Genetic Determined Iron Starvation Signature in Friedreich's Ataxia.

BACKGROUND: Early studies in cellular models suggested an iron accumulation in Friedreich's ataxia (FA), yet findings from patients are lacking. OBJECTIVES: The objective is to characterize systemic iron metabolism, body iron storages, and intracellular iron regulation in FA patients. METHODS: In FA patients and matched healthy controls, we assessed serum iron parameters, regulatory hormones as well as the expression of regulatory proteins and iron distribution in peripheral blood mononuclear cells (PBMCs). We applied magnetic resonance imaging with R2*-relaxometry to quantify iron storages in the liver, spleen, and pancreas. Across all evaluations, we assessed the influence of the genetic severity as expressed by the length of the shorter GAA-expansion (GAA1). RESULTS: We recruited 40 FA patients (19 women). Compared to controls, FA patients displayed lower serum iron and transferrin saturation. Serum ferritin, hepcidin, mean corpuscular hemoglobin and mean corpuscular volume in FA inversely correlated with the GAA1-repeat length, indicating iron deficiency and restricted availability for erythropoiesis with increasing genetic severity. R2*-relaxometry revealed a reduction of splenic and hepatic iron stores in FA. Liver and spleen R2* values inversely correlated with the GAA1-repeat length. FA PBMCs displayed downregulation of ferritin and upregulation of transferrin receptor and divalent metal transporter-1 mRNA, particularly in patients with >500 GAA1-repeats. In FA PBMCs, intracellular iron was not increased, but shifted toward mitochondria. CONCLUSIONS: We provide evidence for a previously unrecognized iron starvation signature at systemic and cellular levels in FA patients, which is related to the underlying genetic severity. These findings challenge the use of systemic iron lowering therapies in FA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Dopamine­iron homeostasis interaction rescues mitochondrial fitness in Parkinson's disease.

Imbalances of iron and dopamine metabolism along with mitochondrial dysfunction have been linked to the pathogenesis of Parkinson's disease (PD). We have previously suggested a direct link between iron homeostasis and dopamine metabolism, as dopamine can increase cellular uptake of iron into macrophages thereby promoting oxidative stress responses. In this study, we investigated the interplay between iron, dopamine, and mitochondrial activity in neuroblastoma SH-SY5Y cells and human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons differentiated from a healthy control and a PD patient with a mutation in the α-synuclein (SNCA) gene. In SH-SY5Y cells, dopamine treatment resulted in increased expression of the transmembrane iron transporters transferrin receptor 1 (TFR1), ferroportin (FPN), and mitoferrin2 (MFRN2) and intracellular iron accumulation, suggesting that dopamine may promote iron uptake. Furthermore, dopamine supplementation led to reduced mitochondrial fitness including decreased mitochondrial respiration, increased cytochrome c control efficiency, reduced mtDNA copy number and citrate synthase activity, increased oxidative stress and impaired aconitase activity. In dopaminergic neurons derived from a healthy control individual, dopamine showed comparable effects as observed in SH-SY5Y cells. The hiPSC-derived PD neurons harboring an endogenous SNCA mutation demonstrated altered mitochondrial iron homeostasis, reduced mitochondrial capacity along with increased oxidative stress and alterations of tricarboxylic acid cycle linked metabolic pathways compared with control neurons. Importantly, dopamine treatment of PD neurons promoted a rescue effect by increasing mitochondrial respiration, activating antioxidant stress response, and normalizing altered metabolite levels linked to mitochondrial function. These observations provide evidence that dopamine affects iron homeostasis, intracellular stress responses and mitochondrial function in healthy cells, while dopamine supplementation can restore the disturbed regulatory network in PD cells.

Recycling of Spent FCC Catalysts: Conversion of Leached Residues to Zeolite ZSM-5.

