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Macrophages are at the center of innate pathogen control and iron recycling. Divalent metal transporter 1 (DMT1) is essential for the uptake of non-transferrin-bound iron (NTBI) into macrophages and for the transfer of transferrin-bound iron from the endosome to the cytoplasm. As the control of cellular iron trafficking is central for the control of infection with siderophilic pathogens such as Salmonella Typhimurium, a Gram-negative bacterium residing within the phagosome of macrophages, we examined the potential role of DMT1 for infection control. Bone marrow derived macrophages lacking DMT1 (DMT1fl/fl LysMCre ) present with reduced NTBI uptake and reduced levels of the iron storage protein ferritin, the iron exporter ferroportin and, surprisingly, of the iron uptake protein transferrin receptor. Further, DMT1-deficient macrophages have an impaired control of (+) Typhimurium infection, paralleled by reduced levels of the peptide lipocalin-2 (LCN2). LCN2 exerts anti-bacterial activity upon binding of microbial siderophores but also facilitates systemic and cellular hypoferremia. Remarkably, nifedipine, a pharmacological DMT1 activator, stimulates LCN2 expression in RAW264.7 macrophages, confirming its DMT1-dependent regulation. In addition, the absence of DMT1 increases the availability of iron for Salmonella Typhimurium infection, paralleled by reduced levels of the peptide lipocalin-2 (LCN2). LCN2 exerts anti-bacterial activity upon binding of microbial siderophores but also facilitates systemic and cellular hypoferremia. Remarkably, nifedipine, a pharmacological DMT1 activator, stimulates LCN2 expression in RAW264.7 macrophages, confirming its DMT1-dependent regulation. In addition, the absence of DMT1 increases the availability of iron for Salmonella infection. This study highlights the importance of DMT1 in nutritional immunity and the significance of iron delivery for the control of infection with siderophilic bacteria.Salmonella infection. This study highlights the importance of DMT1 in nutritional immunity and the significance of iron delivery for the control of infection with siderophilic bacteria.

Iron is an essential nutrient for mammals as well as for pathogens. Inflammation-driven changes in systemic and cellular iron homeostasis are central for host-mediated antimicrobial strategies. Here, we studied the role of the iron storage protein ferritin H (FTH) for the control of infections with the intracellular pathogen Salmonella enterica serovar Typhimurium by macrophages. Mice lacking FTH in the myeloid lineage (LysM-Cre+/+Fthfl/fl mice) displayed impaired iron storage capacities in the tissue leukocyte compartment, increased levels of labile iron in macrophages, and an accelerated macrophage-mediated iron turnover. While under steady-state conditions, LysM-Cre+/+Fth+/+ and LysM-Cre+/+Fthfl/fl animals showed comparable susceptibility to Salmonella infection, i.v. iron supplementation drastically shortened survival of LysM-Cre+/+Fthfl/fl mice. Mechanistically, these animals displayed increased bacterial burden, which contributed to uncontrolled triggering of NF-κB and inflammasome signaling and development of cytokine storm and death. Importantly, pharmacologic inhibition of the inflammasome and IL-1β pathways reduced cytokine levels and mortality and partly restored infection control in iron-treated ferritin-deficient mice. These findings uncover incompletely characterized roles of ferritin and cellular iron turnover in myeloid cells in controlling bacterial spread and for modulating NF-κB and inflammasome-mediated cytokine activation, which may be of vital importance in iron-overloaded individuals suffering from severe infections and sepsis.

