Saliba et al. Journal of Neuroinflammation (2018) 15:322 https://doi.org/10.1186/s12974-018-1362-7 RESEARCH Open Access Anti-neuroinflammatory effects of GPR55 antagonists in LPS-activated primary microglial cells Soraya Wilke Saliba1, Hannah Jauch1, Brahim Gargouri1, Albrecht Keil1, Thomas Hurrle2,3, Nicole Volz2, Florian Mohr2,4, Mario van der Stelt4, Stefan Bräse2,3 and Bernd L. Fiebich1,5* Abstract Background: Neuroinflammation plays a vital role in Alzheimer’s disease and other neurodegenerative conditions. Microglia are the resident mononuclear immune cells of the central nervous system, and they play essential roles in the maintenance of homeostasis and responses to neuroinflammation. The orphan G-protein-coupled receptor 55 (GPR55) has been reported to modulate inflammation and is expressed in immune cells such as monocytes and microglia. However, its effects on neuroinflammation, mainly on the production of members of the arachidonic acid pathway in activated microglia, have not been elucidated in detail. Methods: In this present study, a series of coumarin derivatives, that exhibit GPR55 antagonism properties, were designed. The effects of these compounds on members of the arachidonic acid cascade were studied in lipopolysaccharide (LPS)-treated primary rat microglia using Western blot, qPCR, and ELISA. Results: We demonstrate here that the various compounds with GPR55 antagonistic activities significantly inhibited the release of PGE2 in primary microglia. The inhibition of LPS-induced PGE2 release by the most potent candidate KIT 17 was partially dependent on reduced protein synthesis of mPGES-1 and COX-2. KIT 17 did not affect any key enzyme involved on the endocannabinoid system. We furthermore show that microglia expressed GPR55 and that a synthetic antagonist of the GPR receptor (ML193) demonstrated the same effect of the KIT 17 on the inhibition of PGE2. Conclusions: Our results suggest that KIT 17 is acting as an inverse agonist on GPR55 independent of the endocannabinoid system. Targeting GPR55 might be a new therapeutic option to treat neurodegenerative diseases with a neuroinflammatory background such as Alzheimer’s disease, Parkinson, and multiple sclerosis (MS). Keywords: GPR55, Prostaglandin E2, Cyclooxygenase, Microglia, Neuroinflammation Introduction microglia has been associated with neurodegenerative dis- For many years, neuroinflammation has been known as a eases such as Alzheimer’s disease (AD), Parkinson disease common phenomenon in the pathology of many brain dis- (PD), and traumatic brain injury or aging [4, 5]. eases. Microglia, the principal cells involved in the innate The GPR55 is an orphan G-protein-coupled receptor, immune response in the central nervous system (CNS), play first described by Sawzdargo et al. in 1999 [6], and it is essential roles in the maintenance of homeostasis and re- not only highly expressed in CNS, but also in peripheral sponses to inflammatory stimulus [1–3]. The over-activated tissue [7]. It can be activated by cannabinoids (CB) and non-CB, leading to the hypothesis that it might be a pu- * Correspondence: [email protected] tative “type-3” cannabinoid receptor [8]. However, in 1Neuroimmunology and Neurochemistry Research Group, Department of contrast to the cannabinoid receptors CB1 and CB2, Psychiatry and Psychotherapy, Medical Center - University of Freiburg, GPR55 only couples to Gα proteins which leads to Faculty of Medicine, University of Freiburg, Freiburg, Germany 12,13 5Department of Psychiatry and Psychotherapy, Laboratory of Translational the activation of the ras homolog gene family member A Psychiatry, University Hospital Freiburg, Hauptstr. 