Copper is an essential micronutrient that is necessary for healthy immune function. This requirement is underscored by an increased susceptibility to bacterial infection in copper-deficient animals; however, a molecular understanding of its importance in immune defense is unknown. In this study, we investigated the effect of proinflammatory agents on copper homeostasis in RAW264.7 macrophages. Interferon-␥ was found to increase expression of the high affinity copper importer, CTR1, and stimulate copper uptake. This was accompanied by copper-stimulated trafficking of the ATP7A copper exporter from the Golgi to vesicles that partially overlapped with phagosomal compartments. Silencing of ATP7A expression attenuated bacterial killing, suggesting a role for ATP7A-dependent copper transport in the bactericidal activity of macrophages. Significantly, a copper-sensitive mutant of Escherichia coli lacking the CopA copper-transporting ATPase was hypersensitive to killing by RAW264.7 macrophages, and this phenotype was dependent on ATP7A expression. Collectively, these data suggest that copper-transporting ATPases, CopA and ATP7A, in both bacteria and macrophage are unique determinants of bacteria survival and identify an unexpected role for copper at the hostpathogen interface.

Adult humans have about 25 trillion red blood cells (RBCs) and each second we recycle about five million RBCs by erythrophagocytosis (EP) in macrophages of the reticuloendothelial system. Despite the central role for EP in mammalian iron metabolism, the molecules and pathways responsible for heme trafficking during EP remain unknown. Here, we show that the mammalian homolog of HRG1, a transmembrane heme permease in C. elegans, is essential for macrophage iron homeostasis and transports heme from the phagolysosome to the cytoplasm during EP. HRG1 is strongly expressed in macrophages of the reticuloendothelial system and specifically localizes to the phagolysosomal membranes during EP. Depletion of Hrg1 in mouse macrophages causes attenuation of heme transport from the phagolysosomal compartment. Importantly, missense polymorphisms in human HRG1 are defective in heme transport. Our results reveal HRG1 as the long-sought heme transporter for heme-iron recycling in macrophages and suggest that genetic variations in HRG1 could be modifiers of human iron metabolism.

Copper is an essential nutrient for a variety of biochemical processes; however, the redox properties of copper also make it potentially toxic in the free form. Consequently, the uptake and intracellular distribution of this metal is strictly regulated. This raises the issue of whether specific pathophysiological conditions can promote adaptive changes in intracellular copper distribution. In this study, we demonstrate that oxygen limitation promotes a series of striking alterations in copper homeostasis in RAW264.7 macrophage cells. Hypoxia was found to stimulate copper uptake and to increase the expression of the copper importer, CTR1. This resulted in increased copper delivery to the ATP7A copper transporter and copper-dependent trafficking of ATP7A to cytoplasmic vesicles. Significantly, the ATP7A protein was required to deliver copper into the secretory pathway to ceruloplasmin, a secreted copperdependent enzyme, the expression and activity of which were stimulated by hypoxia. However, the activities of the alternative targets of intracellular copper delivery, superoxide dismutase and cytochrome c oxidase, were markedly reduced in response to hypoxia. Collectively, these findings demonstrate that copper delivery into the biosynthetic secretory pathway is regulated by oxygen availability in macrophages by a selective increase in copper transport involving ATP7A.

J. Neurochem. (2010) 114, 1630–1638. Abstract Alzheimer’s disease (AD) is characterized by progressive neurodegeneration associated with the aggregation and deposition of β‐amyloid (Aβ40 and Aβ42) peptide in senile plaques. Recent studies suggest that copper may play an important role in AD pathology. Copper concentrations are elevated in amyloid plaques and copper binds with high affinity to the Aβ peptide and promotes Aβ oligomerization and neurotoxicity. Despite this connection between copper and AD, it is unknown whether the expression of proteins involved in regulating copper homeostasis is altered in this disorder. In this study, we demonstrate that the copper transporting P‐type ATPase, ATP7A, is highly expressed in activated microglial cells that are specifically clustered around amyloid plaques in the TgCRND8 mouse model of AD. Using a cultured microglial cell line, ATP7A expression was found to be increased by the pro‐inflammatory cytokine interferon‐gamma, but not by TNF‐α or IL‐1β. Interferon‐gamma also elicited marked changes in copper homeostasis, including copper‐dependent trafficking of ATP7A from the Golgi to cytoplasmic vesicles, increased copper uptake and elevated expression of the CTR1 copper importer. These findings suggest that pro‐inflammatory conditions associated with AD cause marked changes in microglial copper trafficking, which may underlie the changes in copper homeostasis in AD. It is concluded that copper sequestration by microglia may provide a neuroprotective mechanism in AD.

Copper is an essential cofactor of enzymes involved in a variety of important metabolic processes includding ATP producting, iron transport, and antioxidant defense. The maintenance of copper homeostasis requires a balance of copper uptake and export as well as the appropriate partitioning of copper between the cytoplasm, mitochondria and secretory compartments. Although many of the proteins involved in copper homeostasis have been identified, it is unknown whether specific pathophysiological conditions lead to compensatory changes in the intracellular copper distribution. In this study, we identify changes in copper homeostasis in response to pro-inflammatory mediators. We also show a novel role for the copper-transporting ATPase, ATP7A, in the bactericidal activity of RAW264.7 macrophage cell. We also identify striking alterations in copper homeostasis in response to hypoxia in RAW264.7 macrophage cells. In response to hypoxia, we observe a change in the heirarchy of intracellular distribution favoring delivery of copper to ATP7A and to the secretory pathway, as evidenced by enhanced activity of the ferroxidase, ceruloplasmin, and by copper-dependent trafficking of ATP7A in hypoxic macrophages in vitro and in vivo. Our study underscores the potential for pathophysiological condititions to regulate adaptive responses involving altered copper distribution to cuproenzymes.