Peptidyl-prolyl cis-trans isomerase Pin1-dependent neutrophil extracellular trap formation (NETosis)

Background Thrombo-occlusive disorders of the arterial and venous system, such as myocardial infarction and venous thromboembolism, are major health issues and their development, progression and outcome is pivotally mediated by Neutrophil Extracellular Trap formation (NETosis). The peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin-1) protein regulates and affects the structure of intermediate filaments, thus regulating nuclear delobulation and cytoskeletal dynamics. However, although NETosis is a strict sequence of cellular processes including intermediate filament changes, the underlying molecular signalling pathways are still ill-defined. Purpose The overall aim is the identification of the Pin1-mediated signalling mechanisms and cellular events in neutrophils during NETosis and associated thrombo-occlusive events. Methods and Results Pharmacological inhibition of Pin1 (KPT-6566) resulted in a significantly impaired in vitro NET formation in primary human neutrophils upon stimulation with the calcium ionophore Ionomycin. This effect was due to a reduced delobulation in KPT-6566 treated neutrophils when compared to the solvent control as revealed by in vitro NET assays. Further immunofluorescence stainings unravelled a markedly increased Ionomycin-dependent clustering and polarisation of the intermediate filament Vimentin. Interestingly, the induction of Vimentin phosphorylation at Ser56 using the acknowledged Vimentin-binding compound Vimentin-IN-1 resulted in enhanced nuclear delobulation in neutrophils and aggravated Ionomycin-dependent NETosis. Furthermore, Ser56 hyperphosphorylation mimicked the observed Vimentin clustering during NETosis, which was significantly reduced after pharmacological inhibition of Pin1 employing KPT-6566. Conclusion The present study unravelled Pin1 as a powerful regulator of NETosis in vitro. Pin1 is at least partially effective in neutrophils by mediating dynamic changes of the intermediate filament Vimentin. Consequently, Pin1 could represent a promising druggable target in NET-associated thrombo-occlusive diseases.