Activated mesenchymal stem cells increase drug susceptibility of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa are major causes of hospital-acquired infections and sepsis. Due to increasing antibiotic resistance, new treatments are needed. Mesenchymal stem cells (MSCs) have antimicrobial effects, which can be enhanced by preconditioning with antibiotics. This study investigated using antibiotics to strengthen MSCs against MRSA and P. aeruginosa. MSCs were preconditioned with linezolid, vancomycin, meropenem, or cephalosporin. Optimal antibiotic concentrations were determined by assessing MSC survival. Antimicrobial effects were measured by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antimicrobial peptide (AMP) gene expression. Optimal antibiotic concentrations for preconditioning MSCs without reducing viability were 1 µg/mL for linezolid, meropenem, and cephalosporin and 2 µg/mL for vancomycin. In MIC assays, MSCs preconditioned with linezolid, vancomycin, meropenem, or cephalosporin inhibited MRSA or P. aeruginosa growth at lower concentrations than non-preconditioned MSCs (p ≤ 0.001). In MBC assays, preconditioned MSCs showed enhanced bacterial clearance compared to non-preconditioned MSCs, especially when linezolid and vancomycin were used against MRSA (p ≤ 0.05). Preconditioned MSCs showed increased expression of genes encoding the antimicrobial peptide genes hepcidin and LL-37 compared to non-preconditioned MSCs. The highest hepcidin expression was seen with linezolid and vancomycin preconditioning (p ≤ 0.001). The highest LL-37 expression was with linezolid preconditioning (p ≤ 0.001). MSCs' preconditioning with linezolid, vancomycin, meropenem, or cephalosporin at optimal concentrations enhances their antimicrobial effects against MRSA and P. aeruginosa without compromising viability. This suggests preconditioned MSCs could be an effective adjuvant treatment for antibiotic-resistant infections. The mechanism may involve upregulation of AMP genes.

Anti-microbial activity and immunomodulation of recombinant hepcidin 2 and NK-lysin from flounder (Paralichthys olivaceus).

Infections due to pathogens impact global aquaculture economy, where diseases caused by bacteria should be in particular focus due to antibiotic resistance. Hepcidin and NK-lysin are important innate immune factors having potential to be exploited as alternatives to antibiotics due to their antimicrobial activity and immunomodulatory capacity. In this study, the recombinant proteins of hepcidin 2 and NK-lysin (rPoHep2 and rPoNKL) from flounder (Paralichthys olivaceus) were obtained via a prokaryotic expression system. The results exhibited that rPoHep2 and rPoNKL killed both gram-negative and gram-positive bacteria mainly via attachment and disruption of the membrane. Interestingly, both peptides could bind to bacterial DNA. The antiviral assay showed that both peptides have antiviral activity against hirame nonvirhabdovirus. They exhibited no cytotoxicity to the mammalian and fish cell lines. PoHep2 was found localized in G-CSFR-positive peritoneal cells. Moreover, rPoHep2 significantly enhanced the phagocytosis of flounder leukocytes in vitro. These findings suggested that neutrophils contained rPoHep2 and may respond to the immunoreaction of neutrophils. In summary, both rPoHep2 and rPoNKL possess antimicrobial activities and may be exploited to replace traditional antibiotics. rPoHep2 possess immune regulatory functions, that can be further investigated as an immunostimulant in aquaculture.

Antibacterial Activity and Mechanisms of TroHepc2-22, a Derived Peptide of Hepcidin2 from Golden Pompano (Trachinotus ovatus).

