Conceived and designed the experiments: LEC HLL DL TWN. Performed the experiments: DL TWN. Analyzed the data: DL HLL OGC LEC. Contributed reagents/materials/analysis tools: HLL. Wrote the paper: DL HLL AR. Scientific advisor of the work who reviewed the manuscript: AR LEC.
The authors have declared that no competing interests exist.
Paracoccidioidomycosis (PCM), an endemic systemic mycosis caused by the fungus
Considering the difficulties to sequentially study the infection in humans, this work was done in mice inoculated intranasally with infective
Results of this study identified both classic and novel patterns corresponding to histopathologic and immunologic responses occurring during the course of experimental PCM.
Paracoccidioidomycosis (PCM), an endemic fungal infection of pulmonary origin resulting in severe disseminated disease, occurs in rural areas of most South American countries and presents several clinical forms. The infection is acquired by inhalation of specific fungal propagules, called conidia. Considering the difficulties encountered when studying the infection in humans, this work was done in mice infected by inhalation of infective fungal conidia thus mimicking the human natural infection. The lungs of mice were sequentially studied by histopathological and multiplex cytokine methods from 2 h to 16 weeks after infection to verify the course of the disease. The mycosis presented different morphologic aspects during the course of time, affecting several pulmonary compartments. Otherwise and based on the analysis of 30 cytokines, the immune response also showed heterogeneous responses, which were up or down regulated depending on the time of infection. By recognizing the different stages that correspond to the evolution of pulmonary lesions, the severity (benign, chronic or fibrotic) of the disease could be predicted and the probable prognosis of the illness be inferred.
The chronic adult form of PCM progresses slowly with minor symptoms and may take months to years to become clinically manifested; consequently, when the patients are finally diagnosed and receive medical treatment, pulmonary lesions are already well established, presenting different stages of development
The primary infection takes place in the lungs where the fungus may persist inactive for years. Tuder
A deeper knowledge of the pattern of lesions and cytokines profiles induced by the fungus and their changes according to the time of infection could contribute to prognosis prediction.
All animals were handled according to the national (Law 84 of 1989, Res No. 8430 of 1993) and international (Council of European Communities and Canadian Council of Animal Care, 1998) guidelines for animal research and the experimental protocols were approved by Corporación para Investigaciones Biológicas (CIB) research ethics committee.
BALB/c mice were originally obtained from Taconic Farms, Inc., Quality Laboratory Animals and Services for Research, New York, USA, with the breeding colony being expanded at the (CIB), Medellin, Colombia. Male mice, 6–7 weeks old and approximately 20 g in weight were used in this study. Mice were divided into 2 groups: non-infected control mice (
Mice were anesthetized by the intramuscular injection of a solution containing -ketamin hydrochloride (Park, Davis & Company, Berlin, Germany; 100 mg/kg) and xylazine (Bayer, Brazil 10 mg/kg)
Ten animals (5 control and 5
Location, shape, size, frequency, cellular composition, fungal budding patterns, and collagen deposits features were evaluated for each inflammatory pattern using scanned slides images and the free ImageScope software (
The total inflammatory area was measured by using one panoramic image of both lungs per mouse. Regions of interest (ROIs), correspondent to the inflammatory regions, were manually drawn and measured. The percentage of pulmonary area with inflammatory reaction was calculated dividing the sum of total ROIs by the total area of lung (excluding the air space).
The Aperio positive pixel count algorithm was used to quantify the amount of a specific stain present in a scan slide image. Red pixels in the PIFG stained slides were measured to quantify collagen, and brown/black pixels in the Grocott's stained slides to quantify yeast cells.
