Transcriptional modulation of trout <i>gpx1</i>, <i>trxr3</i>, and <i>selp</i> isoforms in blood cells (A) and muscle (B) from rainbow trout fed a control diet and three diets enriched with different concentrations (0.5, 4 and 8 g Kg^-1) of Sel-Plex for 2, 6 and 10 weeks.

<p>The expression of gene transcripts was quantified by <i>q</i>PCR and normalized against the geometric mean of three housekeeping genes (<i>ef-1α</i>, <i>drpII</i>, <i>hprt</i>) from the same sample, and then used for statistical analysis. A fold change, calculated as the average expression level of stimulated samples divided by that of the controls, is presented. The results represent the mean + SEM from six fish. The letters above the columns indicate values statistically significant from the controls (<i>p<</i>0.05), with different letters indicating significant differences between the treatments.</p

Transcriptional modulation of trout <i>gpx1</i>, <i>trxr3</i>, and <i>selp</i> isoforms in liver (A) and kidney (B) from rainbow trout fed a control diet and three diets enriched with different concentrations (0.5, 4 and 8 g Kg^-1) of Sel-Plex for 2, 6 and 10 weeks.

<p>The expression of gene transcripts was quantified by <i>q</i>PCR and normalized against the geometric mean of three housekeeping genes (<i>ef-1α</i>, <i>drpII</i>, <i>hprt</i>) from the same sample, and then used for statistical analysis. A fold change, calculated as the average expression level of stimulated samples divided by that of the controls, is presented. The results represent the mean + SEM from six fish. The letters above the columns indicate values statistically significant from the controls (<i>p<</i>0.05), with different letters indicating significant differences between the treatments.</p

Growth performance of rainbow trout given various levels of organic supplemental Se for 10 weeks.

<p>The results represent the mean <b>±</b> SEM from six fish per diet at each time point. On the graph the significance of the difference in body weight between the diets groups at each time point is indicated, whilst in the table underneath the changes in body weight for each diet group across the different time points is reported. Values not statically different are indicated by “n.s.”, whereas values significantly different (<i>p<</i>0.05) are indicated by different letters.</p

Tissue specific assimilation of Se in rainbow trout fed a control diet and three diets enriched with different concentrations (0.5, 4 and 8 g Kg^-1) of Sel-Plex for ten weeks.

<p>The results represent the mean + SEM of four independent measurements. The Greek letters above the columns indicate values statistically significant from the controls (<i>p</i><0.05), with different letters indicating significant differences between the groups fed the Sel-Plex supplemented diet at each time point. The tables below the graphs indicate differences in Se level within the same diet group across the different time points.</p

Concentration factor of Se in rainbow trout fed a control diet and three diets enriched with different concentrations (0.5, 4 and 8 g Kg^-1) of Sel-Plex for ten weeks.

<p>The results represent the mean ± SEM of four independent measurements. The letters above the columns indicate values statistically significant from the controls (<i>p</i><0.05), with different letters indicating significant differences between the treatments. The tables below the graphs indicate differences in Se levels within the same diet group across the different time points.</p

Se concentration in the feed pellets of the four different diets used for this feeding trial.

<p>The results represent the mean and SEM of four independent measurements.</p><p>Se concentration in the feed pellets of the four different diets used for this feeding trial.</p

Selective chemical ablation of splenic red pulp macrophages recapitulates the renal iron accumulation phenotype in the absence of <i>Candida</i> infection.

<p><b>Panel A.</b> Immunohistochemical confirmation of selective ablation of splenic red pulp macrophages by clodronate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat.1003676-VanRooijen1" target="_blank">[41]</a> (100 µL of 5 mg/mL liposome-encapsulated clodronate per animal): for antibodies see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat-1003676-g005" target="_blank">Fig. 5D</a>. <b>Panel B.</b> LA-ICP MS mapping of ⁵⁶Fe distribution reveals medullary shift of renal iron in clodronate-treated mice. Normalised ⁵⁶Fe/¹³C ratios across kidney following 8 h clodronate treatment are presented (see panel <b>A</b>); for colour scale see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat-1003676-g002" target="_blank">Fig. 2</a>. In the bar chart (<b>B</b>, right) bars correspond to the percentage surface area with normalized ⁵⁶Fe/¹³C intensity≥2-fold (left, light coloured bar) and ≥3-fold above background (right, dark coloured bar): error bars, standard deviations from the mean; <i>n</i>, number of biological replicates. In all cases, the experiments represent <i>C. albicans</i> SC5314 infections in BALB/c mice. ‘Control’, experimental control with no primary antibodies. Size bars: 200 µm. Figure in <b>B</b> is representative of three biological replicates.</p

Systemic candidiasis affects host iron homeostasis. A.

