Limits...
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

High-content image acquisition of PMN cultures from healthy donors stained with DAPI (DNA) and anti-elastase antibody (FITC-green).Non-treated PMNs (A), activated with PMA (100ng/mL) for 30 minutes (B), challenged with Pb18 (C) and with Pb265 (D) for two hours (50:1 ratio).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4556621&req=5

pntd.0004037.g006: High-content image acquisition of PMN cultures from healthy donors stained with DAPI (DNA) and anti-elastase antibody (FITC-green).Non-treated PMNs (A), activated with PMA (100ng/mL) for 30 minutes (B), challenged with Pb18 (C) and with Pb265 (D) for two hours (50:1 ratio).

Mentions: To corroborate data obtained by Confocal Microscopy and Scanning Electron Microscopy, we analyzed PMN cultures with the automated microscope ImageXpress Micro XL Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA). Once more, images showed an extracellular material stained with DAPI (DNA) and FITC (elastase), which characterized the presence of NETs in the cultures activated with PMA or challenged with Pb18 or Pb265 (Fig 6B, 6C and 6D, respectively). In Fig 6A, we observed non-treated PMNs, showing few PMNs in NETosis process, ratifying the idea that an activation is essential for NETs release. Whereas, in Fig 6B, when PMA was added to the culture, PMNs were able to release NETs to the extracellular environment. In Fig 6C and 6D, we observed Pb18 and Pb265, respectively, demonstrating the formation of NETs covering the yeast cells, certifying that this defense mechanism is triggered in the infection with P. brasiliensis.


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)

High-content image acquisition of PMN cultures from healthy donors stained with DAPI (DNA) and anti-elastase antibody (FITC-green).Non-treated PMNs (A), activated with PMA (100ng/mL) for 30 minutes (B), challenged with Pb18 (C) and with Pb265 (D) for two hours (50:1 ratio).
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004037.g006: High-content image acquisition of PMN cultures from healthy donors stained with DAPI (DNA) and anti-elastase antibody (FITC-green).Non-treated PMNs (A), activated with PMA (100ng/mL) for 30 minutes (B), challenged with Pb18 (C) and with Pb265 (D) for two hours (50:1 ratio).
Mentions: To corroborate data obtained by Confocal Microscopy and Scanning Electron Microscopy, we analyzed PMN cultures with the automated microscope ImageXpress Micro XL Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA). Once more, images showed an extracellular material stained with DAPI (DNA) and FITC (elastase), which characterized the presence of NETs in the cultures activated with PMA or challenged with Pb18 or Pb265 (Fig 6B, 6C and 6D, respectively). In Fig 6A, we observed non-treated PMNs, showing few PMNs in NETosis process, ratifying the idea that an activation is essential for NETs release. Whereas, in Fig 6B, when PMA was added to the culture, PMNs were able to release NETs to the extracellular environment. In Fig 6C and 6D, we observed Pb18 and Pb265, respectively, demonstrating the formation of NETs covering the yeast cells, certifying that this defense mechanism is triggered in the infection with P. brasiliensis.

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