Morphometry of lung granulomas and analysis of fungal burden in infected animals treated with rPCN.

<p>Each group of mice was either not treated (PBS) or therapeutically treated according to protocols G1–G4 (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003317#s2" target="_blank">Material and Methods</a>). Panel A: Number of granulomas per mm² of tissue. Panel B: Granuloma area, in µm². Panel C: Pulmonary CFU recovery. For morphometric analysis, the Image J program, developed by Wayne Rasband of the National Institute of Mental Health, was used. Bars depict the mean and SD. * p<0.05; ** p<0.01; *** p<0.001 <i>vs.</i> the PBS group.</p

The absence of either CD14 or CD36 does not affect the TLR activation triggered by rPCN.

<p>HEK293T were cotransfected with CD14 and/or CD36 along with TLR2/1 (A) or TLR2/6 (B). The total amount of DNA in each transfection was kept constant by adding empty expression vector. The HEK293T cells were stimulated with rPCN (1.25 µg/mL), which was previously incubated with polymyxin to neutralize LPS. The positive controls were Pam3CysSK4 (P3C) for TLR2/1 and FSL-1 for TLR2/6. Medium was used as negative control for cell stimulation (white bars). Results are representative of three independent experiments. Statistical differences were assessed by comparing the response of cells lacking one of the co-receptors to the response of cells expressing both co-receptors, under similar stimuli. Values are the mean ± S.D. *** p<0.001.</p

Therapeutic groups.

a<p>PBS alone.</p><p>Therapeutic groups.</p

Reduction of r-PCN-induced activation of mammalian cells transfected with TLR2 glycosylation mutants.

<p>HEK293T cells expressing full-glycosylated TLR2 (WT – A6) or mutants of the N-glycosylation sites were stimulated with rPCN. The total amount of DNA in each transfection was kept constant by adding empty expression vector. Reduced activation was detected by comparing the IL-8 levels produced by HEK293Tcells expressing a certain TLR2 mutant with that produced by cells transfected with the full glycosylated TLR2. FSL-1 was used as a positive control. Medium supplemented with polymyxin (white bars) was used as negative control for cell stimulation. The E6 mutant was used as a negative control of the assay. For each transfected TLR2 ectodomain, the mutated site(s) [lacking N-glycans] is (are) represented by traces in red, while the preserved N-glycans are in black. Results are representative of two independent experiments. Values are mean ± S.D. The statistical comparison was done between HEK293T cells expressing full-glycosylated TLR2 (WT – A6) and the mutants of the N-glycosylation sites, stimulated with rPCN. The results were considered significant when p<0.01 (**); p<0.001 (***).</p

TLR2 heterodimerization is not critical for the cell activation triggered by rPCN.

<p>HEK293T cells were transfected with CD14 and CD36 along with TLR2, TLR2/TLR1 or TLR2/TLR6. The total amount of DNA in each transfection was kept constant by adding empty expression vector. After 48 h of transfection, cells were stimulated with the agonists: Pam3CysSK4 (P3C) for TLR2/1 and FSL-1 for TLR2/6. Medium was used as negative control for cell stimulation (white bars). rPCN (1.25 µg/mL), previously incubated with polymyxin to neutralize LPS, was assayed. The cell supernatants were analyzed for IL-8 by ELISA. Panel A: Cells transfected with TLR2 and TLR1. Panel B: Cells transfected with TLR2 and TLR6. Results are representative of five independent experiments. Statistical differences were assessed by comparing the response of cells expressing TLR2 to the response of cells expressing TLR2/TLR1 or TLR2/TLR6. Values are the mean ± S.D. * p<0.05; ** p<0.01; *** p<0.001.</p

Lung histopathology of <i>P. brasiliensis</i>-infected mice treated with rPCN.

<p>The panels show representative lung sections from mice that were not infected (A); infected and not treated (B); or infected and therapeutically treated according to different protocols (C–F). The section were stained with hematoxylin and eosin (H&E) (panels G–L) or with Gomori's methenamine silver (GMS) (panels M–R). Images were captured using a Carl Zeiss Axiophot microscope coupled to a JVC TK1270 camera. Magnification bars = 500 µm for total lung and 200 µm for lung sections.</p

rPCN triggers TLR-mediated cell activation.

<p>HEK293T cells were transfected with CD14 and CD36 along with TLR2/1 (A), TLR2/6 (B), or TLR4 (C). The total amount of DNA in each transfection was kept constant by adding empty expression vector. The cells were stimulated with the indicated concentrations of rPCN, previously incubated with polymyxin to neutralize LPS, at 37°C for 20 h. The agonists used as positive controls were: Pam3CysSK4 (P3C) for TLR2/1, fibroblast stimulating ligand-1 (FSL-1) for TLR2/6, and bacterial lipopolysaccharide (LPS) for TLR4. Medium was used as negative control for cell stimulation (white bars). The cell supernatants were analyzed for IL-8 by ELISA. Statistical differences were determined by comparing transfected cells stimulated with rPCN to transfected cells incubated with medium. The results were considered significant when p<0.01 (**) or p<0.001 (***).</p

Therapeutic administration of rPCN increases proinflammatory cytokine and NO production.

<p>Lung homogenates were analyzed for IL-12p40 (A), IFN-γ (B), TNF-α (C), IL-10 (D), IL-4 (E), and NO (F) concentrations. Data represent the mean and SD of five mice per group; the experiments were performed in triplicate. * p<0.05; ** p<0.01; *** p<0.001 <i>vs.</i> the PBS group.</p

Possible mechanism of the protection against murine paracoccidioidomycosis conferred by paracoccin administration, as suggested by <i>in vivo</i> and <i>in vitro</i> studies.

<p>Once administered to BALB/c mice, before or after inoculation of <i>P. brasiliensis</i> yeasts, rPCN interacts with N-glycans of TLRs on antigen-presenting cells (APC). It triggers IL-12 production, which drives immunity to the Th1 axis. Production of IL-10 is also induced. The consequent balanced Th1 immunity that is developed protects mice against PCM, as manifested by lower incidence of granulomatous lesions and more efficient fungal clearance in the lungs, at day 30 post-infection.</p