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FATP4 deficiency in BMDMs and Kupffer cells led to increased proinflammatory response. Fatp4M−/− mice displayed thrombocytopenia, splenomegaly, and elevated liver enzymes. In response to HFHC feeding, male mutants were prone to hepatic steatosis, whereas female mutants showed exaggerated fibrosis. Our study provides insights into a sex-dimorphic susceptibility to NASH by myeloid-FATP4 deficiency.

Muckenthaler et al describe a novel form of hemochromatosis caused by a constitutional PIGA mutation in 3 children with associated neurologic dysfunction. Hemochromatosis results from decreased hepcidin, which is regulated by HFE, hemojuvelin (HJV), and transferrin receptor 2. HJV is a glycosylphosphatidylinositol-linked protein, so PIGA mutation leads to decreased HJV expression. Interestingly, none of the children had evidence of paroxysmal nocturnal hemoglobinuria. The cause of the novel association with central nervous system manifestations remains to be elucidated.

Iron is mostly devoted to the hemoglobinization of erythrocytes for oxygen transport. Yet, emerging evidence points to a broader role for the metal in hematopoiesis, including the formation of the immune system. Iron availability in mammalian cells is controlled by iron-regulatory proteins (IRP)-1 and −2. We report that global disruption of both IRP1 and IRP2 in adult mice impairs neutrophil development and differentiation in the bone marrow, yielding immature neutrophils with abnormally high glycolytic and autophagic activity, resulting in neutropenia. IRPs promote neutrophil differentiation in a cell intrinsic manner by securing cellular iron supply together with transcriptional control of neutropoiesis to facilitate differentiation to fully mature neutrophils. Unlike neutrophils, monocyte count was not affected by IRP and iron deficiency, suggesting a lineage-specific effect of iron on myeloid output. This study unveils the previously unrecognized importance of IRPs and iron metabolism in the formation of a major branch of the innate immune system.

Hereditary hemochromatosis (HH) is a genetic disorder in which mutations affect systemic iron homeostasis. Most subtypes of HH result in low hepcidin levels and iron overload. Accumulation of iron in various tissues can lead to widespread organ damage and to various complications, including liver cirrhosis, arthritis, and diabetes. Osteoporosis is another frequent complication of HH, and the underlying mechanisms are poorly understood. Currently, it is unknown whether iron overload in HH directly damages bone or whether complications associated with HH, such as liver cirrhosis or hypogonadism, affect bone secondarily. This review summarizes current knowledge of bone metabolism in HH and highlights possible implications of metabolic dysfunction in HH-driven bone loss. We further discuss therapeutic considerations managing osteoporosis in HH.

Polymorphisms of phospholipase A2VIA (iPLA2β or PLA2G6) are associated with body weights and blood C-reactive protein. The role of iPLA2β/PLA2G6 in non-alcoholic steatohepatitis (NASH) is still elusive because female iPla2β-null mice showed attenuated hepatic steatosis but exacerbated hepatic fibrosis after feeding with methionine- and choline-deficient diet (MCDD). Herein, female mice with myeloid- (MPla2g6

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the Alox15 gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation., Haematologica, 105 (4), ISSN:0390-6078, ISSN:1592-8721

Hyperferremia and hyperferritinemia are observed in patients and disease models of type 2 diabetes mellitus (T2DM). Likewise, patients with genetic iron overload diseases develop diabetes, suggesting a tight link between iron metabolism and diabetes. The liver controls systemic iron homeostasis and is a central organ for T2DM. Here, we investigate how the control of iron metabolism in hepatocytes is affected by T2DM.Perls Prussian blue staining was applied to analyze iron distribution in liver biopsies of T2DM patients. To identify molecular mechanisms underlying hepatocyte iron accumulation we established cellular models of insulin resistance by treatment with palmitate and insulin.We show that a subset of T2DM patients accumulates iron in hepatocytes, a finding mirrored in a hepatocyte model of insulin resistance. Iron accumulation can be explained by the repression of the iron exporter ferroportin upon palmitate and/or insulin treatment. While during palmitate treatment the activation of the iron regulatory hormone hepcidin may contribute to reducing ferroportin protein levels in a cell-autonomous manner, insulin treatment decreases ferroportin transcription via the PI3K/AKT and Ras/Raf/MEK/ERK signaling pathways.Repression of ferroportin at the transcriptional and post-transcriptional level may contribute to iron accumulation in hepatocytes observed in a subset of patients with T2DM.

Twenty years ago the discovery of hepcidin deeply changed our understanding of the regulation of systemic iron homeostasis. It is now clear that hepcidin orchestrates systemic iron levels by controlling the amount of iron exported into the bloodstream through ferroportin. Hepcidin expression is increased in situations where systemic iron levels should be reduced, such as in iron overload and infection. Conversely, hepcidin is repressed during iron deficiency, hypoxia or expanded erythropoiesis, to increase systemic iron availability and sustain erythropoiesis. In this review, we will focus on molecular mechanisms of hepcidin regulation and on the pathological consequences of their disruption.

