Victoria Gabayan

IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin

Hypoferremia is a common response to systemic infections or generalized inflammatory disorders. In mouse models, the development of hypoferremia during inflammation requires hepcidin, an iron regulatory peptide hormone produced in the liver, but the inflammatory signals that regulate hepcidin are largely unknown. Our studies in human liver cell cultures, mice, and human volunteers indicate that IL-6 is the necessary and sufficient cytokine for the induction of hepcidin during inflammation and that the IL-6-hepcidin axis is responsible for the hypoferremia of inflammation.

Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading in mice

In response to iron loading, hepcidin synthesis is homeostatically increased to limit further absorption of dietary iron and its release from stores. Mutations in HFE, transferrin receptor 2 (Tfr2), hemojuvelin (HJV), or bone morphogenetic protein 6 (BMP6) prevent appropriate hepcidin response to iron, allowing increased absorption of dietary iron, and eventually iron overload. To understand the role each of these proteins plays in hepcidin regulation by iron, we analyzed hepcidin messenger RNA (mRNA) responsiveness to short and long‐term iron challenge in iron‐depleted Hfe, Tfr2, Hjv, and Bmp6 mutant mice. After 1‐day (acute) iron challenge, Hfe−/− mice showed a smaller hepcidin increase than their wild‐type strain‐matched controls, Bmp6−/− mice showed nearly no increase, and Tfr2 and Hjv mutant mice showed no increase in hepcidin expression, indicating that all four proteins participate in hepcidin regulation by acute iron changes. After a 21‐day (chronic) iron challenge, Hfe and Tfr2 mutant mice increased hepcidin expression to nearly wild‐type levels, but a blunted increase of hepcidin was seen in Bmp6−/− and Hjv−/− mice. BMP6, whose expression is also regulated by iron, may mediate hepcidin regulation by iron stores. None of the mutant strains (except Bmp6−/− mice) had impaired BMP6 mRNA response to chronic iron loading. Conclusion: TfR2, HJV, BMP6, and, to a lesser extent, HFE are required for the hepcidin response to acute iron loading, but are partially redundant for hepcidin regulation during chronic iron loading and are not involved in the regulation of BMP6 expression. Our findings support a model in which acute increases in holotransferrin concentrations transmitted through HFE, TfR2, and HJV augment BMP receptor sensitivity to BMPs. A distinct regulatory mechanism that senses hepatic iron may modulate hepcidin response to chronic iron loading. (HEPATOLOGY 2011;)

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.

A mouse model of anemia of inflammation: complex pathogenesis with partial dependence on hepcidin

Anemia is a common complication of infections and inflammatory diseases, but the few mouse models of this condition are not well characterized. We analyzed in detail the pathogenesis of anemia induced by an injection of heat-killed Brucella abortus and examined the contribution of hepcidin by comparing wild-type (WT) to iron-depleted hepcidin-1 knockout (Hamp-KO) mice. B abortus-treated WT mice developed severe anemia with a hemoglobin nadir at 14 days and partial recovery by 28 days. After an early increase in inflammatory markers and hepcidin, WT mice manifested hypoferremia, despite iron accumulation in the liver. Erythropoiesis was suppressed between days 1 and 7, and erythrocyte destruction was increased as evidenced by schistocytes on blood smears and shortened red blood cell lifespan. Erythropoietic recovery began after 14 days but was iron restricted. In B abortus-treated Hamp-KO compared with WT mice, anemia was milder, not iron restricted, and had a faster recovery. Similarly to severe human anemia of inflammation, the B abortus model shows multifactorial pathogenesis of inflammatory anemia including iron restriction from increased hepcidin, transient suppression of erythropoiesis, and shortened erythrocyte lifespan. Ablation of hepcidin relieves iron restriction and improves the anemia.

Hepcidin Induction by Pathogens and Pathogen-Derived Molecules Is Strongly Dependent on Interleukin-6

ABSTRACT Hepcidin, the iron-regulatory hormone, is increased during infection or inflammation, causing hypoferremia. This response is thought to be a host defense mechanism that restricts iron availability to invading pathogens. It is not known if hepcidin is differentially induced by bacterial versus viral infections, whether the stimulation of pattern recognition receptors directly regulates hepcidin transcription, or which of the proposed signaling pathways are essential for hepcidin increase during infection. We analyzed hepcidin induction and its dependence on interleukin-6 (IL-6) in response to common bacterial or viral infections in mice or in response to a panel of pathogen-derived molecules (PAMPs) in mice and human primary hepatocytes. In wild-type (WT) mice, hepcidin mRNA was induced several hundred-fold both by a bacterial (Streptococcus pneumoniae) and a viral infection (influenza virus PR8) within 2 to 5 days. Treatment of mice and human primary hepatocytes with most Toll-like receptor ligands increased hepcidin mRNA within 6 h. Hepcidin induction by microbial stimuli was IL-6 dependent. IL-6 knockout mice failed to increase hepcidin in response to S. pneumoniae or influenza infection and had greatly diminished hepcidin response to PAMPs. In vitro, hepcidin induction by PAMPs in primary human hepatocytes was abolished by the addition of neutralizing IL-6 antibodies. Our results support the key role of IL-6 in hepcidin regulation in response to a variety of infectious and inflammatory stimuli.

