder common in the Caucasian population. Both iron and alcohol individually cause oxidative stress and lipid peroxidation, which culminates in liver injury. Despite these observations, the underlying mechanisms of iron accumulation and the source of the excess iron observed in alcoholic liver disease remain unclear. Over the last decade, several novel iron-regulatory proteins have been identified and these have greatly enhanced our understanding of iron metabolism. For example, hepcidin, a circulatory antimicrobial peptide synthesized by the hepatocytes of the liver is now known to play a central role in the regulation of iron homeostasis. This review attempts to describe the interaction of alcohol and iron-regulatory molecules. Understanding these molecular mechanisms is of considerable clinical importance because both alcoholic liver disease and genetic hemochromatosis are common diseases, in which alcohol and iron appear to act synergistically to cause liver injury.     

Pathology of hepatic iron overload

Although progress in imaging and genetics allow for a noninvasive diagnosis of most cases of genetic iron overload, liver pathology remains often useful (1) to assess prognosis by grading fibrosis and seeking for associated lesions and (2) to guide the etiological diagnosis, especially when no molecular marker is available.Then, the type of liver siderosis (parenchymal, mesenchymal or mixed) and its distribution throughout the lobule and the liver are useful means for suggesting its etiology HLA-linked hemochromatosis gene (HFE) hemochromatosis or other rare genetic hemochromatosis,nonhemochromatotic genetic iron overload (ferroportin disease, aceruloplasminemia), or iron overload secondary to excessive iron supply, inflammatory syndrome,noncirrhotic chronic liver diseases including dysmetabolic iron overload syndrome, cirrhosis, and blood disorders.