BACKGROUND AND AIMS TGFβ/bone morphogenetic protein (BMP) signaling in the liver plays a critical role in liver disease. Growth factors, such as BMP2, BMP6, and TGFβ1, are released from LSECs and signal in a paracrine manner to hepatocytes and hepatic stellate cells to control systemic iron homeostasis and fibrotic processes, respectively. The misregulation of the TGFβ/BMP pathway affects expression of the iron-regulated hormone hepcidin, causing frequent iron overload and deficiency diseases. However, whether LSEC-secreted factors can act in an autocrine manner to maintain liver homeostasis has not been addressed so far. APPROACH AND RESULTS We analyzed publicly available RNA-sequencing data of mouse LSECs for ligand-receptor interactions and identified members of the TGFβ family (BMP2, BMP6, and TGFβ1) as ligands with the highest expression levels in LSECs that may signal in an autocrine manner. We next tested the soluble factors identified through in silico analysis in optimized murine LSEC primary cultures and mice. Exposure of murine LSEC primary cultures to these ligands shows that autocrine responses to BMP2 and BMP6 are blocked despite high expression levels of the required receptor complexes partially involving the inhibitor FK-506-binding protein 12. By contrast, LSECs respond efficiently to TGFβ1 treatment, which causes reduced expression of BMP2 through activation of activin receptor-like kinase 5. CONCLUSIONS These findings reveal that TGFβ1 signaling is functionally interlinked with BMP signaling in LSECs, suggesting druggable targets for the treatment of iron overload diseases associated with deficiency of the BMP2-regulated hormone hepcidin, such as hereditary hemochromatosis, β-thalassemia, and chronic liver diseases.

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

Hematopoietic stem cells (HSCs) and distinct multipotent progenitor (MPP) populations (MPP1-4) contained within the Lin−Sca-1+c-Kit+ (LSK) compartment have previously been identified using diverse surface-marker panels. Here, we phenotypically define and functionally characterize MPP5 (LSK CD34+CD135−CD48−CD150−). Upon transplantation, MPP5 supports initial emergency myelopoiesis followed by stable contribution to the lymphoid lineage. MPP5, capable of generating MPP1-4 but not HSCs, represents a dynamic and versatile component of the MPP network. To characterize all hematopoietic stem and progenitor cells, we performed RNA-sequencing (RNA-seq) analysis to identify specific transcriptomic landscapes of HSCs and MPP1-5. This was complemented by single-cell RNA-seq analysis of LSK cells to establish the differentiation trajectories from HSCs to MPP1-5. In agreement with functional reconstitution activity, MPP5 is located immediately downstream of HSCs but upstream of the more committed MPP2-4. This study provides a comprehensive analysis of the LSK compartment, focusing on the functional and molecular characteristics of the newly defined MPP5 subset.

Living organisms face the dual challenge of acquiring enough iron to perform biological functions while preventing toxic iron accretion. A study now shows that sensing of iron-catalysed free radicals by a druggable gene-regulatory pathway helps the body avoid iron poisoning.

Iron is an essential micronutrient and a critical cofactor for proteins involved in fundamental processes such as oxygen transport, energy production and DNA synthesis. However, iron levels need to be tightly balanced to avoid pathological consequences of iron overload or deficiency. Genetically engineered mouse models with alterations in systemic or cellular iron handling advanced our knowledge how systemic and cellular iron homeostasis is maintained. Here, we prepared a comprehensive overview of mouse models that provide insight into mechanisms of iron regulation and/or rare or frequent iron-related disorders.

Supplemental Digital Content is available in the text.

Diabetes mellitus is frequently associated with iron overload conditions, such as primary and secondary hemochromatosis. Conversely, patients affected by type 2 diabetes mellitus (T2DM) show elevated ferritin levels, a biomarker for increased body iron stores. Despite these documented associations between dysregulated iron metabolism and T2DM, the underlying mechanisms are poorly understood. Here, we show that T2DM patients have reduced serum levels of hepcidin, the iron-regulated hormone that maintains systemic iron homeostasis. Consistent with this finding, we also observed an increase in circulating iron and ferritin levels. Our analysis of db/db mice demonstrates that this model recapitulates the systemic alterations observed in patients. Interestingly, db/db mice show an overall hepatic iron deficiency despite unaltered expression of ferritin and the iron importer TfR1. In addition, the liver correctly senses increased circulating iron levels by activating the BMP/SMAD signaling pathway even though hepcidin expression is decreased. We show that increased AKT phosphorylation may override active BMP/SMAD signaling and decrease hepcidin expression in 10-week old db/db mice. We conclude that the metabolic alterations occurring in T2DM impact on the regulation of iron homeostasis on multiple levels. As a result, metabolic perturbations induce an "iron resistance" phenotype, whereby signals that translate increased circulating iron levels into hepcidin production, are dysregulated.T2DM patients show increased circulating iron levels. T2DM is associated with inappropriately low hepcidin levels. Metabolic alterations in T2DM induce an "iron resistance" phenotype.

Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the murine ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3’-untranslated region (3’-UTR) composition. The relevance of APA in stem cell hierarchies in vivo remains elusive. Using extensive in vitro and in vivo analysis approaches we show that deregulation of the APA regulator Pabpn1 results in severe hematopoietic stem cell (HSC) defects. Further, we performed low input 3’-sequencing and established bioinformatic pipelines to uncover dynamic APA patterns in numerous genes of HSCs and multipotent progenitors determining the genome-wide APA landscape (APAome). This revealed transcriptome-wide dynamic APA patterns and an overall shortening of 3’-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation (Sommerkamp et al., Cell Stem Cell, 2020). Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching. This process is mediated by Nudt21 and is required for the proper stress response of HSCs. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Inhibition of this metabolic adjustment leads to impaired HSC function and a partial block in differentiation. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal and commitment.

Background: Disease severity in COVID-19 patients is associated with cytokine release syndrome (CRS). Understanding factors linked to disease severity and identifying early markers that are predictive for the need of hospitalization or oxygen supplementation are highly relevant. Therefore we assessed iron metabolism markers in COVID-19 patients for their ability to predict disease severity. Methods: Patients tested positive for SARS-CoV-2 referred to the Heidelberg University Hospital were retrospectively analyzed. Patients were divided into outpatients never admitted to hospital (cohort A, n=204), inpatients (cohort B,n=81), and outpatients later admitted to hospital because of health deterioration (cohort C,n=23). Findings: Iron metabolism parameters were severely altered in patients of cohort B and C compared to cohort A. In multivariate regression analysis including age, gender, CRP and iron-related parameters only serum iron and ferritin were significantly associated with hospitalization. ROC analysis revealed an AUC for serum iron of 0.894 and an iron concentration

Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119(+) cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo. However, Cdk6(-/-) erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeleton organization. Cdk6(-/-) erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We also found that Cdk6 regulates the transcription of a panel of genes involved in actin (de-) polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia - and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors

The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels.

Iron accumulates in the lungs of patients with common respiratory diseases or transfusion-dependent beta-thalassemia. Based on our previous work, we hypothesized that systemic iron overload affects the alveolar region of the lung and in particular the surfactant producing alveolar epithelial type II (AE2) cells. Mice with a point mutation in the iron exporter ferroportin, a model for human hemochromatosis type 4 were compared to wildtype mice (n = 5 each). Lungs were fixed and prepared for light and electron microscopy (EM) according to state-of-the-art protocols to detect subcellular iron localization by scanning EM/EDX and to perform design-based stereology. Iron was detected as electron dense particles in membrane-bound organelles, likely lysosomes, in AE1 cells. AE2 cells were higher in number but had a lower mean volume in mutated mice. Lamellar body volume per AE2 cell was lower but total volume of lamellar bodies in the lung was comparable to wildtype mice. While the volume of alveoli was lower in mutated mice, the volume of alveolar ducts as well as the surface area, volume and the mean thickness and composition of the septa was similar in both genotypes. The thickness of the air–blood barrier was greater in the mutated than in the WT mice. In conclusion, disruption of systemic iron homeostasis affects the ultrastructure of interalveolar septa which is characterized by membrane-bound iron storage in AE1 cells, thickening of the air–blood barrier and hyperplasia and hypotrophy of AE2 cells despite normal total intracellular surfactant pools. The functional relevance of these findings requires further analysis to better understand the impact of iron on intra-alveolar surfactant function.

Aims Whether and how iron affects the progression of atherosclerosis remains highly debated. Here, we investigate susceptibility to atherosclerosis in a mouse model (ApoE−/− FPNwt/C326S), which develops the disease in the context of elevated non-transferrin bound serum iron (NTBI). Methods and results Compared with normo-ferremic ApoE−/− mice, atherosclerosis is profoundly aggravated in iron-loaded ApoE−/− FPNwt/C326S mice, suggesting a pro-atherogenic role for iron. Iron heavily deposits in the arterial media layer, which correlates with plaque formation, vascular oxidative stress and dysfunction. Atherosclerosis is exacerbated by iron-triggered lipid profile alterations, vascular permeabilization, sustained endothelial activation, elevated pro-atherogenic inflammatory mediators, and reduced nitric oxide availability. NTBI causes iron overload, induces reactive oxygen species production and apoptosis in cultured vascular cells, and stimulates massive MCP-1-mediated monocyte recruitment, well-established mechanisms contributing to atherosclerosis. NTBI-mediated toxicity is prevented by transferrin- or chelator-mediated iron scavenging. Consistently, a low-iron diet and iron chelation therapy strongly improved the course of the disease in ApoE−/− FPNwt/C326S mice. Our results are corroborated by analyses of serum samples of haemochromatosis patients, which show an inverse correlation between the degree of iron depletion and hallmarks of endothelial dysfunction and inflammation. Conclusion Our data demonstrate that NTBI-triggered iron overload aggravates atherosclerosis and unravel a causal link between NTBI and the progression of atherosclerotic lesions. Our findings support clinical applications of iron restriction in iron-loaded individuals to counteract iron-aggravated vascular dysfunction and atherosclerosis.

Regulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here, we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptors 2 and 6 (TLR2 and TLR6) and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin messenger RNA and protein expression in bone marrow-derived macrophages, liver, and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin downregulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin downregulation in the major sites of iron recycling may represent a first-line response to restrict iron access for numerous pathogens.

Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state., This work was supported by the FOR2033 and SFB873 funded by the Deutsche Forschungsgemeinschaft (DFG), the Dietmar Hopp Foundation (all to A.T.), and the Wellcome Trust (to A.S.).

Since modern foods are unnaturally enriched in single metabolites, it is important to understand which metabolites are sensed by the human body and which are not. We previously showed that the fatty acid stearic acid (C18:0) signals via a dedicated pathway to regulate mitofusin activity and thereby mitochondrial morphology and function in cell culture. Whether this pathway is poised to sense changes in dietary intake of C18:0 in humans is not known. We show here that C18:0 ingestion rapidly and robustly causes mitochondrial fusion in people within 3 h after ingestion. C18:0 intake also causes a drop in circulating long-chain acylcarnitines, suggesting increased fatty acid beta-oxidation in vivo. This work thereby identifies C18:0 as a dietary metabolite that is sensed by our bodies to control our mitochondria. This could explain part of the epidemiological differences between C16:0 and C18:0, whereby C16:0 increases cardiovascular and cancer risk whereas C18:0 decreases both., Dietary fatty acids have different effects on human health. Here, the authors show that ingestion of the fatty acid C18:0, but not of C16:0, rapidly leads to fusion of mitochondria and fatty acid oxidation in humans, possibly explaining the health benefits of C18:0.

SummaryThe regulatory axis between the iron hormone hepcidin and its receptor, the iron exporter ferroportin (FPN), is central to iron homeostasis. Mutations preventing hepcidin-mediated degradation of FPN cause systemic iron overload. We have introduced a point mutation (C326S) into the murine Fpn locus, resembling human hereditary hemochromatosis type 4, including elevated plasma iron and ferritin levels, high transferrin saturation, hepatic iron overload, and iron depletion of duodenal enterocytes and reticuloendothelial macrophages. Unlike other mouse models of iron overload, homozygous C326S mice die between 7 and 14 months of age. Pancreatic acinar cells display marked iron accumulation, oxidative damage and degeneration, associated with failure of the exocrine pancreas and severe body weight loss. Rescue experiments reveal iron overload and exocrine pancreatic failure as leading causes of death. This work uncovers the critical importance of the hepcidin-ferroportin regulatory axis for life and unveils the sensitivity of the exocrine pancreas to iron overload.

The liver stores iron and senses systemic and tissue iron availability. Hepatocytes control iron homeostasis by producing the peptide hormone hepcidin that controls dietary iron absorption and iron release from intracellular stores. Recent data challenged the exclusive role of hepatocytes in controlling iron levels. Indeed, liver sinusoidal endothelial cells (LSECs) increase BMP2 and BMP6 levels in response to iron, which control hepcidin expression in a paracrine manner. However the molecular mechanism(s) of how BMPs respond to iron levels remain unknown. We established primary murine LSEC cultures and exposed these to iron sources. Unexpectedly, BMP2 mRNA expression is strongly reduced by iron treatment, while BMP6 levels are only mildly increased. This finding suggests that intracellular iron content cannot directly activate BMP2 transcription and only slightly contribute to BMP6 upregulation in LSEC cultures. However, if LSECs are co-cultured with iron-loaded primary hepatocytes the expression of BMP2 and BMP6 is increased and the fold induction of BMP6 is greater compared to LSECs cultured alone, suggesting that the iron status of hepatocytes instructs the LSEC BMP response. These data are supported by findings in a genetic mouse model of iron overload (Slc40a1C326S/C326S). Hepatocytes isolated from Slc40a1C326S/C326S mice display an iron-loaded molecular signature and the expected low mRNA expression of Transferrin Receptor 1 (Tfr1). By contrast, LSECs show high expression of Tfr1, indicating intracellular iron deficiency. Despite this, hepatic BMP levels are increased, suggesting that BMP2 and BMP6 expression are directly related to the intracellular iron content of hepatocytes but not LSECs. RNA-sequencing of isolated hepatic cell populations is ongoing to identify putative hepatocyte regulators involved in the iron-mediated BMP2 and BMP6 regulation. Disclosures Muckenthaler: Silence Therapeutics: Consultancy; Novartis: Research Funding.

