Yunlong Tao

Ferroportin1 deficiency in mouse macrophages impairs iron homeostasis and inflammatory responses

Systemic iron requirements are met predominantly through the recycling of iron from senescent erythrocytes by macrophages, a process in which the iron exporter ferroportin (Fpn1) is considered to be essential. Yet the role of Fpn1 in macrophage iron recycling and whether it influences innate immune responses are poorly understood in vivo. We inactivated Fpn1 in macrophages by crossing Fpn1-floxed animals with macrophage-targeted LysM-Cre or F4/80-Cre transgenic mice. Macrophage Fpn1 deletion mice were overtly normal; however, they displayed a mild anemia and iron accumulation in splenic, hepatic, and bone marrow macrophages when fed a standard diet. Iron loading was exacerbated after the administration of iron dextran or phenylhydrazine. When Fpn1(LysM/LysM) mice were challenged with an iron-deficient diet, they developed a more severe anemia and strikingly higher splenic iron levels than control mice, indicating significantly impaired iron mobilization from macrophages. Because immune responses can be altered by modulating iron status, we also examined the expression of proinflammatory cytokines. We found that expression levels of TNF-α and IL-6 were significantly enhanced in Fpn1(LysM/LysM) macrophages lacking Fpn1. These studies demonstrate that Fpn1 plays important roles in macrophage iron release in vivo and in modulating innate immune responses.

Ferroportin1 in hepatocytes and macrophages is required for the efficient mobilization of body iron stores in mice

The liver is a major site of iron storage where sequestered iron can be actively mobilized for utilization when needed elsewhere in the body. Currently, hepatocyte iron efflux mechanisms and their relationships to macrophage iron recycling during the control of whole‐body iron homeostasis are unclear. We hypothesized that the iron exporter, ferroportin1 (Fpn1), is critical for both iron mobilization from hepatocytes and iron recycling from macrophages. To test this, we generated hepatocyte‐specific Fpn1 deletion mice (Fpn1Alb/Alb) and mice that lacked Fpn1 in both hepatocytes and macrophages (Fpn1Alb/Alb;LysM/LysM). When fed a standard diet, Fpn1Alb/Alb mice showed mild hepatocyte iron retention. However, red blood cell (RBC) counts and hemoglobin (Hb) levels were normal, indicating intact erythropoiesis. When fed an iron‐deficient diet, Fpn1Alb/Alb mice showed impaired liver iron mobilization and anemia, with much lower RBC and Hb levels than Fpn1flox/flox mice on the same diet. Using a strategy where mice were preloaded with differing amounts of dietary iron before iron deprivation, we determined that erythropoiesis in Fpn1Alb/Alb and Fpn1flox/flox mice depended on the balance between storage iron and iron demands. On a standard diet, Fpn1Alb/Alb;LysM/LysM mice displayed substantial iron retention in hepatocytes and macrophages, yet maintained intact erythropoiesis, implying a compensatory role for intestinal iron absorption. In contrast, when Fpn1Alb/Alb;LysM/LysM mice were fed an iron‐deficient diet, they developed severe iron‐deficiency anemia, regardless of their iron storage status. Thus, Fpn1 is critical for both hepatocyte iron mobilization and macrophage iron recycling during conditions of dietary iron deficiency. Conclusion: Our data reveal new insights into the relationships between Fpn1‐mediated iron mobilization, iron storage, and intestinal iron absorption and how these processes interact to maintain systemic iron homeostasis. (HEPATOLOGY 2012;56:961–971)

Perturbed iron distribution in Alzheimer's disease serum, cerebrospinal fluid, and selected brain regions: a systematic review and meta-analysis

BACKGROUND The homeostasis and physiological role of iron in Alzheimers disease (AD) has been debated for decades. Overall, it has been difficult to reach a consensus to prove marked disease-associated changes in the iron content of the AD brain, blood, or cerebrospinal fluid (CSF). OBJECTIVES We sought to contribute to resolve this issue by quantifying the iron content in serum, CSF, and sub-regions of the AD brain. METHODS We conducted a comprehensive systematic meta-analysis and review of multiple observational studies till October 2013 that investigated the iron content in AD serum, CSF, or brain tissue. RESULTS 2,556 publications were screened. Forty-three eligible studies with 1,813 AD patients and 2,401 healthy controls were identified. Twenty-one studies investigated the serum iron in AD while seven and nineteen studies investigated the CSF iron and various brain regions iron respectively. Our meta-analysis showed that serum iron was significant lower in AD than healthy controls. CSF iron appeared not to be affected by AD although more studies are required due to the relative small number of CSF studies reported to date. We critically analyzed iron content in twelve selective brain regions by separated meta-analyses using cross-referenced statistical methods. We found that eight specific brain regions had higher iron concentrations that correlated with the clinical diagnosis of AD in a statistically validated manner. CONCLUSIONS These data provided rigorous statistical support for the model that iron homeostasis was changed in AD patients, including the finding of lower iron in their serum and evidence for iron overload in several specific brain regions.