Hepcidin, a cysteine-rich antimicrobial peptide, has a highly conserved gene structure in teleosts, and it plays an essential role in host immune response against various pathogenic bacteria. Nonetheless, few studies on the antibacterial mechanism of hepcidin in golden pompano (Trachinotus ovatus) have been reported. In this study, we synthesized a derived peptide, TroHepc2-22, from the mature peptide of T. ovatus hepcidin2. Our results showed that TroHepc2-22 has superior antibacterial abilities against both Gram-negative (Vibrio harveyi and Edwardsiella piscicida) and Gram-positive (Staphylococcus aureus and Streptococcus agalactiae) bacteria. Based on the results of a bacterial membrane depolarization assay and propidium iodide (PI) staining assay in vitro, TroHepc2-22 displayed antimicrobial activity by inducing the bacterial membrane depolarization and changing the bacterial membrane permeability. Scanning electron microscopy (SEM) visualization illustrated that TroHepc2-22 brought about membrane rupturing and the leakage of the cytoplasm for the bacteria. In addition, TroHepc2-22 was verified to have hydrolytic activity on bacterial genomic DNA in view of the results of the gel retardation assay. In terms of the in vivo assay, the bacterial loads of V. harveyi in the tested immune tissues (liver, spleen, and head kidney) were significantly reduced in T. ovatus, revealing that TroHepc2-22 significantly enhanced the resistance against V. harveyi infection. Furthermore, the expressions of immune-related genes, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1-ß (IL-1ß), IL-6, Toll-like receptor 1 (TLR1), and myeloid differentiation factor 88 (MyD88) were significantly increased, indicating that TroHepc2-22 might regulate inflammatory cytokines and activate immune-related signaling pathways. To summarize, TroHepc2-22 possesses appreciable antimicrobial activity and plays a vital role in resisting bacterial infection. The observation of our present study unveils the excellent application prospect of hepcidin as a substitute for antibiotics to resist pathogenic microorganisms in teleosts.

Effects of hypoxia-inducible factor-prolyl hydroxylase inhibitors vs. erythropoiesis-stimulating agents on iron metabolism in non-dialysis-dependent anemic patients with CKD: A network meta-analysis.

Objective: To compare the effects of five hypoxia-inducible factor-prolyl hydroxylase domain inhibitors (HIF-PHIs), two erythropoiesis-stimulating agents (ESAs), and placebo on iron metabolism in renal anemia patients with non-dialysis-dependent chronic kidney disease (NDD-CKD). Method: Five electronic databases were searched for studies. Randomized controlled clinical trials comparing HIF-PHIs, ESAs, and placebo in NDD-CKD patients were selected. The statistical program used for network meta-analysis was Stata/SE 15.1. The main outcomes were the change in hepcidin and hemoglobin (Hb) levels. The merits of intervention measures were predicted by the surface under the cumulative ranking curve method. Results: Of 1,589 original titles screened, data were extracted from 15 trials (3,228 participants). All HIF-PHIs and ESAs showed greater Hb level-raising ability than placebo. Among them, desidustat demonstrated the highest probability of increasing Hb (95.6%). Hepcidin [mean deviation (MD) = -43.42, 95%CI: -47.08 to -39.76], ferritin (MD= -48.56, 95%CI: -55.21 to -41.96), and transferrin saturation (MD = -4.73, 95%CI: -5.52 to -3.94) were decreased, while transferrin (MD = 0.09, 95%CI: 0.01 to 0.18) and total iron-binding capacity (MD = 6.34, 95%CI: 5.71 to 6.96) was increased in HIF-PHIs versus those in ESAs. In addition, this study observed heterogeneity in the ability of HIF-PHIs to decrease hepcidin. Compared with darbepoetin, only daprodustat (MD = -49.09, 95% CI: -98.13 to -0.05) could significantly reduce hepcidin levels. Meanwhile, daprodustat also showed the highest hepcidin-lowering efficacy (84.0%), while placebo was the lowest (8.2%). Conclusion: For NDD-CKD patients, HIF-PHIs could ameliorate functional iron deficiency by promoting iron transport and utilization, which may be achieved by decreasing hepcidin levels. Interestingly, HIF-PHIs had heterogeneous effects on iron metabolism. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=242777, Identifier CRD42021242777.

Structural basis of ferroportin inhibition by minihepcidin PR73.