Cellular composition was evaluated using a semi-quantitative approach. A score of − to +++ was given according to the degree of infiltration with (+++) for intense, (++) for moderate, (+) for slight, (±) for very slight and (−) for no reaction
Rabbit immunoglobulin to human fibronectin (DAKO, Cat # A245), known to react with fibronectin-covered
Lungs of five animals per group were individually homogenized in tissue grinder (Tissue tearor, model 985-370, Biospec Products) with a cocktail solution of protease inhibitors (Pepsin 0.1 uM, Leupeptin 0.1 uM, Phenylmethyl sulfonide fluoride 1 mM, N-tosil-L-Phenilalamine choromethyl ketone 0.2 mM, (a)-p-methyl L lisyne choromethyl ketone 0.1 mM from Sigma chemical, plus ethylene diamine tetra-acetic acid EDTA 1 nM from Merck Germany). Homogenized lungs supernatant were collected by centrifugation at 3000 rpm for 15 min at 4°C, aliquoted and stored at −70°C until the analysis day.
All homogenized lung supernatants were normalized to 1 mg/ml of protein. Then, a magnetic bead-based multiplex assay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokines, chemokines, and growth factors measurement according to the manufacturer's instructions (Bio-Plex pro-mouse cytokine 23-plex assay and group II customized 8-plex assay; Bio-Rad Inc., Hercules, CA, USA). Molecules measured were: IL-1-α ,IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), IFN-γ, KC/CXCL1, monocyte chemoattractive protein (MCP-1/CCL2), macrophage inflammatory protein-1 alpha and beta (MIP-1α/CCL3 and MIP-1β/CCL4), RANTES (CCL5), TNF-α, leukocyte inhibitory factor (LIF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGFb), monokine induced by IFN-γ (MIG/CXCL9) and macrophage inflammatory protein-2 (MIP-2/CXCL2).
Cytokine levels were determined using a multiplex array reader from Luminex™ Instrumentation System (Bio-Plex Workstation from Bio-Rad Laboratories). The analyte concentration was calculated using software provided by the manufacturer (Bio-Plex Manager Software) and expressed as pg.
Histopathology results were obtained from five biological replicates at each time point. Multiplex cytokine bioplex assays were analyzed in five additional animals at each time and run in duplicates.
Cytokine data analysis was performed through the use of R language (R Development Core Team, 2010). R: A language and environment for statistical computing. (R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL
None of the animal receiving PBS exhibited histopathologic alterations except transient and slight septal infiltration of neutrophils with some eosinophils at 2 h post-inoculations. In comparison, the lungs of BALB/c mice infected with
The bar charts show the percentage of pulmonary area occupied by each type of histopathologic pattern of lesions at 4, 8, 12 and 16 weeks post
It corresponded to multiple sphere-like or oval-shaped parenchymal granulomas adjacent to terminal bronchioles, sometimes isolated, but becoming confluent with the course of infection (
Nodules (A to D), periarterial sheath inflammation (E to H) and pseudotummoral mass (I to L). (A) Multiple nodules in a panoramic view (3.5×), H&E. (B) Typical granuloma structure with intermixed PMN (arrow), Sirius red pH 10.2, 10×. (C) Granuloma with atheromatous appearance showing multiple cholesterol crystals (arrows) and Touton giant cells (insert, 100×), Masson's trichrome stain, 20×. (D) Xantomatous macrophages with foamy cytoplasm in the central zone of granuloma (63×), H&E. (E) Panoramic view of a complete coronal lung section (12th wk p.i) showing extensive inflammatory consolidation in the left lung with multiple periarterial lesions (0.4×), H&E. (F) Periarterial sheath inflammation showing also several fungi enclosed in perifungal spaces (40×), Sirius red pH 10.2. (G) Large number of plasmocytes and small macrophage aggregates (63×), Lennert's Giemsa. (H) Mott cells containing Russell's bodies (cells in red) (63×), Masson's trichrome stain. (I) Panoramic view of a coronal lung section (16th wk p.i) showing a pseudotumoral mass in the left lung obstructing the bronchial lumen (0.4×), H&E. (J) Detail of pseudotumoral mass presented in figure I showing its large dimension (20×), Sirius red pH 10.2, (K) Bronchial obstruction remarked in F, H&E, 20×. (L) Focus of plasmocytogenesis expressed by plasmoblasts and pro-plasmocytes (arrow) in the periphery of a pseudotumoral mass (100×), Sirius red pH 10.2.
