Fig 1.
Histopathology of cutaneous lesions of patients with PCM showing Hematoxilin filamentous suggestive of NETs (white arrows) (A-20x and B-35x, insert-40x).
The same sample was stained with Gomori-Grocott, confirming the presence of P. brasiliensis in the lesion (black arrows) (C-20x and D-35x). (Bar size: A and C—100μm, B and D—50μm).
Fig 2.
Confocal microscopy of NETs identified in skin lesions of patients with PCM.
Tissue was stained with DAPI (A), labeled with anti-elastase antibody followed by FITC-conjugated secondary antibody (B) and anti-histone H1 secondary antibody followed by Texas Red (C). In the last frame, the overlapping images (D). (Bar size 15μm).
Fig 3.
Confocal microscopy of NETs identified in skin lesions of patients with PCM.
Tissue was stained with DAPI (A), labeled with anti-elastase antibody followed by FITC-conjugated secondary antibody (B) and anti-histone H1 secondary antibody followed by Texas Red (C). In the last frame, the overlapping images (D). The highlights show the process of NETs formation. (Bar size 10μm).
Fig 4.
Scanning electron microscopy from PMNs challenged with P. brasiliensis (50:1 ratio) in different periods of incubation showing NETs release.
(A) PMNs activated with PMA (100ng/mL) for 30 minutes. (B) PMNs challenged with Pb18 for one hour. (C) PMNs challenged with Pb18 for two hours. (D) PMNs challenged with Pb18 and treated with DNAse (100U/mL) for 30 minutes. (PMN–neutrophil; Pb–P. brasiliensis; NETs–Neutrophil Extracellular Traps).
Fig 5.
Scanning electron microscopy from patients’ PMNs challenged with Pb18 (50:1 ratio) (A and B) or Pb265 (50:1 ratio) (C and D).
PMN cultures were challenged for two hours before analysis. (PMN–neutrophil; Pb–P. brasiliensis; NETs–Neutrophil Extracellular Traps).
Fig 6.
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).
Fig 7.
Average of total extracellular DNA structures released by PMN cultures activated with PMA (100 ng/mL) for 30 minutes or challenged with Pb18 or Pb265 (50:1 ratio) for two hours (p = 0.0016).
Fig 8.
Measure of NETs area (μm2) released by PMN cultures challenged with Pb18 or Pb265 (50:1 ratio) for two hours (p = 0.0625).
Fig 9.
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.
Fig 10.
PMNs from healthy donors were stimulated with PMA (100 ng/mL) for 30 minutes or challenged with Pb18 and Pb265 (50:1 ratio) for two hours. Supernatants were collected, and DNA were quantified by Picogreen dsDNA kit (p = 0.0167).