Ferroportin (Fpn) is the only known iron exporter in humans and is essential for maintaining iron homeostasis. Fpn activity is suppressed by hepcidin, an endogenous peptide hormone, which inhibits iron export and promotes endocytosis of Fpn. Hepcidin deficiency leads to hemochromatosis and iron-loading anemia. Previous studies have shown that small peptides that mimic the first few residues of hepcidin, i.e., minihepcidins, are more potent than hepcidin. However, the mechanism of enhanced inhibition by minihepcidins remains unclear. Here, we report the structure of human ferroportin in complex with a minihepcidin, PR73 that mimics the first 9 residues of hepcidin, at 2.7 Å overall resolution. The structure reveals novel interactions that were not present between Fpn and hepcidin. We validate PR73-Fpn interactions through binding and transport assays. These results provide insights into how minihepcidins increase inhibition potency and will guide future development of Fpn inhibitors.

Intracellular labile iron is a key regulator of hepcidin expression and iron metabolism.

BACKGROUND AND AIMS: Liver iron loading can induce hepatic expression of hepcidin and regulate iron metabolism. However, the mechanism by which hepatocyte senses iron loading and further regulates iron metabolism remains unclear. Intracellular labile iron is nonferritin-bound and redox active; it is transitory, and it serves as a crossroads of cellular iron metabolism, the effect of intracellular labile iron in iron metabolism regulation is particularly poorly understood. METHODS: An intracellular labile iron overload cell model was established using ferric ammonium citrate (FAC) and the lipophilic iron chelator 8-hydroxyquinoline (8HQ/FAC). RNA-Seq was performed to screen the genes that were highly expressed exclusively in 8HQ/FAC-treated HepG2 cells. High-iron-diet mice model and Hfe knockout hemochromatosis mice were used to investigate the importance of tumor necrosis factor α (TNFα) in iron metabolism. RESULTS: Intracellular labile iron in hepatocytes had a dual function in iron metabolism: It induced hepatocytes to express hepcidin via endoplasmic reticulum stress-induced transcription factors, and it stimulated expression of bone morphogenic protein 6 (BMP6, regulator of iron metabolism) in liver sinusoidal endothelial cells (LSECs) via promoting the secretion of TNFα by the hepatocytes. Blockade of TNFα dysregulated iron metabolism during iron overload. Furthermore, administration of TNFα could reduce iron burden in Hfe knockout hemochromatosis mice. CONCLUSIONS: Our findings reveal the importance of intracellular labile iron in iron metabolism, and propose that TNFα might be a novel therapeutic target for HFE-associated hemochromatosis.

Hepcidin is potential regulator for renin activity.

An association between genetic variants in the genes HFE, HJV, BMP4 and arterial hypertension has been shown earlier. Proteins encoded by these genes participate in the signalling routes leading eventually to the production of the peptide hormone hepcidin. Mutations in these genes have been associated with the abnormal production of hepcidin in the body. This finding led to studies exploring the possible role of hepcidin in regulating the activity of blood pressure related renin-angiotensin system enzymes. We used molecular modelling to find out if it is possible for hepcidin to bind to the active site of the renin-angiotensin system enzymes, especially renin. Fluorometric assays were used to evaluate the inhibitory effect of hepcidin on renin as well as angiotensin converting enzymes 1 and 2. Finally, bio-layer interferometry technique was used to study hepcidin binding to renin. The molecular modelling showed that hepcidin seems to have similar binding properties to the renin active site as angiotensinogen does. Based on fluorometric enzyme activity assay, hepcidin has an inhibitory effect on renin in vitro, too. However, angiotensin converting enzymes 1 and 2 were not inhibited remarkably by hepcidin-25. In bio-layer interferometry analysis hepcidin-renin binding was concentration dependent. Our results suggest that hepcidin could act as an inhibitor to the renin. Nowadays, there is no known biological inhibitor for renin in vivo and our finding may thus have important clinical implications.

Vitamin A does not influence mRNA expression of hormone hepcidin but other biomarkers of iron homeostasis in young male Wistar rats.

