Grace Jung

Identification of erythroferrone as an erythroid regulator of iron metabolism

Recovery from blood loss requires a greatly enhanced supply of iron to support expanded erythropoiesis. After hemorrhage, suppression of the iron-regulatory hormone hepcidin allows increased iron absorption and mobilization from stores. We identified a new hormone, erythroferrone (ERFE), that mediates hepcidin suppression during stress erythropoiesis. ERFE is produced by erythroblasts in response to erythropoietin. ERFE-deficient mice fail to suppress hepcidin rapidly after hemorrhage and exhibit a delay in recovery from blood loss. ERFE expression is greatly increased in Hbbth3/+ mice with thalassemia intermedia, where it contributes to the suppression of hepcidin and the systemic iron overload characteristic of this disease.

Human α-Defensin 6 Promotes Mucosal Innate Immunity Through Self-Assembled Peptide Nanonets

Netting the Bad Guys Antimicrobial peptides are an evolutionarily conserved component of innate immunity in the intestine. One family, α-defensins, typically exert their antimicrobial effects through microbicidal activity against bacteria. Humans express only two α-defensins, human defensin 5 (HD5) and HD6. HD5 exhibits bactericidal activity and plays a role in shaping the bacterial composition of the gut. HD6, on the other hand, does not show bactericidal activity and its function in the gut is unclear. Now, Chu et al. (p. 477, published online 21 June; see the Perspective by Ouellette and Selsted) show that HD6 protects against bacterial pathogens. Rather than killing them directly, HD6 binds to bacteria surface proteins and, through a process of self-assembly, forms fibrils and nanonets that ensnare invading bacterial pathogens. Rather than killing bacteria directly, a gut antimicrobial peptide forms netlike structures that ensnare invading bacteria. Defensins are antimicrobial peptides that contribute broadly to innate immunity, including protection of mucosal tissues. Human α-defensin (HD) 6 is highly expressed by secretory Paneth cells of the small intestine. However, in contrast to the other defensins, it lacks appreciable bactericidal activity. Nevertheless, we report here that HD6 affords protection against invasion by enteric bacterial pathogens in vitro and in vivo. After stochastic binding to bacterial surface proteins, HD6 undergoes ordered self-assembly to form fibrils and nanonets that surround and entangle bacteria. This self-assembly mechanism occurs in vivo, requires histidine-27, and is consistent with x-ray crystallography data. These findings support a key role for HD6 in protecting the small intestine against invasion by diverse enteric pathogens and may explain the conservation of HD6 throughout Hominidae evolution.

θ-Defensins Prevent HIV-1 Env-mediated Fusion by Binding gp41 and Blocking 6-Helix Bundle Formation

Retrocyclin-1, a θ-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 μm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Erythroferrone contributes to hepcidin suppression and iron overload in a mouse model of β-thalassemia.

Inherited anemias with ineffective erythropoiesis, such as β-thalassemia, manifest inappropriately low hepcidin production and consequent excessive absorption of dietary iron, leading to iron overload. Erythroferrone (ERFE) is an erythroid regulator of hepcidin synthesis and iron homeostasis. Erfe expression was highly increased in the marrow and spleen of Hbb(Th3/+) mice (Th3/+), a mouse model of thalassemia intermedia. Ablation of Erfe in Th3/+ mice restored normal levels of circulating hepcidin at 6 weeks of age, suggesting ERFE could be a factor suppressing hepcidin production in β-thalassemia. We examined the expression of Erfe and the consequences of its ablation in thalassemic mice from 3 to 12 weeks of age. The loss of ERFE in thalassemic mice led to full restoration of hepcidin mRNA expression at 3 and 6 weeks of age, and significant reduction in liver and spleen iron content at 6 and 12 weeks of age. Ablation of Erfe slightly ameliorated ineffective erythropoiesis, as indicated by reduced spleen index, red cell distribution width, and mean corpuscular volume, but did not improve the anemia. Thus, ERFE mediates hepcidin suppression and contributes to iron overload in a mouse model of β-thalassemia.

Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin

θ-defensins are cyclic octadecapeptides encoded by the modified α-defensin genes of certain nonhuman primates. The recent demonstration that human α-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of θ-defensins on Bacillus anthracis (Sterne). We tested rhesus θ-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of θ-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of θ-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that θ-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.

Interactions of α-, β-, and θ-Defensins with Influenza A Virus and Surfactant Protein D

We have reported that the α-defensins human neutrophil peptides (HNP)-1 and HNP-2 neutralize and aggregate influenza A virus (IAV) and promote uptake of IAV by neutrophils. These α-defensins were also shown to bind to surfactant protein (SP)-D and reduce its antiviral activity. In this study, we examined retrocyclin (RC)1 and RC2, humanized versions of the antiviral θ-defensins found in the leukocytes of certain nonhuman primates. RC1 was just as effective as HNP-1–3 in neutralizing IAV, and RC2 and RC101 (an analog of RC1) were more effective. In contrast, human β-defensins (HBDs) showed less neutralizing activity. Human defensins 5 and 6 (mainly produced by intestinal Paneth cells) had viral neutralizing activity similar to HNP-1–3. Like HNP-1–3, RCs induced viral aggregation and promoted the uptake of IAV by neutrophils. We used surface plasmon resonance to evaluate binding of defensins to SP-D. HBDs, HD6, and HNP-4 bound minimally to SP-D. HNP-1–3 and RCs bound SP-D with high affinity; however, unlike HNP-1 and HNP-2, RCs did not inhibit SP-D antiviral activity. HBDs also did not inhibit antiviral activity of SP-D. Given their strong neutralizing activity and compatibility with SP-D, RCs may provide attractive prototypes for designing therapeutics that can prevent or treat respiratory infections caused by IAV.