<p>Immunohistochemical detection of hepcidin in kidneys of healthy (right) and infected (left) animals. <b>B.</b> Perls staining of non-haem iron in livers of healthy and infected animals. The intensity of the blue stain is proportional to the amount of hepatic non-haem iron. <b>C.</b> Immunohistochemical analysis of Kupffer cells in mouse liver with anti-HO1 antibodies. The red arrowheads indicate hepatic Kupffer cells stained dark brown with anti-HO-1 antibodies. 1C1& 1C3 represent negative controls lacking the primary antibody. <b>D.</b> Perls staining of non-haem iron deposits in spleens of healthy and infected animals. The blue arrowheads highlight splenic non-haem iron deposits. <i>C. albicans</i> SC5314 infections were conducted in BALB/c mice and all images are representative of at least three separate biological replicates. Size bars correspond to 100 µm. Experimental details can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#s4" target="_blank">Materials and Methods</a>.</p

Systemic candidiasis disturbs host renal iron homeostasis and affects splenic function.

<p><b>Panels A–C.</b> Immunohistochemical detection of iron homeostasis associated proteins in kidneys of healthy and infected animals reveals correlation with renal iron loading. Ferritin, HO-1, HO-2, and the transferrin receptor (TfR) all increase in infected kidneys in comparison to healthy controls (<b>A</b>) Ferritin is distributed outside areas occupied by <i>C. albicans</i> (<b>A</b>, arrowheads). In healthy tissue (<b>B, left</b>), ferritin content is low and mainly cortical, while increased and mainly medullary in animals with advanced candidiasis (<b>B, right</b>). HO-1 is concentrated in rings encompassing fungal lesions (<b>A,</b> arrowheads) and is produced by cell type(s) other than the Ly-6G or F4/80-producing immune cells (<b>C</b>). <b>Panel D</b>. Immunohistochemical analyses of murine spleens. Blue areas correspond to white pulp (dotted lines), embedded in the red pulp. Red pulp macrophages stain selectively with anti-HO-1 and anti-F4/80 antibodies: the stain for HO-1 is depleted in tissues from infected animals (arrows) (<b>D</b>). In all cases, the experiments represent <i>C. albicans</i> SC5314 infections in BALB/c mice. ‘Pas_h’, sections processed for histology; ‘c’, kidney cortex; ‘m’, medulla; ‘control’, experimental control with no primary antibodies. Size bars: panels <b>A</b> and <b>D</b>, 500 µm; <b>B</b>, 1000 µm; <b>C</b>, 200 µm.</p

<i>C. albicans</i> infection is accompanied by dramatic changes in the renal iron landscape.

<p>LA-ICP MS mapping of iron distribution in transverse mouse kidney sections. Normalised ⁵⁶Fe/¹³C ratios are presented, with the colour scale indicating fold increases in signal intensities relative to background. As the infection progresses, iron loading increases and the iron becomes redistributed from the cortex of healthy kidneys (<b>A</b>), to the medulla in intermediate (<b>B</b>) and advanced infections (<b>C</b>). Histology insets (<b>D</b>) are representative of healthy, intermediate and advanced infections, and correspond to the tissues imaged in panels <b>A</b>–<b>C</b>. The pie charts in (<b>D</b>) present the percentage total tissue area with a given ⁵⁶Fe/¹³C intensity and the colour scale represents increments of 0.5-fold intensity changes from background (black) to 8-fold increase (white). In the bar chart (<b>D</b>) bars correspond to the percentage surface area with normalized ⁵⁶Fe/¹³C intensity ≥2-fold (left, light coloured bars) and ≥3-fold above background (right, dark coloured bars): error bars, standard deviations from the mean; <i>n</i>, number of biological replicates. The effects observed with the virulent <i>C albicans</i> isolate SC5314 (epidemiological clade 1) are replicated with a different clinical isolate AM2003/0191(clade 2) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003676#ppat.1003676-MacCallum2" target="_blank">[30]</a> (panels <b>E</b> and <b>F</b>, bottom). Infections with <i>C. albicans</i> SC5314 stimulate significant immune infiltrates (<b>F</b>, top, dotted line), while AM2003/0191 elicits minimal immune infiltrates (<b>F</b>, bottom, solid line).</p