The effects of an adequate supply of vitamin A and iron, in comparison with diets low or absent in vitamin A and low in iron, on the mRNA expression of some biomarkers of iron homeostasis as hepcidin (Hamp), transferrin receptor-1 (Tfrc), iron regulatory protein-2 (Ireb2) and ferritin (Fth1) in rats were investigated. 35 male Wistar rats were randomly divided into 5 dietary groups: control, sufficient in iron and insufficient in vitamin A (FesvAi), sufficient in iron and depleted in vitamin A (FesvAd), insufficient in iron and sufficient in vitamin A (FeivAs) and insufficient in both iron and vitamin A (FeivAi). After 6 weeks rats showed no significant effects of variations in vitamin A on the expression of Hamp relative to the control group (FesvAi: 1.37-fold; FesvAd: 1.22-fold); however, iron deficiency showed significant reduction on it relative to the control group (FeivAs: 71.4-fold, P = 0.0004; FeivAi: 16.1-fold, P = 0.0008). Vitamin A deficiency (FesvAd) affects expression of Fth1 independent of low dietary iron in spleen (0.29-fold, P = 0.002) and duodenum (5.15-fold, P = 0.02). Variations of dietary iron and vitamin A showed significant effects relative to the control group for expression of Tfrc in spleen (FesvAd: 0.18-fold, P = 0.01; FeivAs: 0.24-fold, P < 0.0001; FeivAi: 0.42-fold, P = 0.014), Ireb2 in spleen (FeivAs: 3.7-fold, P < 0.0001; FeivAi: 2.9-fold, P < 0.0001) and Ireb2 in duodenum (FeivAs: 2.68-fold, P = 0.012; FeivAi: 2.60-fold, P = 0.014). These results show that vitamin A and iron must be supplied together to regulate some of the main biomarkers of iron metabolism as a strategy to reduce prevalence of iron deficiency anemia.

Ineffective erythropoiesis and its treatment.

The erythroid marrow and circulating red blood cells (RBCs) are the key components of the human erythron. Abnormalities of the erythron that are responsible for anemia can be separated into 3 major categories: erythroid hypoproliferation, ineffective erythropoiesis, and peripheral hemolysis. Ineffective erythropoiesis is characterized by erythropoietin-driven expansion of early-stage erythroid precursors, associated with apoptosis of late-stage precursors. This mechanism is primarily responsible for anemia in inherited disorders like ß-thalassemia, inherited sideroblastic anemias, and congenital dyserythropoietic anemias, as well as in acquired conditions like some subtypes of myelodysplastic syndrome (MDS). The inherited anemias that are due to ineffective erythropoiesis are also defined as iron-loading anemias because of the associated parenchymal iron loading caused by the release of erythroid factors that suppress hepcidin production. Novel treatments specifically targeting ineffective erythropoiesis are being developed. Iron restriction through enhancement of hepcidin activity or inhibition of ferroportin function has been shown to reduce ineffective erythropoiesis in murine models of ß-thalassemia. Luspatercept is a transforming growth factor-ß ligand trap that inhibits SMAD2/3 signaling. Based on preclinical and clinical studies, this compound is now approved for the treatment of anemia in adult patients with ß-thalassemia who require regular RBC transfusions. Luspatercept is also approved for the treatment of transfusion-dependent anemia in patients with MDS with ring sideroblasts, most of whom carry a somatic SF3B1 mutation. While the long-term effectiveness and safety of luspatercept need to be evaluated in ß-thalassemia and MDS, defining the molecular mechanisms of ineffective erythropoiesis in different disorders might allow the discovery of new effective compounds.

PTH 1-34 promoted bone formation by regulating iron metabolism in unloading-induced bone loss.

PTH 1-34 (teriparatide) is approved by FDA for the treatment of postmenopausal osteoporosis. Iron overload is a major contributing factor for bone loss induced by unloading. Whether iron metabolism is involved in the regulation of PTH 1-34 on unloading-induced osteoporosis has not yet been reported. Here, we found that PTH 1-34 attenuated bone loss in unloading mice. PTH 1-34 regulated the disturbance of iron metabolism in unloading mice by activating Nrf2 and further promoting hepcidin expression in the liver. In addition, the Nrf2 inhibitor selectively blocked hepcidin expression in the liver of unloading mice, which neutralized the inhibitory effect of PTH 1-34 on bone loss and the recovery of iron metabolism in unloading mice. Finally, we found that PTH 1-34 promoted the differentiation and inhibited apoptosis of osteoblasts by regulating iron metabolism and maintaining redox balance under unloading conditions. Our results suggested that PTH 1-34 promoted bone formation by regulating iron metabolism under unloading conditions.

