Neutrophil extracellular trap formation and citrullination in bronchiectasis

Abstract

Bronchiectasis (BR) patients show risk for developing rheumatoid arthritis (BROS). As citrullination is implicated in rheumatoid arthritis pathogenesis it is possible that neutrophil extracellular trap (NET) formation, which is associated with PAD enzyme activity (i.e. citrullination), may be the mechanism connecting the two diseases. This body of work took 4 separate approaches to study the process of NETosis/PAD activity in the context of BR. Firstly, healthy peripheral blood neutrophils were assayed for changes in NETosis/PAD following stimulation with BR relevant molecules. Both NETosis and PAD activity increased following incubation, suggesting BR related stimuli promote NETosis and citrullination. Secondly, LPS signalling was assessed to determine how inhibition of the two arms of the signalling pathway (TRIF and MyD88) impacted NETosis/PAD activity. Pre-treatment with either inhibitor downregulated both NETosis rates in vitro, suggesting molecular signalling underpinning NETosis is broader and more complex than predicted. Thirdly, the impact of Cl-amidine (a PAD inhibitor) on NETosis, PAD activity and neutrophil function was assessed in vitro. Cl-amidine was shown to significantly reduce NETosis and PAD activity in response to BR stimuli. Superoxide production and phagocytosis was also shown to be inhibited by Cl-amidine, suggesting PAD plays some role these aspects of neutrophil function. Finally, NETosis, PAD activity and neutrophil function was assessed in healthy, BR and BROS neutrophils. Several differences in these results were observed between the groups, however limited sample size and lack of age matched healthy comparators complicate results interpretation. These results imply that the molecular mechanisms underpinning NETosis/PAD activation is likely more complicated than previously suggested, with the results also indicating that PAD activity may play a role in neutrophil function. BR relevant stimuli appear to promote both NETosis and citrullination, with preliminary data suggesting there may be some difference in these processes between healthy, BR and BROS patients.