The commercial recycling of spent FCC catalyst typically focuses on recovering only 1-3 % of rare-earth elements, with the remaining residues often disposed of in landfills. Here, we present a novel method to close a recycling loop for spent FCC catalyst. The method involves a series of leaching steps: Firstly, the spent catalyst material is leached with HNO3 to remove rare-earth elements such as La; second, solvothermal leaching with HCl removes most of Al and impurities like Fe, Ni and V; finally, a third leaching with H2SO4 removes Ti. The solid residues are then used to synthesize ZSM-5 without the addition of any extra silicon or aluminum sources after mild activation. The impurities in the synthesis gel strongly modify the properties of the zeolite, with ZSM-5 crystals containing higher levels of impurities exhibiting lower crystallinities, surface areas, acidities, cracking activities, as well as larger particle sizes.

Quantification of Macrophage Cellular Ferrous Iron (Fe2+) Content Using a Highly Specific Fluorescent Probe in a Plate Reader.

Macrophages are at the center of innate immunity and iron metabolism. In the case of an infection, macrophages adapt their cellular iron metabolism to deprive iron from invading bacteria to combat intracellular bacterial proliferation. A concise evaluation of the cellular iron content upon an infection with bacterial pathogens and diverse cellular stimuli is necessary to identify underlying mechanisms concerning iron homeostasis in macrophages. For the characterization of cellular iron levels during infection, we established an in vitro infection model where the murine macrophage cell line J774A.1 is infected with Salmonella enterica serovar Typhimurium (S.tm), the mouse counterpart to S. enterica serovar Typhi, under normal and iron-overload conditions using ferric chloride (FeCl3) treatment. To evaluate the effect of infection and iron stimulation on cellular iron levels, the macrophages are stained with FerroOrange. This fluorescent probe specifically detects Fe2+ ions and its fluorescence can be quantified photometrically in a plate reader. Importantly, FerroOrange fluorescence does not increase with chelated iron or other bivalent metal ions. In this protocol, we present a simple and reliable method to quantify cellular Fe2+ levels in cultured macrophages by applying a highly specific fluorescence probe (FerroOrange) in a TECAN Spark microplate reader. Compared to already established techniques, our protocol allows assessing cellular iron levels in innate immune cells without the use of radioactive iron isotopes or extensive sample preparation, exposing the cells to stress. Key features • Easy quantification of Fe2+ in cultured macrophages with a fluorescent probe. • Analysis of iron in living cells without the need for fixation. • Performed on a plate reader capable of 540 nm excitation and 585 nm emission by trained employees for handling biosafety level 2 bacteria.

Klebsiella pneumoniae manipulates human macrophages to acquire iron.

Background: Klebsiella pneumoniae (KP) is a major cause of hospital-acquired infections, such as pneumonia. Moreover, it is classified as a pathogen of concern due to sprawling anti-microbial resistance. During infection, the gram-negative pathogen is capable of establishing an intracellular niche in macrophages by altering cellular metabolism. One factor critically affecting the host-pathogen interaction is the availability of essential nutrients, like iron, which is required for KP to proliferate but which also modulates anti-microbial immune effector pathways. We hypothesized, that KP manipulates macrophage iron homeostasis to acquire this crucial nutrient for sustained proliferation. Methods: We applied an in-vitro infection model, in which human macrophage-like PMA-differentiated THP1 cells were infected with KP (strain ATCC 43816). During a 24-h course of infection, we quantified the number of intracellular bacteria via serial plating of cell lysates and evaluated the effects of different stimuli on intracellular bacterial numbers and iron acquisition. Furthermore, we analyzed host and pathogen specific gene and protein expression of key iron metabolism molecules. Results: Viable bacteria are recovered from macrophage cell lysates during the course of infection, indicative of persistence of bacteria within host cells and inefficient pathogen clearing by macrophages. Strikingly, following KP infection macrophages strongly induce the expression of the main cellular iron importer transferrin-receptor-1 (TFR1). Accordingly, intracellular KP proliferation is further augmented by the addition of iron loaded transferrin. The induction of TFR1 is mediated via the STAT-6-IL-10 axis, and pharmacological inhibition of this pathway reduces macrophage iron uptake, elicits bacterial iron starvation, and decreases bacterial survival. Conclusion: Our results suggest, that KP manipulates macrophage iron metabolism to acquire iron once confined inside the host cell and enforces intracellular bacterial persistence. This is facilitated by microbial mediated induction of TFR1 via the STAT-6-IL-10 axis. Mechanistic insights into immune metabolism will provide opportunities for the development of novel antimicrobial therapies.

