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Story of Discovery: Erythroferrone–new regulator of iron balance

The proper maintenance of blood iron levels is complex, and multiple diseases can result when iron balance goes awry. Hepcidin, a small protein made in the liver, has been previously shown to play a key role in preserving proper iron balance. Critically important to the overall understanding of iron balance is the recent discovery of erythroferrone—with potential implications for treating multiple blood disorders and diseases.

Iron is essential to the body’s oxygen-delivery system. Humans need iron to make hemoglobin, the oxygen-carrying molecule in red blood cells. Most of the 3 to 4 grams (0.1 to 0.14 ounces) of iron in adults is in hemoglobin. Much of the remaining iron is stored in the liver, spleen, and bone marrow. Because excess iron damages tissues, total body iron is carefully regulated, with most of it being constantly recycled. While small amounts are absorbed daily via the digestive tract, about 10 times more iron is simply retrieved from aged red blood cells and reused. A protein called transferrin picks up this iron, along with dietary iron that has been absorbed via the digestive tract. Transferrin then carries the iron to the bone marrow, where it is used to produce new red blood cells. Unfortunately, the human body does not seem to have an efficient or regulated way to rid itself of excess iron.

If insufficient iron flows to the bone marrow, normal red blood cell production drops and anemia can result. Thus, an important, and the most common, cause of anemia is iron deficiency, which can be corrected through administration of iron supplements. Another form of anemia, however, is associated with inflammation. Called the anemia of inflammation and chronic diseases, this condition affects people who have infections, chronic inflammatory disorders—such as rheumatoid arthritis—and many other chronic disorders, including cancers. Patients with this form of anemia typically have inadequate red blood cell production, low levels of iron in the blood, and low levels of transferrin; they may also be resistant to the effects of erythropoietin, the hormone that normally stimulates and regulates red blood cell production.

Patients with anemia of inflammation and chronic diseases are usually not iron-deficient. Instead, the iron balance in their bodies has been altered, such that more iron is sequestered in the cells involved in iron recycling and absorption, as well as in liver cells that store iron. The cellular sequestration of iron leaves less available for transport to the bone marrow. Attempts to treat this condition with oral iron supplements typically do not work (a condition referred to as iron-refractory anemia), even though this form of anemia mimics iron deficiency. Anemia of inflammation generally improves if the underlying condition resolves.

An additional form of anemia can arise in people with a condition called thalassemia. Characterized by the under-production of normal hemoglobin, some people with thalassemia are treated with blood transfusions to provide much-needed red blood cells. While improving anemia, these transfusions can also lead to iron overload.

Regulation of Systemic Iron Levels by Hepcidin

NIH-supported research showed that hepcidin, a small protein produced in the liver, is the master regulator of iron absorption and tissue distribution. Hepcidin was identified in 1998 in a search for small molecules active in “innate immunity,” the body’s first line of defense against invading bacteria, fungi, and other microorganisms.

In 2001, a research team in France found that when the levels of hepcidin were disrupted in mice, the animals developed iron overload, while mice that were genetically altered to “turn on” the Hepcidin gene to a higher level than normal were severely anemic and died within hours of birth. In 2002, while studying abnormally high iron storage levels in the liver, using a mouse model of the most common form of inherited iron overload (hereditary hemochromatosis), researchers found that the Hepcidin gene was turned off to a greater extent in the mice with the excess liver iron compared to normal mice. When rats were fed an iron-abundant diet and then switched to an iron-deficient diet, investigators reported that the Hepcidin gene was significantly turned off in the liver while genes encoding iron transporters were significantly turned on in the digestive tract.

From this research, hepcidin emerged as a fundamental regulator of iron balance that inhibits iron absorption and iron release from tissue stores when iron levels in the blood are high, and eases off when blood iron levels decline. When the Hepcidin gene was always turned on, the iron accumulation normally seen in two different mouse models of iron overload was prevented. Mechanistically, hepcidin was shown to bind to the iron transport protein ferroportin and induce its destruction, thereby leading to both decreased iron absorption and release of iron into the blood. In a proof of principle of its systemic action, investigators observed a significant decrease in blood iron levels within an hour when mice were injected with hepcidin. Thus far shown to detect elevated or diminished human hepcidin protein levels in a spectrum of human diseases and conditions, a clinical assay to standardize measurement of human hepcidin is under development for commercial release to the clinical community. NIH investments in discovery research have provided a clear understanding of the role of hepcidin in normal physiology and its role in certain disease conditions.

Discovery of Erythroferrone as New Regulator of Iron Balance

During times of acute blood loss, there is an immediate need for the bone marrow to produce new blood cells, including red blood cells (RBCs), to replenish lost cells. Newly made RBCs demand an ample supply of iron—a component of hemoglobin. Erythropoietin drives RBC production within a few hours following blood loss, and this process continues for several days. Just how the body suppresses the action of hepcidin to allow increased iron absorption and mobilization from stores has been unclear. Recent NIH-supported research has been instrumental in identifying a key factor responsible for controlling the supply of iron needed for RBC production.

