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S87 Bronchiectasis relevant molecules promote NETosis and citrullination in human peripheral blood neutrophils
  1. C Cole1,
  2. J Scott1,
  3. G Davies2,
  4. G Jones2,
  5. K Jiwa2,
  6. J Chalmers3,
  7. J Simpson1,
  8. A De Soyza1
  1. 1Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, UK
  2. 2Freeman Hospital, Newcastle Upon Tyne, UK
  3. 3Dundee University, Dundee, UK

Abstract

Introduction The formation of Neutrophil extracellular traps (NETS) may protect against pathogens but might promote autoimmunity. Bronchiectasis (BR) is a model for autoimmunity with strong links to rheumatoid arthritis. Bacterial antigens may promote NETosis and/or peptidyl arginine deiminase (PAD) enzyme activity leading to protein citrullination. The ability of these bronchiectasis relevant pathogens/molecules to induce NETosis and PAD activity in human derived neutrophils using consistent methodologies has not been assessed.

Aims To undertake multi-modal assessment of NETosis rates and PAD activity in healthy peripheral blood neutrophils following stimulation with BR relevant stimuli.

Methods Neutrophils were isolated from healthy volunteer whole blood by gradient separation then incubated for 4 hours at 37°C in the presence of PMA (used as a positive NET control) or BR relevant stimuli including: LPS, TNF-α, P. aeruginosa whole cell lysates (WCLs) or H. influenzae WCLs. NETosis rates were assessed in neutrophil samples using two methodologies: Microscopy count of immunofluorescent (IF) stained fixed NETs (n=13) and histone-elastase complex ELISAs of collected supernatants (n=16, semi-quantified using a NET containing biological standard). Supernatant PAD activity was measured using a commercial PAD activity assay from Modiquest Research (n=16).

Results IF microscopy counts showed NETosis rates in unstimulated neutrophils of 4% (95% CI: 2.8% to 5.4%). All stimuli (excluding H. influenzae WCLs) caused a significant increase in NETosis, with the highest increase being observed in 50 nM PMA with a mean rate of 53% (95% CI: 46% to 59%) (figure 1). NET ELISAs of supernatants showed significant increase in the NET dependent absorbance for neutrophils stimulated with PMA (p<0.001), LPS (p<0.001) and TNF-α (p<0.05), absorbance increased following P. aeruginosa and H. influenzae WCL stimulation but not significantly. PAD activity increased significantly following stimulation with PMA(p<0.001), LPS(p<0.01), TNF-α(p<0.05) and P. aeruginosa(p<0.05). H. influenzae WCL stimulated supernatants showed a decrease in supernatant PAD activity, however this was not significant.

Abstract S87 Figure 1

NETosis rates in healthy peripheral blood neutrophil stimulated with bronchiectasis relevant stimuli Incubation with 50 nM PMA, 1 µg/ml LPS. 10ng/ml TNF-α, 2.5% (v/v) P. aeruginosa WCLs or 2.5% (v/v) H. infleunzae WCLs resulted in an increase in NETosis rates (determined by immunofluorescent NET staining and counts). Excluding H. influenzae WCL results, all increases in NETosis rates were shown to be statistically significant (n=13±95% CI, ** p<0.01, *** p<0.001)

Conclusion Collectively the NETosis assays show PMA, LPS and TNF-α promote NETosis, however IF counts also suggest P. aeruginosa WCLs induced NETosis. Extracellular PAD activity increased following stimulation with several BR relevant stimuli. These data may explain the links between Bronchiectasis and Rheumatoid arthritis through protein citrullination.

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