Hepcidin promotes L. amazonensis growth in macrophages.

(A) Effect of iron loading and hepcidin on the intracellular replication of L. amazonensis. BMDM were infected with L. amazonensis axenic amastigotes (L.a). L.a, 1 h infection; Hepcidin+L.a, 4 h of hepcidin treatment followed by 1 h infection; Fe-NTA+L.a, overnight incubation with Fe-NTA followed by 1 h infection; Fe-NTA+L.a+hepcidin, overnight incubation with Fe-NTA followed by 1 h infection, parasite removal, and 18 h treatment with hepcidin. After washing, BMDM were cultured at 34°C for another 1, 24 or 48 h, fixed, stained and the number of intracellular parasites was determined microscopically. The values represent the mean +/− SD of 3 independent experiments. (*) p<0.05; (**) p<0.01 (unpaired Student's t test). (B) Representative images of BDMM infected as in (A), after 48 h Top panels, phase contrast; bottom panels, anti-Leishmania antibodies (red) and DAPI (blue) fluorescence. The arrows point to PVs containing replicating parasites; arrowheads point to single, non-replicating parasites. Bar  =  10 µm (applies to all images). (C) Intracellular growth of L. amazonensis in wild type or Hamp^−/− macrophages. BMDM from WT and Hamp^−/− mice were infected with WT axenic amastigotes of L. amazonensis for 1 h followed by washes, incubation at 34°C for 1, 24, 48 h or 72 h, staining with DAPI and quantification of the number of intracellular parasites. The values represent the mean +/− SD of triplicates (***) p<0.001 (unpaired Student's t-test).