Intracellular Iron Sequestration Research Articles

217 Tofacitinib suppresses IL-10/IL-10R signaling and modulates host defense responses in human macrophages

JAK inhibitors are increasingly used in dermatology. Despite broad inhibitory effects on cytokine signaling cascades, they only modestly increase the risk for infectious diseases. To address molecular mechanisms underlying this unexpected clinical observation, we investigated how tofacintib, a first-in-class JAK inhibitor, regulates host defense responses in TLR4-activated human macrophages. Specifically, we asked if tofacitinib inhibits anti-inflammatory IL-10 signaling, thereby counteracting downregulation of inflammatory, host-protective pathways. We found that tofacitinib blocked macrophage responses to IL-10 at the level of STAT3 phosphorylation. Furthermore, TLR4-induced, auto-/paracrine IL-10/IL-10R activation promoted expression of hepcidin, the master regulator of iron metabolism, resulting in intracellular iron sequestration. In contrast, auto-/paracrine IL-10/IL-10R activation repressed expression of cathelicidin antimicrobial peptide, as well as antigen-presenting molecules, thus together, inducing a pathogen-favoring environment. While tofacitinib further repressed cathelicidin, it prevented induction of intracellular hepcidin, and restored expression of antigen-presentation molecules in TLR4-activated macrophages. Our study supports the concept that induction of IL-10/IL-10R signaling drives a complex immune evasion strategy of intracellular microbes. Moreover, we conclude that tofacitinib has diverging effects on macrophage host response pathways, and we identify the TLR4-IL-10-STAT3-hepcidin axis as a potential therapeutic target to counteract immune evasion.

Tofacitinib Suppresses IL-10/IL-10R Signaling and Modulates Host Defense Responses in Human Macrophages

Jak inhibitors are increasingly used in dermatology. Despite broad inhibitory effects on cytokine signaling cascades, they only modestly increase the risk for infectious diseases. To address the molecular mechanisms underlying this unexpected clinical observation, we investigated how tofacintib (tofa), a first-in-class Jak inhibitor, regulates host defense responses in toll-like receptor 4-activated human macrophages. Specifically, we asked whether tofa inhibits anti-inflammatory IL-10 signaling, thereby counteracting the downregulation of inflammatory, host-protective pathways. We found that tofa blocked macrophage responses to IL-10 at the level of signal transducer and activator of transcription 3 phosphorylation. Furthermore, toll-like receptor 4-induced, autocrine/paracrine IL-10/IL-10R activation promoted the expression of hepcidin, the master regulator of iron metabolism, resulting in intracellular iron sequestration. In contrast, autocrine/paracrine IL-10/IL-10R activation repressed the expression of cathelicidin antimicrobial peptide as well as antigen-presenting molecules, thus together, inducing a pathogen-favoring environment. Although tofa further repressed cathelicidin, it prevented the induction of intracellular HAMP and restored the expression of antigen-presentation molecules in toll-like receptor 4-activated macrophages. Our study supports the concept that induction of IL-10/IL-10R signaling drives a complex immune evasion strategy of intracellular microbes. Moreover, we conclude that tofa has diverging effects on macrophage host response pathways, and we identify the toll-like receptor 4-IL-10-signal transducer and activator of transcription 3-HAMP axis as a potential therapeutic target to counteract immune evasion.

Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication.

Salmonellosis and listeriosis together accounted for more than one third of foodborne illnesses in the United States and almost half the hospitalizations for gastrointestinal diseases in 2018 while tuberculosis afflicted over 10 million people worldwide causing almost 2 million deaths. Regardless of the intrinsic virulence differences among Listeria monocytogenes, Salmonella enterica and Mycobacterium tuberculosis, these intracellular pathogens share the ability to survive and persist inside the macrophage and other cells and thrive in iron rich environments. Interferon-gamma (IFN-γ) is a central cytokine in host defense against intracellular pathogens and has been shown to promote iron export in macrophages. We hypothesize that IFN-γ decreases iron availability to intracellular pathogens consequently limiting replication in these cells. In this study, we show that IFN-γ regulates the expression of iron-related proteins hepcidin, ferroportin, and ferritin to induce iron export from macrophages. Listeria monocytogenes, S. enterica, and M. tuberculosis infections significantly induce iron sequestration in human macrophages. In contrast, IFN-γ significantly reduces hepcidin secretion in S. enterica and M. tuberculosis infected macrophages. Similarly, IFN-γ-activated macrophages express higher ferroportin levels than untreated controls even after infection with L. monocytogenes bacilli; bacterial infection greatly down-regulates ferroportin expression. Collectively, IFN-γ significantly inhibits pathogen-associated intracellular iron sequestration in macrophages and consequently retards the growth of intracellular bacterial pathogens by decreasing iron availability.