Iron plays an important role in host-pathogen interactions, in being an essential element for both pathogen and host metabolism, but also by impacting immune cell differentiation and anti-microbial effector pathways. Iron has been implicated to affect the differentiation of T lymphocytes during inflammation, however, so far the underlying mechanism remained elusive. In order to study the role of iron in T cell differentiation we here investigated how dietary iron supplementation affects T cell function and outcome in a model of chronic infection with the intracellular bacterium Salmonella enterica serovar typhimurium ( S. Typhimurium ). Iron loading prior to infection fostered bacterial burden and, unexpectedly, reduced differentiation of CD4 + T helper cells type 1 (Th1) and expression of interferon-gamma (IFNγ), a key cytokine to control infections with intracellular pathogens. This effect could be traced back to iron-mediated induction of the negative immune checkpoint regulator T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), expressed on the surface of this T cell subset. In vitro experiments demonstrated that iron supplementation specifically upregulated mRNA and protein expression of TIM-3 in naïve Th cells in a dose-depdendent manner and hindered priming of those T cells towards Th1 differentiation. Importantly, administration of TIM-3 blocking antibodies to iron-loaded mice infected with S. Typhimurium virtually restored Th1 cell differentiation and significantly improved bacterial control. Our data uncover a novel mechanism by which iron modulates CD4 + cell differentiation and functionality and hence impacts infection control with intracellular pathogens. Specifically, iron inhibits the differentiation of naive CD4 + T cells to protective IFNγ producing Th1 lymphocytes via stimulation of TIM-3 expression. Finally, TIM-3 may serve as a novel drug target for the treatment of chronic infections with intracellular pathogens, specifically in iron loading diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pfeifhofer-Obermair, Tymoszuk, Nairz, Schroll, Klais, Demetz, Engl, Brigo and Weiss.)

We have recently shown that the catecholamine dopamine regulates cellular iron homeostasis in macrophages. As iron is an essential nutrient for microbes, and intracellular iron availability affects the growth of intracellular bacteria, we studied whether dopamine administration impacts the course of Salmonella infections. Dopamine was found to promote the growth of Salmonella both in culture and within bone marrow-derived macrophages, which was dependent on increased bacterial iron acquisition. Dopamine administration to mice infected with Salmonella enterica serovar Typhimurium resulted in significantly increased bacterial burdens in liver and spleen, as well as reduced survival. The promotion of bacterial growth by dopamine was independent of the siderophore-binding host peptide lipocalin-2. Rather, dopamine enhancement of iron uptake requires both the histidine sensor kinase QseC and bacterial iron transporters, in particular SitABCD, and may also involve the increased expression of bacterial iron uptake genes. Deletion or pharmacological blockade of QseC reduced but did not abolish the growth-promoting effects of dopamine. Dopamine also modulated systemic iron homeostasis by increasing hepcidin expression and depleting macrophages of the iron exporter ferroportin, which enhanced intracellular bacterial growth. Salmonella lacking all central iron uptake pathways failed to benefit from dopamine treatment. These observations are potentially relevant to critically ill patients, in whom the pharmacological administration of catecholamines to improve circulatory performance may exacerbate the course of infection with siderophilic bacteria. IMPORTANCE Here we show that dopamine increases bacterial iron incorporation and promotes Salmonella Typhimurium growth both in vitro and in vivo These observations suggest the potential hazards of pharmacological catecholamine administration in patients with bacterial sepsis but also suggest that the inhibition of bacterial iron acquisition might provide a useful approach to antimicrobial therapy., (Copyright © 2019 Dichtl et al.)

Iron is an essential co-factor for several metabolic processes, including mitochondrial respiration, and mitochondria are the major sites of iron-utilization. Cellular iron homeostasis must be tightly regulated, as intracellular iron deficiency can lead to insufficient energy production, whereas iron overload triggers ROS (reactive oxygen species) formation via the Fenton reaction. So far little is known on how iron imbalances affect mitochondrial function in vivo and the impact of the genotype on that, we studied the effects of dietary iron loading on mitochondrial respiratory capacity in liver by comparing two genetically divergent mouse strains, namely C57BL/6N and FVB mice. Both mouse strains differed in their basal iron levels and their metabolic responses to iron loading as determined by expression of iron trafficking proteins (ferritin was increased in livers of animals receiving high iron diet) as well as tissue iron content (2-fold increase, FVB p = 0.0013; C57BL/6N p = 0.0022). Dietary iron exposure caused a significant impairment of mitochondrial oxidative phosphorylation, especially regarding OXPHOS capacity (FVB p = 0.0006; C57BL/6N p = 0.0087) and S-ETS capacity (FVB p = 0.0281; C57BL/6N p = 0.0159). These effects were more pronounced in C57BL/6N than in FVB mice and were paralleled by an iron mediated induction of oxidative stress in mitochondria. The increased susceptibility of C57BL6/N mice to iron loading may be due to reduced expression of anti-oxidant defense mechanisms and altered iron trafficking upon dietary challenge pointing to a role of genetic modifiers for cellular and mitochondrial iron trafficking. Finally, iron-mediated induction of mitochondrial oxidative stress and reduction of oxidative phosphorylation may underlie fatigue in subjects with iron loading diseases.

