<i>Mycobacterium avium</i> Infection Induces H-Ferritin Expression in Mouse Primary Macrophages by Activating Toll-Like Receptor 2

<div><p>Important for both host and pathogen survivals, iron is a key factor in determining the outcome of an infectious process. Iron with-holding, including sequestration inside tissue macrophages, is considered an important strategy to fight infection. However, for intra-macrophagic pathogens, such as <i>Mycobacterium avium</i>, host defence may depend on intracellular iron sequestration mechanisms. Ferritin, the major intracellular iron storage protein, plays a critical role in this process. In the current study, we studied ferritin expression in mouse bone marrow-derived macrophages upon infection with <i>M. avium</i>. We found that H-ferritin is selectively increased in infected macrophages, through an up-regulation of gene transcription. This increase was mediated by the engagement of Toll like receptor-2, and was independent of TNF-alpha or nitric oxide production. The formation of H-rich ferritin proteins and the consequent iron sequestration may be an important part of the panoply of antimicrobial mechanisms of macrophages.</p> </div

TLR-2 activation leads to increased expression of H-ferritin.

<p>A, B – BMM from C57Bl/6 (WT) and TLR-2^-/- mice were left uninfected or infected for 24h with <i>M. avium</i>. The H-ferritin fold increase in infected BMM in comparison with uninfected ones is shown at the protein level (A) and mRNA (B). C – BMM were treated with the TLR-2 agonist FSL-1 for 24h, and the levels of H- and L-ferritin was quantified by ELISA. Results show the average + SD from one experiment performed in triplicate out of three independent experiments. Statistical differences as described in Figure 1.</p

Effect of <i>Mycobacterium avium</i> infection on intramacrophagic ferritin.

<div><p>Bone marrow-derived macrophages were obtained from C57Bl/6 mice and infected with <i>M. avium</i>, as described in Material and Methods, or left uninfected. A - At different time points, macrophages were lysed and the amount of ferritin was quantified by ELISA. Data are presented as ng of ferritin per mg of total protein. The results are shown as average ± SD from one experiment performed in triplicate out of four independent experiments. Superscripts indicate statistical significance between M. avium-infected and uninfected, within the correspondent time-point, as follows: *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. B – BMM uninfected or infected with <i>M. avium</i> for 24h were incubated for 6h with (⁵⁵Fe) ferric ammonium citrate. Total protein (18 µg) was loaded (in duplicates) in native PAGE and exposed to autoradiography to analyze protein-bound iron. A single band was detected corresponding to cytosolic H/L ferritin. The values indicate the average relative band intensity for each condition. C – BMM infected with <i>M. avium</i> for 4h, 1 and 3 days and respective uninfected controls were tested for IRP-IRE binding activity, by gel retardation assay. 2% of 2-mercaptoethanol (2-ME) fully activates IRP binding activity and shows equal loading. BMM treated with iron or deferoxamine (DFO) were tested in a separated gel to confirm the reliability of the assay. D – BMM were treated with the transcriptional inhibitor actinomycin D or with vehicle. After an 8h-infection with <i>M. avium</i>, the BMM were lysed and H- and L-ferritin were quantified by ELISA. Results show the average + SD from one experiment performed in triplicate out of three independent experiments. ***<i>p</i><0.001, NS not significant. </p> <p>E – At different time points, total RNA was collected from macrophages and the expression levels of ferritin genes was quantified by qRT-PCR, and normalized to <i>Hprt1</i>. Results are shown as fold increase in <i>M. avium</i>-infected macrophages in comparison with uninfected ones. Data are presented as average ± SE from one experiment performed in triplicate from a total of two independent experiments. </p></div

Effect of <i>M. avium</i> infection on ferritin content in the absence of TNF-alpha, iNOS and TLR-2.

<p>Bone marrow-derived macrophages were obtained from C57Bl/6 (WT), TLR-2^-/-, TNF-alpha^-/- and NOS2^-/- mice. BMM were infected and the ferritin content was quantified as described in Figure 1. The results are shown as average ± SD from one experiment performed in triplicate out of two independent experiments.</p

Effect of iron overload on the capacity of the mouse to induce the production of key cytokines.