Timing of Interleukin-4 Stimulation of Macrophages Determines Their Anti-Microbial Activity during Infection with Salmonella enterica Serovar Typhimurium.

Priming of macrophages with interferon-gamma (IFNγ) or interleukin-4 (IL-4) leads to polarisation into pro-inflammatory or anti-inflammatory subtypes, which produce key enzymes such as inducible nitric oxide synthase (iNOS) and arginase 1 (ARG1), respectively, and in this way determine host responses to infection. Importantly, L-arginine is the substrate for both enzymes. ARG1 upregulation is associated with increased pathogen load in different infection models. However, while differentiation of macrophages with IL-4 impairs host resistance to the intracellular bacterium Salmonella enterica serovar Typhimurium (S.tm), little is known on the effects of IL-4 on unpolarised macrophages during infection. Therefore, bone-marrow-derived macrophages (BMDM) from C57BL/6N, Tie2Cre+/-ARG1fl/fl (KO), Tie2Cre-/-ARG1fl/fl (WT) mice were infected with S.tm in the undifferentiated state and then stimulated with IL-4 or IFNγ. In addition, BMDM of C57BL/6N mice were first polarised upon stimulation with IL-4 or IFNγ and then infected with S.tm. Interestingly, in contrast to polarisation of BMDM with IL-4 prior to infection, treatment of non-polarised S.tm-infected BMDM with IL-4 resulted in improved infection control whereas stimulation with IFNγ led to an increase in intracellular bacterial numbers compared to unstimulated controls. This effect of IL-4 was paralleled by decreased ARG1 levels and increased iNOS expression. Furthermore, the L-arginine pathway metabolites ornithine and polyamines were enriched in unpolarised cells infected with S.tm and stimulated with IL-4. Depletion of L-arginine reversed the protective effect of IL-4 toward infection control. Our data show that stimulation of S.tm-infected macrophages with IL-4 reduced bacterial multiplication via metabolic re-programming of L-arginine-dependent pathways.

Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

Comparative analysis of oral and intravenous iron therapy in rat models of inflammatory anemia and iron deficiency.

Anemia is a major health issue and associated with increased morbidity. Iron deficiency anemia (IDA) is the most prevalent, followed by anemia of chronic disease (ACD). IDA and ACD often co-exist, challenging diagnosis and treatment. While iron supplementation is the first-line therapy for IDA, its optimal route of administration and the efficacy of different repletion strategies in ACD are elusive. Female Lewis rats were injected with group A streptococcal peptidoglycan-polysaccharide (PG-APS) to induce inflammatory arthritis with associated ACD and/or repeatedly phlebotomized and fed with a low iron diet to induce IDA, or a combination thereof (ACD/IDA). Iron was either supplemented by daily oral gavage of ferric maltol or by weekly intravenous (i.v.) injection of ferric carboxymaltose for up to 4 weeks. While both strategies reversed IDA, they remained ineffective to improve hemoglobin (Hb) levels in ACD, although oral iron showed slight amelioration of various erythropoiesis-associated parameters. In contrast, both iron treatments significantly increased Hb in ACD/IDA. In ACD and ACD/IDA animals, i.v. iron administration resulted in iron trapping in liver and splenic macrophages, induction of ferritin expression and increased circulating levels of the iron hormone hepcidin and the inflammatory cytokine interleukin-6, while oral iron supplementation reduced interleukin-6 levels. Thus, oral and i.v. iron resulted in divergent effects on systemic and tissue iron homeostasis and inflammation. Our results indicate that both iron supplements improve Hb in ACD/IDA, but are ineffective in ACD with pronounced inflammation, and that under the latter condition, i.v. iron is trapped in macrophages and may enhance inflammation.

