Structural and Topographic Dynamics of Pulmonary Histopathology and Local Cytokine Profiles in Paracoccidioides brasiliensis Conidia-Infected Mice
[['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 [1]–[3] .', 'The primary infection takes place in the lungs where the fungus may persist inactive for years .', 'Tuder et al . ( 1985 ) , in twelve patients with chronic PCM , described five main pathologic aspects:', '( a ) pneumonic reaction;', '( b ) early granulomatous formation;', '( c ) mature and healed granulomas;', '( d ) mixed pattern ( early and mature granuloma in the same pulmonary area ) ;', '( e ) pulmonary fibrosis , with the latter being present in all cases [4] .', 'In addition , in chronic human PCM , the lesions may be either diffuse or circumscribed [3] .', 'Human studies , however , do not allow the histopathologic sequential analyses required to build up the sequence of events .', 'Therefore , several experimental models have been applied to compensate this limitation , aiming to better define the pathogenic mechanisms in PCM [5]–[11] .', 'Despite important differences in the experimental designs due to infective forms ( conidia , yeasts ) and fungal isolates used , inoculum concentration , route of inoculation and host genetic background , overall results have contributed to determine some stages of the inflammatory dynamics , delineate cellular and extracellular matrix composition of the granulomas and related aspects [5]–[11] .', 'Nonetheless , a detailed description of the topographic location of the lesions in the lung compartments and the characteristics of other inflammatory patterns , besides the granulomatous reaction , had been incomplete and superficially referred to in the literature .', 'Additionally , the immunological studies reported have been focused on specific cell populations or tested a restricted number of cytokines produced in vivo or in vitro [12]–[14] .', 'Therefore , the aim of this study was to sequentially determine histopathologic patterns of lesions and multiplex-determined cytokine profiles in an experimental model of PCM .', '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 ( n\u200a=\u200a50 ) and infected mice ( n\u200a=\u200a50 ) .', 'Paracoccidioides brasiliensis isolate ATCC-60855 from a Colombian patient registered at the American Type Culture Collection ( Rockville , MD ) , and known to produce abundant conidia was used in all experiments [15] .', 'The techniques applied to grow the mycelial forms with subsequent collection and dislodgement of conidia had been previously reported [15]–[17] .', '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 ) [18] .', 'When deep anesthesia was obtained , 3×106 Pb viable conidia ( in 0 . 06 ml of the inoculum ) were intranasally instilled .', 'Control mice received an intranasal inoculum of 0 . 06 ml of PBS .', 'Ten animals ( 5 control and 5 Pb-infected mice ) were euthanized by thiopental overdose ( Sandoz GmbH .', 'Kundl Austria; 1 ml at 2 . 5% ,', 'i . p ) at 2 hours and 4 , 8 , 12 and 16 weeks post-inoculation in accordance to animal ethical practices .', 'Lungs of mice were intracardiacally perfused with 10% neutral formalin in phosphate-buffered saline , removed and fixed in the same solution for at least 48', "h . Fixed lungs were embedded in paraffin and coronal sections ( 5 µm ) were stained by the following methods: hematoxylin-eosin ( HE ) ; Grocott's methenamine-silver nitrate technique ( Grocott ) for identification of fungi; Lennert's Giemsa for cellular characterization [19]; Sirius red pH 10 . 2 for eosinophils [20] , [21]; Weigert's resorcin-fuchsin for elastic fibers; Masson' trichrome; PAS and PAS-Alcian Blue pH 2 . 5 and 1 . 0 for proteoglycans and neutral glycoproteins; Gomori's silver reticulin for reticulin fibers and picrosirius with fast green ( PIFG ) , for identification of collagen I and III [22] .", 'Slides were automatically scanned by ScanScope® CS ( Aperio , USA ) and also analyzed with Axio Observer . Z1 ( Zeiss ) , coupled with digital camera AxioCam HRc and software AxioVision 4 . 7 . 2 ( Zeiss ) .', 'Representative specimens were also stained with Phosphomolibdic acid-Picrosirius red ( PMA-PS ) [23] for interstitial collagen and analyzed by confocal microscopy ( LSM 510 Meta-Zeiss ) .', '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 ( http://www . aperio . com/download-imagescope-viewer . asp ) .', 'Tissue sections were examined and evaluated blindly by a pathologist .', '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 [24] .', 'Rabbit immunoglobulin to human fibronectin ( DAKO , Cat # A245 ) , known to react with fibronectin-covered Pb yeasts [25] , was used to better observe the yeasts into a “perifungal space” .', 'Indirect immunofluorescence was done on paraffin lung sections as described [25] .', 'All slides were counterstained with Evans blue at a dilution of 1/10 . 000 and mounted in buffered glycerol containing p-phenylenediamine and analyzed by confocal microscopy ( LSM-510-META , Zeiss ) .', '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 http://www . R-project . org ) .', 'Cytokine data were initially analyzed by exploratory data analysis ( EDA ) .', 'The variables were individually plotted with 40% of smoothing by lowess ( local regression ) , aiming to define the behavior of each variable during the infection .', "Pb-infected and control groups were compared by non-parametric Kruskal-Wallis test with Dunn's multiple comparisons post-test ( Prism 5 . 