Modulation of the HIF2-NCOA4 axis in enterocytes attenuates iron loading in a mouse model of hemochromatosis

Intestinal iron absorption is activated during increased systemic iron demand. The best-studied example is iron-deficiency anemia, which increases intestinal iron absorption. Interestingly, the intestinal response to anemia is very similar to that of iron overload disorders, as both the conditions activate a transcriptional program that leads to a hyperabsorption of iron via the transcription factor hypoxia-inducible factor (HIF)2a. However, pathways to selectively target intestinal-mediated iron overload remain unknown. Nuclear receptor co-activator 4 (NCOA4) is a critical cargo receptor for autophagic breakdown of ferritin (FTN) and subsequent release of iron, in a process termed ferritinophagy. Our work demonstrates that NCOA4-mediated intestinal ferritinophagy is integrated to systemic iron demand via HIF2a. To demonstrate the importance of intestinal HIF2a/ferritinophagy axis in systemic iron homeostasis, whole body and intestine-specific NCOA4-null mouse lines were generated and assessed. These analyses revealed that the intestinal and systemic response to iron deficiency was not altered following disruption of intestinal NCOA4. However, in a mouse model of hemochromatosis, ablation of intestinal NCOA4 was protective against iron overload. Therefore, NCOA4 can be selectively targeted for the management of iron overload disorders without disrupting the physiological processes involved in the response to systemic iron deficiency.

Dietary Iron

Iron metabolism differs from the metabolism of other metals in that there is no physiologic mechanism for iron excretion, it is unusual; approximately 90% of daily iron needs are obtained from an endogenous source, the breakdown of circulating RBCs. Additionally humans derive iron from their everyday diet, predominantly from plant foods and the rest from foods of animal origin. Iron is found in food as either haem or non-haem iron. Iron bioavailability has been estimated to be in the range of 14–18% for mixed diets and 5–12% for vegetarian diets in subjects with no iron stores. Iron absorption in humans is dependent on physiological requirements, but may be restricted by the quantity and availability of iron in the diet. Bioavailability of food iron is strongly influenced by enhancers and inhibitors in the diet. Iron absorption can vary from 1 to 40%. A range of iron bioavailability factors that depend on the consumption of meat, fruit, vegetables, processed foods, iron-fortified foods, and the prevalence of obesity. The methods of food preparation and processing influence the bioavailability of iron. Cooking, fermentation, or germination can, by thermal or enzymatic action, reduce the phytic acid and the hexa- and penta-inositol phosphate content. Thus improving bioavailability of non-haem iron. This chapter will elaborate the dietary iron sources and means of enhancing bioavailability.

GENERIC ALGORITHM SOLUTION IN ESTIMATION OF THE IRON ABSORPTION POTENTIAL FROM COMPLEX SCHOOL MEALS

New analytical tools to study iron bioavailability are proposed in this article. An algorithm was devised to predict dietary iron abortion from school meals based on the contents of dietary factors that have the ability to promote or inhibit heme or non-heme iron absorption. The highest absorption rate of iron from representative meals was observed in the presence of ascorbic acid (27,73%) or meat ingredients (27,70%), and the lowest absorption rate – in the presence of Ca (12,40%), tannins (5,83%) and polyphenols (5,36%). Relationships between quantities of total iron intake from foods (dialyzable iron, in vitro assay) and the value of nutritional factors in those are described as exponential equations allowing calculations at any stationary state. In elaborated formula the rate of iron absorbance can be predicted considering one, two or three nutritional factors at the same time. The results emphasize the fact that it is important to understand the meal composition for the correct estimation of iron bioavailability.

Lack of IL6 Improves Recovery from Anemia of Inflammation Which Gets Hampered in Presence of Excess Iron