Hepcidin induces intestinal calcium uptake while suppressing iron uptake in Caco-2 cells.

Abnormal calcium absorption and iron overload from iron hyperabsorption can contribute to osteoporosis as found in several diseases, including hemochromatosis and thalassemia. Previous studies in thalassemic mice showed the positive effects of the iron uptake suppressor, hepcidin, on calcium transport. However, whether this effect could be replicated in other conditions is not known. Therefore, this study aimed to investigate the effects of hepcidin on iron and calcium uptake ability under physiological, iron uptake stimulation and calcium uptake suppression. To investigate the potential mechanism, effects of hepcidin on the expression of iron and calcium transporter and transport-associated protein in Caco-2 cells were also determined. Our results showed that intestinal cell iron uptake was significantly increased by ascorbic acid together with ferric ammonium citrate (FAC), but this phenomenon was suppressed by hepcidin. Interestingly, hepcidin significantly increased calcium uptake under physiological condition but not under iron uptake stimulation. While hepcidin significantly suppressed the expression of iron transporter, it had no effect on calcium transporter expression. This indicated that hepcidin-induced intestinal cell calcium uptake did not occur through the stimulation of calcium transporter expression. On the other hand, 1,25(OH)2D3 effectively induced intestinal cell calcium uptake, but it did not affect intestinal cell iron uptake or iron transporter expression. The 1,25(OH)2D3-induced intestinal cell calcium uptake was abolished by 12 mM CaCl2; however, hepcidin could not rescue intestinal cell calcium uptake suppression by CaCl2. Taken together, our results showed that hepcidin could effectively and concurrently induce intestinal cell calcium uptake while reducing intestinal cell iron uptake under physiological and iron uptake stimulation conditions, suggesting its therapeutic potential for inactive calcium absorption, particularly in thalassemic patients or patients who did not adequately respond to 1,25(OH)2D3.

Advances in understanding the crosstalk between mother and fetus on iron utilization.

A full-term pregnancy comes with significant demand for iron. Not meeting this demand has adverse effects on maternal health and on the intrauterine and postnatal development of the infant. In the infant, some of these adverse effects cannot be reversed by postnatal iron supplementation, highlighting the need to tackle iron deficiency in utero. Achieving this requires sound understanding of the pathways that govern iron transfer at the fetomaternal interface. Two pathways are emerging as key players in this context; the hepcidin/ferroportin axis pathway and the iron regulatory protein (IRPs) pathway. In late gestation, suppression of maternal hepcidin, by as yet unknown factors, is required for increasing iron availability to the growing fetus. In the placenta, the rate of iron uptake by transferrin receptor TfR1 at the apical/maternal side and of iron release by ferroportin FPN at the basal/fetal side is controlled by IRP1. In fetal hepatocytes, build up of fetal iron stores requires post-translational inhibition of FPN by the cell-autonomous action of hepcidin. In the fetal liver, FPN is also subject to additional control at the transcriptional level, possibly by the action of hypoxia-inducible factor HIF2α. The rates of apical iron uptake and basal iron release in the placenta are modulated according to iron availability in the maternal blood and the placenta's own needs. This placental modulation ensures that the amount of iron delivered to the fetal circulation is maintained within a normal range, even in the face of mild maternal iron deficiency or overload. However, when maternal iron deficiency or overload are extreme, placental modulation is not sufficient to maintain normal iron supply to the fetus, resulting in fetal iron deficiency and overload respectively. Thus, the rate of iron transfer at the fetomaternal interface is subject to several regulatory signals operating simultaneously in the maternal liver, the placenta and the fetal liver. These regulatory signals act in concert to maintain normal iron supply to the fetus within a wide range of maternal iron states, but fail to do so when maternal iron deficiency or overload are extreme. The limitations of existing experimental models must be overcome if we are to gain better understanding of the role of these regulatory signals in normal and complicated pregnancy. Ultimately, that understanding could help identify better markers of fetal iron demand and underpin novel iron replacement strategies to treat maternal and fetal iron deficiency.

