Recent advances are furthering our understanding of the role of iron in disease. CLI caught up with Professor Martina U. Muckenthaler and Dr Oriana Marques to learn more about the importance of iron homeostasis.
Iron is needed in the body for many metabolic processes. Can you summarize how normal iron homeostasis is achieved?
Iron is an essential element for numerous biological processes, such as energy production, oxygen transport, cell proliferation and intermediate metabolism. Systemic iron homeostasis has evolved to maintain a plasma iron concentration that secures adequate supplies while preventing organ iron overload. As there is no physiologically regulated pathway for iron elimination, iron uptake from the diet is a tightly controlled process. Iron is absorbed as inorganic non-heme iron, mostly available in vegetables, and heme iron present in meat. In the duodenum it passes through a cellular layer, the enterocytes, and reaches the blood where iron is bound to transferrin. Iron-bound transferrin is transported to all organs with a demand for iron. Approximately 20–25mg of iron are transported in the blood every day, whereby most iron is required to sustain red blood cell synthesis. Iron homeostasis, to a large extent, relies on the recycling of aged red blood cells by macrophages; only 1–2mg/day are absorbed from the diet to compensate for blood loss and sloughing intestinal cells. Despite rapid turnover, plasma iron concentrations are generally stable, indicating that the mechanisms regulating plasma iron must be very tightly controlled. See Fig. 3 in Hentze et al. 2004 for a summary of systemic iron homeostasis  (https://bit.ly/3wpoffO).
Iron absorption by the enterocytes, its release from iron stores (e.g. liver cells) and iron recycling macrophages are mainly controlled by the hepatic hormone hepcidin. In response to iron overload and infection/inflammation, hepcidin synthesis is increased in the liver, and through release in the bloodstream it reaches iron-exporting cells where it binds its receptor, the iron exporter ferroportin (Fpn), promoting its degradation and internalization. As a consequence, plasma iron levels are reduced. Conversely, in situations of higher iron demand, such as during iron deficiency, hypoxia or increased erythropoiesis, hepcidin secretion by the liver is reduced, causing a stabilization of Fpn on the cellular membrane of iron-exporting cell types and, consequently, an increase in plasma iron levels. See Fig. 4 in Hentze et al. 2004 for a summary of the regulation of systemic iron homeostasis by the Hepcidin-Ferroportin axis  (https://bit.ly/3wpoffO).
Disorders of iron metabolism are common. What can happen when normal iron homeostasis is disrupted?
Iron overload and deficiency diseases belong to the most common pathologies across the globe. Malnutrition, chronic inflammation and infection, as well as uncontrolled blood loss (e.g. premenopausal women or Crohn’s disease patients) account for most cases of iron deficiency. In rare cases, iron-deficiency anemias are caused by mutations in genes that affect iron handling, for example, during erythropoiesis or dietary iron uptake. This group of rare microcytic anemias is monitored in the German national registry maintained in the Department of Pediatric Hematology, Immunology and Oncology of the University of Heidelberg, which is linked to the registries of the EuroBloodNet (https://eurobloodnet.eu/) at the European level. An example for this group of rare anemias is the iron-refractory iron-deficiency anemia (IRIDA), a congenital microcytic and hypochromic anemia caused by mutations in the TMPRSS6 gene, a negative regulator of hepcidin. In these patients, hepcidin levels are extremely high, inhibiting not only iron uptake from the diet and iron release from iron recycling macrophages, but also preventing oral and intravenous iron supplementation.