Crucial Role of Silica-Alumina Binder Mixtures for Hydrocarbon Cracking with ZSM-5 Additives.

Alumina-containing binders are widely used for the binding of catalyst particles by spray drying and calcination. As a part of the active matrix, they contribute to the catalytic performance of the resulting catalyst grain during hydrocarbon cracking. In this study, correlations are investigated using different compositions of Al- and Si-based binders (AlCl3 and colloidal silica) together with kaolin as a filler and ZSM-5 zeolite as an active compound. It was demonstrated that the conversion of a 50:50 hexane mixture, the selectivity toward unsaturated hydrocarbons, and the shape-selective conversion of the hexane feed are highly dependent on the amount and distribution of alumina in binder formulations. While silica species are distributed near the outer shell of catalyst grains, the alumina species are distributed evenly as an adhesive between the catalyst compounds ZSM-5 and kaolin. An optimum amount of alumina in binder formulations results in an increasing conversion of hydrocarbon feedstock due to optimum in active-site accessibility but only a slight decrease in shape-selective properties compared to pure ZSM-5, resulting in an optimum yield of light olefins, especially propylene.

Surface ion-imprinted brewer's spent grain with low template loading for selective uranyl ions adsorption from simulated wastewater.

Efficient removal of uranyl ions from wastewater requires excellent selectivity of the adsorbents. Herein, we report a new strategy using a high monomer/template molar ratio of 500:1 to prepare surface ion-imprinted brewer's spent grain (IIP-BSG) for selective U(VI) removal using binary functional monomers (2-hydroxyethyl methacrylate and diethyl vinylphosphonate) with high site accessibility and easy template removal. IIP-BSG exhibits a maximum U(VI) adsorption capacity of 165.7 mg/g, a high selectivity toward U(VI) in the presence of an excess amount of Eu(III) (Eu/U molar ratio = 20), a good tolerance of salinity, and a high reusability. In addition, mechanism studies have revealed electrostatic interaction and a coordination of uranyl ions by carboxyl and phosphoryl groups, the predominant contribution of high-energy (specific) sites during selective adsorption, and internal mass transfer as the rate-controlling step of U(VI) adsorption. Furthermore, IIP-BSG shows great potentials to separate U(VI) from lanthanides in simulated nuclear wastewater (pH0 = 3.5) and selectively concentrate U(VI) from simulated mine water (pH0 = 7.1). This study proves that the ion-imprinting effect can be achieved using a very low template amount with reduced production cost and secondary pollution, which benefits large-scale promotion of the ion-imprinted materials for selective uranyl ions removal.

Airway Epithelial Cells Differentially Adapt Their Iron Metabolism to Infection With Klebsiella pneumoniae and Escherichia coli In Vitro.

Background: Pneumonia is often elicited by bacteria and can be associated with a severe clinical course, respiratory failure and the need for mechanical ventilation. In the alveolus, type-2-alveolar-epithelial-cells (AECII) contribute to innate immune functions. We hypothesized that AECII actively adapt cellular iron homeostasis to restrict this essential nutrient from invading pathogens - a defense strategy termed 'nutritional immunity', hitherto mainly demonstrated for myeloid cells. Methods: We established an in-vitro infection model using the human AECII-like cell line A549. We infected cells with Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), two gram-negative bacteria with different modes of infection and frequent causes of hospital-acquired pneumonia. We followed the entry and intracellular growth of these gram-negative bacteria and analyzed differential gene expression and protein levels of key inflammatory and iron metabolism molecules. Results: Both, K. pneumoniae and E. coli are able to invade A549 cells, whereas only K. pneumoniae is capable of proliferating intracellularly. After peak bacterial burden, the number of intracellular pathogens declines, suggesting that epithelial cells initiate antimicrobial immune effector pathways to combat bacterial proliferation. The extracellular pathogen E. coli induces an iron retention phenotype in A549 cells, mainly characterized by the downregulation of the pivotal iron exporter ferroportin, the upregulation of the iron importer transferrin-receptor-1 and corresponding induction of the iron storage protein ferritin. In contrast, cells infected with the facultative intracellular bacterium K. pneumoniae exhibit an iron export phenotype indicated by ferroportin upregulation. This differential regulation of iron homeostasis and the pathogen-specific inflammatory reaction is likely mediated by oxidative stress. Conclusion: AECII-derived A549 cells show pathogen-specific innate immune functions and adapt their iron handling in response to infection. The differential regulation of iron transporters depends on the preferential intra- or extracellular localization of the pathogen and likely aims at limiting bacterial iron availability.

