Lysosomal iron recycling in mouse macrophages is dependent upon both reductases LcytB and Steap3

Iron that is stored in macrophages as ferritin can be made bioavailable by degrading ferritin in the lysosome and releasing iron back into the cytosol. Iron stored in ferritin is found as Fe3+ and must be reduced to Fe2+ before it can be exported from the lysosome. Here we report that the lysosomal reductase Cyb561a3 (LcytB) and the endosomal reductase Six-transmembrane epithelial antigen of the prostate 3 (Steap3) act as lysosomal ferrireductases in the mouse macrophage cell line RAW264.7 converting Fe3+ to Fe2+ for iron recycling. We determined that when lysosomes were loaded with horse cationic ferritin, reductions or loss of LcytB or Steap3 using CrispR/Cas9-mediated knockout technology resulted in decreased lysosomal iron export. Loss of both reductases was additive in decreasing lysosomal iron export. Decreased reductase activity resulted in increased transcripts for iron acquisition proteins DMT1 and Tfrc1 suggesting cells were iron limited. We show transcript expression of LcytB and Steap3 is decreased in macrophages exposed to Escherichia coli pathogen UTI89 supporting a role for these reductases in regulating iron availability for pathogens. We further show that loss of LcytB and Steap3 in macrophages infected with UTI89, led to increased intracellular UTI89 proliferation suggesting that the endolysosomal system is retaining Fe3+ that can be used for intravesicular pathogen proliferation. Together, our findings reveal an important role for both LcytB and Steap3 in macrophage iron recycling and suggest that limiting iron recycling by decreasing expression of endolysosomal reductases is an innate immune response to protect against pathogen proliferation and sepsis.

Gut mélange à trois: fluctuating selection modulated by microbiota, host immune system, and antibiotics

Iron is critical in host-microbe interactions, and its availability is under tight regulation in the mammalian gut. Antibiotics and inflammation are known to perturb iron availability in the gut, which could subsequently alter host-microbe interactions. Here, we show that an adaptive allele of iscR, encoding a major regulator of iron homeostasis of Escherichia coli, is under fluctuating selection in the mouse gut. In vivo competitions in immune-competent, immune-compromised, and germ-free mice reveal that the selective pressure on an iscR mutant E. coli is modulated by the presence of antibiotics, other members of the microbiota, and the immune system. In vitro assays show that iron availability is an important mediator of the iscR allele fitness benefits or costs. We identify Lipocalin-2, a host's innate immune system protein that prevents bacterial iron acquisition, as a major host mechanism underlying fluctuating selection of the iscR allele. Our results provide a remarkable example of strong fluctuating selection acting on bacterial iron regulation in the mammalian gut.

Geospatial Distribution of Iron in Major Sugarcane Growing Soils of Sivagangai, Tamil Nadu, India

The present study was undertaken to assess the available DTPA iron status in the major sugarcane growing soils of Southern Sivangai district, Tamil Nadu, India. A total of 500 geo referenced surface (0-30 cm) were collected from five blocks viz., Kalaiyarkovil, Padamathur, Sivagangai, Thiruppachetty and Thiruppuvanam and analyzed for basic soil properties and available DTPA iron. Simple correlation was worked out to ascertain the degree of relationship between soil properties and available DTPA iron content of soil study area. The available DTPA iron in the entire sugarcane growing soils ranged from 2.95 to 5.79 mg kg-1, 2.11 to 4.31 mg kg-1, 3.49 to 5.59 mg kg-1, 1.99 to 5.66 mg kg-1 and 3.94 to 6.39 mg kg-1 in soil samples of Kalaiyarkovil, Padamathur, Sivagangai, Thiruppachetty and Thiruppuvanam respectively. In the soil samples from Kalaiyarkovil, Padamathur, Sivagangai, Thiruppachetty, and Thiruppuvanam, the results revealed that 52, 59, 55, 53, and 51 % of the soils were deficient in available iron and 33, 29, 35, 30 and 32 % of the soils were moderate in available iron, and 15, 12, 10,5 and 17 % of the soils were sufficient in available iron. As per the nutrient index study, the soils of study area recorded very low to low fertility rating for available iron and the mean nutrient index value (NIV) ranged from 1.42 to 1.64 in the soil of the study area. SOC and CEC were found to have a beneficial impact on iron availability, whereas EC and CaCO3 levels had a negative impact on DTPA iron availability.

