Peptide Hepcidin Research Articles

Inducers of salmon innate immunity: An in vitro and in vivo approach

Maintaining fish health is one of the most important aims in aquaculture. Prevention of fish diseases therefore is crucial and can be achieved by various different strategies, including most often a combination of different methods such as optimal feed and fish density, as well as strengthening the immune system. Understanding the fish innate immune system and developing methods to activate it, in an effort to prevent infections in the first place, has been a goal in recent years. In this study we choose different inducers of the innate immune system and examined their effects in vitro on the salmon cell line CHSE-214. We found that the butyrate derivatives 4-phenyl butyrate (PBA) and β-hydroxy-β-methyl butyrate (HMB) induce the expression of various innate immune genes differentially over 24–72 h. Similarly, lipids generated from fish oils were found to have an effect on the expression of the antimicrobial peptides cathelicidin and hepcidin, as well as iNOS and the viral receptor RIG-1. Interestingly we found that vitamin D3, similar as in mammals, was able to increase cathelicidin expression in fish cells. The observed induction of these different innate immune factors correlated with antibacterial activity against Aeromonas salmonicida and antiviral activity against IPNV and ISAV in vitro. To relate this data to the in vivo situation we examined cathelicidin expression in juvenile salmon and found that salmon families vary greatly in their basal cathelicidin levels. Examining cathelicidin levels in families known to be resistant to IPNV showed that these QTL-families had lower basal levels of cathelicidin in gills, than non QTL-families. Feeding fish with HMB caused a robust increase in cathelicidin expression in gills, but not skin and this was independent of the fish being resistant to IPNV. These findings support the use of fish cell lines as a tool to develop new inducers of the fish innate immune system, but also highlight the importance of the tissue studied in vivo. Understanding the response of the innate immune system in different tissues and what effect this might have on infections and downstream cellular pathways is an interesting research topic for the future.

Pathogen invasion changes the intestinal microbiota composition and induces innate immune responses in the zebrafish intestine

Numerous bacteria are harbored in the animal digestive tract and are impacted by several factors. Intestinal microbiota homeostasis is critical for maintaining the health of an organism. However, how pathogen invasion affects the microbiota composition has not been fully clarified. The mechanisms for preventing invasion by pathogenic microorganisms are yet to be elucidated. Zebrafish is a useful model for developmental biology, and studies in this organism have gradually become focused on intestinal immunity. In this study, we analyzed the microbiota of normal cultivated and infected zebrafish intestines, the aquarium water and feed samples. We found that the predominant bacteria in the zebrafish intestine belonged to Gammaproteobacteria (67%) and that feed and environment merely influenced intestinal microbiota composition only partially. Intestinal microbiota changed after a pathogenic bacterial challenge. At the genus level, the abundance of some pathogenic intestinal bacteria increased, and these genera included Halomonas (50%), Pelagibacterium (3.6%), Aeromonas (2.6%), Nesterenkonia (1%), Chryseobacterium (3.4‰), Mesorhizobium (1.4‰), Vibrio (1‰), Mycoplasma (0.7‰) and Methylobacterium (0.6‰) in IAh group. However, the abundance of some beneficial intestinal bacteria decreased, and these genera included Nitratireductor (0.8‰), Enterococcus (0.8‰), Brevundimonas (0.7‰), Lactococcus (0.7‰) and Lactobacillus (0.4‰). Additionally, we investigated the innate immune responses after infection. ROS levels in intestine increased in the early stages after a challenge and recovered subsequently. The mRNA levels of antimicrobial peptide genes lectin, hepcidin and defensin1, were upregulated in the intestine after pathogen infection. These results suggested that the invasion of pathogen could change the intestinal microbiota composition and induce intestinal innate immune responses in zebrafish.

