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Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro.

Della Coletta AM, Bachiega TF, de Quaglia e Silva JC, Soares ÂM, De Faveri J, Marques SA, Marques ME, Ximenes VF, Dias-Melicio LA - PLoS Negl Trop Dis (2015)

Bottom Line: The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release.In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro.The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, UNESP - São Paulo State University, Botucatu Medical School, Botucatu, São Paulo, Brazil.

ABSTRACT
Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.

No MeSH data available.


Related in: MedlinePlus

Image of the customized method with MetaXpress Custom Module Editor and measured by Integrated Morphometry Analysis.Images were acquired using the automated microscope ImageXpress Micro XL Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA) with a cooled 16-bit monochromes CMOS PCO camera (2160 × 2160 imaging array, 6.5 × 6.5 μm pixels) using an 20× Super Plan Fluor ELWD, NA 0.45 Nikon objective. Total area (μm2) of the resulting NETs in response to Pb18 and Pb265 was analyzed by thresholding the NETs stained with DAPI and anti-elastase-FITC antibody and measured by Integrated Morphometry Analysis. (A) DAPI and (B) FITC.
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pntd.0004037.g009: Image of the customized method with MetaXpress Custom Module Editor and measured by Integrated Morphometry Analysis.Images were acquired using the automated microscope ImageXpress Micro XL Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA) with a cooled 16-bit monochromes CMOS PCO camera (2160 × 2160 imaging array, 6.5 × 6.5 μm pixels) using an 20× Super Plan Fluor ELWD, NA 0.45 Nikon objective. Total area (μm2) of the resulting NETs in response to Pb18 and Pb265 was analyzed by thresholding the NETs stained with DAPI and anti-elastase-FITC antibody and measured by Integrated Morphometry Analysis. (A) DAPI and (B) FITC.

Mentions: In addition to the obtained images, the same PMN cultures were analyzed in attempt to quantify the number of extracellular DNA structures, which represents NETs released after the culture’s challenge with Pb18, Pb265 or PMA activation. Analysis consisted in thresholding the structures stained with DAPI (DNA). Total DNA structures were quantified using a customized method with MetaXpress Custom Module Editor and measured by Integrated Morphometry Analysis presented as an average of extracellular DNA structures for each well. Corroborating our images, total extracellular DNA structures were substantially higher in the cultures activated with PMA or challenged with Pb18 or Pb265 when compared to non-treated PMNs, showing a significantly increase of extracellular DNA structures staining in cultures challenged mainly with Pb18 (p = 0.0016) (Fig 7). We also observed in these experiments, interesting different NETs patterns from cultures challenged with Pb18 or Pb265. Analyzing NETs area (μm2), that consisted in thresholding the structures stained with DAPI (DNA) colocalized with FITC (elastase), NETs from Pb18 coculture appeared to be bigger and more scattered than those presented by PMNs challenged with Pb265, which seemed to be smaller (p = 0.0625) (Fig 8), even though both are capable of entrapping the yeast cells as shown in Figs 5 and 6. Fig 9 was included to demonstrate how the software selected and performed the analysis.


Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro.

Della Coletta AM, Bachiega TF, de Quaglia e Silva JC, Soares ÂM, De Faveri J, Marques SA, Marques ME, Ximenes VF, Dias-Melicio LA - PLoS Negl Trop Dis (2015)

Image of the customized method with MetaXpress Custom Module Editor and measured by Integrated Morphometry Analysis.Images were acquired using the automated microscope ImageXpress Micro XL Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA) with a cooled 16-bit monochromes CMOS PCO camera (2160 × 2160 imaging array, 6.5 × 6.5 μm pixels) using an 20× Super Plan Fluor ELWD, NA 0.45 Nikon objective. Total area (μm2) of the resulting NETs in response to Pb18 and Pb265 was analyzed by thresholding the NETs stained with DAPI and anti-elastase-FITC antibody and measured by Integrated Morphometry Analysis. (A) DAPI and (B) FITC.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556621&req=5

pntd.0004037.g009: Image of the customized method with MetaXpress Custom Module Editor and measured by Integrated Morphometry Analysis.Images were acquired using the automated microscope ImageXpress Micro XL Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA) with a cooled 16-bit monochromes CMOS PCO camera (2160 × 2160 imaging array, 6.5 × 6.5 μm pixels) using an 20× Super Plan Fluor ELWD, NA 0.45 Nikon objective. Total area (μm2) of the resulting NETs in response to Pb18 and Pb265 was analyzed by thresholding the NETs stained with DAPI and anti-elastase-FITC antibody and measured by Integrated Morphometry Analysis. (A) DAPI and (B) FITC.
Mentions: In addition to the obtained images, the same PMN cultures were analyzed in attempt to quantify the number of extracellular DNA structures, which represents NETs released after the culture’s challenge with Pb18, Pb265 or PMA activation. Analysis consisted in thresholding the structures stained with DAPI (DNA). Total DNA structures were quantified using a customized method with MetaXpress Custom Module Editor and measured by Integrated Morphometry Analysis presented as an average of extracellular DNA structures for each well. Corroborating our images, total extracellular DNA structures were substantially higher in the cultures activated with PMA or challenged with Pb18 or Pb265 when compared to non-treated PMNs, showing a significantly increase of extracellular DNA structures staining in cultures challenged mainly with Pb18 (p = 0.0016) (Fig 7). We also observed in these experiments, interesting different NETs patterns from cultures challenged with Pb18 or Pb265. Analyzing NETs area (μm2), that consisted in thresholding the structures stained with DAPI (DNA) colocalized with FITC (elastase), NETs from Pb18 coculture appeared to be bigger and more scattered than those presented by PMNs challenged with Pb265, which seemed to be smaller (p = 0.0625) (Fig 8), even though both are capable of entrapping the yeast cells as shown in Figs 5 and 6. Fig 9 was included to demonstrate how the software selected and performed the analysis.

Bottom Line: The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release.In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro.The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, UNESP - São Paulo State University, Botucatu Medical School, Botucatu, São Paulo, Brazil.

ABSTRACT
Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.

No MeSH data available.


Related in: MedlinePlus