Immunomodulating effects of 13- and 16-hydroxylated docosahexaenoyl ethanolamide in LPS stimulated RAW264.7 macrophages

PUFA-Derived N-Acylethanolamide Probes Identify Peroxiredoxins and Small GTPases as Molecular Targets in LPS-Stimulated RAW264.7 Macrophages

“…Recently, we reported that the expression of Ifit1 was reduced as a result of incubation with 5 μM 13-HDHEA and 16-HDHEA (hydroxydocosahexaenoyl ethanolamide), products of the interaction between DHEA and COX-2. 18 As a significant Ifit1 interaction with AEA probe 5 was not observed here, it appears that Ifit1 is only involved in the signaling of DHEA or its metabolites. Interestingly, previously described endocannabinoid-interacting proteins, for example, CB1, CB2, and GPR55, as well as TRPV1 and PPAR, 1 , 3 , 4 , 7 , 8 , 19 , 21 are not identified in our model, likely due to poor expression of those proteins in LPS-stimulated RAW264.7 macrophages.…”

“…The effects of DHEA and AEA on ROS regulation were already described above, and other studies have reported antimigratory properties of AEA 3 , 4 , 10 and DHEA, 10 including its oxidized metabolites. 15 , 16 , 18 In addition to small GTPase signaling, several actin and myosin-related proteins were also significantly affected by DHEA probe 3 and AEA probe 5 , suggesting that cytoskeletal remodeling is a prerequisite for migration.…”

“…Identified interactions with control probe 8 are used to filter out nonspecific PUFA-derived probe interactions in the data analysis of the proteomics. Following earlier in vitro studies investigating the anti-inflammatory effect of DHEA and AEA in this and similar models, ,,,,,,, probe concentrations of 10 μM were used, which are of the same order of magnitude or even below those used in comparable studies with similar lipid probes. ,, Lastly, applying 10 μM probe counters the attrition that is associated with different steps in the methodology (i.e., uptake of probes by macrophages, nonquantitative yield of photolabeling, and loss during enrichment) to obtain sufficient levels of labeled protein above the detection limit of the liquid chromatography tandem mass spectrometry (LC–MS/MS).…”

“… 13 Additionally, DHEA and AEA are metabolized by COX-2, 15-LOX, and CYP450 to yield compounds with distinct anti-inflammatory and antitumorigenic properties. 1 , 2 , 14 − 18 Other studies reported interactions between DHEA and the cannabinoid receptors CB 1 /CB 2 , transient receptor potential V1 (TRPV-1), and peroxisome proliferator-activated receptors (PPARs), although the obtained agonistic effects seem to depend on the model that was used. 1 , 9 , 14 , 16 , 19 − 21 Our previous studies on LPS-stimulated RAW264.7 macrophages did not show DHEA agonism with CB 1 /CB 2 or PPARs but rather indicated an important role in reducing COX-2-derived prostaglandins.…”

“…Studies on lipopolysaccharide (LPS)-stimulated mouse-derived RAW264.7 macrophages and microglia cell lines showed that DHEA reduced the formation of nitric oxide (NO), COX-2-derived prostaglandins, and thromboxanes and also lowered expression and production of various inflammatory-regulating cytokines such as monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and IL-1β. , Apart from cytokine regulation in macrophages, DHEA exerted synaptogenic and neuroprotective effects in neural cells, , stimulated reactive oxidation species (ROS) production in head and neck squamous cell carcinoma (HNSCC) cells, and reduced inflammatory and nociceptive pain-related behavior in mice . Additionally, DHEA and AEA are metabolized by COX-2, 15-LOX, and CYP450 to yield compounds with distinct anti-inflammatory and antitumorigenic properties. ,,− Other studies reported interactions between DHEA and the cannabinoid receptors CB 1 /CB 2 , transient receptor potential V1 (TRPV-1), and peroxisome proliferator-activated receptors (PPARs), although the obtained agonistic effects seem to depend on the model that was used. ,,,,− Our previous studies on LPS-stimulated RAW264.7 macrophages did not show DHEA agonism with CB 1 /CB 2 or PPARs but rather indicated an important role in reducing COX-2-derived prostaglandins. , Clearly, many open questions persist about DHEA signaling in LPS-stimulated macrophages.…”