Decreased clearance of Pseudomonas aeruginosa from airways of mice deficient in lysozyme M

Lysozyme is a ubiquitous and abundant, cationic, antimicrobial polypeptide of leukocytes and epithelia, but its biological function in host defense is largely unexplored. To ascertain the role of lysozyme during bacterial infection of murine airways, we exposed the airways of lysozyme M‐deficient (lys M−/−) mice to the pulmonary pathogen Pseudomonas aeruginosa and examined the host’s response to infection. Despite partial compensation as a result of the appearance of lysozyme P in the infected airways of lys M−/− mice, these lys M−/− mice showed decreased clearance of P. aeruginosa compared with their lys M+/− or lys M+/+ littermates. Lysozyme contributes to optimal clearance of P. aeruginosa from the murine airways.

Endogenous hepcidin and its agonist mediate resistance to selected infections by clearing non-transferrin-bound iron

The iron-regulatory hormone hepcidin is induced early in infection, causing iron sequestration in macrophages and decreased plasma iron; this is proposed to limit the replication of extracellular microbes, but could also promote infection with macrophage-tropic pathogens. The mechanisms by which hepcidin and hypoferremia modulate host defense, and the spectrum of microbes affected, are poorly understood. Using mouse models, we show that hepcidin was selectively protective against siderophilic extracellular pathogens (Yersinia enterocolitica O9) by controlling non-transferrin-bound iron (NTBI) rather than iron-transferrin concentration. NTBI promoted the rapid growth of siderophilic but not nonsiderophilic bacteria in mice with either genetic or iatrogenic iron overload and in human plasma. Hepcidin or iron loading did not affect other key components of innate immunity, did not indiscriminately promote intracellular infections (Mycobacterium tuberculosis), and had no effect on extracellular nonsiderophilic Y enterocolitica O8 or Staphylococcus aureus Hepcidin analogs may be useful for treatment of siderophilic infections.

Effects of dietary iron intake and chronic kidney disease on fibroblast growth factor 23 metabolism in wild-type and hepcidin knockout mice

In the setting of normal kidney function, iron deficiency is associated with increased FGF23 production and cleavage, altering circulating FGF23 levels. Our objective was to determine how chronic kidney disease (CKD) and dietary iron intake affect FGF23 production and metabolism in wild-type (WT) and hepcidin knockout (HKO) mice. For 8 wk, the mice were fed diets that contained adenine (to induce CKD) or no adenine (control group), with either low-iron (4 ppm) or standard-iron (335 ppm) concentrations. The low-iron diet induced iron deficiency anemia in both the WT and HKO mice. Among the WT mice, in both the control and CKD groups, a low-iron compared with a standard-iron diet increased bone Fgf23 mRNA expression, C-terminal FGF23 (cFGF23) levels, and FGF23 cleavage as manifested by a lower percentage intact FGF23 (iFGF23). Independent of iron status, CKD was associated with inhibition of FGF23 cleavage. Similar results were observed in the HKO control and CKD groups. Dietary iron content was more influential on FGF23 parameters than the presence or absence of hepcidin. In the CKD mice (WT and HKO, total n = 42), independent of the effects of serum phosphate, iron deficiency was associated with increased FGF23 production but also greater cleavage, whereas worse kidney function was associated with increased FGF23 production but decreased cleavage. Therefore, in both the WT and HKO mouse models, dietary iron content and CKD affected FGF23 production and metabolism.