25 mg of iron are required every day to sustain hematopoiesis in the bone marrow. Most iron is consumed by erythroid cells that take up transferrin-bound iron to satisfy their demand for hemoglobin biogenesis. Unexpectedly, erythroblasts express high levels of the iron exporter Ferroportin (Fpn). Fpn surface expression and activity is controlled by Hepcidin, a small liver peptide hormone, produced in response to elevated systemic and tissue iron availability. Resistance of ferroportin to hepcidin binding is caused by a gain of function mutation in the FpnC326S residue, which is the reason for iron overload in patients with Hereditary Hemochromatosis type 4. Some years ago we generated the corresponding mouse model hallmarked by this constitutively active iron exporter. We now applied bone marrow transplantation to investigate the role of the hepcidin/ferroportin regulatory system in cell types of the bone marrow (BM-FpnC326S). CD45.1 host mice were irradiated twice with 500 cGy 4h apart and injected with 2 million of BM cells obtained from C57BL/6N mice (control) or from C57BL/6N congenic constitutive FpnC326S animals. Mice were analyzed three months after transplantation, whereby only animals with an engraftment higher than 95% were included for further analyses. Analysis of the erythroid hematological parameters revealed mild macrocytosis in the presence of unaltered red blood cell count (RBC), hematocrit (HCT), hemoglobin (Hb) and mean corpuscular hemoglobin (MCH) values. Serum iron content, transferrin saturation and serum hepcidin levels remained unchanged, despite a strong decrease in splenic iron levels within the red pulp, where reticuloendothelial macrophages are located. The latter phenotype is consistent with high ferroportin surface expression in macrophages derived from FpnC326S monocytes that causes increased iron export and cellular iron deficiency. Cytokine production (IL1β, TNFα and IL6) in the spleen was unchanged suggesting that iron deficiency in splenic macrophages did not cause inflammation. In contrast to the spleen iron content was not changed in other organs analyzed, including the liver, suggesting that de novo monocyte infiltration is not a major feature in these organs. Interestingly, histological analysis of the femurs revealed a marked decrease in bone marrow iron content; we were unable to detect a single iron-stained cell in the bone marrow of BM-FpnC326S mice, contrasting results from control mice. To investigate whether iron deficiency in bone marrow cells alters numbers of immune cells, we performed a detailed characterization by FACS. Unexpectedly, iron deficiency in the bone marrow did not cause changes in total bone marrow cellularity, total numbers of Ter119+ erythroid cells and the percentage of different erythroblast subpopulations, the number of hematopoietic stem cells (HSC) and of common lymphoid (CLP), myeloid (CMP) and megakaryocyte/erythroid (MEP) progenitors. Taken together, our data clearly demonstrate that at steady state, the hepcidin/ferroportin regulatory circuitry in the bone marrow is dispensable and that iron deficiency in bone marrow cells is not altering the normal hematopoietic process. Future studies will have to extend the analysis to stress conditions. Disclosures Muckenthaler: Novartis: Research Funding; Silence Therapeutics: Consultancy.

Alternative Polyadenylation (APA) is emerging as an important regulatory mechanism controlling 3’-UTRs and thereby RNA and protein isoform expression. However, the relevance of APA in stem cell hierarchies in vivo remains unexplored. Here, we show that deregulation of the APA regulator Pabpn1 results in severe hematopoietic stem cell (HSC) defects. Furthermore, we performed low input 3’-sequencing and established bioinformatic pipelines to uncover dynamic APA patterns in numerous genes of HSCs and multipotent progenitors. The 3’-UTRs of HSCs undergo shortening upon transition from quiescence to proliferation during homeostasis and in response to inflammation as well as during HSC differentiation. Strikingly, we observe APA-mediated metabolic reprogramming upon HSC activation leading to an increase in glutaminolysis. This increase is mediated by APA regulators and required for proper HSC function. Inhibition of this metabolic adjustment by targeting APA-regulators leads to impaired HSC function and a partial block in differentiation. Our study establishes APA and associated adaptations of the glutamine metabolism as novel layers of regulation that orchestrate HSC behavior and commitment in the hematopoietic cascade.

In the version of this article initially published, affiliation 14 was incorrect, and Deutsche Forschungsgemeinschaft grants SFB1036 and SFB1118 were missing from the Acknowledgements. The errors have been corrected in the HTML and PDF versions of the article.

Contains fulltext : 95939.pdf (Publisher’s version ) (Closed access) Congenital dyserythropoietic anaemias (CDAs) are heterogeneous, hereditary disorders hallmarked by ineffective erythropoiesis and tissue iron overload. Growth differentiation factor 15 (GDF15) was suggested to mediate iron overload in iron-loading anaemias, such as the thalassaemias and CDAI by suppressing hepcidin, the key regulator of iron absorption. Here, we show that serum GDF15 concentrations are elevated in subjects with CDAI and CDAII. Despite similar disease characteristics, CDAI patients present with significantly higher GDF15 concentrations compared to CDAII patients. Hepcidin concentrations are inappropriately low in CDAII patients considering the severe hepatic iron overload associated with this disorder. GDF15 significantly correlates with the degree of anaemia (Hb), the response of erythropoiesis (reticulocyte index) as well as with iron availability in the serum (transferrin saturation). The observation that GDF15 is elevated in CDAII patients is consistent with the proposal that GDF15 is among the erythroid factors down-regulating hepcidin and contributing to iron overload in conditions of dyserythropoiesis.