Ontogeny and modulation after PAMPs stimulation of β-defensin, hepcidin, and piscidin antimicrobial peptides in meagre (Argyrosomus regius)

Antimicrobial peptides (AMPs), components of innate immunity, play an important role in protecting fish. In this study we report the molecular cloning of full open reading frames and characterization of expression of three AMP genes (β-defensin (defb), hepcidin (hep2), piscidin (pisc) in meagre (Argyrosomus regius). A phylogenetic analysis of the expressed sequences obtained shows the defensin isoform forms a clade with the other members of the beta class of this family, hepcidin corresponds to hepcidin 2, and piscidin corresponds to class I of its respective family. Gene expression profiles of AMPs was investigated, by means of quantification of mRNA in nine development stages, from 8 days post-hatching (dph) to accomplishment of juvenile form (120 dph). During development it was demonstrated defb, hep2, pisc were expressed in all stages of larval development and in juvenile tissues (kidney, spleen gut and gill). Moreover, expression patterns suggest the expression levels of theses AMPs are influenced by live prey (rotifer, Artemia) and first intake of commercial diet. Induction experiments in vivo (24 h) and in vitro (4, 12, 24 h) with PAMPs (LPS, poly (I:C), β-glucan) revealed significant changes in gene expression of the three AMP genes, in kidney, spleen, gut and gill. However, expression profiles differed in magnitude and time course response. defb expression shows a similar trend in vivo and in vitro in kidney at 24 h after LPS and β-glucan stimulation. The hep2 expression levels were up-regulated upon β-glucan challenge in vivo, more in gut and gills than kidney, while in vitro hep2 expression was up-regulated in kidney cells by LPS, poly (I:C), β-glucan (4 h). pisc expression was up-regulated in kidney cells, splenocytes by β-glucan, but in gill cells by poly (I:C) and β-glucan in vivo. However, pisc expression was upregulated in kidney cells by β-glucan and gill cells by LPS at 4 post-stimulation in vitro. These data suggest that AMPs play an important role in defense against pathogens, with each AMP having differing efficacies against specific types of microorganisms, although follow-up studies focusing on the biological activities in fish are needed.

Immunostimulatory effects of dietary poly-β-hydroxybutyrate in European sea bass postlarvae

The stable production of high-quality fry in marine aquaculture is still hampered by unpredictable mortality caused by infectious diseases during larval rearing. Consequently, the development of new biocontrol agents is crucial for a viable aquaculture industry. The bacterial energy storage compound poly-β-hydroxybutyrate (PHB) has been shown to exhibit beneficial properties on aquatic organisms such as enhanced survival, growth, disease resistance and a controlling effect on the gastrointestinal microbiota. However, the effect of PHB on the developing immune system of fish larvae has so far not been investigated. In this study, the effect of feeding PHB-enriched Artemia nauplii on survival, growth and immune response in European sea bass (Dicentrarchus labrax) postlarvae was examined. Amorphous PHB was administered to 28-day-old sea bass postlarvae over a period of 10 days. The survival and growth performance were monitored, and the expression of 29 genes involved in immunity, growth, metabolism and stress-response was measured. While the expression of the insulin-like growth factor 1 (igf1), an indicator of relative growth, was upregulated in response to feeding PHB, the larval survival and growth performance remained unaffected. After 10 days of PHB treatment, the expression of the antimicrobial peptides dicentracin (dic) and hepcidin (hep) as well as mhc class IIa and mhc class IIb was elevated in the PHB fed postlarvae. This indicates that PHB is capable of stimulating the immune system of fish early life stages, which may be the cause of the increased resistance to diseases and robustness observed in previous studies.

Hepcidin Protects Neuron from Hemin-Mediated Injury by Reducing Iron.