Research conducted in the mid-2000s strongly suggested that the factor responsible for hepcidin suppression during RBC production arose from the bone marrow. This critical piece of the puzzle came from a study in which drugs were used to interfere with RBC production in mice, and then the mice were subjected to blood loss. These animals lost their ability to turn off the Hepcidin gene, in contrast to animals not treated with these drugs.

NIDDK-supported scientists have conducted seminal studies designed to identify and characterize the factor emanating from the bone marrow that regulates hepcidin. For these studies, they used an animal model (male mice). They found that the time needed for erythropoietin administration or blood loss to significantly turn off the Hepcidin gene in liver or decrease hepcidin protein blood levels was between 4 and 9 hours. Thus, the unknown hepcidin suppressor must be produced in the bone marrow within the first 4 hours following erythropoietin administration or blood loss.

While evaluating the set of bone marrow genes turned on in mice subjected to blood loss, a previously uncharacterized gene was identified that was turned on within 4 hours of blood loss and was predicted to encode for a secreted protein. In this case, the protein must be able to exit the bone marrow cell and travel to the liver to exert its anti-hepcidin activity. This protein was named “erythroferrone” (Erfe), as it functions as a link between production of red blood cells (erythrocytes) and the regulation of iron (which has a Latin name of ferrum). Erythropoietin administration in mice was also shown to significantly turn on the Erfe gene in bone marrow. The bone marrow cell types responsible for turning on Erfe were shown to be the developing RBCs called erythroblasts. Mice genetically engineered to lack a functional Erfe gene were incapable of turning off the Hepcidin gene. To further confirm that Erfe suppressed hepcidin activity, they administered laboratory-made Erfe protein to the mice, and found that it turned off the Hepcidin gene in liver and reduced hepcidin protein levels in blood. In a mouse model of β-thalassemia that recapitulates features of the human disease, including low levels of hepcidin in blood and iron overload in the liver, the Erfe gene was turned on to a significantly higher level in bone marrow compared to normal mice. When the Erfe gene was inactivated in the mouse model, hepcidin expression was restored and iron overload in liver reduced—providing an initial proof of concept that this pathway may be useful to prevent iron overload in patients with thalassemia.

This groundbreaking study, published in 2014, was the first to demonstrate that Erfe is a biologically important regulator of hepcidin, with implications for supplying iron on demand for RBC production during blood loss. In addition, further experiments conducted in this study suggest that Erfe may serve as a target for treatment or prevention of iron overload in patients with β-thalassemia.

Building upon the critical finding that Erfe plays an important role in hepcidin suppression, NIDDK-supported researchers designed experiments to evaluate whether Erfe contributes to recovery from the anemia of inflammation and chronic diseases. Examining male mice with bacterial-induced anemia of inflammation, the researchers found that the Erfe gene was significantly turned on in the bone marrow. To gain insight into Erfe’s role in responding to bacterial-induced anemia of inflammation, the researchers compared normal mice to genetically engineered mice that could not produce Erfe (Erfe-deficient). Compared to normal mice, the Erfe-deficient mice: 1) exhibited a more severe form of anemia of inflammation (greater loss of hemoglobin), 2) turned on the Hepcidin gene to a significantly higher level, 3) were less capable of properly regulating serum hepcidin levels, 4) had significantly lower blood iron levels, and 5) had prolonged production of serum erythropoietin, reflecting the body’s attempt to compensate for low hemoglobin levels. In addition, the Erfe-deficient mice produced greater numbers of immature RBCs, as a step toward replenishing mature RBCs. However, these immature RBCs were smaller than those produced by the normal mice, because less iron was available for blood cell use in the Erfe-deficient mice. Taken together, this set of experiments highlights the important contribution Erfe makes to the recovery of anemia of inflammation by turning down hepcidin and increasing blood iron levels.

Looking to the Future

As described in this story, knowledge gained from studying hepcidin has led to the identification of Erfe as a new regulator of iron balance in mammals. Future studies will determine how Erfe exerts its mechanism of action in liver to suppress hepcidin. If Erfe functions similarly in humans, then future studies may lead to potential therapies targeting Erfe or hepcidin for disorders of iron balance—absorption, storage, and mobilization.

The NIDDK continues to support a robust portfolio of research projects designed to shed new light on iron homeostasis—the body’s establishment and maintenance of iron balance. For example, investigators are exploring various strategies for increasing hepcidin blood levels and bolstering the effectiveness of chelation therapy for iron overload. Other investigators, funded via the Stimulating Hematology Investigation: New Endeavors (SHINE) program, are examining the role of transferrin in red blood cell production and iron balance, and mouse models of iron recycling.​