Hepcidin-Induced Hypoferremia Is a Critical Host Defense Mechanism Against the Siderophilic Bacterium Vibrio vulnificus

Hereditary hemochromatosis, an iron overload disease caused by a deficiency in the iron-regulatory hormone hepcidin, is associated with lethal infections by siderophilic bacteria. To elucidate the mechanisms of this susceptibility, we infected wild-type and hepcidin-deficient mice with the siderophilic bacterium Vibrio vulnificus and found that hepcidin deficiency results in increased bacteremia and decreased survival of infected mice, which can be partially ameliorated by dietary iron depletion. Additionally, timely administration of hepcidin agonists to hepcidin-deficient mice induces hypoferremia that decreases bacterial loads and rescues these mice from death, regardless of initial iron levels. Studies of Vibrio vulnificus growth ex vivo show that high iron sera from hepcidin-deficient mice support extraordinarily rapid bacterial growth and that this is inhibited in hypoferremic sera. Our findings demonstrate that hepcidin-mediated hypoferremia is a host defense mechanism against siderophilic pathogens and suggest that hepcidin agonists may improve infection outcomes in patients with hereditary hemochromatosis or thalassemia.

Multivalent Binding of Carbohydrates by the Human α-Defensin, HD5

Four of the six human α-defensins (human neutrophil peptides 1–3 and human α-defensin 5; HD5) have a lectin-like ability to bind glycosylated proteins. Using HD5 as a model, we applied surface plasmon resonance techniques to gain insights into this property. HD5 bound natural glycoproteins > neoglycoproteins based on BSA > nonglycosylated BSA ≫ free sugars. The affinity of HD5 for simple sugars covalently bound to BSA was orders of magnitude greater than its affinity for the same sugars in solution. The affinity of HD5 for protein-bound carbohydrates resulted from multivalent interactions which may also involve noncarbohydrate residues of the proteins. HD5 showed concentration-dependent self-association that began at submicromolar concentrations and proceeded to dimer and tetramer formation at concentrations below 5 μM. The (R9A, R28A) and (R13A, R32A) analogs of HD5 showed greatly reduced self-association as well as minimal binding to BSA and to BSA-affixed sugars. From this and other evidence, we conclude that the extensive binding of HD5 to (neo)glycoproteins results from multivalent nonspecific interactions of individual HD5 molecules with carbohydrate and noncarbohydrate moieties of the target molecule and that the primary binding events are magnified and enhanced by subsequent in situ assembly and oligomerization of HD5. Self-association and multivalent binding may play integral roles in the ability of HD5 to protect against infections caused by viruses and other infectious agents.

Erythroferrone contributes to recovery from anemia of inflammation

Erythroferrone (ERFE) is an erythropoiesis-driven regulator of iron homeostasis. ERFE mediates the suppression of the iron-regulatory hormone hepcidin to increase iron absorption and mobilization of iron from stores. We examined the role of ERFE in the recovery from anemia of inflammation (AI) induced by injection of heat-killed Brucella abortus. B abortus-treated wild-type mice developed a moderate anemia and reached nadir hemoglobin 14 days after injection and partially recovered by 28 days. We observed that Erfe expression in the bone marrow and the spleen was greatly increased during anemia and peaked at 14 days after injection, a time course similar to serum erythropoietin. To determine whether ERFE facilitates the recovery from anemia, we analyzed Erfe-deficient mice injected with B abortus. Compared with wild-type mice, Erfe-deficient mice exhibited a more severe anemia, had higher hepcidin levels and consequently lower serum iron concentration on days 14 and 21, and manifested impaired mobilization of iron from stores (liver and spleen). Erfe(-/-) mice eventually compensated by further stimulating erythropoiesis and reticulocyte production. Thus, ERFE contributes to the recovery from AI by suppressing hepcidin and increasing iron availability.

Trp-26 imparts functional versatility to human alpha-defensin HNP1.

We performed a comprehensive alanine scan of human α-defensin HNP1 and tested the ability of the resulting analogs to kill Staphylococcus aureus, inhibit anthrax lethal factor, and bind human immunodeficiency virus-1 gp120. By far, the most deleterious mutation for all of these functions was W26A. The activities lost by W26A-HNP1 were restored progressively by replacing W26 with non-coded, straight-chain aliphatic amino acids of increasing chain length. The hydrophobicity of residue 26 also correlated with the ability of the analogs to bind immobilized wild type HNP1 and to undergo further self-association. Thus, the hydrophobicity of residue 26 is not only a key determinant of the direct interactions of HNP1 with target molecules, but it also governs the ability of this peptide to form dimers and more complex quaternary structures at micromolar concentrations. Although all defensin peptides are cationic, their amphipathicity is at least as important as their positive charge in enabling them to participate in innate host defense.