Antarctic meltwater-induced dynamical changes in phytoplankton in the Southern Ocean

IIt has been suggested that the freshwater flux due to the recent melting of the Antarctic ice-sheet/shelf will suppress ventilation in the Southern Ocean. In this study, we performed idealized earth system simulations to examine the impacts of Antarctic meltwater on surface phytoplankton biomass in the Antarctic Ocean. The enhanced stratification due to the meltwater leads to a decrease in the surface nitrate concentration but an increase in the surface dissolved iron concentration. These changes are associated with the reduced upwelling of nitrate-rich deep water and the trapped iron exported from the terrestrial sediment. Because of the limited iron availability in the Southern Ocean, the trapped iron in surface water enhances the chlorophyll concentration in the open ocean. However, in the marginal sea along the Antarctic coastline where the iron is relatively sufficient, a nitrate reduction induces a chlorophyll decrease, indicating a regime shift from iron-limited to nitrate-limited conditions.

Complete Genome Sequences of Two Nosocomiicoccus ampullae Strains and a Growth-Adapted Mutant

Here, we present the circular and complete genome sequences of the Nosocomiicoccus ampullae isolate 19-00310 and type strain DSM 19163. To our knowledge, these represent the first complete, circular chromosomes in the entire genus. Sequencing of a growth-adapted mutant suggests iron availability as a factor for growth improvement.

Iron, Heme Synthesis and Erythropoietic Porphyrias: A Complex Interplay

Erythropoietic porphyrias are caused by enzymatic dysfunctions in the heme biosynthetic pathway, resulting in porphyrins accumulation in red blood cells. The porphyrins deposition in tissues, including the skin, leads to photosensitivity that is present in all erythropoietic porphyrias. In the bone marrow, heme synthesis is mainly controlled by intracellular labile iron by post-transcriptional regulation: translation of ALAS2 mRNA, the first and rate-limiting enzyme of the pathway, is inhibited when iron availability is low. Moreover, it has been shown that the expression of ferrochelatase (FECH, an iron-sulfur cluster enzyme that inserts iron into protoporphyrin IX to form heme), is regulated by intracellular iron level. Accordingly, there is accumulating evidence that iron status can mitigate disease expression in patients with erythropoietic porphyrias. This article will review the available clinical data on how iron status can modify the symptoms of erythropoietic porphyrias. We will then review the modulation of heme biosynthesis pathway by iron availability in the erythron and its role in erythropoietic porphyrias physiopathology. Finally, we will summarize what is known of FECH interactions with other proteins involved in iron metabolism in the mitochondria.