Metalloreductase Steap3 coordinates the regulation of iron homeostasis and inflammatory responses

Background Iron and its homeostasis are intimately related to inflammatory responses, but the underlying molecular mechanisms are poorly understood. We investigated the role of Steap3 in regulating iron homeostasis in macrophages, and the effects of Steap3 depletion on host inflammatory responses. Design and Methods We analyzed bone marrow-derived macrophages and primary cultured hepatocytes from Steap3-/- mouse models to investigate the roles of Steap3 in coordinately regulating iron homeostasis and inflammatory responses. First, we examined iron distribution and iron status in cells deficient in Steap3, as well as the requirement for the Steap3 gene during inflammatory responses. Secondly, we analyzed the regulation of Steap3 expression by inflammatory stimuli and thus, the influence of these stimuli on iron distribution and homeostasis. Results We found that Steap3 mRNA was expressed at high levels in macrophages and hepatocytes. Steap3 deficiency led to impaired iron homeostasis, causing abnormal iron distribution and a decreased availability of cytosolic iron in macrophages. Among STEAP family members, Steap3 mRNA was uniquely down-regulated in macrophages stimulated by lipopolysaccharides. To determine whether Steap3 regulated iron homeostasis during inflammatory stress, we treated Steap3-/- mice with lipopolysaccharide, which produced greater iron accumulation in the vital tissues of these mice compared to in the tissues of wild-type controls. Furthermore, Steap3 depletion led to impaired induction of interferon-β, monocyte chemoattractant protein-5, and interferon induced protein-10 in macrophages via the TLR4-mediated signaling pathway. Conclusions Steap3 is important in regulating both iron homeostasis and TLR4-mediated inflammatory responses in macrophages. Steap3 deficiency causes abnormal iron status and homeostasis, which leads to impaired TLR4-mediated inflammatory responses in macrophages. Following inflammatory stimuli, Steap3 depletion causes dysregulated iron sequestration and distribution. Our results provide important insights into the function of Steap3 as a coordinate regulator of both iron homeostasis and innate immunity.

Screening Identifies the Chinese Medicinal Plant Caulis Spatholobi as an Effective HAMP Expression Inhibitor

Hepcidin, the pivotal regulator of iron metabolism, plays a critical role in multiple diseases including anemia of chronic disease and hemochromatosis. Recent studies have focused on identifying antagonists of hepcidin. We hypothesized that bioactive extracts from Chinese medicinal plants may be efficacious in the inhibition of expression of the hepcidin-encoding gene (HAMP) product, hepcidin. To test this, we measured the level of hepcidin expression in cultured cells treated with 16 different medicinal plant extracts, all of which are used to treat anemia-related disorders in traditional Chinese medicine. Among the extracts tested, that of Caulis Spatholobi (CS; also called Jixueteng, the stem of Spatholobus suberectus Dunn) showed the most potent inhibitory effect on HAMP expression in the Huh7 cell line and was therefore selected for further mechanistic study. In cells treated with 400 μg/mL of extract, phosphorylated mothers against decapentaplegic homolog proteins 1/5/8 levels were 80% less than those of controls (P < 0.001), and the inhibitory effect on interleukin-6-induced HAMP expression (65% inhibition) was weaker than the strong inhibition on bone morphogenetic protein 6-induced HAMP expression (97% inhibition). Seven-week-old C57BL/6 female mice were fed an AIN-76A diet containing 10.8% dried CS and then analyzed on d 0, 5, 10, or 15. On d 5, there was a 60% decrease in hepatic HAMP expression (P < 0.05), an 18% decrease in hepatic iron concentration, and a 100% increase in serum iron concentration (P < 0.05) compared with the d 0 group. In conclusion, we identify the extract of CS as a novel, potent HAMP expression inhibitor, which may be further modified and optimized to become a dietary supplement or a therapeutic option for the amelioration of hepcidin-overexpression-related diseases, including iron deficiency anemia.

MBD5 regulates iron metabolism via methylation‐independent genomic targeting of Fth1 through KAT2A in mice

Ferritin plays important roles in iron metabolism and controls iron absorption in the intestine. The ferritin subunits ferritin heavy chain (Fth1) and ferritin light chain (Ftl1) are tightly regulated at both the transcriptional and post‐transcriptional levels. However, mechanisms of maintaining stable, basal expression of Fth1 are poorly understood. Here, we show that global deletion of Mbd5 in mice induces an iron overload phenotype. Liver and serum iron levels in Mbd5−/− mice were 3·2‐fold and 1·5‐fold higher respectively, than wild‐type littermates; moreover, serum ferritin was increased >5‐fold in the Mbd5−/− mice. Mbd5 encodes a member of the methyl‐CpG binding domain family; however, the precise function of this gene is poorly understood. Here, we found that intestinal Fth1 mRNA levels were decreased in Mbd5−/− mice. Loss of Fth1 expression in the intestine could lead to iron over‐absorption. Furthermore, deleting Mbd5 specifically in the intestine resulted in a phenotype similar to that of conditional deletion of Fth1 mice. An Fth1 promoter‐report luciferase assay indicated that overexpression of Mbd5 enhanced Fth1 transcription in a dose‐dependent manner. Histone H4 acetylation of the Fth1 promoter was reduced in the intestine of Mbd5−/− mice and further analysis showed that histone acetyltransferase KAT2A was essential for MBD5‐induced Fth1 transcription.