Time-dependent hemeoxygenase-1, lipocalin-2 and ferritin induction after non-contusion traumatic brain injury

Traumatic brain injury (TBI) often presents with focal contusion and parenchymal bleeds, activating heme oxygenase (HO) to degrade released hemoglobin. Here we show that diffuse, midline fluid percussion injury causes time-dependent induction of HO-1 and iron binding proteins within both hemorrhagic neocortex and non-hemorrhagic hippocampus. Rats subjected to midline fluid percussion injury (FPI) survived 1-15d postinjury and tissue was collected for Western blot and immunohistochemical assays. HO-1 was elevated 1d after FPI, peaked at 3d, and returned to control baseline 7-15d. Iron management proteins lipocalin 2 (LCN2) and ferritin (FTL) exhibited distinct postinjury time courses, where peak LCN2 response preceded, and FTL followed that of HO-1. LCN2 elevation supported not only its role in iron transport, but also mediation of matrix metalloproteinase 9 (MMP9) activity. Upregulation of FTL for intracellular iron sequestration was delayed relative to both HO-1 and LCN2 induction. In the neocortex IBA-1+ microglia around the injury core expressed HO-1, but astrocytes co-localized with HO-1 in perilesional parenchyma. Non-hemorrhagic dentate gyrus showed predominant HO-1 labeling in hilar microglia and in molecular layer astrocytes. At 1d postinjury, LCN2 and HO-1 co-localized in a subpopulation of reactive glia within both brain regions. Notably, FTL was distributed within cells around injured vessels, damaged subcortical white matter, and along vessels of the hippocampal fissure. Together these results confirm that even the moderate, non-contusional insult of diffuse midline FPI can significantly activate postinjury HO-1 heme processing pathways and iron management proteins. Moreover, this activation is time-dependent and occurs in the absence of overt hemorrhage.

Rusty Microglia: Trainers of Innate Immunity in Alzheimer's Disease.

Alzheimer's disease, the most common form of dementia, is marked by progressive cognitive and functional impairment believed to reflect synaptic and neuronal loss. Recent preclinical data suggests that lipopolysaccharide (LPS)-activated microglia may contribute to the elimination of viable neurons and synapses by promoting a neurotoxic astrocytic phenotype, defined as A1. The innate immune cells, including microglia and astrocytes, can either facilitate or inhibit neuroinflammation in response to peripherally applied inflammatory stimuli, such as LPS. Depending on previous antigen encounters, these cells can assume activated (trained) or silenced (tolerized) phenotypes, augmenting or lowering inflammation. Iron, reactive oxygen species (ROS), and LPS, the cell wall component of gram-negative bacteria, are microglial activators, but only the latter can trigger immune tolerization. In Alzheimer's disease, tolerization may be impaired as elevated LPS levels, reported in this condition, fail to lower neuroinflammation. Iron is closely linked to immunity as it plays a key role in immune cells proliferation and maturation, but it is also indispensable to pathogens and malignancies which compete for its capture. Danger signals, including LPS, induce intracellular iron sequestration in innate immune cells to withhold it from pathogens. However, excess cytosolic iron increases the risk of inflammasomes' activation, microglial training and neuroinflammation. Moreover, it was suggested that free iron can awaken the dormant central nervous system (CNS) LPS-shedding microbes, engendering prolonged neuroinflammation that may override immune tolerization, triggering autoimmunity. In this review, we focus on iron-related innate immune pathology in Alzheimer's disease and discuss potential immunotherapeutic agents for microglial de-escalation along with possible delivery vehicles for these compounds.

