An Optimized Ex Vivo n-3 PUFA Supplementation Strategy for Primary Human Macrophages Shows That DHA Suppresses...

Evidence suggests beneficial effects of long-chain n-3 polyunsaturated fatty acids (PUFAs) in inflammatory diseases. However, the underlying mechanisms are still subject of research. For this purpose, we developed an ex vivo n-3 PUFA supplementation strategy. M2-like macrophages were supplemented for 2-3 days with 20-40 µM docosahexaenoic acid (DHA) during differentiation. Quality parameters include <3% oxylipins for PUFA-preparation, total fatty acids (FAs) <10 mM, and low oxylipins in plasma, n-3 PUFA <0.25 mM for the selection of donors of plasma as well as %n-6 in highly unsaturated fatty acids (HUFAs) ≥70% for donors of cells. Following supplementation, PUFA pattern of cells was shifted toward one described for blood and tissue from subjects with higher n-3 and lower n-6 PUFAs. This was accompanied by a decrease of arachidonic acid-derived oxylipins in a dose- and time-dependent manner in favor of n-3 PUFA ones. Stimulation with LPS resulted in decreased levels of pro-inflammatory prostaglandins in the DHA-supplemented cells, but no changes in cytokines. In vitro supplementation studies with n-3 PUFA need rigorous controls to exclude the background formation of oxylipins. By accounting for these possible confounders the described approach allows the mechanistic investigation of n-3 PUFAs in primary human immune cells, offering an alternative for intervention studies.