Hepcidin is an antimicrobial peptide produced by the liver in response to inflammatory stimuli and iron overload. Hepcidin regulates iron homeostasis by mediating the degradation of the iron export protein ferroportin 1, thereby inhibiting iron absorption from the small intestine and release of iron from macrophages. Here, we examined the expression of hepcidin in macrophages infected with the intracellular pathogens Mycobacterium avium and Mycobacterium tuberculosis. Stimulation of the mouse RAW264.7 macrophage cell line and mouse bone marrow-derived macrophages with mycobacteria and IFN-gamma synergistically induced high levels of hepcidin mRNA and protein. Similar results were obtained using the human THP-1 monocytic cell line. Stimulation of macrophages with the inflammatory cytokines IL-6 and IL-beta did not induce hepcidin mRNA expression. Iron loading inhibited hepcidin mRNA expression induced by IFN-gamma and M. avium, and iron chelation increased hepcidin mRNA expression. Intracellular protein levels and secretion of hepcidin were determined by a competitive chemiluminescence ELISA. Stimulation of RAW264.7 cells with IFN-gamma and M. tuberculosis induced intracellular expression and secretion of hepcidin. Furthermore, confocal microscopy analyses showed that hepcidin localized to the mycobacteria-containing phagosomes. As hepcidin has been shown to possess direct antimicrobial activity, we investigated its activity against M. tuberculosis. We found that hepcidin inhibited M. tuberculosis growth in vitro and caused structural damage to the mycobacteria. In summary, our data show for the first time that hepcidin localizes to the phagosome of infected, IFN-gamma-activated cells and has antimycobacterial activity.

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Intracellular pathogens, including Mycobacterium tuberculosis, obtain iron from the host for their survival. Ferroportin 1 (FPN1; SLC40A1) is the sole iron exporter from mammalian cells and is expressed in the duodenum and macrophages. In the present study, we show that FPN1 mRNA levels in the mouse macrophage cell line RAW264.7 are synergistically induced by treatment with live or gamma-irradiated M. tuberculosis and IFN-gamma. FPN1 mRNA levels were also induced by Mycobacterium avium and IFN-gamma in RAW264.7 cells and the mouse alveolar macrophage cell line AMJ2-C8. Treatment of mouse resident peritoneal macrophages with M. tuberculosis and IFN-gamma resulted in a sixfold increase in FPN1 mRNA expression. In contrast, M. tuberculosis and IFN-gamma inhibited FPN1 mRNA expression in bone marrow-derived macrophages and lung macrophages, which have high basal levels of FPN1 mRNA expression. Using confocal microscopy, FPN1 protein localized rapidly to M. tuberculosis phagosomes after infection in RAW264.7 macrophages. In RAW264.7 cells expressing wild-type natural resistance-associated macrophage protein 1 (Nramp1(Gly169)), FPN1 and Nramp1 partially colocalized in late endosomes/lysosomes prior to infection. After 2 h of infection, Nramp1 and FPN1 were present in M. tuberculosis phagosomes. Our studies provide evidence for transcriptional regulation of FPN1 by pathogenic mycobacteria and IFN-gamma, which is dependent on the macrophage type. The trafficking of FPN1 to the M. tuberculosis phagosome suggests that it is involved in regulating iron availability to the mycobacteria in this locale.