The release of neutrophil extracellular traps (NETs) is a key factor contributing to the severity of acute respiratory distress syndromes (ARDS). Identifying potential new pathways that can be targeted to modulate this pathological process will offer insights and open up new treatment possibilities for managing this condition.

To evaluate if cyclic nucleotides stimulators are capable of modulating NETs release in vitro.

We stimulated healthy neutrophils with phorbol myristate acetate (PMA) or serum from severe COVID-19 patients (n = 15) as a model of ARDS condition in the presence and in absence of three different cyclic nucleotides stimulators drugs: the guanylyl cyclase activator cinaciguat (0.5 μg/mL, 1 μg/mL and 5 μg/mL), the phosphodiesterase 5 (PDE5) inhibitor sildenafil (10 μM, 50μM and 100 μM) or the inhibitor of phosphodiesterase-3 (PDE-3) milrinone (100 μM, 250 μM and 500 μM). Cell incubation images were captured using Incucyte after a duration of 1.5 hours. The number of cells that underwent netosis and those that did not were determined and the percentage of netosis was calculated. Images were analyzed by fluorescence.

In PMA-stimulated neutrophils, cinaciguat reduced NETs release by 35% at 1μM and 49% at 5μM, but not at the lower concentration of 0.5μM. Sildenafil reduced NETs release by 14% at 50 μM and 62% at 100 μM, but showed no effect at the lower concentration of 10 μM. Milrinone reduced NETs release by 51% at 500μM but not at the lower concentrations of 100 μM and 250 μM. When neutrophils were stimulated with COVID-19 serum, cinaciguat at 5μM reduced NETs release by 30%, sildenafil at 100μM by 19%, and milrinone at 500 μM by 24%, but there was no effect at the lower concentrations of all three drugs.

Cinaciguat, sildenafil and milrinone were capable of “in vitro” reducing netosis induced by PMA and COVID-19 serum. Such findings indicate an involvement of cGMP and cAMP in modulating NETs formation and highlight the potential of targeting the production of these cyclic nucleotides as a novel approach for treating ARDS and other inflammatory conditions associated with excessive NETs release.