Figure 1.
Systemic candidiasis affects host iron homeostasis. A.
Immunohistochemical detection of hepcidin in kidneys of healthy (right) and infected (left) animals. 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. C. 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. D. Perls staining of non-haem iron deposits in spleens of healthy and infected animals. The blue arrowheads highlight splenic non-haem iron deposits. C. albicans 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 Materials and Methods.
Figure 2.
C. albicans infection is accompanied by dramatic changes in the renal iron landscape.
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 (A), to the medulla in intermediate (B) and advanced infections (C). Histology insets (D) are representative of healthy, intermediate and advanced infections, and correspond to the tissues imaged in panels A–C. The pie charts in (D) 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 (D) 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; n, number of biological replicates. The effects observed with the virulent C albicans isolate SC5314 (epidemiological clade 1) are replicated with a different clinical isolate AM2003/0191(clade 2) [30] (panels E and F, bottom). Infections with C. albicans SC5314 stimulate significant immune infiltrates (F, top, dotted line), while AM2003/0191 elicits minimal immune infiltrates (F, bottom, solid line).
Figure 3.
Spatial distributions of HBA and haem mirror 56Fe distributions in healthy and infected kidneys.
Tissue sections were analysed with MALDI IMS to map the distributions of HBA and haem B (see Materials and Methods, and Supporting Material). Similarly to iron distribution (Fig. 2), in the healthy tissue (top panels) both HBA and haem are distributed to the cortex, whereas in kidneys obtained from animals with advanced infection (bottom panels) they are medullary. Transverse kidney sections sequential to those presented in Fig. 2A (‘Healthy’) and 2C (‘Infected’), respectively, are shown. The images are representative of at least three biological replicates.
Figure 4.
The medullary iron accumulated in advanced candidiasis is associated with renal tissue.
LA-ICP MS was used to map iron (56Fe) distribution in transverse kidney sections after rough perfusion of the organs with saline. Saline perfusion displaced most iron from healthy tissue (‘Healthyp’, top), in contrast to infected tissue (‘Infectedp’, bottom), suggesting a tissue-associated pool of iron that accompanies systemic Candida infection. Pie charts 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, 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; n, number of biological replicates.
Figure 5.
Systemic candidiasis disturbs host renal iron homeostasis and affects splenic function.
Panels A–C. 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 (A) Ferritin is distributed outside areas occupied by C. albicans (A, arrowheads). In healthy tissue (B, left), ferritin content is low and mainly cortical, while increased and mainly medullary in animals with advanced candidiasis (B, right). HO-1 is concentrated in rings encompassing fungal lesions (A, arrowheads) and is produced by cell type(s) other than the Ly-6G or F4/80-producing immune cells (C). Panel D. 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) (D). In all cases, the experiments represent C. albicans 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 A and D, 500 µm; B, 1000 µm; C, 200 µm.
Figure 6.
Selective chemical ablation of splenic red pulp macrophages recapitulates the renal iron accumulation phenotype in the absence of Candida infection.
Panel A. Immunohistochemical confirmation of selective ablation of splenic red pulp macrophages by clodronate [41] (100 µL of 5 mg/mL liposome-encapsulated clodronate per animal): for antibodies see Fig. 5D. Panel 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 A); for colour scale see Fig. 2. In the bar chart (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; n, number of biological replicates. In all cases, the experiments represent C. albicans SC5314 infections in BALB/c mice. ‘Control’, experimental control with no primary antibodies. Size bars: 200 µm. Figure in B is representative of three biological replicates.
Figure 7.
Systemic candidiasis impacts upon iron homeostasis of both the fungus and the host.
Panels A and B, disturbed host iron homeostasis affects iron acquisition strategies of C. albicans. Fungal cells from either early (panel A, smaller inset) or advanced (panel A, larger image) stages of infection were dissected via laser capture microscopy, RNA was extracted, amplified, and abundances of the specific transcripts assessed by qRT-PCR (panel B). The expression of genes from the three major C. albicans iron acquisition pathways was assessed: (1) xenosiderophore acquisition – SIT1 (orf19.2179) [42] (no signal, not shown); (2) reductive iron acquisition – ALS3 (orf19.1816), FTR1 (orf19.7219) [43]; (3) haem-iron acquisition –CSA1 (orf19.7114), CSA2 (orf19.3117), PGA10 (orf19.5674), and HMX1 (orf19.6073) [14]. The differences in gene expression patterns between the centre and rim of advanced lesions probably reflect population heterogeneity within fungal lesions [53], with the cells in the centre tightly knit and in contact with each other rather than the host, and not as metabolically active as the cells from the rim exposed to the host tissue. (B). The data presented are normalised to ACT1 expression and represent means from at least three independent biological (lesion material) replicates. Greyed gene names, no transcript detection; error bars, standard deviations from the mean. Normalised transcript abundances were compared to the ‘early’ sample, differences were considered statistically significant (asterisks) for p≤0.05 in two-tailed t-test using Mann-Whitney U statistics. In panel A, the size bars correspond to 100 µm. Panel C, the effect of systemic candidiasis on the iron homeostasis of the host. During advanced candidiasis, the splenic red blood cell recycling function is perturbed presumably due to active clearance of fungal infection from that organ, while the renal involvement in host iron homeostasis increases. Blue colour intensity represents the renal iron distribution and relative concentration in the tissue, with C. albicans lesions represented by purple circles. See text for details.