<i>Leishmania</i>-Mediated Inhibition of Iron Export Promotes Parasite Replication in Macrophages

<div><p><i>Leishmania</i> parasites infect macrophages, cells that play an important role in organismal iron homeostasis. By expressing ferroportin, a membrane protein specialized in iron export, macrophages release iron stored intracellularly into the circulation. Iron is essential for the intracellular replication of <i>Leishmania</i>, but how the parasites compete with the iron export function of their host cell is unknown. Here, we show that infection with <i>Leishmania amazonensis</i> inhibits ferroportin expression in macrophages. In a TLR4-dependent manner, infected macrophages upregulated transcription of hepcidin, a peptide hormone that triggers ferroportin degradation. Parasite replication was inhibited in hepcidin-deficient macrophages and in wild type macrophages overexpressing mutant ferroportin that is resistant to hepcidin-induced degradation. Conversely, intracellular growth was enhanced by exogenously added hepcidin, or by expression of dominant-negative ferroportin. Importantly, dominant-negative ferroportin and macrophages from flatiron mice, a mouse model for human type IV hereditary hemochromatosis, restored the infectivity of mutant parasite strains defective in iron acquisition. Thus, inhibition of ferroportin expression is a specific strategy used by <i>L. amazonensis</i> to inhibit iron export and promote their own intracellular growth.</p></div

Hepcidin promotes <i>L. amazonensis</i> growth in macrophages.

<p>(<b>A</b>) Effect of iron loading and hepcidin on the intracellular replication of <i>L. amazonensis</i>. BMDM were infected with <i>L. amazonensis</i> axenic amastigotes (<i>L.a</i>). <i>L.a</i>, 1 h infection; <i>Hepcidin+L.a</i>, 4 h of hepcidin treatment followed by 1 h infection; <i>Fe-NTA+L.a</i>, overnight incubation with Fe-NTA followed by 1 h infection; <i>Fe-NTA+L.a+hepcidin</i>, 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>B</b>) Representative images of BDMM infected as in (A), after 48 h Top panels, phase contrast; bottom panels, anti-<i>Leishmania</i> 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). (<b>C</b>) Intracellular growth of <i>L. amazonensis</i> in wild type or <i>Hamp</i>^−/− macrophages. BMDM from WT and <i>Hamp</i>^−/− mice were infected with WT axenic amastigotes of <i>L. amazonensis</i> 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).</p

<i>L. amazonensis</i> infection inhibits Fpn1 expression in macrophages in a TLR4 dependent manner.

<p>BMDM were left untreated (Control) or incubated with Fe-NTA overnight, washed and infected or not with <i>L. amazonensis</i> axenic amastigotes (<i>L.a</i>) for 1 h. BMDM were washed to remove extracellular parasites, cultured at 34°C for another 24 or 48 h and <i>Fpn1</i> or hypoxantine phosphoribosyltransferase (<i>HPRT1</i>) mRNA was quantified by qPCR. (<b>A</b>) Effect of <i>L. amazonensis</i> infection on ferroportin (<i>Fpn1</i>) mRNA in wild type (WT) BMDM. (<b>B</b>) Effect of <i>L. amazonensis</i> infection on ferroportin (<i>Fpn1</i>) mRNA in TLR4^−/− BMDM. The values represent <i>Fpn1</i> mRNA levels normalized to <i>HRPT1</i> (mean +/− SD of triplicates). (**) p<0.01; (***) p<0.001 (unpaired Student's t test). (<b>C</b>) Fpn1 protein levels in WT and TLR4⁻/⁻ BMDM treated as in (A, B) and infected or not with <i>L. amazonensis</i> axenic amastigotes (<i>L.a</i>) for 48 h. BMDM lysates were analysed by Western Blot with anti-Fpn1 antibodies. Anti-actin antibodies were used as loading controls. Fpn1/actin ratios were determined by quantitative digital imaging. The data are representative of at least 2 independent experiments.</p

Macrophages from flatiron mice, a model for hereditary hemochromatosis type IV, are more susceptible to infection by <i>L. amazonensis</i>.

<p><b>A</b>) L-ferritin protein levels in wild-type (<i>C3H</i>) and flatiron (<i>ffe/+</i>) macrophages. BMDM from <i>C3H</i> or <i>ffe/+</i> mice were solubilized and analysed by SDS-PAGE and Western Blot with anti-L-ferritin antibodies followed by peroxidase-conjugated secondary antibodies. Anti-actin antibodies were used as loading controls. L-ferritin/actin ratios were determined by quantitative digital imaging. <b>B</b>) Total iron levels in wild-type (<i>C3H</i>) and flatiron (<i>ffe/+</i>) macrophages. BMDM from <i>C3H</i> or <i>ffe/+</i> mice were washed, frozen, lysed in 50 mM NaOH, and subjected to total protein and ferrozine iron quantification assays. The values represent the mean +/− SD of triplicates. (***) p<0.001 (unpaired Student's t-test). (<b>C</b>) Intracellular growth of <i>L. amazonensis</i> in <i>C3H</i> and <i>ffe/+</i> macrophages. BMDM from <i>C3H</i> and <i>ffe/+</i> mice were infected with <i>WT</i>, <i>Δlit1</i> and <i>Δlfr1</i> axenic amastigotes of <i>L. amazonensis</i> for 1 h followed by washes, incubation at 34°C for 1, 24 or 48 h, staining and quantification of the number of intracellular parasites. The values represent the mean +/− SD of triplicates, and the results are representative of 2 independent experiments. (***) p<0.001 (unpaired Student's t-test). (<b>D</b>) Representative confocal fluorescence images of BMDMs infected for 72 h with the <i>L. amazonensis</i> (<i>L.a.</i>)strains as described in (C). Red, anti-<i>Leishmania</i> antibodies; Blue, DAPI.Bar = 10 µm (applies to all images).</p

Effect of <i>L. amazonensis</i> infection on the total iron content of macrophages.