Role of the hepcidin-ferroportin axis in pathogen-mediated intracellular iron sequestration in human phagocytic cells

AbstractUpon infection, pathogen and host compete for the same iron pool, because this trace metal is a crucial micronutrient for all living cells. Iron dysregulation in the host is strongly associated with poor outcomes in several infectious diseases, including tuberculosis, AIDS, and malaria, and inefficient iron scavenging by pathogens severely affects their virulence. Hepcidin is the master regulator of iron homeostasis in vertebrates, responsible for diminishing iron export from macrophages during iron overload or infection. Hepcidin regulation in hepatocytes is well characterized and mostly dependent on interleukin-6 signaling during inflammation, although in myeloid cells, hepcidin induction and the mechanisms leading to intracellular iron regulation remain elusive. Here we show that activation of different Toll-like receptors (TLRs) by their respective ligands leads to increased iron sequestration in macrophages. By measuring the transcriptional levels of iron-related proteins (eg, hepcidin, ferroportin, and ferritin), we observed that TLR signaling can induce intracellular iron sequestration in macrophages through 2 independent but redundant mechanisms. Interestingly, TLR2 ligands or infection with Listeria monocytogenes lead to direct ferroportin transcriptional downregulation, whereas TLR4 ligands, such as lipopolysaccharide, induce hepcidin expression. Infection with Mycobacterium bovis Bacillus Calmette-Guerin promotes intracellular iron sequestration through both hepcidin upregulation and ferroportin downregulation. This is the first study in which TLR1-9–mediated iron homeostasis in human macrophages was evaluated, and the outcome of this study elucidates the mechanism of iron dysregulation in macrophages during infection.

Heparin inhibits intracellular Mycobacterium tuberculosis bacterial replication by reducing iron levels in human macrophages

Iron is a crucial micronutrient for both mammals and their associated pathogens, and extensive literature has shown that Mycobacterium tuberculosis (Mtb) bacilli inhibited from acquiring iron from the host are severely attenuated. In contrast, increased dietary iron concentrations or patients with hemochromatosis have long been associated with a more severe tuberculosis (TB) disease outcome. We have observed that upon macrophage infection, Mtb bacilli strongly promote intracellular iron sequestration, both through increased expression of hepcidin, a key mammalian iron regulatory protein, and downregulation of the iron exporter protein, ferroportin. Heparin is a highly sulfated glycosaminoglycan released by mast cells and basophils at sites of tissue injury. During Mtb infection, heparin alters intracellular trafficking in alveolar epithelial cells and decreases extrapulmonary dissemination but recently, heparin also has been reported to inhibit hepcidin expression in hepatocytes, decreasing intracellular iron availability. In this report, we demonstrate that heparin significantly reduces hepcidin expression in macrophages infected with Mtb bacilli. Heparin-treated macrophages have higher ferroportin expression compared to untreated macrophages, promoting iron export and decreasing iron availability to intracellular bacilli. Thus, here we describe a novel immunomodulatory effect and potential therapeutic role for heparin against mycobacterial infection in human macrophages.

Hepcidin, an Iron Regulatory Hormone of Innate Immunity, is Differentially Expressed in Premature Fetuses with Early-Onset Neonatal Sepsis.

Hepcidin, a mediator of innate immunity, binds the iron exporter ferroportin, leading to functional hypoferremia through intracellular iron sequestration. We explored hepcidin-ferroportin interactions in neonates clinically diagnosed with early-onset neonatal sepsis (EONS). Hepcidin and interleukin (IL)-6 were quantified by enzyme-linked immunosorbent assay (ELISA) in 92 paired cord blood-maternal blood samples in the following groups: "Yes" EONS (n = 41, gestational age [GA] 29 ± 1 weeks) and "No" EONS (n = 51, GA 26 ± 1 weeks). Placental hepcidin and ferroportin expression were evaluated by immunohistochemistry and real-time-polymerase chain reaction (RT-PCR). Liver hepcidin and ferroportin expression patterns were ascertained in autopsy specimens of neonates (n = 8) who died secondary to culture-proven sepsis. Cord blood hepcidin was significantly elevated (GA corrected, p = 0.018) and was positively correlated with IL-6 (r = 0.379, p = 0.001) in EONS. Hepcidin localized at syncytiotrophoblast and fetal vascular endothelium. Placental ferroportin, but not hepcidin mRNA correlated with cord blood hepcidin levels (r = 0.46, p = 0.039) and funisitis severity (r = 0.50, p = 0.018). Newborns who died from sepsis (n = 4) had higher hepatic hepcidin and iron sequestration, but lower ferroportin staining than those who died of nonsepsis causes (n = 4). Premature fetuses with EONS have elevated circulating hepcidin, likely related to lower placenta and liver ferroportin expression. Fetal hepcidin-ferroportin interaction appears to play a role in EONS pathophysiology independent of maternal response to intrauterine inflammation.