Anemia of inflammation (AI) is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine interleukin 6 (IL6) and the hormone hepcidin (HAMP). IL6 is an inflammatory cytokine which also limits iron absorption by inducing HAMP, which promotes the degradation of the iron exporter ferroportin. We hypothesized that knocking down both HAMP and IL6 simultaneously will help us to understand if their mode of action in AI is uniquely limited to iron absorption and erythroid iron intake or if they also have independent roles. Henceforth, we generated IL6/HampKO (DKO) mice and, unexpectedly, observed that IL6KO mice showed the best recovery in bone marrow (BM) erythropoiesis (using flow cytometry analysis and looking at the absolute number of erythroid progenitors) after BA administration when compared to wild type (WT), HampKO and DKO mice. The best differences were observed at 14 days post BA administration. In contrast, the extramedullary erythropoiesis in the spleen was more pronounced in HampKO and DKO mice compared to WT and IL6KO animals, indicating that the mechanism impairing erythropoiesis in the BM did not affect erythroid progenitors in the spleen. These observations suggest that HAMP and IL6 proteins contribute independently to AI, with IL6 having some effect on the erythropoiesis in the BM independent from the IL6-HAMP axis leading to iron restriction. Furthermore, these observations raised the question why both HampKO and DKO mice showed reduced BM erythropoiesis compared to IL6KO animals. We investigated inflammatory cytokines and altered iron parameters as potential mediators of impaired erythropoiesis. We compared several inflammatory cytokines, including IL6, TNFa and INFg following BA administration: cytokine levels were elevated 6 hrs, reduced 48hrs after BA administration and moderately increased again two weeks later. Interestingly, among all the cytokines the levels of IL1b were significantly attenuated in IL6KO mice at day 14 compared to WT and HampKO animals. Moreover, transferrin saturation and NTBI levels were higher in HampKO and DKO animals compared to IL6KO mice. These observations strongly suggested that BM erythropoiesis is more sensitive to inflammatory insult in presence of an excess of iron, while extramedullary erythropoiesis is mildly affected and can eventually thrive under supra-physiological transferrin saturation levels. To test if increased iron affects BM erythropoiesis in presence of inflammation, we treated both WT and IL6KO mice with combination of iron dextran and BA. Both WT and IL6KO mice were treated with a combination of BA and iron at day 0 followed by alternate day of iron injections showed the poorest erythropoiesis in the BM and became rapidly sick, although the effect was significantly more pronounced in WT animals, as suggested by their survival curve. Since mycobacterium infections lead to NLPR3 inflammasome activation and Caspase1 upregulation (Marim et al. Semin Immunopathology 2017), we investigated how erythroid progenitors were affected. By flow cytometry analyses, we observed a significantly higher upregulation of the Caspase1 protein in WT and DKO mice compared to IL6KO animals. This was also reproduced by culturing WT or IL6KO BM progenitor erythroid cells in presence of mouse serum derived from WT or IL6KO mice treated with BA. Most importantly, IL6KO mice treated with BA and iron showed the highest levels of Caspase1 compared to only BA treated IL6KO mice, indicating that excess of iron abrogates the beneficial effect of IL6 deficiency on erythropoiesis under conditions of AI. Furthermore, using flow cytometry, we observed in WT mice treated with BA or BA and iron a significant increase in mitochondrial mass, which is an indicator of mitochondrial stress. The mitochondrial mass was reduced in IL6KO mice treated with BA, but again increased in IL6KO mice treated with BA and iron. We have also observed an increase of mitochondrial superoxide by confocal microscopy in WT mice compared to IL6KO mice treated with BA. Altogether, these data support a model where inflammation in presence of an excess of iron impairs BM erythropoiesis through mechanisms at least in part mediated by Caspase1 and mitochondrial dysfunction, while iron excess itself is sufficient to boost extramedullary erythropoiesis to compensate and sustain RBC production. Disclosures Vinchi: PharmaNutra: Research Funding; Vifor Pharma: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Hemochromatosis classification: update and recommendations by the BIOIRON Society

Hemochromatosis (HC) is a genetically heterogeneous disorder in which uncontrolled intestinal iron absorption may lead to progressive iron overload responsible for disabling and life-threatening complications such as arthritis, diabetes, heart failure, hepatic cirrhosis, and hepatocellular carcinoma. The recent advances in the knowledge of pathophysiology and molecular basis of iron metabolism have highlighted that HC is caused by mutations in at least five genes, resulting in insufficient hepcidin production or, rarely, resistance to hepcidin action. This has led to an HC classification based on different molecular subtypes, mainly reflecting successive gene discovery. This scheme was difficult to adopt in clinical practice and therefore needs revision. Here we present recommendations for unambiguous HC classification developed by a working group of the International Society for the Study of Iron in Biology and Medicine (BIOIRON Society) including both clinicians and basic scientists during a meeting in Heidelberg, Germany. We propose to deemphasize the use of the molecular subtype criteria in favor of a classification addressing both clinical issues and molecular complexity. Ferroportin Disease (former type 4a) has been excluded because of its distinct phenotype. The novel classification aims to be of practical help whenever a detailed molecular characterization of HC is not readily available.

Resveratrol improves the iron deficiency adaptation of Malus baccata seedlings by regulating iron absorption

Background Resveratrol (Res), a phytoalexin, has been widely reported to participate in plant resistance to fungal infections. However, little information is available on its role in abiotic stress, especially in iron deficiency stress. Malus baccata is widely used as apple rootstock in China, but it is sensitive to iron deficiency. Results In this study, we investigated the role of exogenous Res in M. baccata seedings under iron deficiency stress. Results showed that applying 100 μM exogenous Res could alleviate iron deficiency stress. The seedlings treated with Res had a lower etiolation rate and higher chlorophyll content and photosynthetic rate compared with the apple seedlings without Res treatment. Exogenous Res increased the iron content in the roots and leaves by inducing the expression of MbAHA genes and improving the H+-ATPase activity. As a result, the rhizosphere pH decreased, iron solubility increased, the expression of MbFRO2 and MbIRT1 was induced, and the ferric-chelated reductase activity was enhanced to absorb large amounts of Fe2+ into the root cells under iron deficiency conditions. Moreover, exogenous Res application increased the contents of IAA, ABA, and GA3 and decreased the contents of DHZR and BL for responding to iron deficiency stress indirectly. In addition, Res functioned as an antioxidant that strengthened the activities of antioxidant enzymes and thus eliminated reactive oxygen species production induced by iron deficiency stress. Conclusion Resveratrol improves the iron deficiency adaptation of M. baccata seedlings mainly by regulating iron absorption.