<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

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

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>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

Fe and Zn concentrations in the heart of 13 FA^a patients and 8 normal controls (bulk digests).

<p>^aAbbreviations: FA, Friedreich ataxia; Fe, iron; LVW, left ventricular wall; RVW, right ventricular wall; VS, ventricular septum; Zn, zinc</p><p>^bResults of Fe and Zn are expressed as mean μg metal/g wet tissue ± standard deviation. Number of FA patients and normal controls are given in parentheses. In one FA patient, only LVW tissue was available.</p><p>^cp-values are based on statistical comparison by two-tailed t-test at α = 0.05, assuming unequal variances</p><p>Fe and Zn concentrations in the heart of 13 FA<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116396#t003fn001" target="_blank">^a</a> patients and 8 normal controls (bulk digests).</p

Fiber hypertrophy in FA cardiomyopathy.

<p>A Tukey box-and-whisker plot together with the raw data points to the left of each box, shows fiber size distribution in the LVW of 15 patients with FA (FA1-FA15, corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116396#pone.0116396.t001" target="_blank">Table 1</a>) and 10 normal controls (N1-N10). FA cases are shown in red, normal controls in blue. In the box plot, the central rectangle spans the first quartile to the third quartile (the interquartile range), and the horizontal line within the rectangle marks the median. The diamond symbol indicates the mean. The "whiskers" above and below the box are drawn to the furthest point within 1.5 x IQR from the box (the non-outlier range). The range of fiber sizes in FA is much larger than in normal controls. All medians and means in FA are higher than in normal controls. See text for statistical analysis of the data.</p

Hepcidin and cytosolic ferritin in FA myocarditis.

<p>LVW of patient FA7 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116396#pone.0116396.t001" target="_blank">Table 1</a>). Double-label laser scanning confocal immunofluorescence of hepcidin (Alexa 488 green, a and d), α-actinin (Cy3 red, b), and cytosolic ferritin (Cy3 red, e); (c) and (f) are merged images of (a-b) and (d-e), respectively. (a-c) Invasion of a cardiomyocyte by hepcidin-reactive cells. The circles in (a-c) indicate hepcidin-reactive processes among remaining α-actinin-reactive Z-discs. (d-f) Inflammatory cells in the endomysium. Most inflammatory cells contain both hepcidin and cytosolic ferritin (d-f). The interrupted lines indicate hepcidin-reactive cells that display no ferritin fluorescence. Bars: 10 μm.</p

XRF mapping and quantitative <i>in situ</i> measurements of Fe.

<p>(a) LVW in FA (patient FA5, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116396#pone.0116396.t001" target="_blank">Table 1</a>), (b) LVW in a normal control. Pseudocolors represent the intensity of Fe XRF. White indicates maximal emission. Red, orange, green, light blue, and dark blue represent progressively lower fluorescence, respectively. The distribution of Fe in FA and normal heart is heterogeneous. Circles with an area of 1 mm² were placed over regions of strongest Fe XRF, and 25 point measurements were made inside the outlined area. Fe concentrations in μg/ml tissue volume were calculated by reference to PEG-encapsulated Fe-III- and Zn-II-mesoporphyrin as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116396#pone.0116396.ref006" target="_blank">6</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116396#pone.0116396.ref008" target="_blank">8</a>]. In FA (a), the Fe-rich regions appear larger and show a more extended gradient into the surrounding tissue than the control (b). In FA (a), 25 repeated measurements in the circular area of 1 mm² yields an average Fe concentration of 131.8 μg/ml tissue volume; in the illustrated normal control (b), the averaged Fe level is 33.7 μg/ml tissue volume. Quantitative Zn levels in the same outlined regions were obtained after switching to the Zn XRF "map". Levels are 24.3 μg/ml in FA (a) and 20.7 μg/ml in the control sample (b). Bars, 5 mm.</p

<i>In situ</i> quantification of Fe and Zn in the hearts of 15 patients with FA^a and 10 normal controls (XRF).

<p>^aAbbreviations: FA, Friedreich ataxia; Fe, iron; LVW, left ventricular wall; RVW, right ventricular wall; VS, ventricular septum; Zn, zinc</p><p>^bResults of Fe and Zn are expressed as mean μg metal/ml tissue volume ± standard deviation. Number of FA patients and normal controls are given in parentheses.</p><p>^cp-values based on statistical comparison by two-tailed t-test at α = 0.05, assuming unequal variances</p><p><i>In situ</i> quantification of Fe and Zn in the hearts of 15 patients with FA<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116396#t002fn001" target="_blank">^a</a> and 10 normal controls (XRF).</p

The inflammatory infiltrate in FA cardiomyopathy.

<p>Immunohistochemistry of CD68 (a-c) and hepcidin (d-f). All sections derive from LVW of FA patients in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116396#pone.0116396.t001" target="_blank">Table 1</a>: FA5 (a-b), FA8 (c), FA7 (d-e), FA13 (f). The microphotographs in (c) and (f) were taken under differential interference optics to improve contrast, visualize cross-striations of cardiac muscle, and highlight fiber invasion by pseudopods of monocytes (arrows). The cellular infiltrate may be restricted to the endomysium (a) but is most intense following fiber invasion (b and e). Fiber invasion seems to begin with close attachment and breaching of the plasma membrane by delicate CD68- or hepcidin-positive processes, respectively (c and f, arrows). Bars: (a) and (d), 50 μm; (b) and (e), 20 μm; (c) and (f), 10 μm (oil immersion).</p