0 software , Graph Pad , USA ) dividing the groups up to four and after eight weeks of infection .", 'p values less than 0 . 05 were considered statistically significant; p values less than 0 . 01 were considered statistically highly significant .'], ['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 Pb conidia exhibited , from the 4 weeks onwards , pulmonary lesions with three main histopathologic patterns: the classical and commonly reported nodular or granulomatous pattern , followed by inflammatory peri-arterial sheaths and pseudotumoral masses .', 'A quantitative evaluation of those inflammatory infiltrates is illustrated in the Figure 1 .', '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 Figure 3 .', '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 Table 1 .', '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 Pb-infected group was compared with those of the control group by the Kruskal-Wallis test using two time periods consisting of two hours up to four weeks and eight to sixteen weeks of infection ( Figure 4 ) .', 'These analyses enabled us to identify five different patterns of inflammatory mediators , as follows:', '( a ) Pattern 1: molecules significantly up regulated until the 4 week and then becoming normalized ( IL-1α , IL-1β , IL-4 , IL-6 , IL-10 , IL-12p70 , IL-17 , Eotaxin , G-CSF , MCP1 and MIP1α ) ;', '( b ) Pattern 2: molecules significantly up regulated up to 4 weeks and then significantly down regulated ( IL-5 , IL-13 , GM-CSF , IFN-γ , MIP1β and TNFα ) ;', '( c ) Pattern 3: molecules without significant changes up to 4 weeks but significantly down-regulated after 8 weeks p . i . ( IL-2 , IL-3 , IL-9 , IL-15 ) ;', '( d ) Pattern 4: molecules significantly up-regulated at all times p . i . ( MIG , RANTES , and IL-12p40 ) ; and', '( e ) Pattern 5: composed only by one molecule ( PDGF ) without significant changes up to 4 weeks but significantly up-regulated after 8 weeks p . i . In the control group , cytokine levels showed no significant variation with time except for IL15 that increased with observation period .'], ['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 P . b conidia , as follows: In the first stage ( 2 hours p . i . ) , a mild septal neutrophilic and macrophagic infiltration accompanied by an intense “cytokine burst” was observed in lung tissues as shown by a significant increase of IL-1α , IL-1β , IL-4 , IL-5 , IL-6 , IL-10 , IL-12p70 , IL-13 , IL-17 , Eotaxin , G-CSF , MCP1 , MIP1α , GM-CSF , IFN-γ , MIP1β and TNFα ( patterns 1 and 2 ) .', '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: In the course of these studies we have noticed certain histopathological findings that due to their novelty deserve further exploration , as discussed below: These overall results complement previous work by our group [51] , where noninvasive conventional medical X-ray tomography allowed the in vivo follow-up of the three main pathological patterns described above .', '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 .']]
['Introduction', 'Materials and Methods', 'Results', 'Discussion']
['Paracoccidioidomycosis ( PCM ) , an endemic systemic mycosis caused by the fungus Paracoccidioides brasiliensis ( Pb ) , usually results in severe lung damage in patients .', 'Considering the difficulties to sequentially study the infection in humans , this work was done in mice inoculated intranasally with infective Pb-conidia .', 'Lungs of control and Pb-infected mice were studied after 2-hours , 4 , 8 , 12 and 16-weeks post-infection ( p . i ) in order to define histopathologic patterns of pulmonary lesions , multiplex-cytokine profiles and their dynamics during the course of this mycosis .', 'Besides the nodular/granulomatous lesions previously informed , results revealed additional non-formerly described lung abnormalities , such as periarterial sheath inflammation and pseudotumoral masses .', 'The following chronologic stages occurring during the course of the experimental infection were defined: Stage one ( 2-hours p . i ) : mild septal infiltration composed by neutrophils and macrophages accompanied by an intense “cytokine burst” represented by significant increases in IL-1α , IL-1β , IL-4 , IL-5 , IL-6 , IL-10 , IL12p70 , IL-13 , IL-17 , Eotaxin , G-CSF , MCP1 , MIP1α , GM-CSF , IFN-γ , MIP1β and TNFα levels .', 'Stage two ( 4-weeks p . i ) : presence of nodules , evidence of incipient periarterial- and intense but disperse parenchymal- inflammation , abnormalities that continued to be accompanied by hyper-secretion of those cytokines and chemokines mentioned in the first stage of infection .', 'Stages three and four ( 8 and 12-weeks p . i . ) : fungal proliferation , inflammation and collagenesis reached their highest intensity with particular involvement of the periarterial space .', 'Paradoxically , lung cytokines and chemokines were down-regulated with significant decreases in IL-2 , IL-3 , IL-5 , IL-9 , IL-13 , IL-15 , GM-CSF , IFN-γ , MIP1β and TNFα .', 'Stage five ( 16-weeks p . i . ) : inflammation decreased becoming limited to the pseudotumoral masses and was accompanied by a “silent” cytokine response , except for PDGF , MIG , RANTES and IL12p40 which remained up-regulated for the duration of the experiment .', '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 .']
['medicine', 'histology', 'infectious diseases', 'model organisms', 'neglected tropical diseases', 'immunology', 'biology', 'microbiology', 'fungal diseases', 'infectious disease modeling', 'respiratory medicine', 'pulmonology']