Characterization of Endocannabinoid-Metabolizing Enzymes in Human Peripheral Blood Mononuclear Cells under Inflammatory Conditions
Brittany N. Szafran, Jung Hwa Lee, Abdolsamad Borazjani, Peter Morrison, Grace Zimmerman, Kelly L. Andrzejewski, Matthew K. Ross and Barbara L.F. Kaplan
Molecules, 2018, 23, 3167
DOI : 10.3390/molecules23123167
Abstract:
Endocannabinoid-metabolizing enzymes are downregulated in response to lipopolysaccharide (LPS) induced inflammation in mice, which may serve as a negative feedback mechanism to increase endocannabinoid levels and reduce inflammation. Increased plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) and decreased fatty acid amide hydrolase (FAAH) activity in peripheral lymphocytes from individuals diagnosed with Huntington’s disease (HD) suggests that a similar negative feedback system between inflammation and the endocannabinoid system operates in humans. We investigated whether CpG- (unmethylated bacterial DNA) and LPS-induced IL-6 levels in peripheral blood mononuclear cells (PBMCs) from non-HD and HD individuals modulated the activities of endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and carboxylesterase (CES). Baseline plasma IL-6 levels and 2-arachidonoylglycerol (2-AG) hydrolytic activity in PBMC lysates were not different in HD and non-HD individuals. Inhibition of MAGL and CES1 activity in PBMCs using the inhibitors JZL184 and WWL113, respectively, demonstrated that MAGL was the dominant 2-AG hydrolytic enzyme in PBMCs, regardless of disease state. Correlative analyses of 2-AG hydrolytic activity versus enzyme abundance confirmed this conclusion. Flow cytometric analysis of PBMCs showed that MAGL and CES1 were primarily expressed in monocytes and to a lesser extent in lymphocytes. In conclusion, these data suggest that IL-6 did not influence 2-AG hydrolytic activity in human PBMCs; however, monocytic MAGL was shown to be the predominant 2-AG hydrolytic enzyme.
Keywords : endocannabinoids; MAGL; CES; Huntington’s disease
1. Introduction
The endocannabinoid system comprises arachidonoyl-containing endocannabinoids, their cognate G-protein coupled receptors, and biosynthetic and catabolic enzymes that regulate the levels of these lipid signaling molecules. Engagement of the cannabinoid 1 or 2 receptors by plant-derived cannabinoids or endogenous cannabinoids is known to produce anti-inflammatory effects [1,2]. 2-Arachidonoylglycerol (2-AG) and anandamide (AEA) are the best characterized endocannabinoids and are hydrolytically catabolized by monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively, yielding pro-inflammatory arachidonic acid (AA) [3,4]. Other enzymes known to hydrolyze and inactivate 2-AG include carboxylesterases (CES1 and CES2 in human, Ces2g in mice; human and mouse gene symbols for carboxylesterases are CES and Ces, respectively), , -hydrolase domain (ABHD) 6, and ABHD12 [5–7]. It should be noted that FAAH is also capable of hydrolyzing 2-AG [8]. Inactivation of one or more of these enzymes can increase steady-state endocannabinoid levels and is one proposed mechanism to limit or reduce inflammation [9–11].
Previous work from our lab demonstrated that lipopolysaccharide (LPS) treatment of mice induced pro-inflammatorymediators such as interleukin (IL)-6 and reduced themetabolismof endocannabinoids in the spleen by Ces2g [9]. Several studies using in vivo and ex vivo approaches have shown that 2-AG can suppress pro-inflammatory cytokine levels such as tumor necrosis factor- (TNF- ), IL-6, and IL-1 by decreasing their production or release [4,11]. For example, mouse macrophages and rat microglial cells treated with exogenous 2-AG exhibited decreased production or release of these cytokines in response to LPS treatment [10,12]. 2-AG was also shown to have a protective role in several disease processes in humans and rodents, such as experimental autoimmune encephalomyelitis (mouse model of multiple sclerosis), colitis, and autoimmune disease [11,13,14]. Treatment of animal models with small-molecule inhibitors that block endocannabinoid-metabolizing enzymes was shown to reduce disease-associated inflammation [3,15–17]. For example, inhibition of MAGL by the inhibitor JZL184 following a muscle contusion injury in rats decreased TNF- , IL-6, and IL-1 levels three days post-injury [18]. Within the context of neuroinflammation, JZL184 decreased inflammatory responses of microglia in the APdE9mousemodel of Alzheimer’s diseasewhen exposed to LPS, IFN- , and A 42 [15]. Interestingly, another study found that IL-6 suppressed CES1 and CES2 mRNA and enzyme activity in primary human hepatocytes [19], providing evidence to support the hypothesis we proposed in our earlier publication [9] that this may be a negative feedback mechanism to regulate 2-AG catabolism and inflammation resolution.
Huntington’s disease (HD) is a progressive neurodegenerative disease caused by a polyglutamine expansion in the human HTT gene, which is located on chromosome 4. This mutation results in the aggregation of mutated huntingtin protein in many neuronal cell types, including medium spiny neurons of the striatum [20]. Mutant protein aggregation leads to progressive cell loss and development of motor, cognitive, and psychiatric manifestations of HD over time [21]. Chronic inflammation is observed in both the central and peripheral nervous system in HD, and IL-6 levels have been reported to be elevated in the plasma and cerebrospinal fluid of HD individuals and a mouse model of HD [22–26]. It was also reported that FAAH activity was reduced in blood lymphocytes from HD individuals compared to those from non-HD individuals [27]. Another study in the R6/2 mouse model of HD found a decreased level of FAAH activity in the striatum and increased 2-AG levels in whole brain as compared to control mice. However, no differences in peripheral lymphocyte FAAH activity were observed between R6/2 and control mice [28]. These studies suggest that inflammatory diseases might be associated with decreased activities of endocannabinoid-metabolizing enzymes and that HD disease could be a potential model to determine whether IL-6 can suppress endocannabinoid-metabolizing enzyme activity. Therefore, the goals of this study were to (i) examine the role of inflammation, specifically IL-6, on endocannabinoid-metabolizing enzyme activity in peripheral blood mononuclear cells (PBMCs) obtained from non-HD and HD individuals; (ii) identify the primary 2-AG hydrolytic enzymes in human PBMCs; and (iii) determine the cell specificity of MAGL and CES1 expression in human PBMCs. We hypothesized that HD individuals would have elevated plasma IL-6 levels and decreased PBMC endocannabinoid-metabolizing activity as compared to non-HD individuals. We examined IL-6 levels in plasma obtained from HD and non-HD individuals, assessed IL-6 levels and endocannabinoid-metabolizing enzyme activity in PBMCs isolated from HD and non-HD individuals that were stimulated with inflammogens, and characterized the expression of MAGL and CES1 in PBMCs using immunoblot and flow cytometry.
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molecules-23-03167