Oral ferroportin inhibitor vamifeport for improving iron homeostasis and erythropoiesis in ß-thalassemia: current evidence and future clinical development.

INTRODUCTION: In ß-thalassemia, imbalanced globin synthesis causes reduced red blood cell survival and ineffective erythropoiesis. Suppressed hepcidin levels increase ferroportin-mediated iron transport in enterocytes, causing increased iron absorption and potentially iron overload. Low hepcidin also stimulates ferroportin-mediated iron release from macrophages, increasing transferrin saturation (TSAT), potentially forming non-transferrin-bound iron, which can be toxic. Modulating the hepcidin-ferroportin axis is an attractive strategy to improve ineffective erythropoiesis and limit the potential tissue damage resulting from iron overload. There are no oral ß-thalassemia treatments that consistently ameliorate anemia and prevent iron overload. AREAS COVERED: The preclinical and clinical development of vamifeport (VIT-2763), a novel ferroportin inhibitor, was reviewed. PubMed, EMBASE and ClinicalTrials.gov were searched using the search term 'VIT-2763'. EXPERT OPINION: Vamifeport is the first oral ferroportin inhibitor in clinical development. In healthy volunteers, vamifeport had comparable safety to placebo, was well tolerated and rapidly decreased iron levels and reduced TSAT, consistent with observations in preclinical models. Data from ongoing/planned Phase II studies are critical to define its potential in ß-thalassemia and other conditions associated with iron overabsorption and/or ineffective erythropoiesis. If vamifeport potentially increases hemoglobin and reduces iron-related parameters, it could be a suitable treatment for non-transfusion-dependent and transfusion-dependent ß-thalassemia.

The Acute Phase Protein Hepcidin Is Cytotoxic to Human and Mouse Myeloma Cells.

BACKGROUND/AIM: Hepcidin is a cationic acute phase reactant synthesized by the liver. It has bactericidal properties and is a major regulator of iron homeostasis. Cationic antimicrobial peptides represent an innate antimicrobial defense system. We hypothesized that, like other cationic antimicrobial peptides, hepcidin is cytotoxic to cancer cells. MATERIALS AND METHODS: The cytotoxicity of human hepcidin against myeloma cells was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and DNA fragmentation assays. Plasma membrane damage was quantified by propidium iodide (PI) staining. Cell membrane changes were visualized by scanning electron microscopy. RESULTS: Hepcidin impaired myeloma cell survival and induced DNA fragmentation. PI staining and scanning electron microscopy revealed hepcidin-induced disruption of the plasma membrane. CONCLUSION: Human hepcidin is an anti-cancer peptide that induces myeloma cell lysis, and therefore may play a role in innate anticancer immunity. To our knowledge, this is the first biological function ascribed to human hepcidin that is not related to its antimicrobial and iron-regulatory properties.

Medium cut-off dialyzer improves erythropoiesis stimulating agent resistance in a hepcidin-independent manner in maintenance hemodialysis patients: results from a randomized controlled trial.