Their cellular composition varied according to time of infection. At 4 weeks, the granulomas were composed by a central core of macrophages with fungi and intermixed neutrophils sometimes presenting an apoptotic aspect (
The most peripheral zone often consisted of non-continuous halo of small lymphocytes, at times forming pseudo-follicular lymphocyte aggregates.
At 8 weeks post-infection (p.i.), the nodules showed an increase in their number and a higher confluence among them. The most significant change in the central zone was the appearance of some xantomatous cells and cholesterol crystals. Additionally, the number of neutrophils significantly decreased and a large number of mature plasma cells appeared at the granuloma periphery.
At 12 weeks p.i., the xantomatous aspect of the macrophages and the number of cholesterol crystals were intensified (
At 16 weeks p.i., nodules presented a significant increase in the number of xantomatous and Touton cells.
At 4 weeks, the periarterial spaces were globally preserved but presented lymphatic dilatation and edema. The inflammatory cells consisted in only focal accumulation of small lymphocytes and some macrophages with neutral glycoproteins (PAS positive stain).
At 8 weeks, the involvement of this pulmonary compartment was more diffuse and defined periarterial inflammatory sheaths (
The same aspects were also seen at 12 weeks p.i. except for presenting a higher number of eosinophils, plasmocytes and Mott cells (
Peri-venous inflammation occurred less frequently than arterial involvement with the former being more prominent at 12 weeks p.i. The perivenular cellular composition was similar to that in arteries.
Pseudotumoral masses were 5 to 6 times larger (2000±194 µm of length) than the nodules (
The aspect and quantity of extracellular matrix were dependent on the type of lesion and time of infection. Collagen fibers gradually increased up to 12 weeks, with further decrease at 16 weeks.
Comparative features between extracellular matrix arrangements in the lesions are presented in the
(A to C) PMA-picrosirius stain for collagen. (D to F) Weigert's resorcin-fuchsin stain for elastic fibers. Lower insert in A and B shows fibronectin expression observed by confocal indirect Immunofluorescence. (A) Nodule presenting a thin and concentric pseudo-capsule (10×), better exemplified in upper insert (100×). Lower insert in (A) shows fibronectin expression. (B) Periarterial space showing more abundant and thicker collagen fibers than the ones surrounding nodules (10×), detailed in the upper insert (100×). Lower insert in B shows a slight and diffuse fibronectin expression. (C) Pseudotumoral mass illustrating interstitial collagens with slight intensification at the periphery and around small cocoons (10×) (insert 100×). A large central area is devoid of collagen fibers. (D) Three adjacent nodules with intense elastolysis (10×). Insert shows tiny residual elastic fibers in the periphery of nodule (100×). (E) Periarterial space with radial elastogenesis (10×), detailed in the insert (100×). (F) Focal intimal vein thickening (10×).
The morphology and quantity of the fungi in the lung tissue also varied depending on the location of the lesions and the time of the infection with a tendency to increase fungal numbers at 8 and 12 weeks post-infection.
In nodules, fungal cells were restricted to the central zone with predominance of large yeasts (10–20 µm). In comparison with the nodular pattern, fungi in perivascular lesions were more dispersed and frequently presenting a perifungal space surrounding a large mother cell with multiple small oval buds, less than 2 µm. These buds formed concentric aggregates around the mother cell and/or were dispersed in or attached to the external border of the perifungal space. Fungal cells were present in high amounts in pseudotumoral masses.
A summary of the main histopathologic aspects for each pulmonary lesion is represented in
Nodular | Peri-arterial sheath | Pseudotumoral masses | |
Location | Parenchyma, adjacent to terminal bronchiole | Around arteries | Upper third of lung, mainly hilar |
Shape | Sphere-like or oval-shaped | Sheath-like | Sphere-like or oval-shaped |
Size | 360.2±SD 103.7 µm in diameter/length | Variable | 2000±194 µm of length |
Time of maximum appearance | 8th week post-infection | 12th week post-infection | 16th week post-infection |
Plasmocytes | +/++ | +++ | +++ |
Eosinophils | + | +++ | +++ |
Yeasts with perifungal space | + | +++ | + |
Interstitial Collagen | + | +++ | ++ |
Elastic fibers | Elastolysis | Elastogenesis | Elastolysis |
By means of exploratory data analysis, the kinetic of each cytokine was plotted against time of infection using Lowess smoothing (40%). The observed trajectories (data not showed) clearly showed a switch of some cytokine levels up to 4- and from 8- weeks afterwards. To test this statistical hypothesis, each cytokine in the
Control and
The overall results of this study, once integrated, have allowed to differentiate five chronological stages occurring during the course of experimental PCM induced by inhalation of infective
In the second stage (4 wks p.i.), nodules and incipient periarterial inflammatory reaction, together with disperse and intense parenchymal inflammation became evident. In addition, hyper-secretion of the cytokines and chemokines grouped in patterns 1 and 2 was also recorded.
