Nanoformulated linseed oil differentially modulates ex vivo monocyte inflammation of patients with coronary artery disease: identification of responders and non-responders
S Hmadeh, R B Basso, A Q Qureshi, A M Mroueh, S A Amissi, A T Trimaille, A G Granier, G N Ndiaye, E T Tureli, P K Kerth, M S Schneider, M B Back, O M Morel, V S K Schini-KerthAbstract
Introduction
Atherosclerosis underlying coronary artery disease (CAD) is driven by inflammatory cells such as pro-inflammatory macrophages. Omega-3 fatty acids mitigate inflammation by promoting macrophage polarization toward an anti-inflammatory phenotype.
Aim
To study the impact of stabilized nanoformulated (NF) linseed oil (25% w/w) containing 13.75% (w/w) alpha-linolenic acid (ALA), a vegetal source of omega-3 fatty acids, on resolution of pro-inflammatory macrophages using monocytes from healthy donors and CAD patients.
Methods
Monocytes isolated from blood samples of healthy donors (n = 14) or CAD patients (n = 30) were studied. Gene expression was measured by RT-qPCR, protein levels by Western blot, cytokine release by ELISA, and reactive oxygen species (ROS) by dihydroethidium (DHE) staining. Cellular glucose uptake and intracellular calcium levels were also assessed.
Results
TNF-α and IL-6 polarized healthy donor monocytes into pro-inflammatory macrophages, marked by increased CD86, iNOS, ROS, and pro-inflammatory cytokines. NF linseed oil—unlike NF corn oil—significantly reduced ROS in a concentration-dependent manner and decreased major inflammatory, adhesion, pro-coagulant, remodeling, and calcification markers. CAD monocytes displayed markedly elevated basal ROS levels (54.4 ± 4.2 A.U.) versus TNF-α and IL-6 activated healthy donor monocytes (40.8 ± 2.8 A.U.). In CAD patients with elevated circulating pro-inflammatory cytokines, NF linseed oil (0.003%, 24 h) reduced IL-6, TNF-α, CD86, tissue factor, p47phox, ACE1, and MMP-9 expression in monocytes from 19 “Responders,” while monocytes from 11 patients showed no response and were classified as “Non-responders”. NF linseed oil also decreased ROS generation and suppressed metabolic activity (glucose uptake) and Ca²⁺ signaling in TNF-α and IL-6-polarized healthy monocytes and responder CAD monocytes, these effects were absent in non-responder cells. In responder CAD monocytes, its inhibitory actions were blocked by the FPR2 antagonist WRW4, but not by the GPR18 antagonist O1918 or an FPR1 antagonist, demonstrating a selective FPR2-dependent mechanism. Furthermore, FPR2 gene expression was significantly downregulated in non-responder CAD monocytes, consistent with their lack of responsiveness to NF linseed oil.
Conclusion
NF linseed oil exerts potent anti-inflammatory, antioxidant, and anti-remodeling effects in polarized pro-inflammatory monocytes from healthy donors and in CAD responder monocytes. The identification of distinct responder and non-responder subgroups, evident across molecular, inflammatory, and functional assays, highlights substantial inter-individual variability in monocyte responsiveness. These findings suggest that NF linseed oil may serve as a promising immunomodulatory strategy for targeting monocyte-driven inflammation in cardiovascular disease, while underscoring the need to define the determinants of patient-specific responsiveness.