To better understand the tissue iron overload and anemia previously reported in a human patient and mice that lack heme oxygenase-1 (HO-1), we studied iron distribution and pathology in HO-1(Hmox1) ؊/؊ mice. We found that resident splenic and liver macrophages were mostly absent in HO-1 ؊/؊ mice. Erythrophagocytosis caused the death of HO-1 ؊/؊ macrophages in in vitro experiments, supporting the hypothesis that HO-1 ؊/؊ macrophages died of exposure to heme released on erythrophagocytosis. Rupture of HO-1 ؊/؊ macrophages in vivo and release of nonmetabolized heme probably caused tissue inflammation. In the spleen, initial splenic enlargement progressed to red pulp fibrosis, atrophy, and functional hyposplenism in older mice, recapitulating the asplenia of an HO-1-deficient patient. We postulate that the failure of tissue macrophages to remove senescent erythrocytes led to intravascular hemolysis and increased expression of the heme and hemoglobin scavenger proteins, hemopexin and haptoglobin. Lack of macrophages expressing the haptoglobin receptor, CD163, diminished the ability of haptoglobin to neutralize circulating hemoglobin, and iron overload occurred in kidney proximal tubules, which were able to catabolize heme with HO-2. Thus, in HO-1 ؊/؊ mammals, the reduced function and viability of erythrophagocytosing macrophages are the main causes of tissue damage and iron redistribution. IntroductionHumans and mice contain 2 well-characterized heme oxygenase (HO) enzymes: HO-1, which is inducible, and HO-2, which is constitutively expressed in most tissues. 1,2 HO metabolizes heme and releases free iron, carbon monoxide, and biliverdin, which quickly undergoes conversion to bilirubin. Red blood cells (RBCs) contain very high concentrations of hemoglobin (Hb), 3,4 but HO allows efficient recycling of the iron that is bound to Hb molecules in RBCs. On phagocytosis of senescent RBCs, macrophages increase their expression of HO-1 to efficiently degrade heme, and iron returns to the circulation through the iron exporter ferroportin. 5 Excess free heme is highly toxic in the circulation, 4 and protective systems exist that enable animals to avoid toxicity caused by free heme and free Hb. Hemopexin (Hpx) is a heme-binding serum protein that scavenges free heme in the circulation, 6,7 and haptoglobin (Hp) binds free Hb,8,9 whereupon the Hb-Hp complex is endocytosed through the CD163 receptor 10 and is metabolized by macrophages.Previous work in mouse models has shown that the lack of both HOs is embryonically lethal, whereas work on an HO-1 Ϫ/Ϫ mouse model 11,12 and a single HO-1-deficient human patient revealed that both the HO-1 Ϫ/Ϫ mouse and the human patient were anemic. 13 However, splenomegaly was described in the mouse model, 11,12 whereas hyposplenia was present in the human patient. 14 Hepatic and renal iron overload was observed in the patient and in mouse models, but the mechanism for iron redistribution was not clear. HO-1 deficiency was discovered in a single patient with hemolytic anemia when his physicians ...

Glutaredoxin 5 (GLRX5) deficiency has previously been identified as a cause of anemia in a zebrafish model and of sideroblastic anemia in a human patient. Here we report that GLRX5 is essential for iron-sulfur cluster biosynthesis and the maintenance of normal mitochondrial and cytosolic iron homeostasis in human cells. GLRX5, a mitochondrial protein that is highly expressed in erythroid cells, can homodimerize and assemble [2Fe-2S] in vitro. In GLRX5-deficient cells, [Fe-S] cluster biosynthesis was impaired, the iron-responsive element-binding (IRE-binding) activity of iron regulatory protein 1 (IRP1) was activated, and increased IRP2 levels, indicative of relative cytosolic iron depletion, were observed together with mitochondrial iron overload. Rescue of patient fibroblasts with the WT GLRX5 gene by transfection or viral transduction reversed a slow growth phenotype, reversed the mitochondrial iron overload, and increased aconitase activity. Decreased aminolevulinate δ, synthase 2 (ALAS2) levels attributable to IRP-mediated translational repression were observed in erythroid cells in which GLRX5 expression had been downregulated using siRNA along with marked reduction in ferrochelatase levels and increased ferroportin expression. Erythroblasts express both IRP-repressible ALAS2 and non-IRP-repressible ferroportin 1b. The unique combination of IRP targets likely accounts for the tissue-specific phenotype of human GLRX5 deficiency.

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesiclelike structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ϳ4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.All known positive-strand RNA viruses replicate their genomes in association with membranous structures that are formed after the synthesis of viral proteins in infected cells by remodeling membranes from existing intracellular organelles. Different viruses target different organelle membranes (e.g., endoplasmic reticulum [ER], Golgi, endosomes, and mitochondria) and generate different morphological structures on which the replication complexes assemble (37).Among the Picornavirus family members, RNA replication complexes from poliovirus-infected HeLa cells have been the best studied. Heterogeneously sized, vesicle-like structures that cluster in the perinuclear space in infected cells were observed by electron microscopy and described more than 40 years ago. Viral RNA replication occurs on the cytosolic surfaces of the vesicles, which aggregate into clusters (7-9). Little is known about how these structures are formed, although some recent observations have suggested that components of the cellular secretory and/or autophagy pathways are involved (24,39,41).The process of membrane traffic begins at the ER, where polio proteins appear to be synthesized. Rust et al. (36) have visualized, by three-dimensional reconstruction of serial confocal microscope sections, poliovirus protein 2B sequences, and presumably other viral proteins, budding from multiple sites on the ER and showed extensive colocalization of the 2B sequences with COPII coatamer protein.