A regulatory loop involving the cytochrome P450-soluble epoxide hydrolase axis and TGF-b signaling

Fatty acid metabolites generated by the sequential actions of cytochrome P450 enzymes and the soluble epoxide hydrolase (sEH) regulate inflammation. Here we report that the TGF-?-induced polarization of macrophages to a pro-resolving phenotype requires the activation of Alk5 and Smad2 to increase sEH expression and activity. Macrophages lacking sEH failed to fully repolarize, were less efficient at phagocytosis, and retained a pro-inflammatory gene expression profile. 11,12-Epoxyeicosatrienoic acid (EET) was one of the fatty acid metabolites altered in sEH-/- macrophages and was able to reproduce the effect of sEH deletion on gene expression. Importantly, 11,12-EET also attenuated the expression of Alk5 to inhibit the TGF-?-induced phosphorylation of Smad2 by eliciting the cytosolic translocation of the E3 ligase Smurf2. These results indicate that the expression of sEH is not only controlled by TGF-? but that the activity of the enzyme, which keeps 11,12-EET levels low, actually promotes TGF-? signaling by preventing the proteolytic degradation of Alk5. Thus, an autocrine loop between the sEH/11,12-EET and TGF-?1 determines macrophage function. Overall design: Monocytes were isolated from bone marrow and then differenciated into macrophages with a combination of M-CSF and GM-CSF for 7 days. M1 polarization was carried out with a combination of IFN-? and LPS and after 24 hours TGF-ß1 was added on top for additional 48 hours.