WWL70 attenuates PGE2 production derived from 2-arachidonoylglycerol in microglia by ABHD6-independent mechanism

Abstract

Backgroundα/β-Hydrolase domain 6 (ABHD6) is one of the major enzymes for endocannabinoid 2-arachidonoylglycerol (2-AG) hydrolysis in microglia cells. Our recent studies have shown that a selective ABHD6 inhibitor WWL70 has anti-inflammatory and neuroprotective effects in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 in the neuroinflammatory response and the mechanisms by which WWL70 suppresses inflammation has not yet been elucidated in reactive microglia.MethodsThe hydrolytic activity and the levels of 2-AG in BV2 cells were measured by radioactivity assay and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthases in microglia treated with lipopolysaccharide (LPS) with/without WWL70 was determined by western blot and quantitative RT-PCR. The conversion of 2-AG to PGE2 or PGE2-glyceryl ester (PGE2-G) was assessed by enzyme-linked immunoassay (EIA) or LC-MS/MS. The involvement of ABHD6 in PGE2 production was assessed using pharmacological inhibitors and small interfering RNA (siRNA). The effect of WWL70 on PGE2 biosynthesis activity in the microsome fraction from BV2 cells and experimental autoimmune encephalopathy (EAE) mouse brain was also examined.ResultsWe found that WWL70 suppressed PGE2 production in LPS-activated microglia via cannabinoid receptor-independent mechanisms, although intracellular levels of 2-AG were elevated by WWL70 treatment. This reduction was not attributable to WWL70 inhibition of ABHD6, given the fact that downregulation of ABHD6 by siRNA or use of KT182, an alternative ABHD6 inhibitor failed to suppress PGE2 production. WWL70 attenuated the expression of COX-2 and PGES-1/2 leading to the downregulation of the biosynthetic pathways of PGE2 and PGE2-G. Moreover, PGE2 production from arachidonic acid was reduced in the microsome fraction, indicating that WWL70 also targets PGE2 biosynthetic enzymes, which are likely to contribute to the therapeutic mechanisms of WWL70 in the EAE mouse model.ConclusionsWWL70 is an anti-inflammatory therapeutic agent capable of inhibiting PGE2 and PGE2-G production, primarily due to its reduction of COX-2 and microsomal PGES-1/2 expression and their PGE2 biosynthesis activity in microglia cells, as well as in the EAE mouse brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0783-4) contains supplementary material, which is available to authorized users.

The α/β–hydrolase domain 6 inhibitor WWL70 decreases endotoxin‐induced lung inflammation in mice, potential contribution of 2‐arachidonoylglycerol, and lysoglycerophospholipids

“…Finally, we also investigated the effect of WWL70 on PGE 2 levels. Indeed, recent studies suggested that the antiinflammatory effects of WWL70 were partially due to an inhibition of mPGES1 and thus an attenuation of PGE 2 production (34). Surprisingly, in our hands, WWL70 administration increased PGE 2 levels in the lungs.…”

“…Besides the substrates of ABHD6, we thought to quantify PGE 2 in the lungs of mice treated with WWL70. Indeed, a recent study suggested ABHD6-independent effects for WWL70 via the inhibition of mPGES 1 and 2 (mPGES1/2) and thus decreased production of PGE 2 (34). Because in the LPS-treated animal the antiinflammatory effects of WWL70 would have masked a potential direct effect of WWL70 on mPGES1/2, we decided to assess whether administration of WWL70 (20 mg/kg, i.p.)…”

Endocannabinoid Modulation of Microglial Phenotypes in Neuropathology

“…Thus, the two eCBdegrading enzyme inhibitors demonstrated therapeutic efficacy and the potential to modulate microglial phenotype. Our recent report shows that WWL70 inhibits not only ABHD6 but also prostaglandin E synthesis in BV2 microglia (126); these results suggest that eCB-independent mechanisms might also contribute to the therapeutic effect of WWL70 in the TBI mouse model. Another study examined the effects of SMM-189, a CB2R inverse agonist, on microglial phenotype in a TBI mouse model.…”

WWL70 protects against chronic constriction injury-induced neuropathic pain in mice by cannabinoid receptor-independent mechanisms

“…It has been reported that activation of NF-κB is involved in the induction and maintenance of inflammatory pain, which can be weakened by the use of NF-κB inhibitors [ 33 , 34 ]. Our previous study also demonstrated that WWL70 inhibited NF-κB transcriptional activity in BV2 cells and attenuated NF-κB phosphorylation in the EAE mouse spinal cord [ 21 , 24 ]. To elucidate the potential mechanisms underlying the reduced pro-inflammatory cytokines in WWL70 treated CCI mice, we first examined NF-κB phosphorylation in the DRG and sciatic nerve.…”

“…The production and release of inflammatory mediators, such as interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α), and prostaglandin E 2 (PGE 2 ) from activated microglia, lead to activation of adjacent astrocytes [ 23 ], which then act in concert to excite nociceptive neurons causing persistent pain [ 3 ]. We recently reported that the ABHD6 inhibitor WWL70 suppressed PGE 2 production and the expression of its synthetic enzymes cyclooxygenase-2 (COX-2) and prostaglandin E synthase (PGES) in reactive microglia [ 24 ]. In agreement with these results, we find in this study that WWL70 significantly reduced neuropathic pain through interference with arachidonic acid (AA) metabolism and PGE 2 production rather than inhibition of 2-AG breakdown.…”