DMT1 Protects Macrophages from Salmonella Infection by Controlling Cellular Iron Turnover and Lipocalin 2 Expression.

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/flLysMCre(+)) 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 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 upon infection and leads to increased bacterial proliferation and persistence within macrophages. Accordingly, mice harboring a macrophage-selective DMT1 disruption demonstrate reduced survival following 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.

Binder Selection to Modify Hydrocarbon Cracking Properties of Zeolite-Containing Composites.

Activity, selectivity, and deactivation behavior of catalyst materials determine their efficiency in hydrocarbon conversion processes. For hydrocarbon cracking, the industrial catalyst is an important parameter in reaction technology to produce valuable compounds, e.g., light olefins (C3-C5) and gasoline from crude oil fractions with high molecular weight (C16+). One strategy to enhance the catalytic activity for precracking is increasing the matrix activity, which depends on the used binder and additives. In this work, three binders (water glass, aluminum chloride, and a mixture of colloidal silica with aluminum dihydrogen phosphate) were used in combination with active zeolite Y, kaolin as filler, and ZSM-5 as additive to produce composite materials. Specific surface area and surface acidity measurements were combined with catalytic testing of the formulated samples in order to find the relation between the catalyst morphology and its activity. In addition, constraint index was used as a control parameter for the determination of the shape-selective properties and their correlation with the catalytic activity. The results show that the binders determine the porosity of the matrix and so the accessibility to zeolite pores and active sites. Matrixes with low porosity and activity enhance coke production and deactivate faster than matrixes with mesopores. Furthermore, ZSM-5 modifies the individual morphological and catalytic effects of the binders. Everything considered, the small crystals of ZSM-5 together with mesopores increase the olefins yield, reduce coking, and therefore enhance the performance of the final grain.

One-pot synthesis of brewer's spent grain-supported superabsorbent polymer for highly efficient uranium adsorption from wastewater.

High-efficient and fast adsorption of uranium is important to reduce the hazards caused by the uranium contamination of water environment due to the increased human activities. Herein, brewer's spent grain (BSG)-supported superabsorbent polymers (SAP) with different cross-linking densities are prepared as cheap and eco-friendly adsorbents for the first time via one-pot swelling and graft polymerization. A 7 wt% NaOH solution is used to swell BSG before grafting and subsequently neutralize the acrylic acid to control the reaction rate without producing alkaline wastewater. Compared with the traditional methods, swelling improves the grafting density and the utilization of raw materials due to the increased disorder degree of the BSG fibers. This results in the grafting of abundant carboxyl and amide groups onto the BSG backbone, forming a strongly hydrophilic polymer network of the BSG-SAP. Compared with the reference polymers without BSG, BSG-SAP presents higher adsorption capacity and enhanced reusability. The highly cross-linked BSG-SAP (BSG-SAP-H) shows an outstanding adsorption capacity of U(VI) (1465 mg/g at pH0 = 4.6), a fast adsorption rate (81% of equilibrium adsorption capacity in 15 min), and a high selectivity in the presence of competing ions. Adsorption mechanism studies reveal the involvement of amide groups, a bidentate binding structure between UO22+ and the carboxyl groups, and a cation exchange between Na+ and UO22+. More importantly, the adsorption capacity of BSG-SAP-H reaches 254.4 mg/g in the fixed-bed column experiment at a low initial concentration (c0(U) = 30 mg/L) and keeps 80% of the adsorption capacity after four cycles, indicating a great potential for uranium removal from wastewater. This work shows a suitable approach to explore the untreated biomass to prepare SAP with enhanced adsorption performance via a general and low-cost strategy.