<p>BALB/c mice were i.p. injected with saline solution or 10 mg of iron (-dextran, in a single dose) and were infected 15 days later by the i.v. route, with 2×10⁷<i>L. infantum</i> stationary promastigotes (inf) or were left uninfected (non-inf). Mice were sacrificed 60 days later and liver (100 mg) and spleen (50 mg) samples were collected and homogenised in protein lysis buffer with 2 mM PMSF (Bio-plex Cell Lysis Kit, Biorad). The levels of IL (interleukin)-1β, IL-2, IL-4, IL-10, IL-13, IL-6, IL-12p70, IFNγ (interferon-gamma), TNF (tumour necrosis factor) and IL-17 were quantified in liver and spleen homogenates of mice without (white bars) or with (black bars) iron overload, accordingly to 10-Plex assay instructions (Biorad). The results express the average+standard deviation of the cytokine levels, expressed in pg/mg of protein (<i>n</i> = 3–5). Student's <i>t</i>-test was performed to determine the statistical significance of the differences between the different groups (*p<0.05; **p<0.01). The results of one representative experiment are shown.</p

Effect of genetically determined iron overload on the growth of <i>L. infantum</i> in mice.

<p><b>A. B.</b> Wild-type (WT) and Hfe^−/− mice were i.v. infected with 2×10⁷ stationary promastigotes of <i>L. infantum</i> and were sacrificed 60 days after infection. <b>A.</b> Non-heme iron content in both organs was quantified in WT (white bars) and Hfe^−/− (black bars) groups. The data shows the average+standard deviation of the non-heme iron content, expressed in µg/organ (<i>n</i> = 4–6). Student's <i>t</i>-test was performed to determine the statistical significance of the differences between WT and Hfe^−/− groups (***p<0.001). <b>B.</b> The parasite burden in the liver and spleen of wild-type (WT; white bars) and Hfe^−/− (black bars) mice was determined by limiting dilution (<i>n</i> = 5–6). <b>C–F:</b> Perl's blue staining was performed in liver sections of uninfected (<b>C</b>) and infected (<b>E</b>) WT and uninfected (<b>D</b>) and infected (<b>F</b>) Hfe^−/− mice. Black bar corresponds to 50 µm. The results of one representative experiment are shown. Two experiments were performed with similar results.</p

Effect of different forms of iron on the axenic growth of <i>L. infantum</i> pro- and amastigotes.

<p>Promastigotes in the exponential phase of growth (2×10⁶/well; <b>A, B, C</b>) or amastigotes (4×10⁵/well; <b>D, E, F</b>) were incubated in RPMI (25°C) or MAA20 (37°C) medium, respectively, with iron-dextran (<b>A</b>, <b>D</b>), iron citrate (<b>B</b>, <b>E</b>) and iron sulphate (<b>C</b>, <b>F</b>) (black circles) in the concentrations of 0.018, 0.035, 0.070, 0.14, 0.28, 0.56, 1.1, 2.2, 4.5, 9 and 18 mM. Equivalent concentrations of dextran (<b>A</b>, <b>D</b>), tri-sodium citrate (<b>B</b>, <b>E</b>) and magnesium sulphate (<b>C</b>, <b>F</b>) were used as controls (white circles). Resazurin dye was added after 24 h of culture. The fluorescence was measured 24 h (for amastigotes) or 48 h (for promastigotes) after resazurin addition. The results show the average ± standard deviation of the percentage viability in relation to the non-treated control (<i>n</i> = 3). One-way ANOVA, followed by a Newman-Keuls multiple comparison <i>post-hoc</i> test, was performed to determine the statistical significance of the differences between control (white circles) and iron treated (black circles) groups (*p<0.05, **p<0.01; ***p<0.001). The results of one representative experiment are shown. Three experiments were performed with similar results.</p

Effect of different iron doses and periods of administration on the growth of <i>L. infantum</i>. A.