Chronic inflammatory diseases, such as cancer or autoimmune conditions, may cause anemia of inflammation – a disease arising from a pro-inflammatory cytokine-mediated increase of hepcidin and as a consequence, persistently decreased iron absorption from the diet and iron release from stores. In this disease, iron is sequestered in macrophages causing hypoferremia (decreased iron levels in the plasma). If inflammation and hypoferremia persists over an extended period of time, anemia will develop. The fact that iron homeostasis is altered in response to inflammation reveals an important crosstalk between the control of systemic iron metabolism and immune responses. Iron is not only essential for humans, but also for most pathogenic organisms. It is, therefore, part of our innate immune response to withhold iron from pathogenic microorganisms by iron sequestration in macrophages. An impressive example are malaria infections, where the risk of adverse health outcomes increases upon iron supplementation. Furthermore, higher iron stores in patients with thalassemia increases susceptibility to infections. Of note, balanced iron levels need to be maintained as low serum iron levels impair primary and memory immune responses due to T-cell iron requirements for proliferation and function. On the other end of the spectrum, iron overload is also common and seen in iron overload disorders of genetic origin (e.g. hereditary hemochromatosis, iron-loading anemias) and as a result of repeated blood transfusions (e.g. to treat hemoglobinopathies and congenital hemolytic anemias). Iron overload is also observed in aceruloplasminemia, a disease caused by mutations in the gene encoding ceruloplasmin (CP), which is associated with brain iron deposition, retinal degeneration, ataxia and dementia. In addition, atransferrinemia, hallmarked by mutations in the gene encoding transferrin (TF) and resulting in a microcytic, hypochromic anemia, is also associated with tissue iron overload.
It is now well accepted that dysregulation of iron levels contributes to the pathogenesis of several multifactorial diseases. We have recently shown that atherosclerosis is exacerbated by iron overload, causing lipid profile alterations, endothelial dysfunction, and inflammation. These alterations are associated with the occurrence of a toxic form of iron – non-transferrin bound iron that induces reactive oxygen species. In addition, iron overload is frequently seen in liver diseases, such as diabetes and non-alcoholic fatty liver disease. Conversely, iron deficiency also is a risk factor for cardiovascular disease.
Balancing iron homeostasis is also critical for brain function, as iron deficiency is associated with cognitive deficits, while excess iron contributes to neurodegeneration. Furthermore, iron availability is linked to cancer development and metastasis, as cancer cells are addicted to essential nutrients such as iron to sustain proliferation.
The examples mentioned here demonstrate the critical importance of balancing iron levels.
Iron overload causes hemochromatosis, which is perhaps more common than we realize. What is hemochromatosis, how is it caused, and how can it be diagnosed and treated?
Hemochromatosis is the most frequently inherited genetic disease in a population of Caucasian descent. It is hallmarked by increased dietary iron absorption due to inappropriately low hepcidin levels. Progressive iron loading causes complications, such as cirrhosis, arthritis, heart disease. Genes mutated in hemochromatosis are upstream activators of hepcidin levels and are involved in monitoring systemic iron levels. Diagnostic parameters include increased transferrin saturation (>45%) and serum ferritin (>200μg/L in females and >300μg/L in males), evidence of liver iron overload (either by MRI or biopsy) and absence of hematological signs of a primary red blood cell disorder or acquired risk factors for hepcidin deficiency.
By far, the most common subtype of hemochromatosis (HFE-related hemochromatosis) is caused by p.C282Y mutations in the HFE gene. HFE codes for an MHC class I-type transmembrane protein, hereditary hemochromatosis protein. The p.C282Y mutation disrupts the formation of a disulfide bond in hereditary hemochromatosis protein, impairing its capacity to interact with β2-microglobulin and reach the cell surface to participate in signalling to increase hepcidin expression. It is important to diagnose HFE mutations as early as possible to prevent organ damage due to iron accumulation. Treatment of hemochromatosis is accomplished through phlebotomies. More rare subtypes of hemochromatosis are caused by additional genes that are involved in iron sensing and contribute to hepcidin regulation, or in hepcidin itself. These hemochromatosis subtypes are generally more severe, with early adult or juvenile onset, and include rare pathogenic mutations in the genes for hepcidin (HAMP), the bone morphogenic protein (BMP) co-receptor hemojuvelin (HJV), the second receptor for transferrin (TFR2) and ferroportin (SLC40A1). Of note, in some patients with hemochromatosis causative mutations cannot be identified. Control of hepcidin expression is complex and mutations in these regulatory pathways may be disease causing for hemochromatosis. BMPs constitute interesting candidates, owing to their recently described role in the regulation of hepcidin expression. BMPs are cytokines belonging to the transforming growth factor beta (TGF-β) family that, through interaction with BMP receptors on the cell membrane of hepatocytes, can activate the BMP/SMAD pathway and increase hepcidin expression. Deficiency in BMP2 and 6 in mice causes iron overload. Consistently, BMP6 mutations in humans may cause hemochromatosis.