Contrasting roles of fungal siderophores in maintaining iron homeostasis in Epichloë festucae

The symbiosis between Epichloë festucae and its host perennial ryegrass (Lolium perenne) is a model system for mutualistic interactions in which the fungal endophyte grows between plant shoot cells and acquires host nutrients to survive. E. festucae synthesises the siderophore epichloënin A (EA) via SidN, a non-ribosomal peptide synthetase (NRPS). EA is involved in the acquisition of iron, an essential micronutrient, as part of the process of maintaining a stable symbiotic interaction. Here, we mutated a different NRPS gene sidC and showed that it is required for production of a second siderophore ferricrocin (FC). Furthermore mutations in sidA, encoding an l-ornithine N5-monooxygenase, abolished both EA and FC production. Axenic growth phenotypes of the siderophore mutants were altered relative to wild-type (WT) providing insights into the roles of E. festucae siderophores in iron trafficking and consequently in growth and morphogenesis. During iron-limitation, EA is the predominant siderophore and in addition to its role in iron acquisition it appears to play roles in intracellular iron sequestration and oxidative stress tolerance. FC in contrast is exclusively located intracellularly and is the dominant siderophore under conditions of iron sufficiency when it is likely to have roles in iron storage and iron transport. Intriguingly, EA acts to promote but may also moderate E. festucae growth (depending on the amount of available iron). We therefore hypothesise that coordinated cellular iron sequestration through FC and EA may be one of the mechanisms that E. festucae employs to manage and restrain its growth in response to iron fluxes and ultimately persist as a controlled symbiont.

Adamantyl- and other polycyclic cage-based conjugates of desferrioxamine B (DFOB) for treating iron-mediated toxicity in cell models of Parkinson’s disease

The death of dopaminergic neurons is a major pathological hallmark of Parkinson’s disease (PD). Elevated iron within the substantia nigra of the PD brain is thought to catalyze this neuronal death through hydroxyl radical-derived oxidative damage. Removing this excess iron presents a potential therapeutic strategy for PD. Seventeen derivatives of the non-toxic iron chelator desferrioxamine B (DFOB) were prepared by the conjugation of adamantyl- (1–4, 8–12), deconstructed adamantyl units (5–7), norborna(e)ne- (13–16) or bicyclo[2.2.2]octane-based (17) ancillary fragments to the terminal amine group. The range of experimental logP values of 1–17 (logP=0.15–2.82) was greater than water soluble DFOB (logP −2.29), with the increased hydrophobicity designed to improve cell membrane carriage to facilitate intracellular iron sequestration. The first activity screen showed compounds with methyl-substituted adamantyl (1–3), noradamantyl (5), or 1-pentylbicyclo[2.2.2]octane (17) ancillary groups significantly rescued iron-mediated oxidative stress in confluent PD-relevant SK-N-BE2-M17 neuroblastoma cells (M17 cells) exposed to 1,1ʹ-dimethyl-4,4ʹ-bipyridinium (paraquat, PQ) or H2O2. The second dose-dependence screen ranked 1–3 and 17 as the top candidates (EC50 ∼10µM) in the rescue of PQ-treated M17 cells. The ancillary fragments of 1–3 and 17 clustered in a region defined by a close-to-zero dipole moment, logP values of 2–2.8 and a surface area:volume ratio of 0.60–0.61. Results of iron leaching studies indicate that the compounds may be operating via mechanisms beyond solely removing intracellular iron. The DFOB conjugates with methyl-substituted adamantyl ancillary groups (1–3) were the top and most consistent performers in this class of compound designed for PD.

Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evolution.