The response to erythropoiesis stimulating agents (ESAs) is affected by inflammation linked to middle molecules in hemodialysis (HD) patients. We evaluated the effect of a medium cut-off (MCO) dialyzer on ESA resistance in maintenance HD patients. Forty-nine patients who underwent high-flux HD were randomly allocated to the MCO or high-flux group. The primary outcome was the changes of erythropoietin resistance index (ERI; U/kg/wk/g/dL) between baseline and 12 weeks. The MCO group showed significant decrease in the ESA dose, weight-adjusted ESA dose, and ERI compared to the high-flux group at 12 weeks (p < 0.05). The generalized estimating equation models revealed significant interactions between groups and time for the ESA dose, weight-adjusted ESA dose, and ERI (p < 0.05). Serum iron and transferrin saturation were higher in the MCO group at 12 weeks (p < 0.05). The MCO group showed a greater reduction in TNF-α and lower serum TNF-α level at 12 weeks compared to the high-flux group (p < 0.05), whereas no differences were found in the reduction ratio of hepcidin and serum levels of erythropoietin, erythroferrone, soluble transferrin receptor and hepcidin between groups. HD with MCO dialyzer improves ESA resistance over time compared to high-flux HD in maintenance HD patients. The MCO dialyzer provides superior removal of the inflammatory cytokine and thus improves iron metabolism in a hepcidin-independent manner.

Immune Regulation, but Not Antibacterial Activity, Is a Crucial Function of Hepcidins in Resistance against Pathogenic Bacteria in Nile Tilapia (Oreochromis niloticus Linn.).

In this study, the functions of a recombinant propeptide (rProOn-Hep1) and the synthetic FITC-labelled mature peptides sMatOn-Hep1 and sMatOn-Hep2 were analyzed. Moreover, sMatOn-Hep1 and sMatOn-Hep2 were mildly detected in the lymphocytes of peripheral blood mononuclear cells (PBMCs) and strongly detected in head kidney macrophages. The in vitro binding and antibacterial activities of these peptides were slightly effective against several pathogenic bacteria. Immune regulation by sMatOn-Hep1 was also analyzed, and only sMatOn-Hep1 significantly enhanced the phagocytic index in vitro (p < 0.05). Interestingly, intraperitoneal injection of sMatOn-Hep1 (10 or 100 µg) significantly elevated the phagocytic activity, phagocytic index, and lysozyme activity and clearly decreased the iron ion levels in the livers of the treated fish (p < 0.05). Additionally, sMatOn-Hep1 enhanced the expression levels of CC and CXC chemokines, transferrin and both On-Hep genes in the liver, spleen and head kidney, for 1-96 h after injection, but did not properly protect the experimental fish from S. agalactiae infection after 7 days of treatment. However, the injection of S. agalactiae and On-Heps indicated that 100 µg of sMatOn-Hep1 was very effective, while 100 µg of rProOn-Hep1 and sMatOn-Hep2 demonstrated moderate protection. Therefore, On-Hep is a crucial iron-regulating molecule and a key immune regulator of disease resistance in Nile tilapia.

Maternal hepcidin determines embryo iron homeostasis in mice.

Iron disorders are associated with adverse pregnancy outcomes, yet iron homeostatic mechanisms during pregnancy are poorly understood. In humans and rodents, the iron-regulatory hormone hepcidin is profoundly decreased in pregnant mothers, which is thought to ensure adequate iron availability for transfer across placenta. However, the fetal liver also produces hepcidin, which may regulate fetal iron endowment by controlling placental iron export. To determine the relative contribution of maternal vs embryo hepcidin to the control of embryo iron endowment in iron-sufficient or iron-overloaded mice, we generated combinations of mothers and embryos that had or lacked hepcidin. We found that maternal, but not embryonic, hepcidin determined embryo and placental iron endowment in a healthy pregnancy. We further determined that inflammation can counteract pregnancy-dependent suppression of maternal hepcidin. To establish how essential maternal hepcidin suppression is for embryo iron homeostasis, we mimicked the range of maternal hepcidin activity by administering a hepcidin peptide mimetic to pregnant mice. This also allowed us to determine the effect of isolated maternal hepcidin excess on pregnancy, in the absence of other confounding effects of inflammation. Higher doses of hepcidin agonist caused maternal iron restriction and anemia, lower placenta and embryo weight, embryo anemia, and increased embryo mortality. Low agonist doses did not cause maternal anemia but still adversely affected the embryo, causing anemia, tissue iron deficiency (including in the brain), and decreased weight. Our studies demonstrate that suppression of maternal hepcidin during pregnancy is essential for maternal and embryo iron homeostasis and health.