Accumulation of excess iron in tissues causes organ damage and dysfunction and may lead to serious clinical consequences including liver cirrhosis, diabetes, growth retardation and heart failure. Iron overload is a major health threat in iron loading anemias, like beta-thalassemia, myelodysplastic syndrome and in hereditary hemochromatosis. In patients with beta-thalassemia major, iron overload develops due to frequent blood transfusions to control the severe anemia. In addition, iron overload also occurs in patients with beta thalassemia intermedia (non-transfusion dependent beta-thalassemia). In the later cases, iron overload develops through gastrointestinal iron hyperabsorption due to stressed and ineffective erythropoiesis. Importantly, expression of the peptide hormone hepcidin, which is the key modulator in iron homeostasis, is abnormally low and unable to block ferroportin-mediated intestinal iron absorption. In hereditary hemochromatosis, gene defects in the hepcidin-ferroportin axis controlling iron homeostasis, lead to hepatic iron overload. Therefore, in these indications, iron overload is caused by dysregulation or dysfunction of the hepcidin-ferroportin axis. Hepcidin is predominantly produced by the liver and is induced by activation of the BMP/SMAD signaling pathway. Furthermore, hepcidin is under the negative control of the transmembrane protease matriptase-2, encoded by the TMPRSS6 gene. RNA interference is a natural mechanism and a powerful approach for inhibiting the expression of disease-associated genes. Silence Therapeutics has developed short interfering RNA (siRNA) conjugate technology for the selective inhibition of target gene expression in the liver. GalNAc-conjugated siRNAs bind efficiently to the asialoglycoprotein (ASGP) receptor expressed predominantly by hepatocytes thereby providing a highly specific, safe and efficient delivery technology to enable a new class of therapeutic use. Here we present the pharmacological characterization of SLN124, our GalNAc-siRNA conjugate targeting TMPRSS6 expression, in preclinical models. A single subcutaneous administration is sufficient to achieve significant modulation of target gene expression in mice and in non-human primates over several weeks. SLN124 treatment reduces systemic iron levels, transferrin saturation and tissue iron levels in a rodent model for hereditary hemochromatosis type 1. In addition, we report for the first time the therapeutic efficacy of iron restriction by SLN124 in mice with established iron overload both as monotherapy and in combination with an oral iron chelator - current standard of care- over an extended treatment period. The effects of these treatments on red blood cell parameters and tissue iron levels will be presented. In addition, we assessed the therapeutic effects of SLN124 in an animal model for beta-thalassemia intermedia, showing dose-dependent and long-lasting effects on target gene expression as well as on modulation of iron stores and normalization of erythropoiesis and anemia. Safety and tolerability studies in relevant preclinical models confirmed that SLN124 is well tolerated and shows promise as an effective and safe treatment for unmet medical need in iron loading anemias, such as beta-thalassemia. SLN124 is currently in preclinical development. The first in human study is planned to commence in 2019 in patients with beta-thalassemia and in patients with myelodysplastic syndrome. Disclosures Muckenthaler: Novartis: Research Funding. Dames:Silence Therapeutics GmbH: Employment. Frauendorf:Silence Therapeutics GmbH: Employment. Schubert:Silence Therapeutics GmbH: Employment. Aleku:Silence Therapeutics GmbH: Employment. Zügel:Silence Therapeutics GmbH: Employment.

Liver is a heterogeneous organ central for the control of systemic iron homeostasis. Among the different cell types, only hepatocytes (parenchymal cells) produce the iron regulatory hormone hepcidin. Hepcidin binds to the iron exporter ferroportin and triggers its degradation , thus modulating iron absorption from the diet and iron release from macrophagesdistrib. The iron-mediated hepcidin response is controlled via the SMAD1/5/8 pathway which is activated by bone morphogenetic proteins (BMPs). In contrast to hepcidin, BMPs are mainly produced by liver non-parenchymal cells, questioning the exclusive role of hepatocytes in regulating iron homeostasis. In particular, liver sinusoidal endothelial cells (LSECs) are emerging as cells that sense iron availability and modulate hepcidin levels through the production of BMP2 and BMP6 - the main regulators of hepcidin. However, how LSECs sense iron and which signals control BMP2 and BMP6 production have to be clarified. We have generated a mouse model with heterozygous depletion of the ferroportin allele (Slc40a1wt/trp). These mice show normal haematological parameters, serum iron levels and transferrin saturation. However, the liver is iron deficient and expresses low levels of hepcidin. The phosphorylation of SMAD1/5/8 proteins is lower in Slc40a1wt/trp compared to wild type littermates, explaining the hepcidin phenotype. To further investigate the molecular mechanism underlying hepcidin downregulation mediated by hepatic iron deficiency, we established a protocol to isolate hepatocytes and LSECs from total mouse liver and analyzed expression of iron-related genes. BMP6 is downregulated in LSECs of Slc40a1wt/trp mice, explaining the diminished activity of the SMAD1/5/8 signaling pathway. We next examined intracellular iron levels in hepatocytes and LSECs by measuring ferritin (Ft) and transferrin receptor 1 expression (Tfr1), two genes whose expression is regulated via the IRE/IRP system according to intracellular iron stores. Hepatocytes have an iron-deficient molecular signature, with low levels of Ft and a high expression of Tfr1. Surprisingly in LSECs both Ft and Tfr1 are unchanged, suggesting that this cellular population has unaltered iron levels. Taken together, our results show for the first time that decreased hepatic iron content is self-sufficient to cause a dramatic reduction in hepcidin expression and secretion. The hepcidin downregulation in the Slc40a1wt/trp mouse model is caused by a reduction in BMP6 levels that correlates with hepatocyte iron content. BMP6 modulation cannot be explained either by differences in circulating iron or by altered intracellular iron levels of LSECs, suggesting that BMP6 is not regulated by LSEC-mediated iron sensing. In our mouse model BMP6 seems to be linked with the intracellular iron content of hepatocytes. Therefore, we hypothesize that hepatocytes are the sensor of liver iron content which controls BMP6 expression in LSECs. RNAseq analysis on hepatocytes from Slc40a1wt/trp mice has been performed to identify underlying mechanisms. Disclosures Muckenthaler: Novartis: Research Funding.

