Unravelling the interaction targets and metabolic fate of docosahexaenoyl ethanolamide

Poly-unsaturated fatty acids (PUFAs) and their metabolites have well-described immune regulating functions. In general, n-6 PUFAs and related metabolites possess pro-inflammatory functionality whereas n-3 PUFAs and related metabolites possess less pro-inflammatory or even anti-inflammatory properties. In this thesis the metabolism and anti-inflammatory properties of the endogenously produced n-3 PUFA derivative docosahexaenoyl ethanolamide (DHEA) are described. In chapter 2 a literature overview of the metabolic and immune regulating properties of PUFAs and their acyl derivatives is given. It is described that DHEA levels in murine and human plasma and tissues depend on the dietary intake of n-3 PUFAs. Herein DHEA is not the end-product but oxygenation of DHEA by enzymes like CYP450 and LOX-15 is described. This observation showed that studying the metabolism and immune regulatory roles of n-3 PUFAs and n-3 PUFA derivatives is complex. Recent studies revealed that the use of chemical probes, which are synthetic chemical PUFA or PUFA related compounds with bio-orthogonal handles, can provide an interesting alternative to conventional immunological tools to investigate the immune regulating effects of the PUFAs.In chapter 3, bi-functional synthetic chemical probes were used to map the interactions of the potent anti-inflammatory regulator DHEA and the n-6 PUFA ethanolamine derivative arachidonoyl ethanolamide (AEA) in 1.0 μg/mL LPS stimulated murine RAW264.7 macrophages. Diazirine and alkyne moieties were installed into DHEA and AEA related structures to allow for covalent coupling to proteins using 366 nm UV-light, and subsequent CuAAC click coupling to a fluorescent probe or biotin. Despite the small chemical alterations present in the probe when compared to native DHEA, the bi-functional chemical derived DHEA probes displayed similar anti-inflammatory properties compared to the natural DHEA, on IL-6 and prostaglandin E2 (PGE2) reduction. Uptake of the PUFA derived probes was successful and resulted in cytosolic localisation in the suggested regions of the ER, Golgi-system, and showed vesicular compartmentalisation. Chemical proteomic interactome mapping showed that both AEA and DHEA interacted with the oxygenation enzyme cyclooxygenase 2 (COX-2), but also with peroxiredoxin I, peroxiredoxin IV, different proteins involved in the Rho GTPase signalling pathway, and Rac1 and its related interactome proteins. Immunolabelling studies showed co-localisation as prerequisite for molecular interaction between the PUFA derived amides with COX-2 and Rac1. Bio-informatic analysis of the proteome interactors suggested regulatory roles for DHEA in cytoskeletal remodelling and cell migration, and also in ROS scavenging. Future studies are required to interpret the immunological consequences of the chemical proteomic interactions obtained.To study the interaction between DHEA and COX-2, chapter 4 describes the development of a cell free COX-2 enzymatic assay. First, the enzymatic assay is verified using the known COX-2 substrates arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). After validation of the assay the N-acyl ethanolamine derived PUFAs AEA, eicosapentaenoyl ethanolamide (EPEA), and DHEA were added to the enzymatic assay. For AEA production of prostaglandin ethanolamide E2 PGE2-EA, PGD2-EA, 11-hydroxyeicosatetraenoyl ethanolamide (11-HETE-EA), and 15-HETE-EA were identified, verifying the validity of the methodology. For EPEA the novel products prostaglandin E3 ethanolamide (PGE3-EA), and 11-hydroxypentaenoyl ethanolamide 11-HEPE-EA (and the postulated products 14- and 18-HEPE-EA) were obtained, and DHEA conversion resulted in 13- and 16-hydroxydocosahexaenoyl ethanolamide (13- and 16-HDHEA). Metabolic conversion of EPEA and DHEA was limited resulting in synthetic yields of 0.7% for PGE3-EA, 1.3% for 16-HDHEA, and 1.4% for 13-HDHEA. Production of 13- and 16-HDHEA was also confirmed in 1.0 μg/mL LPS-stimulated RAW264.7 macrophages, in which it was shown that both DHEA addition and LPS-stimulation are required for the production of 13- and 16-HDHEA. Addition of the selective COX-2 inhibitor celecoxib resulted in blocking of 13- and 16-HDHEA synthesis, proving that 13- and 16-HDHEA synthesis in vitro was mediated by COX-2. Incubations with 10 μM DHEA in 1.0 μg/mL LPS-stimulated RAW264.7 macrophages resulted in production of 7.3 (± 2.3) pmol of 13-HDHEA and 8.6 (± 3.5) pmol of 16-HDHEA per 106 RAW macrophage cells.In chapter 5 the immune regulating effect of 13-HDHEA and 16-HDHEA were tested. It was shown that the novel compounds 13- and 16-HDHEA reduced the production of inflammatory cytokines TNFα and IL-1β in 1.0 μg/mL LPS-stimulated RAW264.7 macrophages, but did not affect levels of nitric oxide (NO), IL-6, and the prostaglandins PGE2 and PGD2. Transcriptomic analysis revealed a shift in upregulation of several anti-inflammatory related genes and reduction of pro-inflammatory related genes, especially downstream of toll-like receptor 4 (TLR4) and its regulators. Furthermore, 13- and 16-HDHEA showed distinct anti-inflammatory gene regulation compared to DHEA, but the anti-inflammatory regulation of DHEA was more potent than its COX-2 metabolites in 1.0 μg/mL LPS-stimulated RAW264.7 macrophages. It was therefore proposed that COX-2 metabolism of DHEA could also acts as a regulatory mechanism to limit the anti-inflammatory properties of DHEA.In chapter 6 the effects of intraperitoneally injected (i.p.) DHEA were tested in a DSS induced colitis model in C57Bl/6 mice. The mice received 2% DSS in drinking water (ad libitum) for five days and were injected with a vehicle, 10 mg/kg DHEA, or 15 mg/kg DHEA each day, starting one day before DSS administration and ending one day after the final DSS administration. It was shown that DHEA reduced phenotypic colitis markers like body weight and rectal bleeding, and improved stool consistency. Pathophysiological markers in the colon such as colon length, cellular infiltration, and neutrophil activity were not significantly affected by the DHEA treatment. DHEA metabolism in the liver of these mice was investigated showing that i.p. DHEA treatment resulted in increased DHEA levels in the liver, but DHEA metabolites of COX-2 were not identified. AEA levels and oxylipin levels were not affected in the livers of those mice, which is most likely explained by the absence of COX-2 mRNA gene expression. In conclusion, i.p. DHEA treatment reduced phenotypic colitis markers, but did not affect classical markers of colon damage. Apart from hepatic DHEA levels, no significant metabolic effects were observed in the livers.The thesis is concluded by a general discussion and summary regarding the findings.