Hemin plays a key role in mediating secondary neuronal injury after intracerebral hemorrhage (ICH) and the cell toxicity of hemin is thought to be due to iron that is liberated when hemin is degraded. In a recent study, we demonstrated the iron regulatory hormone hepcidin reduces brain iron in iron-overloaded rats. Therefore, we hypothesized that hepcidin might be able to reduce iron and then protect neurons from hemin or iron-mediated neurotoxicity in hemin-treated neuronal cells. Here, we tested the hypothesis and demonstrated that ad-hepcidin and hepcidin peptide both have the ability to suppress the hemin-induced increase in LDH release and apoptotic cell numbers, to reduce cell iron and ferritin contents, and to inhibit expression of transferrin receptor 1, divalent metal transporter 1, and ferroportin 1 in hemin-treated neurons. We conclude that hepcidin protects neuron from hemin-mediated injury by reducing iron via inhibition of expression of iron transport proteins.

Comparative study of interleukin-17C (IL-17C) and IL-17D in large yellow croaker Larimichthys crocea reveals their similar but differential functional activity

Interleukin 17 (IL-17) family members are key players in regulating the immune response in mammals. Here, we identified the IL-17C and IL-17D homologs from large yellow croaker (Larimichthys crocea), named LcIL-17C and LcIL-17D, respectively. The deduced LcIL-17C and LcIL-17D proteins possessed the typical IL-17 domain and shared a conserved arrangement of eight cysteine residues. Both LcIL-17C and LcIL-17Dc genes were constitutively expressed in all tissues examined, although at different levels. After challenge with Aeromonas hydrophila, the expression of LcIL-17C and LcIL-17D was significantly increased in gills, head kidney, and spleen. In the peripheral blood leukocytes (PBLs), the recombinant LcIL-17C (rLcIL-17C) could strongly promote the expression of chemokines (CXCL8, CXCL12, and CXCL13), proinflammatory factors (TNF-α, IL-1β, IL-6, and IFNg), and antibacterial peptide hepcidin, whereas rLcIL-17D induced a weaker expression of these chemokines. Consistently, the culture supernatants from the PBLs treated by rLcIL-17C showed a stronger ability to induce the migration of PBLs than those treated by rLcIL-17D. Furthermore, both rLcIL-17C and rLcIL-17D could activate the NF-κB signalling in the epithelioma papulosum cyprini (EPC) cells. Taken together, these results indicated that LcIL-17C and LcIL-17D, although differing in their ability to mediate chemotaxis for PBLs, may promote the inflammatory response and host defence via activating NF-κB signalling. To our knowledge, this is the first report on functional identification of a IL-17C in teleost.

Fish skeletal muscle tissue is an important focus of immune reactions during pathogen infection

Skeletal muscle in mammals can express and secrete immune-related molecules during pathogen infection. Despite in fish is known that classical immune tissues participate in innate immunity, the role of skeletal muscle in this function is poorly understood. To determine the immunocompetence of fish skeletal muscle, juvenile fine flounder (Paralichthys adpersus) were challenged with Vibrio ordalii. Different Toll-like receptors, pro-inflammatory cytokines (TNFα, Il-1β, and IL-8), and immune-effector molecules (NKEF and the antimicrobial peptides hepcidin and LEAP-2) were analyzed. Infection initially triggered IL-1β upregulation and P38-MAPK/AP-1 pathway activation. Next, the NFĸB pathway was activated, together with an upregulation of intracellular Toll-like receptor expressions (tlr3, tlr8a tlr9, and tlr21), TNFα production, and leap-2 expression. Finally, transcriptions of il-1β, il-8, tnfα, nkef-a, and hepcidin were also upregulated. These results suggest that fish skeletal muscle is an immunologically active organ that could play an important role against pathogens.

Anaemia of Chronic Kidney Disease: What We Know Now

Our understanding of the pathophysiology of the anaemia of chronic kidney disease (CKD) has improved considerably in the last decade with the discovery of the iron regulatory peptide hepcidin. Reduced clearance of hepcidin and the presence of a chronic inflammatory state contribute to elevated hepcidin levels in kidney disease. The recent discovery of the various factors and signalling pathways regulating hepcidin has opened up an exciting avenue for research into the development of newer agents that could treat anaemia of CKD. This review highlights our current understanding of iron metabolism in health, the regulators of hepcidin, issues associated with the current available therapies for the treatment of anaemia in CKD and potential novel therapies that could be available in the near future targeting the various factors that regulate hepcidin.