Transferrin receptor 2 (TFR2), the type 3 hemochromatosis gene, is an activator of the iron hormone hepcidin in the liver and a partner of erythropoietin (EPO) receptor in erythroid cells. The loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity inducing erythrocytosis in mice (Nai et al, Blood 2015). We explored whether deletion of BM Tfr2 improves anemia and ineffective erythropoiesis in β-thalassemias, iron-loading anemias due to recessive β-globin gene mutations. We generated thalassemic mice (Hbbth3/+) with selective BM inactivation of Tfr2 (Tfr2BMKO/Hbbth3/+) through BM transplantation (BMT). Deletion of BM Tfr2 ameliorates RBC morphology with consistent and persistent increase of RBC count and Hb levels in thalassemic mice, accompanied by reduced iron accumulation. Around 22 weeks after BMT the improvement fades in double mutantanimals: Hb levels return comparable to those of Hbbth3/+mice, while RBC count persists higher. Anemia improvement in double mutant mice reduces serum EPO levels and improves erythropoiesis, in particular 22 weeks after BMT. Overall these data prove that the loss of the beneficial effect of deleting Tfr2 is not accounted for by erythropoiesis failure, but likely by exhaustion of splenic iron consumed by the enhanced erythropoiesis. In order to elucidate the molecular mechanisms of the phenotype improvement, we investigated whether the EPO-EPOR signaling pathway is overactive, as occurs in Tfr2 null erythroid cells (Nai et al, Blood 2015). Taking into account that Tfr2BMKO/Hbbth3/+ mice have lower serum EPO than Hbbth3/+, the expression levels of target genes of the EPOR-JAK2-STAT5 (Erfe and Bcl-xl) and of EPOR-PI3K-AKT pathway (Fasl, Epor and Ccng2) is consistent with the signaling being inappropriately active in double mutant mice. To start unraveling the global molecular/cellular processes underlying the remarkable phenotype amelioration, we performed RNAseq analysis on spleen samples from double mutant and Hbbth3/+ control mice at the time point of maximal erythropoiesis improvement (22 weeks post BMT). Spleens are enlarged in both genotypes with about 80% Ter119+ (erythroid) cells in both. In total we identified 2796 genes (1997 protein coding) differentially regulated between the two genotypes. The analysis of iron-related genes reveals a strong reduction of the expression of the iron exporter Fpn, Hmox1 and Alas2, suggestive of decreased hemolysis and/or of free heme accumulation in double mutants. Gene ontology analysis reveals enrichment of genes involved in cell cycle and proliferation, mitochondrial function, as well as proteasome activity and of most of the antioxidant targets (Sod1, Sod2, Fth1, Txn1, Txn2, Gstpi) of the canonical NF-kB pathway. Underrepresented genes are those involved in lipid handling, leukocyte/lymphocyte differentiation and coagulation. In summary, the RNAseq patterns indicate an increased spleen erythroid commitment and a mitochondrial metabolic shift, similar to the shift occurring during hematopoietic development to sustain erythroid proliferation and differentiation. We speculate that this effect is mediated by the enhanced EPO sensitivity. Interestingly EPO directly stimulates mitochondrial genes expression in adipocytes. Also the increased proteasome activity may significantly contribute to the improved erythropoiesis, since proteasomal degradation is required in the process of erythroblast enucleation. Finally, the activation of the NF-kB antioxidant response may contrast ROS increase and limit ineffective erythropoiesis. In conclusion, targeting erythroid TFR2 might become a novel erythropoiesis stimulating approach, worth to be tested in other forms of anemia. Disclosures Muckenthaler: Novartis: Research Funding. Camaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Hepcidin is a 25-residue hepatic peptide that regulates iron absorption from the diet and tissue iron distribution. Inappropriately low Hepcidin expression is implicated in the pathogenesis of hereditary hemochromatosis and iron-loading anemias, like the thalassemias. Increased hepcidin expression mediates iron retention in the anemias of inflammation and plays a pathogenic role in iron-refractory iron-deficiency anemia (IRIDA). Because of its clinical importance, Hepcidin is expected to be a useful biomarker for diagnosis and management of iron-related disorders. So far an ELISA for human hepcidin and SELDI-TOF-MS based approaches have been applied to monitor urinary and/or serum hepcidin levels. Here we report a modified protocol for SELDI-TOF based detection of human, urinary hepcidin. We show that CM10 Proteinchips are superior to NP20 Proteinchips commonly used in previously reported protocols to sensitively and accurately detect urinary hepcidin. Application of this modified hepcidin assay accurately detects increased hepcidin levels in the urine of sepsis patients.