<p>WT and TLR4⁻/⁻ BMDM were infected or not with axenic <i>L. amazonensis</i> amastigotes (<i>L.a</i>) at a MOI = 5 for 1 h, washed to remove extracellular parasites, cultured at 34°C for another 2, 24 or 48 h, washed, frozen, lysed in 50 mM NaOH, and subjected to total protein and iron quantification assays. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003901#s2" target="_blank">Results</a> from independent experiments (Exp) are shown.</p

The intracellular growth of <i>L. amazonensis</i> mutants defective in iron uptake is rescued by dominant-negative Fpn1.

<p>(<b>A</b>) Effect of dominant-negative Fpn1 expression on the intracellular growth of <i>L. amazonensis</i>. BMDM were mock-transfected or transfected with Fpn1(H32R)-GFP, followed by infection with axenic amastigotes of wild type (WT), Δ<i>lit1</i> and Δ<i>lfr1 L. amazonensis</i> for 1 h, followed by washes, incubation at 34°C for 1, 24 or 48 h, fixation, staining and microscopic quantification of intracellular parasites. The values represent the mean +/− SD of triplicates, and the results are representative of 2 independent experiments. (***) p<0.001 (unpaired Student's t test). (<b>B</b>) Representative images of BMDM infected for 48 h as described in (A). Left panels, phase contrast; right panels, anti-<i>Leishmania</i> antibodies (red), DAPI (blue) fluorescence. Arrows point to replicating parasites; arrowheads point to single, non-replicating parasites. Bars = 10 µm.</p

<i>L. amazonensis</i> infection enhances hepcidin expression in macrophages in a TLR4 dependent manner.

<p>WT and TLR4⁻/⁻ BMDM were infected or not with <i>L. amazonensis</i> axenic amastigotes (<i>L.a</i>) for 1 h and <i>Hamp</i> mRNA was quantified by qPCR. (<b>A</b>) Effect of <i>L. amazonensis</i> infection on hepcidin (<i>Hamp</i>) transcript levels. The values represent <i>Hamp</i> mRNA levels normalized to <i>HRPT1</i> (mean +/− SD of triplicates). The data are representative of at least 3 independent experiments. (***) p<0.001 (unpaired Student's t test). (<b>B,C</b>) Livers and footpads from uninfected or 9-week infected mice were homogenized and <i>Hamp</i> transcripts were quantified by real time PCR. The values represent <i>Hamp</i> mRNA levels normalized to tubulin (mean +/− SD of triplicates). The values represent the mean +/− SD of assays performed with tissues of 3 independent mice. (*) p<0.05; (**) p<0.01 (unpaired Student's t test).</p

<i>L. amazonensis</i> infection causes iron accumulation and ferritin upregulation in macrophages.

<p>(<b>A</b>) Effect of <i>L. amazonensis</i> infection on H-ferritin (<i>FTH1</i>) transcript levels. BMDM were infected or not with axenic <i>L. amazonensis</i> amastigotes (<i>L.a</i>) for 1 h, washed and cultured at 34°C for another 24 or 48 h, followed by quantification of <i>FTH1</i> and <i>HPRT1</i> mRNA by qPCR. The values represent <i>FTH1</i> mRNA levels normalized to <i>HRPT1</i> (mean +/− SD of triplicates). (***) p<0.001 (unpaired Student's t test). (<b>B</b>) Effect of <i>L. amazonensis</i> infection on L-ferritin protein levels. BMDM infected or not with <i>L. amazonensis</i> axenic amastigotes (<i>L.a</i>) were solubilized after 24 or 48 h and analysed by Western Blot with anti-L-ferritin antibodies. Anti-actin antibodies were used as loading controls. L-ferritin/actin ratios were determined by quantitative digital imaging. The results shown are representative of at least 2 independent experiments.</p

Proposed model for the <i>L. amazonensis</i>-induced downregulation of ferroportin expression in macrophages.

<p>Macrophages infected by <i>Leishmania amazonensis</i> produce hepcidin, a peptide hormone that binds to the iron exporter ferroportin, inducing its internalization and degradation. This causes an increase in the macrophage cytosolic iron content, which includes the bioavailable labile iron pool and iron stored as a complex with ferritin. By an unknown mechanism, Fpn1 internalization and elevated cytosolic iron increases iron availability inside <i>Leishmania</i>-containing parasitophorous vacuoles, stimulating parasite growth.</p

Fpn1 removal from the cell surface stimulates the intracellular growth of <i>L. amazonensis</i>.

<p>BMDM were mock transfected or transfected with Wild type Fpn1-GFP, Fpn1(H32R)-GFP or Fpn1(N144H)-GFP, and after 24 h were treated with hepcidin for 4 h or infected or not with axenic amastigotes of <i>L. amazonensis</i>-RFP for 1 h followed by washes and additional incubation at 34°C for 48 h. The images shown are representative of several live confocal images acquired in 2 independent experiments. The arrows point to Fpn1 on the plasma membrane of BMDM. Bar = 10 µm (applies to all images).</p