BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158908 Available online 19 February 2021 1388-1981/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Immunomodulating effects of 13- and 16-hydroxylated docosahexaenoyl ethanolamide in LPS stimulated RAW264.7 macrophages Ian de Bus a, b , Sandra van Krimpen a , Guido J. Hooiveld a , Mark V. Boekschoten a , Mieke Poland a , Renger F. Witkamp a , Bauke Albada b, *, 1 , Michiel G.J. Balvers a, *, 1 a Division of Human Nutrition and Health, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, the Netherlands b Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, the Netherlands A R T I C L E INFO Keywords: Docosahexaenoyl ethanolamide Transcriptome Immunomodulation Cyclooxygenase Endocannabinoid Macrophage ABSTRACT Docosahexaenoyl ethanolamide (DHEA), the ethanolamine conjugate of the n-3 long chain polyunsaturated fatty acid docosahexaenoic acid, is endogenously present in the human circulation and in tissues. Its immunomodu- lating properties have been (partly) attributed to an interaction with the cyclooxygenase-2 (COX-2) enzyme. Recently, we discovered that COX-2 converts DHEA into two oxygenated metabolites, 13- and 16-hydroxylated- DHEA (13- and 16-HDHEA, respectively). It remained unclear whether these oxygenated metabolites also display immunomodulating properties like their parent DHEA. In the current study we investigated the immunomo- dulating properties of 13- and 16-HDHEA in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The compounds reduced production of tumor necrosis factor alpha (TNFα), interleukin (IL)-1β and IL-1Ra, but did not affect nitric oxide (NO) and IL-6 release. Transcriptome analysis showed that the compounds inhibited the LPS- mediated induction of pro-infammatory genes (InhbA, Ift1) and suggested potential inhibition of regulators such as toll-like receptor 4 (TLR4), MyD88, and interferon regulatory factor 3 (IRF3), whereas anti-infammatory genes (SerpinB2) and potential regulators IL-10, sirtuin 1 (Sirt-1), futicasone propionate were induced. Addi- tionally, transcriptome analysis of 13-HDHEA suggests a potential anti-angiogenic role. In contrast to the known oxylipin-lowering effects of DHEA, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses revealed that 13- and 16-HDHEA did not affect oxylipin formation. Overall, the anti-infammatory effects of 13-HDHEA and 16-HDHEA are less pronounced compared to their parent molecule DHEA. There- fore, we propose that COX-2 metabolism of DHEA acts as a regulatory mechanism to limit the anti-infammatory properties of DHEA. 1. Introduction Long chain n-3 polyunsaturated fatty acids (LC-PUFAs) are essential for neural development and functioning, and have been linked to certain benefcial health effects. For example, n-3 LC-PUFAs have been associ- ated with neuroprotective and anti-depressant effects, improved endo- thelial functioning, lowered triglyceride levels, functional fetal and infant development, and proper cardiovascular and immune functioning [1–3]. Moreover, n-3 LC-PUFAs are described to inhibit propagation of many (chronic) infammatory diseases like infammatory bowel disease [4], cardiovascular disease [5,6], rheumatoid arthritis [7–9], and asthma [10]. Notwithstanding this, the potential health effects of n-3 LC- PUFAs are continuously being challenged by new studies. Suggested explanations for these apparent discrepancies are differences in the level of intake or administered dose, and the study population [1]. One of the most studied n-3 LC-PUFAs is docosahexaenoic acid (DHA; C22:6-n3). DHA exerts immunomodulating effects through various mechanisms [1]. First, DHA can directly bind to receptors or key regulators of infammatory processes [11], such as peroxisome proliferator-activated receptor gamma (PPARγ) [12] or the G-protein coupled receptor 120 (GPR120) [13,14]. Second, increased dietary DHA intake alters the cell membrane composition leading to a higher n-3 content. This change in membrane composition leads to a decreased production of pro-infammatory n-6 oxylipins and increases the pro- duction of potent infammation resolving n-3 oxylipins including resolvins, protectins, and maresins [1,11,15–19]. Third, DHA is * Corresponding authors. E-mail addresses: bauke.albada@wur.nl (B. Albada), michiel.balvers@wur.nl (M.G.J. Balvers). 1 Both authors contributed equally to this manuscript. Contents lists available at ScienceDirect BBA - Molecular and Cell Biology of Lipids journal homepage: www.elsevier.com/locate/bbalip https://doi.org/10.1016/j.bbalip.2021.158908 Received 2 November 2020; Received in revised form 23 January 2021; Accepted 15 February 2021