Eicosapentaenoic acid (EPA) modulates inflammasome activation in monocyte-derived macrophages isolated from individuals with and without established atherosclerotic cardiovascular disease (ASCVD)

Abstract Background Icosapent ethyl is a highly purified form of eicosapentaenoic acid (EPA) prescribed as an adjunct to statin therapy to individuals with established atherosclerotic cardiovascular disease (ASCVD) with persistent hypertriglyceridaemia. It can reduce the risk of myocardial infarction through benefits which extend beyond its triglyceride lowering capacity, the mechanisms of which remain to be fully elucidated. Purpose Monocyte derived macrophages (MDMs) drive the progression of ASCVD through P2X7 receptor mediated activation of the nod-like receptor protein-3 (NLRP3) inflammasome and impaired autophagy, attenuation of which reduces cardiovascular risk. Here we determine if EPA prevents extracellular ATP release and NLRP3 inflammasome activation in MDMs isolated from individuals with and without established ASCVD. Methods Cluster of differentiation-14+ (CD14+) monocytes isolated from healthy donors (n=14) or individuals with ASCVD (n=30) were differentiated into MDMs using macrophage colony stimulating factor (MCSF: 20ng/mL) for 6 days. An ATP lite luminescence assay measured ATP release from lipopolysaccharide (LPS: 0.1μg/mL) stimulated MDMs pre-incubated with EPA (100uM) for 24hrs or the connexin hemichannel blocker Tonabersat (50μM) for 2hrs. NLRP3 activation was assessed by a caspase-1 inflammasome assay in LPS (0.1ug/mL) + ATP (2mM) stimulated MDMs pre-treated for 24hrs and 2hrs with EPA (100uM) or the autophagy inhibitor 3-methyladenine (3MA: 5mM) respectively. Immunoblotting determined mammalian target of rapamycin (mTOR) expression on stimulated MDM lysates. Results MDMs isolated from individuals with ASCVD exhibit a significant increase in ATP release (721.3±129.8 RLu; P0.001) and caspase 1 activation (2511±734.2 RLu; P0.01) as compared to healthy donor MDMs. Pre-incubation with Tonabersat determined that ATP release is in part, connexin hemichannel mediated with ATP release reduced by 19.7%±3.16% (P0.001) as compared to LPS stimulated control. Importantly, EPA reduced ATP release and caspase 1 activation in stimulated healthy donor (P0.01 0.05 respectively) and ASCVD-patient (P0.05 0.001 respectively) isolated MDMs. Inhibition of autophagy increased caspase 1 activation in MDMs isolated from ASCVD patients, whilst EPA reduced expression of p-mTOR (15%±5% P0.05), a negative regulator of autophagy. Conclusion We present novel preliminary evidence that EPA may confer anti-inflammatory protection by modulating the ATP-P2X7 axis and downstream NLRP3 inflammasome activation in monocyte derived macrophages. These events may arise from a favourable effect of EPA on cell autophagy.