Erythroferrone and matriptase-2 independently regulate hepcidin expression

To the Editor: The production of the iron-regulatory hormone hepcidin is physiologically suppressed during increased erythropoietic activity. Hepcidin suppression increases iron availability to meet the additional iron demand of expanded hemoglobin synthesis. Under the influence of erythropoietin (EPO), the hormone erythroferrone (ERFE) is secreted by erythroid precursors in the bone marrow and spleen, and suppresses hepcidin synthesis to facilitate the recovery from anemia. However, the mechanism by which ERFE suppresses hepcidin is unknown. In contrast with forms of anemia in which hepcidin is suppressed, patients with iron-refractory iron deficiency anemia (IRIDA), a disease caused by mutations in transmembrane serine protease 6 (TMPRSS6) which result in pathological activation of the BMP/Smad signaling, exhibit increased hepcidin production despite a severe anemia and elevated EPO levels. Previous studies have shown that hepcidin production was not affected by EPO in Tmprss6 mutant mice. More recently, Nai and colleagues suggested that matriptase-2 may act downstream of EPO to dampen the signaling through the BMP-Smad regulatory pathway and allow ERFE to repress hepcidin production. We therefore examined the crosstalk between ERFE and matriptase-2 in mice. In agreement with Nai et al, we observed that Erfe mRNA expression was highly increased in the bone marrow and spleen of Tmprss6 mice (Supporting Information Figure 1). While wild-type mice (WT) mice have undetectable plasma ERFE, plasma ERFE concentration was elevated in Tmprss6 to levels comparable to those of WT animals 24 h after phlebotomy ( 3 ng/mL) but was lower than ERFE levels in thalassemic mice ( 10 ng/mL) (Figure 1A). To assess the contribution of ERFE to the phenotype of Tmprss6 mice, we generated Tmprss6 mice with disrupted Erfe (Erfe Tmprss6; Erfe Tmprss6, and Erfe Tmprss6). Ablation of Erfe in Tmprss6 mice did not result in any change in hematological parameters compared to Tmprss6 animals at 6 weeks of age (Table 1). Mice deficient for Tmprss6 had higher RBC and lower hemoglobin, hematocrit, and MCV compared to WT, regardless of the Erfe genotype. Liver hepcidin mRNA (Figure 1B) and serum hepcidin concentration (Figure 1C) were higher in Tmprss6 mice compared to WT mice and no difference was observed after manipulation of Erfe. Consistent with chronically increased hepcidin production, liver iron content (Figure 1D) was decreased in Tmprss6 mice compared to WT mice, but this was not influenced by the ablation of Erfe. No statistically significant difference in spleen iron content was observed between the genotypes (Figure 1E). Erfe-deficient Tmprss6 mice and Tmprss6 mice showed similarly increased spleen weight compared to WT mice (Supporting Information Figure 2A). However, we observed a decrease in body weight in double mutant animals at 6 weeks compared to Tmprss6 mice (Supporting Information Figure 2B) suggesting that ERFE may be important for normal development of Tmprss6 mice. As a consequence of smaller body size, spleen index was higher in Erfe, Tmprss6 mice compared to Tmprss6 mice (Supporting Information Figure 2C). Together, these results indicate that the disruption of Erfe has at most a very minor effect on the phenotype of Tmprss6 deficiency in mice. To determine whether ERFE requires TMPRSS6 to regulate hepcidin production, we treated freshly isolated hepatocytes from WT or Tmprss6 mice with conditioned medium from cells expressing recombinant ERFE or from control cells. Consistent with stimulated BMP/Smad signaling, baseline hepcidin expression was higher in Tmprss6 hepatocytes compared to WT hepatocytes in serumcontaining and serum-free conditions (Figure 1F). Hepcidin mRNA expression was reduced more than 12-fold in both WT and Tmprss6 hepatocytes after treatment with ERFE in serum-containing media. Similarly, treatment of hepatocytes with ERFE in serum-free media (ie, in the absence of any soluble matriptase-2) resulted in a 2.5 and 5-fold decrease in hepcidin expression in WT and Tmprss6 hepatocytes, respectively. These results demonstrate in vitro that ERFE represses hepcidin independently of TMPRSS6. Interestingly, Id1 mRNA expression (Supporting Information Figure 3A) was also reduced in primary mouse hepatocytes from WT and Tmprss6 mice after treatment with ERFE in serum-containing and serum-free conditions. Similarly, Atoh8 and Smad7 mRNA expression (Supporting Information Figure 3B and 3C) was decreased in ERFE-treated WT and Tmprss6 hepatocytes in serum-containing conditions. In serum-free conditions, the decrease in Atoh8 and Smad7 mRNA expression was observed only in ERFE-treated Tmprss6 hepatocytes but not in WT hepatocytes. This suggests a potential crosstalk between BMP and ERFE signaling that is independent of TMPRSS6. The reason for the impairment of erythropoietic suppression of hepcidin in patients and mice with IRIDA may lie in the intense stimulation of hepcidin by the BMP pathway in vivo, opposing the effect of ERFE. In accordance with the findings by Nai and colleagues, we previously demonstrated that hepcidin suppression after phlebotomy is blunted in iron-overloaded WT mice and Tfr2 mice where the BMP pathway is strongly stimulated by iron. These

Isocitrate treatment of acute anemia of inflammation in a mouse model.

Acute and severe anemia of inflammation (AI) is a common complication of various clinical syndromes, including fulminant infections, critical illness with multiorgan failure, and exacerbations of autoimmune diseases. Building on recent data showing beneficial results with isocitrate treatment for chronic low-grade AI in a rat model, we used a mouse model of acute and severe AI induced by intraperitoneal heat-killed Brucella abortus to determine if isocitrate would be effective in this more stringent application. Inflamed mice treated with isocitrate developed an early but transient improvement in hemoglobin compared to solvent-treated controls, with a robust improvement on day 7, and only a trend towards improvement by day 14. Reticulocyte counts were increased in treated mice transiently, with no significant difference by day 21. Serum erythropoietin (EPO) levels were similar in treated versus control mice, indicating that isocitrate increased sensitivity to EPO. Serum and tissue iron levels showed no significant differences between the treated and control mice, ruling out improved iron availability as the cause of the increased response to endogenous EPO. Compared to the milder rat model, much higher doses of isocitrate were required for a relatively modest benefit.