Genetically encoding the synthesis of functional nanomaterials such as magnetic nanoparticles enables sensitive and non-invasive biological sensing and control. Via directed evolution of the natural iron-sequestering ferritin protein, we discovered key mutations that lead to significantly enhanced cellular magnetism, resulting in increased physical attraction of ferritin-expressing cells to magnets and increased contrast for cellular magnetic resonance imaging (MRI). The magnetic mutants further demonstrate increased iron biomineralization measured by a novel fluorescent genetic sensor for intracellular free iron. In addition, we engineered Escherichia coli cells with multiple genomic knockouts to increase cellular accumulation of various metals. Lastly to explore further protein candidates for biomagnetism, we characterized members of the DUF892 family using the iron sensor and magnetic columns, confirming their intracellular iron sequestration that results in increased cellular magnetization.

Alterations in the iron homeostasis network: A driving force for macrophage-mediated hepatitis C virus persistency

ABSTRACTMechanisms that favor Hepatitis C virus (HCV) persistence over clearance are unclear, but involve defective innate immunity. Chronic infection is characterized by hepatic iron overload, hyperferraemia and hyperferittinaemia. Hepcidin modulates iron egress via ferroportin and its storage in ferritin. Chronic HCV patients have decreased hepcidin, while HCV replication is modified by HAMP silencing. We aimed to investigate interactions between HCV and hepcidin, during acute and chronic disease, and putative alterations in cellular iron homeostasis that enhance HCV propagation and promote viral persistence. Thus, we used HCV JFH-1-infected co-cultures of Huh7.5 hepatoma and THP-1 macrophage cells, HCV patients’ sera and Huh7 hepcidin-expressing cells transfected with HCV replicons. Hepcidin levels were elevated in acutely infected patients, but correlated with viral load in chronic patients. HAMP expression was up-regulated early in HCV infection in vitro, with corresponding changes in ferritin and FPN. Hepcidin overexpression enhanced both viral translation and replication. In HCV-infected co-cultures, we observed increased hepcidin, reduced hepatoma ferritin and a concurrent rise in macrophaghic ferritin over time. Altered iron levels complemented amplified replication in hepatoma cells and one replication round in macrophages. Iron-loading of macrophages led to enhancement of hepatic HCV replication through reversed ferritin “flow.” Viral transmissibility from infected macrophages to naïve hepatoma cells was induced by iron. We propose that HCV control over iron occurs both by intracellular iron sequestration, through hepcidin, and intercellular iron mobilisation via ferritin, as means toward enhanced replication. Persistence could be achieved through HCV-induced changes in macrophagic iron that enhances viral replication in these cells.

591: Hepcidin, an iron regulatory hormone of innate immunity, is differentially expressed in premature fetuses with early onset neonatal sepsis (EONS)

Hepcidin impacts iron metabolism by binding ferroportin. This process leads to ferroportin degradation, intracellular iron sequestration and reduction of circulating iron. At the expense of reduced iron bioavailability to vital organs, this functional hypoferremia is a key regulatory process in fighting infection. We explored the hepcidin-ferroportin interaction in neonates with EONS.

β-Carotene can reverse dysregulation of iron protein in an in vitro model of inflammation.

Anemia of chronic disease is frequently seen in chronic inflammatory conditions. Its hallmark is disrupted iron homeostasis, with increased uptake and retention of iron in cells of the reticuloendothelial system. Using the Caco-2 cell line as an in vitro model for iron absorption, local intestinal iron-related protein dynamics were evaluated during interleukin (IL)1β/iron-induced inflammation, confirmed by IL8 release, and following β-carotene and vitamin A supplementation. Time- and dose-dependent iron administration to the cells was then studied. The effects on heavy and light ferritin, ferroportin, transferrin receptor and intracellular iron levels were compared in inflamed Caco-2 cells with and without application of the anti-inflammatory agents β-carotene and vitamin A. IL1β treatment led to IL8 release, a surge in both ferritins' expressions and suppression of ferroportin and transferrin receptor expression. β-Carotene significantly reduced IL8 (1,306.2-253.75 pg/ml), decreased light and heavy ferritin by 77.8 and 45.8%, respectively, and increased ferroportin by 59.9% (P < 0.05). Increasing iron concentrations and incubation periods resulted in increased IL8 release. A strong correlation was found between the levels of IL8 and the ferritins. Intracellular iron sequestration was induced by IL1β and iron and alleviated by β-carotene. β-Carotene normalized the main iron-related proteins' levels, reduced IL8 production, and released intracellular trapped iron. These results highlight local mucosal control of iron regulation and suggest that by applying anti-inflammatory compounds, less iron is locked in inflamed intestinal epithelial cells, leading to its increased bioavailability. This suggests a possible approach to combating anemia associated with chronic inflammatory conditions.

