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ZIP8 (SLC39A8) is a transmembrane divalent metal ion importer that is most highly expressed in the lung and is inducible by inflammatory stimuli. In addition to zinc and manganese, ZIP8 can transport iron, but its specific roles in iron regulation during homeostatic and pathologic processes remain poorly understood. Using a novel global inducible ZIP8 knockout (KO) mouse, we analyzed the role of ZIP8 in steady-state iron homeostasis and during inflammation and infection. We observed an unexpected phenotype of elevated spleen iron levels and decreased serum iron in ZIP8 KO mice, suggesting that ZIP8 plays a role in iron recycling. We also showed that ZIP8 is expressed on lung distal airspace epithelial cells and transports iron from the airway into lung tissue. LPS-induced inflammation induced ZIP8 expression in the lung, but ZIP8 deletion had no detrimental effect on the severity of LPS-induced acute lung injury or on the outcomes of Klebsiella pneumoniae lung infection. Thus, ZIP8 plays a role in systemic iron homeostasis but does not modulate the severity of inflammatory lung injury or the host defense against a common bacterial cause of pneumonia.

•ZIP8 knockout mice have elevated spleen iron levels and relative hypoferremia, suggesting that ZIP8 plays a role in iron recycling.•Although lung ZIP8 is induced with inflammation, ZIP8 deletion in mice did not exacerbate acute lung injury or bacterial pneumonia.

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 Fpn1in macrophages by crossing Fpn1-floxed animals with macrophage-targeted LysM-Creor F4/80-Cretransgenic mice. Macrophage Fpn1deletion 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 Fpn1LysM/LysMmice 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 Fpn1LysM/LysMmacrophages lacking Fpn1. These studies demonstrate that Fpn1 plays important roles in macrophage iron release in vivo and in modulating innate immune 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 Fpn1LysM/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 Fpn1LysM/LysM macrophages lacking Fpn1. These studies demonstrate that Fpn1 plays important roles in macrophage iron release in vivo and in modulating innate immune responses.

The expression of ferroportin1 (FPN1) in reticuloendothelial macrophages supports the hypothesis that this iron-export protein participates in iron recycling from senescent erythrocytes. To gain insight into FPN1's role in macrophage iron metabolism, we examined the effect of iron status and erythrophagocytosis on FPN1 expression in J774 macrophages. Northern analysis indicated that FPN1 mRNA levels decreased with iron depletion and increased on iron loading. The iron-induced induction of FPN1 mRNA was blocked by actinomycin D, suggesting that transcriptional control was responsible for this effect. After erythrophagocytosis, FPN1 mRNA levels were also up-regulated, increasing 8-fold after 4 hours and returning to basal levels by 16 hours. Western analysis indicated corresponding increases in FPN1 protein levels, with maximal induction after 10 hours. Iron chelation suppressed FPN1 mRNA and protein induction after erythrophagocytosis, suggesting that FPN1 induction results from erythrocyte-derived iron. Comparative Northern analyses of iron-related genes after erythrophagocytosis revealed a 16-fold increase in FPN1 levels after 3 hours, a 10-fold increase in heme oxygenase-1 (HO-1) after 3 hours, a 2-fold increase in natural resistance macrophage-associated protein 1 (Nramp1) levels after 6 hours, but no change in divalent metal ion transporter 1 (DMT1) levels. The rapid and strong induction of FPN1 expression after erythrophagocytosis suggests that FPN1 plays a role in iron recycling. (Blood. 2003;102:4191-4197)

The expression of ferroportin1 (FPN1) in reticuloendothelial macrophages supports the hypothesis that this iron-export protein participates in iron recycling from senescent erythrocytes. To gain insight into FPN1's role in macrophage iron metabolism, we examined the effect of iron status and erythrophagocytosis on FPN1 expression in J774 macrophages. Northern analysis indicated that FPN1 mRNA levels decreased with iron depletion and increased on iron loading. The iron-induced induction of FPN1 mRNA was blocked by actinomycin D, suggesting that transcriptional control was responsible for this effect. After erythrophagocytosis, FPN1 mRNA levels were also up-regulated, increasing 8-fold after 4 hours and returning to basal levels by 16 hours. Western analysis indicated corresponding increases in FPN1 protein levels, with maximal induction after 10 hours. Iron chelation suppressed FPN1 mRNA and protein induction after erythrophagocytosis, suggesting that FPN1 induction results from erythrocyte-derived iron. Comparative Northern analyses of iron-related genes after erythrophagocytosis revealed a 16-fold increase in FPN1 levels after 3 hours, a 10-fold increase in heme oxygenase-1 (HO-1) after 3 hours, a 2-fold increase in natural resistance macrophage-associated protein 1 (Nramp1) levels after 6 hours, but no change in divalent metal ion transporter 1 (DMT1) levels. The rapid and strong induction of FPN1 expression after erythrophagocytosis suggests that FPN1 plays a role in iron recycling. (Blood. 2003;102:4191-4197)

The measurement of ethane and pentane in breath offers a sensitive and noninvasive means to assessin vivolipid peroxidation in animals and humans. However, numerous technical obstacles inherent in collecting and concentrating air-breath samples have limited the wider application of these measurements for the assessment ofin vivolipid peroxidation. We have developed a relatively simple, inexpensive, rapid, and reliable method to collect, concentrate, and measure breath ethane and total-body pentane from rats. This method, which concentrates alkanes from 4 liters of collected air-breath on adsorbant cooled to −174°C, was found to be superior to similar cryofocusing techniques at −130°C, which fail to effectively trap highly volatile ethane from large volumes of air. We found ethane evolves predominantly through breath, whereas a significant amount of pentane evolves from sources other than breath. Mean evolution rate for ethane was 1.08 pmol/100 g body wt/min. Pentane evolution rates displayed more inter-rat and day-to-day variability with a mean of 0.52 pmol/100 g body wt/min. We also found that excreted rat feces exude large amounts of ethane and pentane.

We investigated the influence of the macrophage iron exporter ferroportin and its ligand hepcidin on intracellular Salmonella growth. Elevated ferroportin expression inhibited bacterial multiplication; hepcidin-induced ferroportin down-regulation enhanced it. Expression analysis of iron-responsive Salmonella genes indicated ferroportin-mediated iron deprivation. These results demonstrate a role for ferroportin in antimicrobial resistance.

ABSTRACTWe investigated the influence of the macrophage iron exporter ferroportin and its ligand hepcidin on intracellular Salmonellagrowth. Elevated ferroportin expression inhibited bacterial multiplication; hepcidin-induced ferroportin down-regulation enhanced it. Expression analysis of iron-responsive Salmonellagenes indicated ferroportin-mediated iron deprivation. These results demonstrate a role for ferroportin in antimicrobial resistance.