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.

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.

24-Norursodeoxycholic acid ameliorates experimental alcohol-related liver disease and activates hepatic PPARγ.

Background & Aims: Alcohol-related liver disease (ALD) is a global healthcare challenge with limited treatment options. 24-Norursodeoxycholic acid (NorUDCA) is a synthetic bile acid with anti-inflammatory properties in experimental and human cholestatic liver diseases. In the present study, we explored the efficacy of norUDCA in experimental ALD. Methods: NorUDCA was tested in a preventive and therapeutic setting in an experimental ALD model (Lieber-DeCarli diet enriched with ethanol). Liver disease was phenotypically evaluated using histology and biochemical methods, and anti-inflammatory properties and peroxisome proliferator-activated receptor gamma activation by norUDCA were evaluated in cellular model systems. Results: NorUDCA administration ameliorated ethanol-induced liver injury, reduced hepatocyte death, and reduced the expression of hepatic pro-inflammatory cytokines including tumour necrosis factor (Tnf), Il-1ß, Il-6, and Il-10. NorUDCA shifted hepatic macrophages towards an anti-inflammatory M2 phenotype. Further, norUDCA administration altered the composition of the intestinal microbiota, specifically increasing the abundance of Roseburia, Enterobacteriaceae, and Clostridum spp. In a therapeutic model, norUDCA also ameliorated ethanol-induced liver injury. Moreover, norUDCA suppressed lipopolysaccharide-induced IL-6 expression in human peripheral blood mononuclear cells and evoked peroxisome proliferator-activated receptor gamma activation. Conclusions: NorUDCA ameliorated experimental ALD, protected against hepatic inflammation, and affected gut microbial commensalism. NorUDCA could serve as a novel therapeutic agent in the future management of patients with ALD. Impact and implications: Alcohol-related liver disease is a global healthcare concern with limited treatment options. 24-Norursodeoxycholic acid (NorUDCA) is a modified bile acid, which was proven to be effective in human cholestatic liver diseases. In the present study, we found a protective effect of norUDCA in experimental alcoholic liver disease. For patients with ALD, norUDCA could be a potential new treatment option.

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.

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.

Nifedipine Potentiates Susceptibility of Salmonella Typhimurium to Different Classes of Antibiotics.

The calcium channel blocker nifedipine induces cellular iron export, thereby limiting the availability of the essential nutrient iron for intracellular pathogens, resulting in bacteriostatic activity. To study if nifedipine may exert a synergistic anti-microbial activity when combined with antibiotics, we used the mouse macrophage cell line RAW267.4, infected with the intracellular bacterium Salmonella Typhimurium, and exposed the cells to varying concentrations of nifedipine and/or ampicillin, azithromycin and ceftriaxone. We observed a significant additive effect of nifedipine in combination with various antibiotics, which was not observed when using Salmonella, with defects in iron uptake. Of interest, increasing intracellular iron levels increased the bacterial resistance to treatment with antibiotics or nifedipine or their combination. We further showed that nifedipine increases the expression of the siderophore-binding peptide lipocalin-2 and promotes iron storage within ferritin, where the metal is less accessible for bacteria. Our data provide evidence for an additive effect of nifedipine with conventional antibiotics against Salmonella, which is partly linked to reduced bacterial access to iron.

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.