In the third and fourth stages (8 and 12 wks p.i., respectively) fungal growth, inflammation and collagenesis reached their highest intensity with particular involvement of the periarterial space. Paradoxically at this point, lung cytokines and chemokines were down-regulated as revealed by significant decreases in IL-5, IL-13, GM-CSF, IFN-γ, MIP1β, TNFα, IL-2, IL-3, IL-9 and IL-15 (patterns 2 and 3).
Finally, in the fifth stage (16 wks p.i.), the inflammation decreased and became practically restricted to the pseudotumoral masses which were now accompanied by a “silent” cytokine response, except for PDGF, and MIG, RANTES and IL12p40, all of which remained up- regulated during the whole experimental period.
Some associations among morphological changes and cytokine profiles could be attempted, as follows:
High levels of IL-1α, IL-1β, IL-6, IL-17, TNFα and chemokines such as eotaxin, MCP1 and MIP1α indicated a highly proinflammatory milieu at 2 h and 4 wks p.i. and would explain the recruitment of monocytes, PMNs and eosinophils
During the first 4 wks p.i, high levels of both Th2 cytokines (IL-4, IL-5, IL-13) and Th1 cytokines (IFN-γ, IL-12p70) were detected, indicating that during this particular infectious stage no polarization of these molecules had occurred in the lungs of
IFN-γ is considered to mediate resistance to PCM
For the first 4 wks p.i., pro-inflammatory cytokines, chemokines and growth factors co-existed with high concentrations of immune-regulators such as IL-10 and IL-12p40. IL-10 hinder full activation of monocytes/macrophages and T cells decreasing their fungicidal activity
MIG (CXCL9), RANTES (CCL5) and IL-12p40 were up-regulated at all times during the experimental period, including most chronic times (8, 12 and 16 wks) when the remaining cytokines decreased. The molecules referred to above could be considered as markers when discriminating PCM cases from controls and could also be useful when treatment follow-up is considered. Indeed, in a study with human PCM patients, serum levels of MIG/CXCL9 were higher in all untreated cases and decreased progressively with treatment
In the course of these studies we have noticed certain histopathological findings that due to their novelty deserve further exploration, as discussed below:
Foamy cells were frequent in the granuloma central areas, as were Touton cells and large cholesterol crystals suggesting participation of lipids in
Another peculiar feature in experimental PCM was the involvement of the periaterial space. Although this compartment has been regularly neglected, its involvement has been reported in several animal models, such as respiratory syncycial virus, bacteria including
We reported pseudotumoral masses in the lungs of
These overall results complement previous work by our group
In conclusion, this study has revealed different patterns of pulmonary lesions according to time post-infection, as well as different cytokine trajectories both of which have allowed the description of five stages in the development of experimental PCM. Recognizing those stages in the course of the mycosis could help us to predict the severity (benign, chronic or fibrotic) of this disease and infer the probable prognosis of the illness.
Finally, this study has open up future possibilities to investigate the mechanisms involved in the different morphological patterns described, as well as gaining new insights into the pathogenesis of PCM.
The authors thank Beatriz E. Finkel-Jimenez, Director, Immunology Core, Stanley S. Scott Cancer Center, LSU HSC - New Orleans, USA, for her cooperation in the multiplex cytokine testing. The authors also thank the personnel of the Laboratório de Patologia, Instituto Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil, for their assistance with histologic methods, immunofluorescence and confocal laser scanning microscopy.