Lipocalin-2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron-laden bacterial siderophores, chemo-attracts neutrophils and has immunomodulatory and apoptosis-regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella-infected macrophages, which reduces cellular iron content and enhances the generation of pro-inflammatory cytokines. Lcn2 represses IL-10 production while augmenting Nos2, TNF-α, and IL-6 expression. Lcn2(-/-) macrophages have elevated IL-10 levels as a consequence of increased iron content. The crucial role of Lcn-2/IL-10 interactions was further demonstrated by the greater ability of Lcn2(-/-) IL-10(-/-) macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2(-/-) counterparts. Overexpression of the iron exporter ferroportin-1 in Lcn2(-/-) macrophages represses IL-10 and restores TNF-α and IL-6 production to the levels found in wild-type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL-10 production, and that both functions are linked to its ability to shuttle iron from macrophages., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Due to its multiple roles for the proliferation and pathogenicity of many microbes on the one hand and via modulation of immune effector functions on the other hand the control over iron homeostasis is thought to play a decisive role in the course of infections. Diversion of cellular iron traffic is considered as an important defense mechanism of macrophages to reduce metal availability for intracellular bacteria residing in the phagosome. However, evidence is lacking whether such alterations of iron homeostasis also become evident upon infection with bacteria gaining access to the cytosol like Listeria monocytogenes. Here we show that infection of macrophages with L. monocytogenes triggers the expression of the major cellular iron exporter ferroportin1 and induces cellular iron egress. As the growth of Listeria within macrophages is promoted by iron, stimulation of ferroportin1 functionality limits the availability of the metal for Listeria residing in the cytoplasm, whereas ferroportin1 degradation upon hepcidin treatment increases intracellular bacterial growth. In parallel to an increase of ferroportin1 expression, infected macrophages induce anti-microbial immune effector mechanisms such as TNFα formation or NO expression which are aggravated upon iron deficiency. These adaptive changes of iron homeostasis and immune response pathways are only found in macrophages infected with Listeria which express listeriolysin O and are therefore able to escape from the phagosome to the cytoplasm. Listeriolysin O deficient Listeria which are restricted to the phagosome are even killed by excess iron which may be based on "iron intoxification" via macrophage radical formation, because iron supplementation in that setting is paralleled by increased ROS formation. Our results indicate that ferroportin1 mediated iron export is a nutritional immune effector pathway to control infection with Listeria residing in the cytoplasm, whereas a different strategy is observed in mutant Listeria restricted to the phagosome, where iron remains in the macrophages likewise contributing to ROS mediated intoxification of bacteria.