Our group has also recently investigated three male patients who combined neurological deficits with early systemic iron overload, as reflected by the increased transferrin saturation and low plasma hepcidin levels. In these patients we could not detect mutations in known hepcidin-regulatory genes. Exome sequencing identified missense constitutional mutations in the PIGA gene. PIGA is located on the X-chromosome and codes for a crucial protein involved in the biosynthesis of GPI-anchors (phosphatidylinositol N-acetylglucosaminyltransferase subunit A; PIG-A). These are extremely important for the dynamics and cell membrane attachment of several human proteins. Somatic PIGA mutations have been described in patients with paroxysmal nocturnal hemoglobinuria (PNH), a clonal hemolytic disorder caused by the lack of certain GPI-anchored proteins on blood cells. Constitutional PIGA mutations are rather rare and cause PIG-A deficiency – a condition with a highly heterogeneous phenotypical spectrum, hallmarked by early onset epilepsy and developmental delay. As the hemoch-romatosis protein HJV is GPI-anchored, we hypothesized that a failure to attach GPI anchors to HJV and a subsequent inability to appropriately express hepcidin may explain the iron overload described in these three patients.
We validated this hypothesis, by CRISPR-Cas-mediated deletion of PIGA, and demonstrated that, in PIG-A deficient hepatocytes, HJV is no longer directed to the cell membrane. PIG-A deficiency decreases downstream signalling pathways and reduces hepcidin expression, which will contribute to the iron overload observed in our patients. These results identified the molecular mechanism by which PIGA mutations cause a novel form of hemochromatosis and raises awareness for the need of clinical assessment of potential iron overload in children with constitutional PIGA mutations.
In 2016, the European Hematology Association (EHA) published “The EHA Research Roadmap: Anemias” to highlight achievements in the diagnosis and treatment of blood disorders and an updated version has been published earlier this year . In your opinion, what have been the main advances in diagnostics of iron-related disease?
The following breakthroughs are important propellers in advancing diagnosis and treatment of iron-related diseases:
• next-generation sequencing that allowed the discovery of new disease entities and genetic disease modifiers;
• precision diagnostics; and
• development of novel treatment options and targeted therapies tailored to each patient.
We are currently rolling out a mass vaccination campaign against COVID-19. Does an individual’s iron status have an effect on their response to vaccination?
Vaccination programmes keep the burden of infectious diseases low. Despite that, there is significant heterogeneity in vaccine efficacy in different populations. And although the reasons for this remain unresolved, there is an epidemiological association between lower vaccine efficacy and prevalence of iron deficiency and anemia. The initial observation that a person’s iron status affects vaccine responses was brought forward by analyses of Kenyan children where anemia and iron deficiency predicted a poor response to vaccination for diphteria, pertussis and pneumococcus. This concept has been further supported by pre-clinical studies demonstrating that iron availability is crucial for the development of proper T- and B-cell responses to infection and immunization, as well as prospective studies describing a significantly improved immune-humoral response upon vaccination in iron-deficient children following iron supplementation. Regardless of the complexity of anemia, where correction of serum iron levels may be difficult to achieve, these studies point towards the potential of correcting serum iron levels to improve vaccination efficiency, particularly in populations with a high prevalence of iron-deficiency anemia. For COVID-19 vaccines, recent results have shown that vaccine efficacy is very high even in those groups where anemia is more prevalent (e.g. premenopausal women, elders). Whether this holds true in populations of developing countries where iron-deficiency anemia is far more severe, and what is the threshold for iron deficiency to be detrimental for immunization responses remains unanswered. Further cross-disciplinary research efforts are necessary to determine exactly for which vaccines the response is hindered by the individual’s iron status – and whether this holds true for both true iron-deficiency anemia and anemia related to underlying inflammatory conditions. Furthermore, the best type of iron delivery/formulation needs to be explored and which populations to target specifically. Nonetheless, these studies alert us that iron deficiency may affect vaccine efficacy beyond its well-recognized contributions to anemia and cognitive deficits. Thus the burden of iron deficiency on global health may be far higher than anticipated. To increase awareness that iron deficiency and anemia are global health problems, the EHA has taken an important step to highlight achievements in the diagnosis and treatment of blood disorders in “The EHA Research Roadmap: Anemias” in 2016 and an updated version that has been published earlier this year.