Dietary alpha-linolenic acid/linoleic acid ratios modulate immune response, physical barrier and related signaling molecules mRNA expression in the gills of juvenile grass carp (Ctenopharyngodon idella)

This study was conducted to explore the possible effects of dietary ALA/LNA ratios on the gill immunity, tight junction and antioxidant capacity, and the related signaling factor mRNA levels of juvenile grass carp (Ctenopharyngodon idella). Fish were fed diets with different ALA/LNA ratios (0.01, 0.34, 0.68, 1.03, 1.41, 1.76 and 2.15) for 60 days. The present results showed that ALA/LNA ratio of 1.03 significantly enhanced lysozyme and acid phosphatase activities, complement 3 contents, promoted mRNA levels of antimicrobial peptides (Hepcidin and liver expression antimicrobial peptide-2), anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1) and inhibitor protein κBα, whereas suppressed pro-inflammatory cytokines (interleukin 1β, interleukin 8, tumor necrosis factor a and interferon γ2), and signal molecules (IκB kinase β, IκB kines γ and nuclear factor κB p65) mRNA levels in the gill, indicating that optimal dietary ALA/LNA ratio improve gill immunity of juvenile fish. Besides, ALA/LNA ratio of 1.03 increased mRNA levels of the barrier functional proteins (occludin, zonula occludens-1, claudin-b, -c and −3), and reduced the pore-formation proteins (claudin-15a) and myosin light-chain kinase mRNA abundance in the gill of juvenile grass carp, indicating optimum ALA/LNA ratio strengthen gill tight junction of juvenile fish. Additionally, ALA/LNA ratio of 1.03 increased glutathione contents, copper/zinc superoxide dismutase, glutathione peroxidase, glutathione S-transferase and glutathione reductase activities and mRNA abundance, and nuclear factor erythoid 2-related factor 2 mRNA levels in the gill of fish, suggesting that optimal ALA/LNA ratio ameliorate gill antioxidant status of juvenile fish. Interestingly, dietary ALA/LNA ratios had no effect on IκB kinase α and catalase activities in fish gills. Collectively, optimal dietary ALA/LNA ratio could improve gill immunity and strengthen physical barrier of juvenile fish. Based on the quadratic regression analysis of complement 3 content in the gill, optimal dietary ALA/LNA ratio for maximum growth of juvenile grass carp was estimated to be 1.12.

DNA methylation regulates expression of the iron regulatory peptide hepcidin in hepatic cell lines

DNA methylation regulates expression of the iron regulatory peptide hepcidin in hepatic cell lines

Production and characterization of functional recombinant hybrid heteropolymers of camel hepcidin and human ferritin H and L chains.

Hepcidin is a liver-synthesized hormone that plays a central role in the regulation of systemic iron homeostasis. To produce a new tool for its functional properties the cDNA coding for camel hepcidin-25 was cloned at the 5'end of human FTH sequence into the pASK-IBA43plus vector for expression in Escherichia coli The recombinant fusion hepcidin-ferritin-H subunit was isolated as an insoluble iron-containing protein. When alone it did not refold in a 24-mer ferritin molecule, but it did when renatured together with H- or L-ferritin chains. We obtained stable ferritin shells exposing about 4 hepcidin peptides per 24-mer shell. The molecules were then reduced and re-oxidized in a controlled manner to allow the formation of the proper hepcidin disulfide bridges. The functionality of the exposed hepcidin was confirmed by its ability to specifically bind the mouse macrophage cell line J774 that express ferroportin and to promote ferroportin degradation. This chimeric protein may be useful for studying the hepcidin-ferroportin interaction in cells and also as drug-delivery agent.

