PB0788 - Serotonin Exhibits an Inhibitory Effect on NETosis

Background: NETosis is the process, by which neutrophils expel their deoxyribonucleic acid (DNA) decorated with granule proteins (e.g. myeloperoxidase (MPO)) to the extracellular space to form neutrophil extracellular traps (NETs). This process has been shown to play a significant role in the pathophysiology of numerous diseases. Moreover, it is well established that platelet-derived serotonin is a key modulator of various immune cells.

Aims: To investigate the potential direct effect of serotonin on NETosis.

Methods: Upon informed written consent, human neutrophils were isolated from whole blood of healthy individuals using immunomagnetic bead-based negative selection. NETosis was induced by phorbol 12-myristate 13-acetate (PMA, 10 nM) and different concentrations of serotonin (1, 10, 100 µM) were co-incubated for different durations (2h, 3h, 4h). Neutrophils were preincubated (15 min) with serotonin receptor antagonists SB-269970 hydrochloride (5-HTR7) or piboserod (5-HTR4) (200 µM). NETs were immunostained for MPO and visualized by fluorescence microscopy. Simultaneously, NETs were quantified by extracellular DNA staining with SYTOX Orange and fluorescence spectroscopy. In addition, the surface expression of neutrophil activation markers (CD66b, CD11b, CD162, CD16) was analysed using flow cytometry.

Results: In qualitative and quantitative approaches, serotonin showed a significant, dose-dependent inhibitory effect on PMA-induced NETosis (2h, n=5, 100 μM P< 0.0001, 10 μM P< 0.05, 1 μM P=0.18). This was reproducible despite longer coincubation periods (n=5, 100 µM, 3h P< 0.001, 4h P< 0.01). Furthermore, serotonin’s inhibitory effect was significantly blocked by preincubation with SB-269970 hydrochloride (n=5, 100 µM, 3h P< 0.05) but not with piboserod (P=0.26). However, the regulation of neutrophil surface activation markers by PMA was not affected by coincubation with serotonin.

Conclusion(s): Serotonin inhibits NETosis via 5-HTR7 irrespective of the coincubation period, and in a mechanism independent of surface activation markers. Our ongoing project provides novel results of serotonin’s inhibitory effect on NETosis and potentially a wide spectrum of major clinical implications.