Antibodies against the erythroferrone N-terminal domain prevent hepcidin suppression and ameliorate murine thalassemia

Abstract

Erythroferrone (ERFE) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating ERFE in conditions of stress erythropoiesis, such as in patients with β-thalassemia, promotes the tissue iron accumulation that substantially contributes to morbidity in these patients. Here we developed antibodies against ERFE to prevent hepcidin suppression and to correct the iron loading phenotype in a mouse model of β-thalassemia [Hbb(th3/+) mice] and used these antibodies as tools to further characterize ERFE’s mechanism of action. We show that ERFE binds to BMP6 with nanomolar affinity and binds BMP2 and BMP4 with somewhat weaker affinities. We found that BMP6 binds the N-terminal domain of ERFE, and a polypeptide derived from the N terminus of ERFE was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in wild-type mice. Anti-ERFE antibodies targeting the N-terminal domain prevented hepcidin suppression in ERFE-treated Huh7 cells and in EPO-treated mice. Finally, we observed a decrease in splenomegaly and serum and liver iron in anti–ERFE-treated Hbb(th3/+) mice, accompanied by an increase in red blood cells and hemoglobin and a decrease in reticulocyte counts. In summary, we show that ERFE binds BMP6 directly and with high affinity, and that antibodies targeting the N-terminal domain of ERFE that prevent ERFE–BMP6 interactions constitute a potential therapeutic tool for iron loading anemias.

The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry

“…Given that osteoblasts secrete ERFE that is known to inhibit hepcidin ( Kautz et al, 2014 ) by sequestering BMPs ( Arezes et al, 2018 ; Wang et al, 2020 ; Arezes et al, 2020 ) that are skeletal anabolics ( Hogan, 1996 ), we measured serum BMP2 concentration to find elevated BMP2 levels in Erfe -/- relative to wild-type mice ( Figure 3A ). Given the specific importance of BMP2 in bone remodeling ( Salazar et al, 2016 ), these results are consistent with the previously demonstrated sequestration of BMP2, along with BMP6, by ERFE ( Wang et al, 2020 )—namely, loss of ERFE led to decreased BMP sequestration.…”

Differentiating iron-loading anemias using a newly developed and analytically validated ELISA for human serum erythroferrone

“…In order to correctly assess ERFE levels in pathologies and after blood donation, appropriate analytical validated quantification of ERFE must be ensured. This will also contribute to correctly evaluate the success of recently suggested anti-ERFE therapies for pathologies with aberrantly high ERFE levels, such as iron-loading anemias [20,21].…”

The Interplay between Drivers of Erythropoiesis and Iron Homeostasis in Rare Hereditary Anemias: Tipping the Balance

“…Compensatory increased erythropoietic activity leads to drastically elevated ERFE levels in both NTDT patients and beta-thalassemic mice, especially due to ongoing ineffective erythropoiesis, which is reflected by the increased levels of GDF15 in patients [ 23 , 41 , 42 ]. Based on this, ERFE is currently being investigated as a potential pharmacological target in NTDT [ 28 , 43 ]. Moreover, the increased erythropoietic drive may lead reticulocytosis and increased sTfR levels [ 40 , 44 ].…”