Mitochondrial Respiration in Response to Iron Deficiency Anemia: Comparison of Peripheral Blood Mononuclear Cells and Liver.

Iron is an essential component for metabolic processes, including oxygen transport within hemoglobin, tricarboxylic acid (TCA) cycle activity, and mitochondrial energy transformation. Iron deficiency can thus lead to metabolic dysfunction and eventually result in iron deficiency anemia (IDA), which affects approximately 1.5 billion people worldwide. Using a rat model of IDA induced by phlebotomy, we studied the effects of IDA on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) and the liver. Furthermore, we evaluated whether the mitochondrial function evaluated by high-resolution respirometry in PBMCs reflects corresponding alterations in the liver. Surprisingly, mitochondrial respiratory capacity was increased in PBMCs from rats with IDA compared to the controls. In contrast, mitochondrial respiration remained unaffected in livers from IDA rats. Of note, citrate synthase activity indicated an increased mitochondrial density in PBMCs, whereas it remained unchanged in the liver, partly explaining the different responses of mitochondrial respiration in PBMCs and the liver. Taken together, these results indicate that mitochondrial function determined in PBMCs cannot serve as a valid surrogate for respiration in the liver. Metabolic adaptions to iron deficiency resulted in different metabolic reprogramming in the blood cells and liver tissue.

Dietary Iron Overload and Hfe-/- Related Hemochromatosis Alter Hepatic Mitochondrial Function.

Iron is an essential co-factor for many cellular metabolic processes, and mitochondria are main sites of utilization. Iron accumulation promotes production of reactive oxygen species (ROS) via the catalytic activity of iron species. Herein, we investigated the consequences of dietary and genetic iron overload on mitochondrial function. C57BL/6N wildtype and Hfe-/- mice, the latter a genetic hemochromatosis model, received either normal diet (ND) or high iron diet (HI) for two weeks. Liver mitochondrial respiration was measured using high-resolution respirometry along with analysis of expression of specific proteins and ROS production. HI promoted tissue iron accumulation and slightly affected mitochondrial function in wildtype mice. Hepatic mitochondrial function was impaired in Hfe-/- mice on ND and HI. Compared to wildtype mice, Hfe-/- mice on ND showed increased mitochondrial respiratory capacity. Hfe-/- mice on HI showed very high liver iron levels, decreased mitochondrial respiratory capacity and increased ROS production associated with reduced mitochondrial aconitase activity. Although Hfe-/- resulted in increased mitochondrial iron loading, the concentration of metabolically reactive cytoplasmic iron and mitochondrial density remained unchanged. Our data show multiple effects of dietary and genetic iron loading on mitochondrial function and linked metabolic pathways, providing an explanation for fatigue in iron-overloaded hemochromatosis patients, and suggests iron reduction therapy for improvement of mitochondrial function.

Baseline iron status and presence of anaemia determine the course of systemic Salmonella infection following oral iron supplementation in mice.

BACKGROUND: Iron deficiency anaemia (IDA) is a major health concern. However, preventive iron supplementation in regions with high burden of infectious diseases resulted in an increase of infection related morbidity and mortality. METHODS: We fed male C57BL/6N mice with either an iron deficient or an iron adequate diet. Next, they received oral iron supplementation or placebo followed by intraperitoneal infection with Salmonella Typhimurium (S.Tm). FINDINGS: We found that mice with IDA had a poorer clinical outcome than mice on an iron adequate diet. Interestingly, iron supplementation of IDA mice resulted in higher bacterial burden in organs and shortened survival. Increased transferrin saturation and non-transferrin bound iron in the circulation together with low expression of ferroportin facilitated the access of the pathogen to iron and promoted bacterial growth. Anaemia, independent of iron supplementation, was correlated with reduced neutrophil counts and cytotoxic T cells. With iron supplementation, anaemia additionally correlated with increased splenic levels of the cytokine IL-10, which is suggestive for a weakened immune control to S.Tm infection. INTERPRETATION: Supplementing iron to anaemic mice worsens the clinical course of bacterial infection. This can be traced back to increased iron delivery to bacteria along with an impaired anti-microbial immune response. Our findings may have important implications for iron supplementation strategies in areas with high endemic burden of infections, putting those individuals, who potentially profit most from iron supplementation for anaemia, at the highest risk for infections. FUNDING: Financial support by the Christian Doppler Laboratory for Iron Metabolism and Anemia Research.