Vitamin E deficiency depressed fish growth, disease resistance, and the immunity and structural integrity of immune organs in grass carp (Ctenopharyngodon idella): Referring to NF-κB, TOR and Nrf2 signaling

This study investigated the effects of dietary vitamin E on growth, disease resistance and the immunity and structural integrity of head kidney, spleen and skin in grass carp (Ctenopharyngodon idella). The fish were fed six diets containing graded levels of vitamin E (0, 45, 90, 135, 180 and 225 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila. The results showed that compared with optimal vitamin E supplementation, vitamin E deficiency caused depressed growth, poor survival rates and increased skin lesion morbidity in grass carp. Meanwhile, vitamin E deficiency decreased lysozyme and acid phosphatase activities, complement component 3 and complement component 4 contents in the head kidney, spleen and skin of grass carp (P < 0.05). Moreover, vitamin E deficiency down-regulated antimicrobial peptides (Hepcidin, liver-expressed antimicrobial peptide-2A, -2B, β-defensin), IL-10, TGFβ1, IκBα, TOR and S6K1 mRNA levels (P < 0.05) and up-regulated IL-1β, IL-6, IL-8, IFN-γ2 and TNFα, NF-κB p65, IKKα, IKKβ and 4EBP1 (not in the head kidney) mRNA levels (P < 0.05). In addition, vitamin E deficiency caused oxidative damage, decreased superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) activities, and down-regulated the mRNA levels of antioxidant enzymes and signaling molecules Nrf2 (P < 0.05). Vitamin E deficiency also induced apoptosis by up-regulating capase-2, -3, -7, and -8 mRNA levels in the head kidney, spleen and skin of grass carp. In conclusion, this study indicated that dietary vitamin E deficiency depressed fish growth, impaired the immune function and disturbed the structural integrity of the head kidney, spleen and skin in grass carp, but optimal vitamin E supplementation can reverse those negative effects in fish. The optimal vitamin E requirements for young grass carp (266.39–1026.63 g) to achieve optimal growth performance and disease resistance based on the percent weight gain (PWG) and skin lesion morbidity were estimated to be 116.2 and 130.9 mg/kg diet, respectively. Meanwhile, based on immune indicator (LA activity in the head kidney) and antioxidant indicator (protection of spleen against MDA), the optimal vitamin E requirements for young grass carp were estimated to be 123.8 and 136.4 mg/kg diet, respectively.

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model.

Piscirickettsia salmonis is a facultative intracellular bacterium that causes the disease called “salmon rickettsial syndrome”. Attempts to control this disease have been unsuccessful, because existing vaccines have not achieved the expected effectiveness and the antibiotics used fail to completely eradicate the pathogen. This is in part the product of lack of scientific information that still lacks on the mechanisms used by this bacterium to overcome infected–cell responses and survive to induce a productive infection in macrophages. For that, this work was focused in determining if P. salmonis is able to modify the expression and the imbalance of IL-12 and IL-10 using an in vitro model. Additionally, we also evaluated the role the antimicrobial peptide hepcidin had in the control of this pathogen in infected cells. Therefore, the expression of IL-10 and IL-12 was evaluated at earlier stages of infection in the RTS11 cell line derived from Oncorhynchus mykiss macrophages. Simultaneously, the hepcidin expression and location was analyzed in the macrophages infected with the pathogen. Our results suggest that IL-10 is clearly induced at early stages of infection with values peaking at 36 hours post infection. Furthermore, infective P. salmonis downregulates the expression of antimicrobial peptide hepcidin and vesicles containing this peptide were unable to merge with the infective bacteria. Our results suggest that P. salmonis is able to manipulate the behavior of host cytokines and likely might constitute a virulence mechanism that promotes intracellular bacterial replication in leukocytes cells lines of trout and salmon. This mechanism involves the generation of an optimum environment for the microorganism and the downregulation of antimicrobial effectors like hepcidin.

Inclusion of ancient Latin-American crops in bread formulation improves intestinal iron absorption and modulates inflammatory markers.