Bmp6 expression can be regulated independently of liver iron in mice.

The liver is the primary organ for storing iron and plays a central role in the regulation of body iron levels by secretion of the hormone Hamp1. Although many factors modulate Hamp1 expression, their regulatory mechanisms are poorly understood. Here, we used conditional knockout mice for the iron exporter ferroportin1 (Fpn1) to modulate tissue iron in specific tissues in combination with iron-deficient or iron-rich diets and transferrin (Tf) supplementation to investigate the mechanisms underlying Hamp1 expression. Despite liver iron overload, expression of bone morphogenetic protein 6 (Bmp6), a potent-stimulator of Hamp1 expression that is expressed under iron-loaded conditions, was decreased. We hypothesized that factors other than liver iron must play a role in controlling Bmp6 expression. Our results show that erythropoietin and Tf-bound iron do not underlie the down-regulation of Bmp6 in our mice models. Moreover, Bmp6 was down-regulated under conditions of high iron demand, irrespective of the presence of anemia. We therefore inferred that the signals were driven by high iron demand. Furthermore, we also confirmed previous suggestions that Tf-bound iron regulates Hamp1 expression via Smad1/5/8 phosphorylation without affecting Bmp6 expression, and the effect of Tf-bound iron on Hamp1 regulation appeared before a significant change in Bmp6 expression. Together, these results are consistent with novel mechanisms for regulating Bmp6 and Hamp1 expression.

Hemojuvelin regulates the innate immune response to peritoneal bacterial infection in mice

Hereditary hemochromatosis and iron imbalance are associated with susceptibility to bacterial infection; however, the underlying mechanisms are poorly understood. Here, we performed in vivo bacterial infection screening using several mouse models of hemochromatosis, including Hfe (Hfe−/−), hemojuvelin (Hjv−/−), and macrophage-specific ferroportin-1 (Fpn1fl/fl;LysM-Cre+) knockout mice. We found that Hjv−/− mice, but not Hfe−/− or Fpn1fl/fl;LysM-Cre+ mice, are highly susceptible to peritoneal infection by both Gram-negative and Gram-positive bacteria. Interestingly, phagocytic cells in the peritoneum of Hjv−/− mice have reduced bacterial clearance, IFN-γ secretion, and nitric oxide production; in contrast, both cell migration and phagocytosis are normal. Expressing Hjv in RAW264.7 cells increased the level of phosphorylated Stat1 and nitric oxide production. Moreover, macrophage-specific Hjv knockout mice are susceptible to bacterial infection. Finally, we found that Hjv facilitates the secretion of IFN-γ via the IL-12/Jak2/Stat4 signaling pathway. Together, these findings reveal a novel protective role of Hjv in the early stages of antimicrobial defense.

Fine-Mapping and Genetic Analysis of the Loci Affecting Hepatic Iron Overload in Mice

The liver, as the major organ for iron storage and production of hepcidin, plays pivotal roles in maintaining mammalian iron homeostasis. A previous study showed that Quantitative Trait Loci (QTLs) on chromosome 7 (Chr7) and 16 (Chr16) may control hepatic non-heme iron overload in an F2 intercross derived from C57BL/6J (B6) and SWR/J (SWR) mice. In this study, we aimed to validate the existence of these loci and identify the genes responsible for the phenotypic variations by generating congenic mice carrying SWR chromosome segments expanding these QTLs (D7Mit68-D7Mit71 and D16Mit125-D16Mit185, respectively). We excluded involvement of Chr7 based on the lack of iron accumulation in congenic mice. In contrast, liver iron accumulation was observed in Chr16 congenic mice. Through use of a series of subcongenic murine lines the interval on Chr16 was further fine-mapped to a 0.8 Mb segment spanning 11 genes. We found that the mRNA expression pattern in the liver remained unchanged for all 11 genes tested. Most importantly, we detected 4 missense mutations in 3 candidate genes including Sidt1 (P172R), Spice1(R708S), Boc (Q1051R) and Boc (S450-insertion in B6 allele) in the liver of SWR homozygous congenic mice. To further delineate potential modifier gene(s), we reconstituted seven candidate genes, Sidt1, Boc, Zdhhc23, Gramd1c, Atp6v1a, Naa50 and Gtpbp8, in mouse liver through hydrodynamic transfection. However, we were unable to detect significant changes in liver iron levels upon reconstitution of these candidate genes. Taken together, our work provides strong genetic evidence of the existence of iron modifiers on Chr16. Moreover, we were able to delineate the phenotypically responsible region to a 0.8 Mb region containing 11 coding genes, 3 of which harbor missense mutations, using a series of congenic mice.