Background: The formation of Neutrophil Extracellular Traps (NETosis) is a strict sequence of cellular processes including nuclear delobulation and cytoskeletal rearrangements. Initially described as pivotal part of the innate immunity, NETosis also mediates the development and progression of venous thrombosis. The peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) isomerizes Serin/Theronine-Proline motifs and thus affects cytoskeletal dynamics and nuclear delobulation processes in a wide variety of cells. However, the role of Pin1 in neutrophils and especially its effect on molecular mechanisms underlying NETosis and venous thrombosis is still unknown.
Objective: The present study investigated the effect of Pin1 and associated downstream signaling pathways on neutrophil function during NETosis and its impact on venous thrombus formation.
Methods and Results: Employing immunofluorescence-based in vitro NET assays in primary neutrophils, pharmacological perturbation of Pin1 using a selective and covalent inhibitor (KPT-6566) resulted in a significantly decreased NETosis, which was at least in part due to an impaired delobulation of the neutrophil nucleus. Since the intermediate filament vimentin is known to protect cells against nuclear rupture, in a next series of experiments immunofluorescence stainings of vimentin in neutrophils were performed and revealed an activation-dependent phosphorylation, aggregation and polarisation of the intermediate filament vimentin upon NET induction. As shown by immunofluorescence and immunoblots, pharmacological Pin1 inhibition did not affect the phosphorylation of vimentin, but significantly decreased Vimentin aggregation during NETosis. More intriguingly, treatment of primary neutrophils with the acknowledged Vimentin-phosphorylating compound Vimentin-IN-1 evoked nuclear delobulation and NETosis, an effect which was significantly attenuated in the presence of the Pin1 inhibitor KPT-6566. To address the physiological relevance of our findings, we employed an established in vivo model of deep vein thrombosis (DVT) in neutrophil-specific Pin1-deficient (Pin1MRP8Δ/MRP8Δ) and corresponding wildtype (Pin1lox/lox) mice. Of note, not only the incidence of thrombus development in Pin1MRP8Δ/MRP8Δ mice was markedly reduced when compared to wildtype mice, but developing thrombi were also significantly smaller in Pin1MRP8Δ/MRP8Δ animals.
Conclusion: The present study reveals the cis/trans isomerase Pin1 as a powerful regulator of nuclear delobulation in neutrophils and thus of NETosis. Pin1 is at least partially effective in neutrophils by mediating dynamic changes of the intermediate filament Vimentin and crucially affects venous thrombus formation in vivo. Finally, Pin1 could represent a promising druggable target in NET-associated thrombo-occlusive diseases such as venous thromboembolism.
