Hepcidin-Mediated Hypoferremia Disrupts Immune Responses to Vaccination and Infection.

BACKGROUND: How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear. METHODS: We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells in vitro and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant TMPRSS6. We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency. FINDINGS: We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity. CONCLUSIONS: Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders. FUNDING: Medical Research Council, UK.

A single cycle influenza virus coated in H7 haemagglutinin generates neutralizing antibody responses to haemagglutinin and neuraminidase glycoproteins and protection from heterotypic challenge.

A non-replicating form of pseudotyped influenza virus, inactivated by suppression of the haemagglutinin signal sequence (S-FLU), can act as a broadly protective vaccine. S-FLU can infect for a single round only, and induces heterotypic protection predominantly through activation of cross-reactive T cells in the lung. Unlike the licensed live attenuated virus, it cannot reassort a pandemic haemagglutinin (HA) into seasonal influenza. Here we present data on four new forms of S-FLU coated with H7 HAs from either A/Anhui/1/2013, A/Shanghai/1/2013, A/Netherlands/219/2003 or A/New York/107/2003 strains of H7 virus. We show that intranasal vaccination induced a strong local CD8 T cell response and protected against heterosubtypic X31 (H3N2) virus and highly virulent PR8 (H1N1), but not influenza B virus. Intranasal vaccination also induced a strong neutralizing antibody response to the encoded neuraminidase. If given at higher dose in the periphery with intraperitoneal administration, H7 S-FLU induced a specific neutralizing antibody response to H7 HA coating the particle. Polyvalent intraperitoneal vaccination with mixed H7 S-FLU induced a broadly neutralizing antibody response to all four H7 strains. S-FLU is a versatile vaccine candidate that could be rapidly mobilized ahead of a new pandemic threat.

Pseudotyped influenza A virus as a vaccine for the induction of heterotypic immunity.

There is a need for vaccines that can protect broadly across all influenza A strains. We have produced a pseudotyped influenza virus based on suppression of the A/PR/8/34 hemagglutinin signal sequence (S-FLU) that can infect cells and express the viral core proteins and neuraminidase but cannot replicate. We show that when given by inhalation to mice, S-FLU is nonpathogenic but generates a vigorous T cell response in the lung associated with markedly reduced viral titers and weight loss after challenge with H1 and H3 influenza viruses. These properties of S-FLU suggest that it may have potential as a broadly protective A virus vaccine, particularly in the setting of a threatened pandemic before matched subunit vaccines become available.

Hepcidin regulation by innate immune and infectious stimuli.

Hepcidin controls the levels and distribution of iron, an element whose availability can influence the outcome of infections. We investigated hepcidin regulation by infection-associated cytokines, pathogen-derived molecules, and whole pathogens in vitro and in vivo. We found that IL-22, an effector cytokine implicated in responses to extracellular infections, caused IL-6-independent hepcidin up-regulation in human hepatoma cells, suggesting it might represent an additional inflammatory hepcidin agonist. Like IL-6, IL-22 caused phosphorylation of STAT3 and synergized with BMP6 potentiating hepcidin induction. In human leukocytes, IL-6 caused potent, transient hepcidin up-regulation that was augmented by TGF-ß1. Pathogen-derived TLR agonists also stimulated hepcidin, most notably the TLR5 agonist flagellin in an IL-6-dependent manner. In contrast, leukocyte hepcidin induction by heat-killed Candida albicans hyphae was IL-6-independent, but partially TGF-ß-dependent. In a murine acute systemic candidiasis model, C albicans strongly stimulated hepcidin, accompanied by a major reduction in transferrin saturation. Similarly, hepcidin was up-regulated with concomitant lowering of serum iron during acute murine Influenza A/PR/8/34 virus (H1N1) infection. This intracellular pathogen also stimulated hepcidin expression in leukocytes and hepatoma cells. Together, these results indicate that hepcidin induction represents a component of the innate immune response to acute infection, with the potential to affect disease pathogenesis.

Regulation of type II transmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors: new link between hypoxia signaling and iron homeostasis.

Hepcidin is a liver-derived hormone with a key role in iron homeostasis. In addition to iron, it is regulated by inflammation and hypoxia, although mechanisms of hypoxic regulation remain unclear. In hepatocytes, hepcidin is induced by bone morphogenetic proteins (BMPs) through a receptor complex requiring hemojuvelin (HJV) as a co-receptor. Type II transmembrane serine proteinase (TMPRSS6) antagonizes hepcidin induction by BMPs by cleaving HJV from the cell membrane. Inactivating mutations in TMPRSS6 lead to elevated hepcidin levels and consequent iron deficiency anemia. Here we demonstrate that TMPRSS6 is up-regulated in hepatic cell lines by hypoxia and by other activators of hypoxia-inducible factor (HIF). We show that TMPRSS6 expression is regulated by both HIF-1α and HIF-2α. This HIF-dependent up-regulation of TMPRSS6 increases membrane HJV shedding and decreases hepcidin promoter responsiveness to BMP signaling in hepatocytes. Our results reveal a potential role for TMPRSS6 in hepcidin regulation by hypoxia and provide a new molecular link between oxygen sensing and iron homeostasis.