Mycobacterium avium Infection Induces H-Ferritin Expression in Mouse Primary Macrophages by Activating Toll-Like Receptor 2

Important for both host and pathogen survivals, iron is a key factor in determining the outcome of an infectious process. Iron with-holding, including sequestration inside tissue macrophages, is considered an important strategy to fight infection. However, for intra-macrophagic pathogens, such as Mycobacterium avium, host defence may depend on intracellular iron sequestration mechanisms. Ferritin, the major intracellular iron storage protein, plays a critical role in this process. In the current study, we studied ferritin expression in mouse bone marrow-derived macrophages upon infection with M. avium. We found that H-ferritin is selectively increased in infected macrophages, through an up-regulation of gene transcription. This increase was mediated by the engagement of Toll like receptor-2, and was independent of TNF-alpha or nitric oxide production. The formation of H-rich ferritin proteins and the consequent iron sequestration may be an important part of the panoply of antimicrobial mechanisms of macrophages.

Tumor Necrosis Factor-α–Induced Iron Sequestration and Oxidative Stress in Human Endothelial Cells

Tumor necrosis factor (TNF)-alpha-induced endothelial injury, which is associated with atherosclerosis, is mediated by intracellular reactive oxygen species. Iron is essential for the amplification of oxidative stress. We tested whether TNF-alpha accelerated iron accumulation in vascular endothelium, favoring synthesis of hydroxyl radical. Diverse iron transporters, including iron import proteins (transferrin receptor [TfR] and divalent metal transporter 1 [DMT1]) and an iron export protein (ferroportin 1 [FP1]) coexist in human umbilical endothelial cells (HUVECs). TNF-alpha caused upregulation of TfR and DMT1 and downregulation of FP1, which were demonstrated in mRNA as well as protein levels. These changes in iron transporters were accompanied by accumulation of iron that was both transferrin-dependent and transferrin-independent. Modifications of these mRNAs were regulated post-transcriptionally, and were coordinated with activation of binding activity of iron regulatory protein 1 to the iron responsive element on transporter mRNAs. Using a salicylate trap method, we observed that only simultaneous exposure of endothelial cells to iron and TNF-alpha accelerated hydroxyl radical production. TNF-alpha could cause intracellular iron sequestration, which may participate importantly in the pathophysiology of atherosclerosis and cardiovascular disease.

Transferrin ensures survival of ovarian carcinoma cells when apoptosis is induced by TNFalpha, FasL, TRAIL, or Myc.

The activation of Myc induces apoptosis of human ovarian adenocarcinoma N.1 cells when serum factors are limited. However, the downstream mechanism that is triggered by Myc is unknown. Myc-activation and treatment with the proapoptotic ligands TNFalpha, FasL, and TRAIL induced H-ferritin expression under serum-deprived conditions. H-ferritin chelates intracellular iron and also intracellular iron sequestration by deferoxamine-induced apoptosis of N.1 cells. Supplementation of serum-free medium with holo-transferrin blocked apoptosis of N.1 cells that was induced by Myc-activation or by treatment with TNFalpha, FasL, and TRAIL, whereas apotransferrin did not prevent apoptosis. This suggests that intracellular iron depletion was a trigger for apoptosis and that transferrin-bound iron rescued N.1 cells. Furthermore, apoptosis of primary human ovarian carcinoma cells, which was induced by TNFalpha, FasL, and TRAIL, was also inhibited by holo-transferrin. The data suggest that Myc-activation, FasL, TNFalpha, and TRAIL disturbed cellular iron homeostasis, which triggered apoptosis of ovarian carcinoma cells and that transferrin iron ensured survival by re-establishing this homeostasis.