DOI: 10.1002/mnfr.202400716 ISSN: 1613-4125

An Optimized Ex Vivo n–3 PUFA Supplementation Strategy for Primary Human Macrophages Shows That DHA Suppresses Prostaglandin E2 Formation

Rebecca Kirchhoff, Nadja Kampschulte, Carina Rothweiler, Nadine Rohwer, Karsten‐Henrich Weylandt, Nils Helge Schebb

ABSTRACT

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.

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