Iron homeostasis and macrophage biology are closely interconnected. On the one hand, iron exerts multiple effects on macrophage polarization and functionality. On the other hand, macrophages are central for mammalian iron homeostasis. The phagocytosis of senescent erythrocytes and their degradation by macrophages enable efficient recycling of iron and the maintenance of systemic iron balance. Macrophages express multiple molecules and proteins for the acquisition and utilization of iron and many of these pathways are affected by inflammatory signals. Of note, iron availability within macrophages has significant effects on immune effector functions and metabolic pathways within these cells. This review summarizes the physiological and pathophysiological aspects of macrophage iron metabolism and highlights its relevant consequences on immune function and in common diseases such as infection and atherosclerosis., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Objective: Hypoxia affects body iron homeostasis; however, the underlying mechanisms are incompletely understood., Design: Using a standardised hypoxia chamber, 23 healthy volunteers were subjected to hypoxic conditions, equivalent to an altitude of 5600 m, for 6 h. Subsequent experiments were performed in C57BL/6 mice, CREB-H knockout mice, primary hepatocytes and HepG2 cells., Results: Exposure of subjects to hypoxia resulted in a significant decrease of serum levels of the master regulator of iron homeostasis hepcidin and elevated concentrations of platelet derived growth factor (PDGF)-BB. Using correlation analysis, we identified PDGF-BB to be associated with hypoxia mediated hepcidin repression in humans. We then exposed mice to hypoxia using a standardised chamber and observed downregulation of hepatic hepcidin mRNA expression that was paralleled by elevated serum PDGF-BB protein concentrations and higher serum iron levels as compared with mice housed under normoxic conditions. PDGF-BB treatment in vitro and in vivo resulted in suppression of both steady state and BMP6 inducible hepcidin expression. Mechanistically, PDGF-BB inhibits hepcidin transcription by downregulating the protein expression of the transcription factors CREB and CREB-H, and pharmacological blockade or genetic ablation of these pathways abrogated the effects of PDGF-BB toward hepcidin expression., Conclusions: Hypoxia decreases hepatic hepcidin expression by a novel regulatory pathway exerted via PDGF-BB, leading to increased availability of circulating iron that can be used for erythropoiesis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Both, mammalian cells and microbes have an essential need for iron, which is required for many metabolic processes and for microbial pathogenicity. In addition, cross-regulatory interactions between iron homeostasis and immune function are evident. Cytokines and the acute phase protein hepcidin affect iron homeostasis leading to the retention of the metal within macrophages and hypoferremia. This is considered to result from a defense mechanism of the body to limit the availability of iron for extracellular pathogens while on the other hand the reduction of circulating iron results in the development of anemia of inflammation. Opposite, iron and the erythropoiesis inducing hormone erythropoietin affect innate immune responses by influencing interferon-gamma (IFN-y) mediated (iron) or NF-kB inducible (erythropoietin) immune effector pathways in macrophages. Thus, macrophages loaded with iron lose their ability to kill intracellular pathogens via IFN-y mediated effector pathways such as nitric oxide (NO) formation. Accordingly, macrophages invaded by the intracellular bacterium Salmonella enterica serovar Typhimurium increase the expression of the iron export protein ferroportin thereby reducing the availability of iron for intramacrophage bacteria while on the other side strengthening anti-microbial macrophage effector pathways via increased formation of NO orTNF-a. In addition, certain innate resistance genes such as natural resistance associated macrophage protein function (Nramp1) or lipocalin-2 exert part of their antimicrobial activity by controlling host and/or microbial iron homeostasis. Consequently, pharmacological or dietary modification of cellular iron trafficking enhances host resistance to intracellular pathogens but may increase susceptibility to microbes in the extracellular compartment and vice versa. Thus, the control over iron homeostasis is a central battlefield in host-pathogen interplay influencing the course of an infectious disease in favor of either the mammalian host or the pathogenic invader. [ABSTRACT FROM AUTHOR]

Iron is an essential co-factor for several metabolic processes, including the Krebs cycle and mitochondrial oxidative phosphorylation. Therefore, maintaining an appropriate iron balance is essential to ensure sufficient energy production and to avoid excessive reactive oxygen species formation. Iron overload impairs mitochondrial fitness; however, little is known about the associated metabolic changes. Here we aimed to characterize the metabolic signature triggered by dietary iron overload over time in a mouse model, where mice received either a standard or a high-iron diet. Metabolic profiling was assessed in blood, plasma and liver tissue. Peripheral blood was collected by means of volumetric absorptive microsampling (VAMS). Extracted blood and tissue metabolites were analyzed by liquid chromatography combined to high resolution mass spectrometry. Upon dietary iron loading we found increased glucose, aspartic acid and 2-/3-hydroxybutyric acid levels but low lactate and malate levels in peripheral blood and plasma, pointing to a re-programming of glucose homeostasis and the Krebs cycle. Further, iron loading resulted in the stimulation of the urea cycle in the liver. In addition, oxidative stress was enhanced in circulation and coincided with increased liver glutathione and systemic cysteine synthesis. Overall, iron supplementation affected several central metabolic circuits over time. Hence, in vivo investigation of metabolic signatures represents a novel and useful tool for getting deeper insights into iron-dependent regulatory circuits and for monitoring of patients with primary and secondary iron overload, and those ones receiving iron supplementation therapy. [ABSTRACT FROM AUTHOR]