Introduction: Neutrophils activated by bacteria release cytotoxic neutrophil extracellular traps (NETs) that can collaterally kill airway epithelial cells. However, molecular mechanisms that mitigate such damage are not clearly understood. Bacterial components such as lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), a diacylglycerol mimetic, activate neutrophils to generate superoxide via NADPH oxidase (NOX) 2. Superoxide is eventually converted to hypochlorous acid (HOCl) by myeloperoxidase (MPO), subsequently to other reactive oxygen species (ROS), and induces NOX-dependent NET formation. Single pulmonary neuroendocrine cells (PNECs) or PNEC clusters (neuroepithelial bodies) present in airways secrete serotonin (5-hydroxytryptamine or 5-HT), a direct inhibitor and a competitive substrate of MPO. We hypothesized that serotonin secreted by PNECs into the lumen inhibits NET formation and protects epithelial cells against NET-mediated cell death.

Methods: To test this hypothesis, neutrophils isolated from the peripheral blood of healthy human donors were stimulated to undergo NETosis or induce ROS production, measured by Sytox Green fluorescent dye and dihydrorhodamine123 (DHR123) respectively. These processes were measured in the absence or presence of exogenous serotonin or serotonin released by H727 neuroendocrine cells. Immunofluoresence and confocal microscopy were performed to supplement the assays' results. Cell death and detachment assays and immunofluorescence were used to measure viability of H727 or A549 epithelial cells in the presence of NETs.

Results: Both exogenous serotonin and serotonin released by H727 neuroendocrine cells suppressed NOX-dependent NETosis induced by PMA and LPS. By contrast, serotonin did not suppress NOX-independent NETosis induced by calcium ionophores A23187 and ionomycin. Both sources of serotonin also inhibited ROS production in NOX-dependent NETosis. A549 cells are highly susceptible to NET-mediated killing unlike H727 cells.

Conclusion: Therefore, we conclude that serotonin inhibits NOX-dependent NETosis by suppressing ROS production and protects epithelial cells from NET-mediated damage.