Nanoformulated omega-3 rich linseed oil reversed IL-6+TNF-a-induced M1 polarized THP-1 cells and monocytes of healthy donors and the activated monocytes of patients with coronary artery disease

Abstract Introduction The development of atherosclerosis underlying coronary artery disease (CAD, a major cause of death worldwide) is promoted by infiltrating inflammatory cells including M1 macrophages and subtypes of T and B lymphocytes. Omega-3 fatty acids have shown promise in mitigating inflammation and enhancing the resolution of inflammatory responses, in part, by reducing M1 polarization of macrophages. Native linseed oil contains 55 % (v/v) of alpha linolenic acid, a plant derived omega-3 fatty acid. Aim Study the impact of stabilized nanoformulated (NF) linseed oil (25% w/w) containing 13.75% (w/w) alpha linolenic acid on the resolution of M1 pro-inflammatory macrophages using THP-1 cells and monocytes from healthy donors and CAD patients. Methods THP-1 cells were polarized towards M1 macrophages by TNF-α+IL-6 (10 ng/ml) for 48 h. Monocytes isolated from blood samples of either healthy donors (n=10) or CAD patients (n=15) were also studied. Gene expression levels were evaluated using RT-qPCR, protein levels by Western blot analysis, cytokines release by ELISA, and the level of reactive oxygen species (ROS) formation using dihydroethidium (DHE) staining. Results THP-1 cells and freshly isolated monocytes from healthy donors exposed to TNF-α+IL-6 (10 ng/ml) for 24-48 h were polarized towards M1 macrophages with increased protein levels of M1 markers (CD86, iNOS), levels of ROS (83.8 ± 2.8 and 40.9 ± 2.8 A.U., respectively) and release of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α). Exposure of polarized M1 THP-1 cells and human monocytes to NF linseed oil but not NF corn oil for 24 h resulted in concentration-dependent reductions in ROS formation, mRNA and protein expression levels of markers of inflammation (IL-6, IL-1β, CD86), adhesion molecules (VCAM-1, ICAM-1), procoagulant responses (tissue factor), remodeling (MMP-9), and calcification (RUNX2). Monocytes isolated from CAD patients showed higher levels of ROS (54.4 ± 4.2 A.U.) under basal conditions compared to TNF-a+IL-6 activated monocytes from healthy donors that was reduced by NF linseed oil (0.003%). NF linseed oil (0.003%) treatment of monocytes from CAD patients (n=8), expressing high levels of circulating proinflammatory cytokines, had reduced levels of markers of inflammation (IL-6, TNF-α, CD86), procoagulant (tissue factor), pro-oxidant (p47phox, ACE1) and pro-remodeling (MMP-9) responses after 24 h. Conclusion The findings indicate that M1 polarization of THP-1 cells and healthy human monocytes is associated with increased levels of ROS and induction of pro-inflammatory responses. Both effects were reversed by NF linseed oil. Monocytes isolated from CAD patients showed an activated state with increased levels of ROS and pro-inflammatory markers that were reversed by NF linseed oil. Thus, NF linseed oil appears as an interesting approach to reverse M1 pro-inflammatory macrophages, hence, may contribute to reduce the burden of low-grade inflammation in patients with CAD.