Hamp Type-1 Promotes Antimicrobial Defense via Direct Microbial Killing and Regulating Iron Metabolism in Grass Carp (Ctenopharyngodon idella).

Hepcidin is an antimicrobial peptide and regulator of iron homeostasis which has two isoforms in most fishes and some mammals. Previous studies have reported that the two hepcidin isoforms have different roles. Hamp type-1 plays a regulatory role in iron metabolism and hamp type-2 mostly performs an antimicrobial role. In this study, we found that Ctenopharyngodon idella (C. idella) have only one hepcidin isoform (hamp type-1), which showed both broad-spectrum antibacterial and iron regulatory functions. C. idella hepcidin mature peptide (hepcidin-25) and truncated peptide (hepcidin-20) exhibited bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner in part through membrane rupture and binding to bacterial genomic DNA. The data from challenge tests demonstrated that the administration of hepcidin-25 significantly reduced mortality rates of C. idella by A. hydrophila infection, probably due to direct bactericidal activities of the peptide and a reduction of iron content in the fish serum. In addition, a comparison between hepcidin-20 and -25 suggests that the N terminal 5 amino acids play a critical role in reducing iron content in fish serum. Our findings revealed an important role of hamp type-1 in maintaining iron homeostasis and fighting against bacterial infections, suggesting the hepcidin has implications for the prevention and control of bacterial infection in aquaculture.

Immunomodulatory function of antimicrobial peptide EC-Hepcidin1 modulates the induction of inflammatory gene expression in primary cells of Caspian Trout (Salmo trutta caspius Kessler, 1877).

Hepcidins, a group of antimicrobial peptides (AMPs), play a key role in the innate immune system of fishes and act against different pathogens. In this study, antimicrobial and immune-inflammatory activity of a synthetic EC-hepcidin1, previously identified from orange-spotted grouper, were evaluated. EC-hepcidin1 showed weak activity against the zoonotic fish pathogen Streptococcus iniae (MIC 100 µg mL-1 and MBC 150 µg mL-1). To study the effect of AMPs in general, and EC-hepcidin1 in particular, a primary cell culture (SC) from the fin tissue of the Caspian Trout (Salmo trutta caspius) was established. The neutral Red method on SC cells revealed that EC-hepcidin1 has no or very low cytotoxic properties. Treatment of cells with either EC-hepcidin1 (150 µg mL-1) or fish pathogen Streptococcus iniae (MOI = 10) and a mixture of both resulted in the up-regulation of gene expression of MHC-UBA, IL-6, and TNFα indicating the modulatory function on inflammatory processes. These findings indicate that EC-hepcidin1 might act as a candidate for modulation of the innate immune system in S. iniae-based infection.

Iron and hepcidin mediate human colorectal cancer cell growth.

High dietary iron intake is a risk factor for the development of colorectal cancer. However, how iron subsequently impacts the proliferation of colorectal cancer cells remains unclear. This study determined the expression of six iron regulatory genes in twenty-one human colorectal cancer (CRC) biopsies and matched normal colonic tissue. The results show that only hepcidin and ferritin heavy chain expression were increased in CRC biopsies as compared to matched normal tissues. Four established human CRC cell lines, HT-29, HCT-116, SW-620 and SW-480 were subsequently examined for their growth in response to increasing concentrations of iron, and iron depletion. Real time cell growth assay showed a significant inhibitory effect of acute iron loading in HCT-116 cells (IC50 = 258.25 µM at 72 h), and no significant effects in other cell types. However, ten week treatment with iron significantly reduced HT-29 and SW-620 cell growth, whereas no effect was seen in HCT-116 and SW-480 cells. Intracellular labile iron depletion induced the complete growth arrest and detachment of all of the CRC cell types except for the SW-620 cell line which was not affected in its growth. Treatment of starved CRC cells with hepcidin, the major regulator of iron metabolism, induced a significant stimulation of HT-29 cell growth but did not affect the growth of the other cell types. Collectively these results show that iron is central to CRC cell growth in a manner that is not identical between acute and chronic loading, and that is specific to the CRC cell type.