Figure 6.

F. alocis pre-treatment causes a significant decrease in NET formation induced by PMA. Neutrophils were challenged with PMA for 180 min, or pre-treated with opsonized CFSE-labeled F. alocis (MOI 10 or 100) for 60 min and then exposed to PMA for 180 min. Following infection, cells were fixed and immunostained using Abs directed against MPO (AlexaFluor647), DNA stained with DAPI, and imaged for NET immunofluorescence by confocal microscopy. (a) Representative confocal images (from 3 independent experiment of 100 quantified cells per experiment) of neutrophils stimulated with PMA only (Alone), or pretreated for 60 min with op-CFSE-labeled F. alocis followed by PMA stimulation for 180 min. Neutrophil nucleus/DNA-DAPI (shown in blue); neutrophil MPO (AlexaFluor647 shown in red), Merge image: NET formation and CFSE-F. alocis (shown in green). (b, c) Quantification of percentage of NETs formed using ImageJ analysis. Data are expressed as means of % NETs ± SEM from four independent experiments in (b) and three independent experiments in (c) * P < 0.05. (d) Neutrophils were challenged with PMA only (Alone) for 180 min, or pre-treated with F. alocis culture supernatants for 60 min and then exposed to PMA for 180 min. Quantification of percentage of NETs formed used ImageJ analysis. Data are expressed as means of % NETs ± SEM from three independent experiments. ns, non-significant.