Inactivation of CES1 Blocks Prostaglandin D₂ Glyceryl Ester Catabolism in Monocytes/Macrophages and Enhances Its Anti-inflammatory Effects, Whereas the Pro-inflammatory Effects of Prostaglandin E₂ Glyceryl Ester Are Attenuated

Abstract

Human monocytic cells in blood have important roles in host defense and express the enzyme carboxylesterase 1 (CES1). This metabolic serine hydrolase plays a critical role in the metabolism of many molecules, including lipid mediators called prostaglandin glyceryl esters (PG-Gs), which are formed during cyclooxygenase-mediated oxygenation of the endocannabinoid 2-arachidonoylglycerol. Some PG-Gs have been shown to exhibit anti-inflammatory effects; however, they are unstable compounds, and their hydrolytic breakdown generates pro-inflammatory prostaglandins. We hypothesized that by blocking the ability of CES1 to hydrolyze PG-Gs in monocytes/macrophages, the beneficial effects of anti-inflammatory prostaglandin D2-glyceryl ester (PGD2-G) could be augmented. The goals of this study were to determine whether PGD2-G is catabolized by CES1, evaluate the degree to which this metabolism is blocked by small-molecule inhibitors, and assess the immunomodulatory effects of PGD2-G in macrophages. A human monocytic cell line (THP-1 cells) was pretreated with increasing concentrations of known small-molecule inhibitors that block CES1 activity [chlorpyrifos oxon (CPO), WWL229, or WWL113], followed by incubation with PGD2-G (10 μM). Organic solvent extracts of the treated cells were analyzed by liquid chromatography with tandem mass spectrometry to assess levels of the hydrolysis product PGD2. Further, THP-1 monocytes with normal CES1 expression (control cells) and “knocked-down” CES1 expression (CES1KD cells) were employed to confirm CES1’s role in PGD2-G catabolism. We found that CES1 has a prominent role in PGD2-G hydrolysis in this cell line, accounting for about 50% of its hydrolytic metabolism, and that PGD2-G could be stabilized by the inclusion of CES1 inhibitors. The inhibitor potency followed the rank order: CPO > WWL113 > WWL229. THP-1 macrophages co-treated with WWL113 and PGD2-G prior to stimulation with lipopolysaccharide exhibited a more pronounced attenuation of pro-inflammatory cytokine levels (interleukin-6 and TNFα) than by PGD2-G treatment alone. In contrast, prostaglandin E2-glyceryl ester (PGE2-G) had opposite effects compared to those of PGD2-G, which appeared to be dependent on the hydrolysis of PGE2-G to PGE2. These results suggest that the anti-inflammatory effects induced by PGD2-G can be further augmented by inactivating CES1 activity with specific small-molecule inhibitors, while pro-inflammatory effects of PGE2-G are attenuated. Furthermore, PGD2-G (and/or its downstream metabolites) was shown to activate the lipid-sensing receptor PPARγ, resulting in altered “alternative macrophage activation” response to the Th2 cytokine interleukin-4. These findings suggest that inhibition of CES1 and other enzymes that regulate the levels of pro-resolving mediators such as PGD2-G in specific cellular niches might be a novel anti-inflammatory approach.

Effects of Chlorpyrifos on Serine Hydrolase Activities, Lipid Mediators, and Immune Responses in Lungs of Neonatal and Adult Mice

“…Ces/CES enzymes have the ability to metabolize 2-AG and other immunomodulatory lipids, such as prostaglandin glyceryl esters (PG-Gs), 34,35,37,75 thus their inactivation or overexpression could alter immune responses. 98,99 Although CPF treatment inhibited Ces isoforms in lung (between 40% and 60% reduction), this did not alter the levels of LPS-induced cytokines and the immunophenotype of lung cells (except for a small increase in the percentage of LPS-induced dendritic cells in female lungs).…”

“…In a separate experiment, the FP-TAMRA probe was used to examine serine hydrolase activities in lung microsomes following their preincubation with increasing concentrations of CPO (30 min, 37 °C), as previously described. 37 Activity-Based Protein Profiling (ABPP-Mass Spectrometry). Lung proteomes (microsomes) were adjusted to a concentration of 1 mg/mL protein in a volume of 1 mL of 50 mM Tris-HCl (adult female lung was <1 mg/mL due to limited protein amounts).…”

“…34,36 Thus, it is possible that Ces/CES enzymes have an important backup role as a 2-AG hydrolytic enzyme in tissues where Magl/MAGL is either not expressed or present at low levels. Because CES1 is expressed in human monocytes/macrophages and can metabolize immunomodulatory lipids, such as eCBs and prostaglandin glyceryl esters, 34,35,37 the CES enzymes might have underappreciated roles in lipid mediator metabolism and immune responses. Indeed, our previous study demonstrated that 2-AG hydrolytic activity in mouse spleen was downregulated during lipopolysaccharide (LPS)-induced inflammation.…”

Pig Liver Esterases Hydrolyze Endocannabinoids and Promote Inflammatory Response

“…AEA and 2-AG, 2-AG in particular, are two major molecular species in the cannabinoid signaling and exert a wide range of pathophysiological functions including inflammatory responses (23)(24)(25)(26). We next tested potential roles of PLEs in the inflammation.…”

“…Sequence alignment identifies only a total of 18 amino acid substitutions between PLE1 and PLE6 (Figure S6), and half of these substitutions are identical between PLE6 and CES1 (human). CES1 (human) has been shown to effectively hydrolyze endocannabinoids (25,30). The amino acids shared by PLE6 and CES1 differ markedly from those in PLE1 in terms of charge, hydrophilicity and aromatic side-chain (Figure S6).…”