<p>BALB/c mice were injected i.p. with 1 or 10 mg of iron (-dextran), in a single dose, 12 days before the infection. <b>B.</b> BALB/c mice were injected i.p. with 10 mg of iron (-dextran), in a single dose, 15 days before or 1 and 15 days after the infection (−15, +1 and +15 days, respectively). <b>A. B.</b> Control mice received saline solution by the same route. Mice were sacrificed 30 (<b>A</b>) or 60 (<b>B</b>) days after the i.v. infection with 2×10⁷ stationary promastigotes of <i>L. infantum</i>. The parasite burden in the liver and spleen of mice without (white bar) and with (gray - black bars) iron administration was determined by limiting dilution. The data shows the average+standard deviation of the log₁₀ number of parasites per organ (<i>n</i> = 5). One-way ANOVA, followed by a Dunnett's multiple comparison <i>post-hoc</i> test, was performed to determine the statistical significance of the differences between each of the iron treated groups and control group (*p<0.05; **p<0.01). The results of one representative experiment are shown. Two experiments were performed with similar results.</p

Effect of iron overload on the capacity of the mouse to induce the expansion of splenic cell populations.

<p>BALB/c mice were i.p. injected with saline solution or 10 mg of iron (-dextran, in a single dose) and were infected 15 days later by the i.v. route, with 2×10⁷<i>L. infantum</i> stationary promastigotes (inf) or were left uninfected (non-inf). Mice were sacrificed 60 days later and the spleen was collected. The levels of CD3⁺, CD3⁻DX5⁺, CD19⁺, CD11c⁺, CD11b⁺F4/80⁺ and CD11b⁺GR1⁺⁺ cells were quantified in the spleen of mice without (white bars) or with (black bars) iron overload. The results express the average+standard deviation of the total number of cells in the spleen (<i>n</i> = 3–5). One-way ANOVA, followed by a Newman-Keuls multiple comparison <i>post-hoc</i> test, was performed to determine the statistical significance of the differences between all groups (*p<0.05). The results of one representative experiment are shown. Two experiments were performed with similar results.</p

Effect of iron deprivation or overload on the growth of <i>L. infantum</i> in the mouse. A. B.

<p>BALB/c mice were kept on a control or iron deficient diet for 120 days prior to their i.v. infection with 2×10⁷ stationary promastigotes of <i>L. infantum</i>. Mice were sacrificed 60 days after infection. <b>A.</b> Non-heme iron content of liver and spleen was quantified in mice fed the control (white bars) or iron deficient diet (black bars). The graph shows the average+standard deviation of the non-heme iron content, expressed as µg/organ (<i>n</i> = 6). <b>B.</b> The parasite burden in the liver and spleen of groups fed the control (white bars) or iron deficient (black bars) diet was quantified by limiting dilution. The graph shows the average+standard deviation of the log₁₀ number of parasites per organ (<i>n</i> = 6). <b>C.</b> BALB/c mice were i.p. injected with 1 mg of iron (given as dextran), in 10 alternate days, from day −20 to day −2 of infection (a total of 10 mg of iron per animal). Control mice received equivalent amounts of dextran by the same route. Mice were infected i.v. with 2×10⁷ stationary promastigotes of <i>L. infantum</i> and were sacrificed at 7, 15, 30 and 60 days of infection. The parasite burden in the liver and spleen of control (white circles) or iron overloaded (black circles) groups was quantified by limiting dilution (<i>n</i> = 5). <b>D.</b> Non-heme iron content in the liver and spleen was quantified in control (white bars) and iron overloaded (black bars) groups at 60 days after infection (<i>n</i> = 4–5). Student's <i>t</i>-test was performed to determine the statistical significance of the differences between groups (**p<0.01; ***p<0.001). <b>E. F. G. H.</b> Perl's blue staining was performed in liver sections of uninfected (<b>E</b>), uninfected iron overloaded (<b>F</b>), 60 days-infected (<b>G</b>) and 60 days-infected iron overloaded (<b>H</b>) mice. Black bar corresponds to 50 µm. The results of one representative experiment are shown. Two experiments were performed with similar results.</p