Erythroferrone Modulates Iron Distribution for Fetal Erythropoiesis

Erythroferrone (ERFE) is an erythroblast-derived regulator of iron metabolism, and its production increases during stress erythropoiesis. ERFE decreases expression of the iron-regulatory hormone hepcidin to enhance iron availability for erythropoiesis 1. Pregnancy requires a substantial increase in iron availability to sustain a dramatic increase in maternal RBC volume and support fetal development. Whether maternal or fetal ERFE plays a role in regulating iron homeostasis during pregnancy is unknown. In humans, maternal ERFE concentrations were elevated in anemic pregnancies at mid gestation and delivery 2. To define the role of ERFE during iron-replete or iron-deficient pregnancy, we utilized Erfe transgenic (ETg) 3 and Erfe knockout (EKO) 1 mice. Maternal iron status of ETg, WT and EKO mice was altered by placing animals on adequate iron (100ppm) or low iron (4ppm) diet 2 weeks prior to and throughout pregnancy. ETg and WT dams were mated with WT sires to generate ETg and WT embryos while EKO dams were mated with EKO sires to generate EKO embryos. Analysis was performed at embryonic day 18.5. To examine the effect of pregnancy on ERFE expression, we compared non-pregnant females to WT dams at E18.5. Serum ERFE was mildly elevated from 0.01 to 0.2 ng/mL in iron-replete dams, but substantially elevated from 0.01 to 3.1 ng/mL in iron-deficient dams, similarly to human pregnancy 2. We next assessed iron and hematological parameters in pregnant dams with different Erfe genotypes. Under iron-replete conditions, all three groups had similar serum hepcidin, serum iron and hemoglobin concentrations, but ETg dams had 3-fold higher liver iron than WT and EKO dams, presumably because they are mildly iron-overloaded before pregnancy. On iron-deficient diet, maternal hepcidin was decreased in all three genotypes but more so in ETg dams; however, all three Erfe genotypes had similarly depleted liver iron stores, hypoferremia and anemia. MCV was the only parameter that was decreased in EKO compared to WT dams under both iron conditions. Overall, maternal ERFE played a minor role in regulation of maternal erythropoiesis and iron homeostasis, with the lack of ERFE resulting in smaller RBCs but not anemia. Among embryos, we observed a significant effect of Erfe genotype on embryo hepcidin. ETg embryos had significantly lower liver hepcidin compared to WT embryos under both iron-replete and iron-deficient conditions. Conversely, Erfe KO embryos had higher hepcidin compared to WTs under iron-deficient conditions, indicating that embryo ERFE regulates embryo hepcidin during pregnancy. Under iron-replete conditions however, all three embryo genotypes had similar hematologic parameters, and embryo liver iron was dependent on maternal iron levels, with both ETg and WT embryos from ETg dams having increased liver iron concentrations, indicating that embryo ERFE does not regulate placental iron transfer. Under iron-deficient conditions, there was no difference between ETg and WT embryos in hematological or iron parameters, and both genotypes developed iron deficiency and anemia. However, Erfe KO embryos, which had elevated hepcidin, had maldistribution of iron and worse anemia. EKO embryo liver iron concentrations were 6-fold higher compared to WT iron-deficient embryos, whereas hemoglobin was significantly decreased compared to WT iron-deficient embryos. These findings indicate that under iron-limiting conditions, embryo ERFE is important for the suppression of embryo hepcidin to ensure iron redistribution for embryo erythropoiesis. In summary, during iron replete pregnancy, ERFE plays a minor role in maternal and fetal iron homeostasis and erythropoiesis. However, in response to iron-deficiency anemia during pregnancy, ERFE is important for the redistribution of iron within the embryo to support embryo erythropoiesis. 1Kautz L et al, Nat Genet, 2014 2Delaney K et al, Curr Dev Nutr, 2020 3Coffey R et al, Blood, 2020 Disclosures Ganz: Ambys: Consultancy; Sierra Oncology: Consultancy, Research Funding; Rockwell: Consultancy; Pharmacosmos: Consultancy; Ionis: Consultancy; Protagonist: Consultancy; Intrinsic LifeSciences: Consultancy; RallyBio: Consultancy; Silence Therapeutics: Consultancy; Silarus Pharma: Consultancy; Alnylam: Consultancy; American Regent: Consultancy; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZenecaFibrogen: Consultancy; Global Blood Therapeutics: Consultancy; Gossamer Bio: Consultancy; Akebia: Consultancy, Honoraria. Nemeth: Silarus Pharma: Consultancy; Intrinsic LifeSciences: Consultancy; Protagonist: Consultancy; Vifor: Consultancy; Ionis: Consultancy.