In vitro binding of HFE to the cation-independent mannose-6 phosphate receptor.

Hereditary hemochromatosis is most frequently associated with mutations in HFE, which encodes a class Ib histocompatibility protein. HFE binds to the transferrin receptor-1 (TfR1) in competition with iron-loaded transferrin (Fe-Tf). HFE is released from TfR1 by increasing concentrations of Fe-Tf, and free HFE may then regulate iron homeostasis by binding other ligands. To search for new HFE ligands we expressed recombinant forms of HFE in the human cell line 293T. HFE protein was purified, biotinylated and made into fluorescently labelled tetramers. HFE tetramers bound to TfR1 in competition with Tf, but in addition we detected a binding activity on some cell types that was not blocked by Fe-Tf or by mutations in HFE that prevent binding to TfR1. We identified this second HFE ligand as the cation independent mannose-6-phosphate receptor (CI-MPR, also known as the insulin-like growth factor-2 receptor, IGF2R). HFE:CI-MPR binding was mediated through phosphorylated mannose residues on HFE. Recombinant murine Hfe also bound to CI-MPR. HFE bound to TfR1 was prevented from binding CI-MPR until released by increasing concentrations of Fe-Tf, a feature consistent with an iron sensing mechanism. However, it remains to be determined whether endogenous HFE in vivo also acquires the mannose-6 phosphate modification and binds to CI-MPR.

Regulation of growth differentiation factor 15 expression by intracellular iron.

Growth differentiation factor 15 (GDF15) is a divergent member of the transforming growth factor-beta superfamily and has been identified in different contexts as a hypoxia-inducible gene product and as a molecule involved in hepcidin regulation. The biology of iron and oxygen is closely related, and known regulatory pathways involving hypoxia-inducible factor (HIF) and iron-regulatory proteins (IRPs) are responsive to both these stimuli. We therefore sought to characterize the regulation of GDF15 by iron and oxygen and to define the involvement or otherwise of HIF and IRP pathways. Here we show that GDF15 is strongly up-regulated by stimuli that deplete cells of iron and that this response is specifically antagonized by the reprovision of iron. GDF15 exhibits greater sensitivity to iron depletion than hypoxia, and responses to hypoxia and iron depletion are independent of HIF and IRP activation, suggesting a novel mechanism of regulation. We also report significant induction of serum GDF15 in iron-deficient subjects and after administration of an iron chelator to normal subjects. These findings indicate that GDF15 can be induced by pathophysiologic changes in iron availability, raising important questions about the mechanism of regulation and its role in iron homeostasis.

Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals.

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses.

Ferroportin: lack of evidence for multimers.

Ferroportin is a multi-transmembrane glycoprotein that mediates iron export from cells. Mutations in ferroportin are linked to type IV hemochromatosis, a dominantly inherited disorder of iron metabolism. Multimers of ferroportin, whose existence may relate to the dominant inheritance pattern of disease, have been detected in some studies but not others. We looked for evidence of multimerization in several different types of experiment. We assayed the maturation of mutant and wild-type ferroportin and found that loss-of-function mutants had a reduced half-life but did not alter the stability of coexpressed wild-type. Using bioluminescence resonance energy transfer analysis, we tested how mature wild-type ferroportin behaved in intact live cell membranes. Ferroportin-ferroportin interactions gave the very low acceptor/donor ratio-independent energy transfer levels characteristic of random protein-protein interactions, consistent with ferroportin behaving as a monomer. Consistent with these experiments, we were unable to detect a dominant negative functional effect of mutant ferroportin on wild-type, even when expression of wild-type protein was titrated to low levels. These data suggest that dominantly inherited ferroportin disease does not result from the direct action of a mutated protein inhibiting a wild-type protein within multimers. We propose other possible mechanisms of disease.

Resistance to hepcidin is conferred by hemochromatosis-associated mutations of ferroportin.

Ferroportin (FPN) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.

In vitro functional analysis of human ferroportin (FPN) and hemochromatosis-associated FPN mutations.

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1 gene encoding ferroportin (FPN). Known as the "ferroportin disease," this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopus oocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162delta, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently "turned on" iron exporter.

The hemochromatosis protein HFE inhibits iron export from macrophages.

Hereditary hemochromatosis (HH) is a disorder of iron metabolism caused by common mutations in the gene HFE. The HFE protein binds to transferrin receptor-1 (TfR1) in competition with transferrin, and in vitro, reduces cellular iron by reducing iron uptake. However, in vivo, HFE is strongly expressed by liver macrophages and intestinal crypt cells, which behave as though they are relatively iron-deficient in HH. These latter observations suggest, paradoxically, that expression of wild-type HFE may lead to iron accumulation in these specialized cell types. Here we show that wild-type HFE protein raises cellular iron by inhibiting iron efflux from the monocytemacrophage cell line THP-1, and extend these results to macrophages derived from healthy individuals and HH patients. In addition, we find that the HH-associated mutant H41D has lost the ability to inhibit iron release despite binding to TfR1 as well as wild-type HFE. Finally, we show that the ability of HFE to block iron release is not competitively inhibited by transferrin. We conclude that HFE has two mutually exclusive functions, binding to TfR1 in competition with Tf, or inhibition of iron release.