Figure 2
mDCs interact with NETotic PMNs and are loaded with proteins from NET. (A) NETs persistently interact with mDCs. In this live imaging experiment, agar-PMNs were seeded onto culture dishes and allowed to adhere 30 minutes before adding mDCs previously labeled with the membrane vital dye PKH-26. The cocultures were also performed in the presence of the DNA dye SYTOX green, which stains only PMNs undergoing NETosis. The coculture was maintained o/n in a humidified chamber under confocal microscope, acquiring a picture every 10 minutes to highlight any interaction between PMNs and mDCs. PMNs in the cellular mixture form NETs (turquoise arrows) and NETs interact with mDCs (red arrows). Black arrows indicate naive neutrophils. Scale bars represent 5 μm. (B) Uploading of mDCs with NET components. After NET interaction, mDCs are uploaded with the neutrophil protein PR3 and MPO. In this live imaging experiment, labeled PKH-26 mDCs were added to agar-PMN in the presence of a mAb to PR3 or MPO directly conjugated with Alexa-488 dye. After 18 hours, cells were fixed with PFA 4% and observed under a confocal microscope. Scale bars represent 10 μm. One representative experiment of 5 performed. (C) Uploading of PR3 and MPO by mDCs tested in the presence of different type of PMN death. Apoptotic PMNs, generated by Fas triggering Ab and necrotic PMNs obtained by freeze and thaw, were compared with NETotic PMNs for their capability to transfer PR3 and MPO to mDCs in coculture experiments. Representative confocal IF analysis showing that necrotic PMNs almost failed to transfer neutrophil PR3 or MPO to mDCs; on the contrary, PR3 and MPO were detectable as dotted green fluorescence in mDCs that interacted with NETotic PMNs and, to a lesser extent, in mDCs cultured with apoptotic PMNs. (D-E) Software-assisted micrograph quantification of mDCs uploading of PR3 (D) and MPO (E) performed on a total of 200 cells that were imaged by confocal microscopy. DCs cultured with naive PMNs, maintained alive by incubating them in the presence of 30% of FCS and DCs alone. were the negative controls for PR3 transfer and MPO. Scale bars represent 30 μm. (F) Appearance of PR3+ apoptotic bodies that are taken up by mDCs cocultured with apoptotic cells.

mDCs interact with NETotic PMNs and are loaded with proteins from NET. (A) NETs persistently interact with mDCs. In this live imaging experiment, agar-PMNs were seeded onto culture dishes and allowed to adhere 30 minutes before adding mDCs previously labeled with the membrane vital dye PKH-26. The cocultures were also performed in the presence of the DNA dye SYTOX green, which stains only PMNs undergoing NETosis. The coculture was maintained o/n in a humidified chamber under confocal microscope, acquiring a picture every 10 minutes to highlight any interaction between PMNs and mDCs. PMNs in the cellular mixture form NETs (turquoise arrows) and NETs interact with mDCs (red arrows). Black arrows indicate naive neutrophils. Scale bars represent 5 μm. (B) Uploading of mDCs with NET components. After NET interaction, mDCs are uploaded with the neutrophil protein PR3 and MPO. In this live imaging experiment, labeled PKH-26 mDCs were added to agar-PMN in the presence of a mAb to PR3 or MPO directly conjugated with Alexa-488 dye. After 18 hours, cells were fixed with PFA 4% and observed under a confocal microscope. Scale bars represent 10 μm. One representative experiment of 5 performed. (C) Uploading of PR3 and MPO by mDCs tested in the presence of different type of PMN death. Apoptotic PMNs, generated by Fas triggering Ab and necrotic PMNs obtained by freeze and thaw, were compared with NETotic PMNs for their capability to transfer PR3 and MPO to mDCs in coculture experiments. Representative confocal IF analysis showing that necrotic PMNs almost failed to transfer neutrophil PR3 or MPO to mDCs; on the contrary, PR3 and MPO were detectable as dotted green fluorescence in mDCs that interacted with NETotic PMNs and, to a lesser extent, in mDCs cultured with apoptotic PMNs. (D-E) Software-assisted micrograph quantification of mDCs uploading of PR3 (D) and MPO (E) performed on a total of 200 cells that were imaged by confocal microscopy. DCs cultured with naive PMNs, maintained alive by incubating them in the presence of 30% of FCS and DCs alone. were the negative controls for PR3 transfer and MPO. Scale bars represent 30 μm. (F) Appearance of PR3+ apoptotic bodies that are taken up by mDCs cocultured with apoptotic cells.

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