Cytokine-Mediated Regulation of ARG1 in Macrophages and Its Impact on the Control of Salmonella enterica Serovar Typhimurium Infection.

Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre+/-ARG1fl/fl and NRAMPG169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre+/-ARG1fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.

Pharmacological Targeting of BMP6-SMAD Mediated Hepcidin Expression Does Not Improve the Outcome of Systemic Infections With Intra-Or Extracellular Gram-Negative Bacteria in Mice.

Introduction: Hepcidin is the systemic master regulator of iron metabolism as it degrades the cellular iron exporter ferroportin. In bacterial infections, hepcidin is upregulated to limit circulating iron for pathogens, thereby increasing iron retention in macrophages. This mechanism withholds iron from extracellular bacteria but could be of disadvantage in infections with intracellular bacteria. We aimed to understand the role of hepcidin in infections with intra- or extracellular bacteria using different hepcidin inhibitors. Methods: For the experiments LDN-193189 and oversulfated heparins were used, which interact with the BMP6-SMAD pathway thereby inhibiting hepcidin expression. We infected male C57BL/6N mice with either the intracellular bacterium Salmonella Typhimurium or the extracellular bacterium Escherichia coli and treated these mice with the different hepcidin inhibitors. Results: Both inhibitors effectively reduced hepcidin levels in vitro under steady state conditions and upon stimulation with the inflammatory signals interleukin-6 or lipopolysaccharide. The inhibitors also reduced hepcidin levels and increased circulating iron concentration in uninfected mice. However, both compounds failed to decrease liver- and circulating hepcidin levels in infected mice and did not affect ferroportin expression in the spleen or impact on serum iron levels. Accordingly, both BMP-SMAD signaling inhibitors did not influence bacterial numbers in different organs in the course of E.coli or S.Tm sepsis. Conclusion: These data indicate that targeting the BMP receptor or the BMP-SMAD pathway is not sufficient to suppress hepcidin expression in the course of infection with both intra- or extracellular bacteria. This suggests that upon pharmacological inhibition of the central SMAD-BMP pathways during infection, other signaling cascades are compensatorily induced to ensure sufficient hepcidin formation and iron restriction to circulating microbes.

Recycling of Brewer's Spent Grain as a Biosorbent by Nitro-Oxidation for Uranyl Ion Removal from Wastewater.

Developing biosorbents derived from agro-industrial biomass is considered as an economic and sustainable method for dealing with uranium-contaminated wastewater. The present study explores the feasibility of oxidizing a representative protein-rich biomass, brewer's spent grain (BSG), to an effective and reusable uranyl ion adsorbent to reduce the cost and waste generation during water treatment. The unique composition of BSG favors the oxidation process and yields in a high carboxyl group content (1.3 mmol/g) of the biosorbent. This makes BSG a cheap, sustainable, and suitable raw material independent from pre-treatment. The oxidized brewer's spent grain (OBSG) presents a high adsorption capacity of U(VI) of 297.3 mg/g (c 0(U) = 900 mg/L, pH = 4.7) and fast adsorption kinetics (1 h) compared with other biosorbents reported in the literature. Infrared spectra (Fourier transform infrared), 13C solid-state nuclear magnetic resonance spectra, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and thermogravimetric analysis were employed to characterize the biosorbents and reveal the adsorption mechanisms. The desorption and reusability of OBSG were tested for five cycles, resulting in a remaining adsorption of U(VI) of 100.3 mg/g and a desorption ratio of 89%. This study offers a viable and sustainable approach to convert agro-industrial waste into effective and reusable biosorbents for uranium removal from wastewater.