High Mobility Group A (HMGA) is a family of architectural nuclear factors which play an important role in neoplastic transformation. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes including transcription. HMGA localization is exclusively nuclear but, to date, the mechanism of nuclear import for these proteins remains unknown. Here, we report the identification and characterization of a nuclear localization signal (NLS) for HMGA2, a member of the HMGA family. The NLS overlaps with the second of the three AT-hooks, the DNA-binding domains characteristic for this group of proteins. The functionality of this NLS was demonstrated by its ability to target a heterologous beta-galactosidase/green fluorescent protein fusion protein to the nucleus. Mutations to alanine of basic residues within the second AT-hook resulted in inhibition of HMGA2 nuclear localization and impairment of its function in activating the cyclin A promoter. In addition, HMGA2 was shown to directly interact with the nuclear import receptor importin-alpha2 via the second AT-hook. HMGA proteins are overexpressed and rearranged in a variety of tumors; our findings can thus help elucidating their role in neoplastic transformation.

All cell types of the lung, including airways, parenchyma and inflammatory cells need iron for metabolic processes. However, they must prevent iron overload which causes cellular damage due to the generation of reactive oxygen species (ROS). The risk for oxidative damage in the lung is exacerbated by its continuous exposure to high oxygen levels making iron detoxification within airway epithelial cells and alveolar macrophages an essential mechanism to prevent lung failure. The aim of our study is to determine the (patho)physiological consequences of unbalanced iron homeostasis in the lung. For this purpose, we analyzed a mouse model with a disruption in the hepcidin/ferroportin circuitry (FPN C326S mice), the regulatory system that controls systemic iron levels. Biochemical and histological analysis of the lung demonstrates that FPN C326S mice exhibit an age dependent increase in pulmonary iron content localized in ciliated airway epithelial cells, alveolar macrophages, alveolar type II cells and smooth muscle cells surrounding pulmonary arteries. Increased pulmonary iron levels correlate with an increase in ROS-mediated lipid peroxidation, suggesting that iron-mediated oxidative stress could contribute to the pathogenesis of lung diseases. To investigate this hypothesis, we performed measurements of lung function and blood oxygen saturation. These analyses revealed signs of restrictive lung disease in FPN C326S mice, with a decrease in total lung capacity and lung compliance. Interestingly, when compared with wild-type littermates, FPN C326S mice present a significant reduction in blood oxygen saturation. Experiments are ongoing to better understand the development of restrictive lung disease and decreased blood oxygen saturation. *Presenting author

Elevated serum ferritin contributes to treatment-related morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The multicenter DE02 trial assessed the safety, efficacy and impact of deferasirox on iron homeostasis after allogeneic HSCT. Deferasirox was administered at a starting dose of 10 mg/kg per day to 76 recipients of allogeneic HSCT, with subsequent dose adjustments based on efficacy and safety. Deferasirox was initiated at a median of 168 days after HSCT, with 84% of patients still on immunosuppression. Baseline serum ferritin declined from 2045 to 957 ng/mL. Deferasirox induced a negative iron balance in 84% of patients. Hemoglobin increased in the first 3 months, and trough serum cyclosporine levels were stable. Median exposure was 330 days, with a median compliance rate of >80%. The most common investigator-reported drug-related adverse events (AEs) were increased blood creatinine (26.5%), nausea (9.0%) and abdominal discomfort (8.3%). Fifty-four (71.1%) patients experienced drug-related AEs, which occasionally resulted in discontinuation (gastrointestinal (n=6), skin (n=3), elevated transaminases (n=1) and creatinine (n=1)). The incidence of AEs appeared to be dose related, with 7.5 mg/kg per day being the best-tolerated dose. Low-dose deferasirox is an effective chelation therapy after allogeneic HSCT, with a manageable safety profile, even in patients receiving cyclosporine.

Low iron availability enhances hypoxic pulmonary vasoconstriction (HPV). Considering that reduced serum iron is caused by increased erythropoiesis, insufficient reabsorption, or elevated hepcidin levels, one might speculate that exaggerated HPV in high-altitude pulmonary edema (HAPE) is related to low serum iron. To test this notion we measured serum iron and hepcidin in blood samples obtained in previously published studies at low altitude and during 2 days at 4,559 m (HA1, HA2) from controls, individuals with HAPE, and HAPE-susceptible individuals where prophylactic dexamethasone and tadalafil prevented HAPE. As reported, at 4,559 m pulmonary arterial pressure was increased in healthy volunteers but reached higher levels in HAPE. Serum iron levels were reduced in all groups at HA2. Hepcidin levels were reduced in all groups at HA1 and HA2 except in HAPE, where hepcidin was decreased at HA1 but unexpectedly high at HA2. Elevated hepcidin in HAPE correlated with increased IL-6 at HA2, suggesting that an inflammatory response related to HAPE contributes to increased hepcidin. Likewise, platelet-derived growth factor, a regulator of hepcidin, was increased at HA1 and HA2 in controls but not in HAPE, suggesting that hypoxia-controlled factors that regulate serum iron are inappropriately expressed in HAPE. In summary, we found that HAPE is associated with inappropriate expression of hepcidin without inducing expected changes in serum iron within 2 days at HA, likely due to too short time. Although hepcidin expression is uncoupled from serum iron availability and hypoxia in individuals developing HAPE, our findings indicate that serum iron is not related with exaggerated HPV.