Fig 1.
Progression of murine chromoblastomycosis induced by different F. pedrosoi fungal forms.
BALB/c mice were infected in the footpad with 1x106 conidia (FC), hyphae (FH), muriform cells (MC) or a combination of hyphal fragments and conidia (FP) in the ratio of 3:1 (A). Morphometric (B) and CFU data (C) showed a fast clearance of inoculated conidia, while infection with MCs was reflected in the persistence of the fungus in the tissue up to 45 days after infection. 400x magnification (A). *P<0.05 and ***P<0.001 compared to FH group.
Fig 2.
Quantification of F. pedrosoi fungal cells in the tissue and production of pro-inflammatory cytokines in the course of murine CBM.
Fungal cells were counted on twenty fields chosen at random in histopathological slides with the aid of a counting reticle (A-D). In all groups muriform cells (red arrows) could be identified after 15 days after infection. At the same time, hyphal fragments (brown arrow) were also present in animals infected either with hyphae (FH) (B) or muriform cells (MC) (C). Few conidia (black arrow) are observed after 15 days of infection in animals infected with F. pedrosoi conidia (FC) (A). Cell counts were expressed as cells per mm2 of injured tissue. 1000x magnification (A-D). After 15 days of infection, high levels of TNF-α (E), IL-1β (F), IL-6 (G) and MCP-1 (H) were observed in groups with higher numbers of hyphae and muriform cells in the tissue. Cytokine production was measured by ELISA from homogenized footpad tissue. *P<0.05, **P<0.01 and ***P<0.001 compared to FC group.
Fig 3.
Differential expression of genes in peritoneal macrophages after co-cultivation with conidia or muriform cells.
Global heatmap displaying all differentially expressed genes (considering each replicate) showed profound changes in peritoneal macrophages (PM) gene expression profile when stimulated with muriform cells (MC), while conidia (FC) and macrophage co-culture showed similar patterns to that found in unstimulated cells (A). Heatmap was built based on z-score ranking considering all genes considered as differentially expressed. Venn diagram (B) shows high number of differentially expressed genes (3672 genes) when macrophages were co-cultured with muriform cells, while only 47 genes were differentially expressed in conidia and macrophage co-culture. GO enrichment analysis in FC-PM interaction showed few differentially expressed genes correlated to immune response (C). On the other hand, MC-PM interaction promoted the up-regulation of a large number of genes related to the immune system, especially those concerning the inflammatory response (D).
Fig 4.
Toll-like receptor signaling pathway overview.
Analysis of genes related to the Toll-like receptor signaling pathway showed they were mostly up-regulated in peritoneal macrophage (PM) co-culture with muriform cells (B), but not conidia (A), leading to the expression of pro-inflammatory cytokines and chemokines genes.
Fig 5.
In vitro cytokine and chemokine production.
The production of cytokines and chemokines by macrophage in conidia (FC) or muriform cell (MC) co-culture supernatants was assessed by ELISA (A-E). NO2 concentration in culture supernatants was used as an indicator of NO generation and measured using Griess reagent (F). High levels of TNF-α (A), IL-1β (B) and IL-6 (C) were observed in co-culture with muriform cells, but not with conidia. To assess IL-1β production after 24 hours, peritoneal macrophages required LPS co-stimulation (B). Muriform cells also induced higher levels of MCP-1 after 48h when compared to macrophages infected with conidia (E). Production of IL-12 (D) and NO2 (F) was strongly inhibited by muriform cells. **P<0.01 and ***P<0.001.
Fig 6.
Phagocytosis gene expression in peritoneal macrophages co-culturing with conidia.
Peritoneal macrophages (PM) co-culturing with conidia (FC) did not induce much gene expression related to phagocytosis.
Fig 7.
Phagocytosis gene expression in peritoneal macrophages co-culturing with muriform cells and phagocytosis index.
Peritoneal macrophages (PM) co-culturing with muriform cells (MC) (A) induced the expression of several genes related to phagocytosis. Muriform cells displayed an elevated expression of Dectin-1 gene while reducing the expression of FcγR gene (A). By blocking Dectin-1 and FcγR with laminarin and FcγR Block, respectively, only muriform cells phagocytosis was impaired (C), while conidia (FC) phagocytosis was not affected (B). *P<0.05, compared to medium control.
Fig 8.
CBM progression in a Zymosan-induced inflammation model.
After 15 days post infection with fungal propagules (FP), animals were treated intra lesionally (i.l.) in the footpad with 20μl of a suspension containing 5 mg/ml of zymosan (ZYM) or PBS, until 15 days post treatment start (d.p.t) (A). Animals treated with ZYM displayed intense inflammatory response up to 30 days post infection (d.p.i) (B). Animals facing prolonged inflammation showed no reduction in fungal load over time, as observed for those animals treated with PBS (C). **P<0.01 and ***P<0.001.