This study compares iron (Fe) absorption in Fe-deficient animals from bread formulations prepared by substitution of white wheat flour (WB) by whole wheat flour (WWB), amaranth flour (Amaranthus hypochondriacus, 25%) (AB) and quinoa flour (Chenopodium quinoa, 25%) (QB), or chia flour (Salvia hispanica L, 5%) (ChB). Hematological parameters of Fe homeostasis, plasmatic active hepcidin peptide production (LC coupled to Ms/Ms), and liver TfR-2 and IL-6 expression (RT-qPCR) were determined. The different bread formulations increased Fe content between 14% and 83% relative to white bread. Only animals fed with WWB, AB and ChB increased haemoglobin concentrations significantly. Feeding the different bread formulations did not increase hepcidin levels, but down-regulated transferrin receptor 2 (TfR2) (apart from WWB) and IL-6 (apart from QB) expression levels. Only AB and ChB had a significant influence on Fe bioavailability at the investigated level of substitution. The potential contribution of these flours would not differ considerably from that of WWB.

The three isoforms of hepcidin in human serum and their processing determined by liquid chromatography-tandem mass spectrometry (LC-tandem MS).

Hepcidin, the iron regulatory hormone, has three isoforms; -20, -22 and -25. While hepcidin-25 has been studied extensively, the physiological significance of other isoforms remains poorly understood. Using a quantitative method based on liquid chromatography-tandem mass spectrometry (LC-tandem MS) developed by our group, we quantified hepcidin isoforms in human serum to elucidate their characteristics, and investigated the role of hepatocytes in isoform processing. Hepcidin isoforms in serum obtained from 40 healthy volunteers were quantified. Synthetic hepcidin peptides were added to healthy serum, and to HepG2 culture media, and hepcidin isoform concentrations determined. All three hepcidin isoforms were detected in human serum; however, hepcidin-25 concentrations were highest. The three hepcidin isoforms showed a strong positive correlation with each other and with serum ferritin. Additionally, while hepcidin-20 was strongly correlated with serum creatinine, the other isoforms were not. Hepcidin-20 and -25 levels were also increased in chronic kidney disease (CKD) serum. Hepcidin-22 rapidly degraded into hepcidin-20, whereas hepcidin-25 remained relatively stable. Finally, hepcidin-22 degradation into hepcidin-20 was accelerated in the presence of HepG2. This method has enabled us to reveal fundamental characteristics of the three hepcidin isoforms in serum and may be a powerful tool for quantifying hepcidin isoform expression and processing.

Driving Up the Dose: Implications for High-Dose Vitamin D Therapy

Driving Up the Dose: Implications for High-Dose Vitamin D Therapy

Iron dysregulation and anemias

Most iron in the body is utilized as a component of hemoglobin that delivers oxygen to the entire body. Under normal conditions, the iron balance is tightly regulated. However, iron dysregulation does occasionally occur; total iron content reductions cause iron deficiency anemia and overexpression of the iron regulatory peptide hepcidin disturbs iron utilization resulting in anemia of chronic disease. Conversely, the presence of anemia may ultimately lead to iron overload; for example, thalassemia, a common hereditary anemia worldwide, often requires transfusion, but long-term transfusions cause iron accumulation that leads to organ damage and other poor outcomes. On the other hand, there is a possibility that iron overload itself can cause anemia; iron chelation therapy for the post-transfusion iron overload observed in myelodysplastic syndrome or aplastic anemia improves dependency on transfusions in some cases. These observations reflect the extremely close relationship between anemias and iron metabolism.