Rebalancing Iron Homeostasis and Erythropoiesis through Tfr2 Inhibition for Correction of Anemia in CKD

Background Transferrin Receptor 2 (TFR2) is a protein expressed in the liver and in the erythroid compartment. Hepatic TFR2 activates the transcription of hepcidin, the master regulator of iron homeostasis, and its inactivation causes iron overload. Erythroid TFR2 interacts with Erythropoietin (EPO) receptor and its deletion enhances erythropoiesis increasing EPO sensitivity of erythroid cells. We recently demonstrated that bone marrow (BM) Tfr2 loss improves anemia in a murine model of β-thalassemia. We hypothesized that the same approach might represent a therapeutic option also for anemias due to insufficient EPO production, as anemia of Chronic Kidney Disease (CKD). Indeed, anemia is a common complication of CKD, since EPO production is inhibited by progressive renal failure. In addition, chronic inflammation that parallels renal damage decreases EPO responsiveness of erythroid cells and enhances the production of hepcidin. Increased hepcidin levels limit iron absorption from the diet and release from the stores, reducing iron supply to erythropoiesis. All these factors contribute to anemia development. Replacement therapy with erythropoiesis stimulating agents (ESA), usually combined with iron supplementation, is effective but may lead to cardio-vascular side effects. Thus, novel and more specific strategies are needed. Aims Here, we exploit different murine models of selective Tfr2 inactivation to test whether Tfr2 targeting corrects anemia of CKD. In this context, BM Tfr2 inhibition is expected to stimulate erythropoiesis and the simultaneous downregulation of hepatic Tfr2 to correct iron deficiency. Methods We induced CKD using an adenine-rich diet in mice with: (1) BM-specific Tfr2 deletion (Tfr2 BMKO), generated through BM transplantation; (2) reduced Tfr2 hepatic expression, obtained treating wild-type mice with anti-Tfr2 antisense oligonucleotides (Tfr2-ASO); (3) germline Tfr2 inactivation in the whole organism (Tfr2KO). Results Renal damage was comparable among all the mice analyzed, excluding a differential effect of the diet in the various groups. Tfr2BMKO mice showed enhanced erythropoiesis relative to controls, due to the increased EPO responsiveness of erythroid cells lacking Tfr2, as suggested by the over-activation of the EPO-EPOR signaling pathway despite comparable EPO levels. Tfr2BMKO mice maintained higher red blood cell (RBC) count than controls for the entire protocol. Hemoglobin (Hb) levels, higher in Tfr2BMKO mice for 6 weeks, reached levels of controls at 8 weeks, concomitant with relative hypoferremia. These results indicate that BM Tfr2 deletion transiently prevents anemia until iron availability is adequate to the enhanced erythropoiesis. Then we investigated the potential beneficial effect of increasing iron availability through Tfr2-ASOs treatment, which efficiently decreased hepatic (95-97%) but not erythroid Tfr2. As expected, circulating iron levels were increased in Tfr2-ASO mice, maintaining RBC count and Hb levels in the normal range for the first 2 weeks of treatment. However, Hb and RBCs reverted to control levels at 6 weeks, before the end of the protocol. These results show that increased iron availability alone, due to hepatic Tfr2 downregulation, delays anemia development but is not sufficient to boost erythropoiesis on a long term. In agreement, Tfr2KO mice, with Tfr2 inactivation both in the liver and in the erythroid compartment, maintained higher RBC count and Hb levels compared to controls until the end of the protocol. Conclusions The concomitant targeting of hepatic and erythroid Tfr2, here obtained through Tfr2 germline genetic inactivation, is necessary and sufficient to ameliorate anemia of CKD. On the contrary selective BM Tfr2 deletion that enhances EPO responsiveness of erythroid cells, or hepatic Tfr2 downregulation that increases iron availability, do not correct anemia in a long term. Therefore, a specific approach able to inhibit both hepatic and erythroid Tfr2 could adjust iron availability according to the enhanced erythropoiesis, correcting both drivers of anemia development in CKD. The development of a pharmacologic tool to downregulate Tfr2 might become an alternative to the standard treatment with ESAs plus iron supplementation with limited off-target effects due to the restricted TFR2 expression. Disclosures Aghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.