Background/Purpose: Increased propensity for neutrophil extracellular trap generation has been described in autoimmune diseases such as rheumatoid arthritis. Neutrophil extracellular traps are thought to contribute to disease propensity. Calcium signalling is known to be important for neutrophil extracellular trap generation. Thus, controlling this calcium signalling pathway may allow for regulation of autoimmune disease pathology. Apremilast is a selective phosphodiesterase 4 inhibitor that is currently approved for the treatment of psoriasis and psoriatic arthritis with a positive phase 3 on Behcet`s disease. Inhibition of phosphodiesterase 4 can elevate cAMP levels, decrease adenylate cyclase levels and thus modulate calcium release and store-operated calcium signalling. We hypothesised that by inhibiting phosphodiesterase 4 with apremilast, calcium release can be controlled and in turn decrease the propensity for neutrophil extracellular trap generation.

Methods: Healthy controls were recruited at the Blood Bank of the Swiss Red Cross, Basel. Patients matching American College of Rheumatology criteria for rheumatoid arthritis were recruited at the University Hospital of Basel (n = 13).

Whole blood was collected into heparin tubes and neutrophils isolated using Dextran-Ficoll density centrifugation. Neutrophils were resuspended in RPMI + 10mM Hepes and were separately treated with apremilast, PDE7 inhibitor (0.1 – 100uM), rolipram (10 uM) and IBMX (300 uM).

For healthy control neutrophils, PMA (20nM) and calcium ionophore (2.5 uM) were used to induce neutrophil extracellular traps. Spontaneous neutrophil extracellular trap formation was measured for rheumatoid arthritis neutrophils. Samples were fixed, then prepared for immunohistochemistry using mouse anti-human MPO antibody (Abcam) followed by incubation with goat anti-mouse IgG AF555 (Invitrogen Life Technologies). DNA was counterstained with DAPI. Neutrophil extracellular traps were then quantified using NETQUANT and a Nikon EclipseTi-E.

Treated neutrophils were loaded with DHR123 (25uM) for 15 min at 37^οC. Once loaded, ROS production was monitored using a Biotek Synergy H1 Hybrid Reader (Biotek) at 0, 15 and 30 minutes (excitation 485 nm, emission 570 nm). In the case healthy control neutrophils, ROS production was induced using PMA (20nM) while for rheumatoid arthritis neutrophils, spontaneous ROS production was monitored.

Results: Reduction in PMA induced ROS production for healthy control neutrophils when treated with apremilast was observed. A decrease in PMA and calcium ionophore-induced neutrophil extracellular trap generation was also observed. Apremilast treatment of rheumatoid neutrophils also displayed a decrease in spontaneous ROS generation and neutrophil extracellular trap generation. We also investigated the effect of phosphodiesterase 7 inhibition. Interestingly, this displayed an opposite effect to phosphodiesterase 4 inhibition.

Conclusion: By reducing neutrophil extracellular traps through modulation of calcium signalling by phosphodiesterase 4 inhibition, the possibility of controlling neutrophil extracellular trap generation in certain autoimmune conditions might be possible.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-neutrophil-extracellular-traps-by-apremilast-phosphodiesterase-4-inhibition/