Molecular and functional identification of three interleukin-17A/F (IL-17A/F) homologues in large yellow croaker (Larimichthys crocea)

The interleukin-17 (IL-17) cytokine family plays a central role in the coordination of inflammatory responses. In fish species, three genes that have a similar homology to both IL-17A and IL-17F were designated IL-17A/F1, 2, and 3. In this study, we identified three IL-17A/F homologues (LycIL-17A/F1, 2, and 3) from large yellow croaker (Larimichthys crocea). The deduced LycIL-17A/F1 and 3 had four cysteine residues conserved in teleost IL-17A/F1 and 3 homologues and shared a domain similar to the B chain of human IL-17F. The deduced LycIL-17A/F2 possessed the unique arrangement of six cysteine residues as teleost IL-17A/F2 (except Fugu IL-17A/F2) and higher vertebrate IL-17A and F, and shared a domain similar to the D/E chain of human IL-17A. Phylogenetic analysis showed that teleost IL-17A/F1 and 3 fall into a major clade, whereas IL-17A/F2 forms a separated clade and is clustered with IL-17N. Based on structural and phylogenetic analyses, we suggest that teleost IL-17A/Fs may be classified into two subgroups: one consisting of IL-17A/F1 and 3, and the other composed of IL-17A/F2. The three LycIL-17A/Fs were constitutively expressed in all tissues examined although at a different level. Following challenge with Aeromonas hydrophila, expression of these three LycIL-17A/Fs was rapidly increased in head kidney and gills. The in vivo assays showed that recombinant LycIL-17A/F1, 2, and 3 all were able to enhance the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α2), chemokines (CXCL8 and CXCL13), and antimicrobial peptide hepcidin in head kidney. Furthermore, LycIL-17A/Fs appeared to mediate pro-inflammatory responses via NF-κB signalling. These results therefore reveal similar functions between the two subgroup members，LycIL-17A/F1 and 3 and LycIL-17A/F2, in promoting inflammation and host defences.

Hepcidin as a Major Component of Renal Antibacterial Defenses against Uropathogenic Escherichia coli.

The iron-regulatory peptide hepcidin exhibits antimicrobial activity. Having previously shown hepcidin expression in the kidney, we addressed its role in urinary tract infection (UTI), which remains largely unknown. Experimental UTI was induced in wild-type (WT) and hepcidin-knockout (Hepc-/-) mice using the uropathogenic Escherichia coli CFT073 strain. Compared with infected WT mice, infected Hepc-/- mice showed a dramatic increase in renal bacterial load. Moreover, bacterial invasion was significantly dampened by the pretreatment of WT mice with hepcidin. Infected Hepc-/- mice exhibited decreased iron accumulation in the renal medulla and significant attenuation of the renal inflammatory response. Notably, we demonstrated in vitro bacteriostatic activity of hepcidin against CFT073. Furthermore, CFT073 repressed renal hepcidin, both in vivo and in cultured renal cells, and reduced phosphorylation of SMAD kinase in vivo, suggesting a bacterial strategy to escape the antimicrobial activities of hepcidin. In conclusion, we provide new mechanisms by which hepcidin contributes to renal host defense and suggest that targeting hepcidin offers a strategy to prevent bacterial invasion.

Differential effects of basolateral and apical iron supply on iron transport in Caco-2 cells.

Iron homeostasis in the human body is maintained primarily through regulation of iron absorption in the duodenum. The liver peptide hepcidin plays a central role in this regulation. Additionally, expression and functional control of certain components of the cellular iron transport machinery can be influenced directly by the iron status of enterocytes. The significance of this modulation, relative to the effects of hepcidin, and the comparative effects of iron obtained directly from the diet and/or via the bloodstream are not clear. The studies described here were performed using Caco-2 cell monolayers as a model of intestinal epithelium, to compare the effects of iron supplied in physiologically relevant forms to either the apical or basolateral surfaces of the cells. Both sources of iron provoked increased cellular ferritin content, indicating iron uptake from both sides of the cells. Supply of basolateral transferrin-bound iron did not affect subsequent iron transport across the apical surface, but reduced iron transport across the basolateral membrane. In contrast, the apical iron supply led to subsequent reduction in iron transport across the apical cell membrane without altering iron export across the basolateral membrane. The apical and basolateral iron supplies also elicited distinct effects on the expression and subcellular distribution of iron transporters. These data suggest that, in addition to the effects of cellular iron status on the expression of iron transporter genes, different modes and direction of iron supply to enterocytes can elicit distinct functional effects on iron transport.