5, 79104 Freiburg, Germany (RhoA) and Rho-associated protein kinase (ROCK). The Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Saliba et al. Journal of Neuroinflammation (2018) 15:322 Page 2 of 13 phospholipase C pathway is triggered by these proteins, members of the arachidonic acid pathway in activated which increase the intercellular Ca2+ and extracellular microglia, have not been elucidated in detail. We therefore signal-regulated kinase (ERK) phosphorylation [9]. studied the effects of novel synthesized GPR55 antagonists GPR55 is expressed in immune cells, such as monocytes, in LPS-activated microglia by examining prostaglandin E2 natural killer (NK) cells [10], and microglia [11], and its in- (PGE2) production, COX/mPGES-1 mRNA, and protein volvement in inflammation has been reported [10, 12, 13]. levels. Furthermore, we evaluated the effect of KIT17 on Activation of GPR55 by the agonist O-1602 increased key enzymes involved in the endocannabinoid system such pro-inflammatory cytokines and cell cytotoxicity in mono- as diacylglycerol lipase-α(DAGLα), monoacylglycerol lipase cytes and NK cells stimulated with LPS [10]. Corroborat- (MAGL), α,β-hydrolase domain-containing 6 and 12 ing with this, an antagonist of GPR55, CID16020046, and (ABHD6 and ABHD12), and fatty acid amide hydrolase − − GPR55 / knockout mice decreased the pro-inflammatory (FAAH). cytokines in colitis mice models comparable to human in- flammatory bowel disease (IBD) [12]. In hyperalgesia asso- Methods ciated with inflammatory and neuropathic pain, it was Ethics statement observed to increase anti-inflammatory cytokines, IL-4 Animals were obtained from the Center for Experimental − − and IL-10, and also pro-inflammatory IFN-γ in GPR55 / Models and Transgenic Services-Freiburg (CEMT-FR). All knockout mice [13]. On the other hand, the GPR55 agon- the experiments were approved and conducted according ist O-1602 decreased IL-6 and TNF-α in a model of ex- to the guidelines of the ethics committee of the University perimental acute pancreatitis [14]. of Freiburg Medical School under protocol no. X-13/06A, The roles of GPR55 in the pathophysiology of the CNS and the study was carefully planned to minimize the are still not clear. In an excitotoxicity in vitro model of rat number of animals used and their suffering. For the organotypic hippocampal slice cultures (OHSC), the activa- activity-based protein profiling (ABPP), the experiments tion of GPR55 receptor by L-α-Lysophosphatidylinositol were performed at Leiden University according to guide- (LPI) mediated neuroprotection through microglia [15]. In lines approved by the ethical committee of Leiden Univer- amousemodelofPD,theexpressionofGPR55wasdown- sity (DEC#13191). regulated in the striatum and the treatment with an agonist of GPR55, abnormal-cannabidiol, improved the motor be- Chemicals havior by neuroprotection of dopaminergic neuron cell Synthetic GPR55 antagonists (Fig. 1) were synthesized at bodies [16]. A molecular, anatomical, electrophysiological, the Institute for Organic Chemistry Karlsruhe - KIT − − and behavioral study of GPR55 / knockout mice demon- (Karlsruhe, Germany) [18] and dissolved in DMSO. The strated a normal development of brain structure and did number of the corresponding compounds in the Rempel not affect the endocannabinoid system nor muscle strength et al. 2013 paper are: KIT3 is 14, KIT17 is 37, and and motor learning. However, these mice presented deficits KIT21 is 41. ML193 and O-1602 were obtained from in motor coordination and thermal sensitivity [17]. Tocris Biosciences. LPS from Salmonella typhimurium These studies suggest that GPR55 signaling can be (Sigma Aldrich, Deissenhofen, Germany) was resus- involved in neurodegeneration and modulate certain pended in sterile phosphate-buffered saline (PBS, 5 mg/ cytokines and thus inflammation. However, its effects on mL) as stock and subsequently used at a final concentra- neuroinflammation, especially on the production of tion (10 ng/mL) in the cultures. A OMe O O 5-isopropyl-3-(2-methoxybenzyl)-8-methyl-2H-chromen-2-one KIT-3 B C HO HO O O O O 3-benzyl-6-hydroxy-5,7,8-trimethyl-2H-chromen-2-one 6-hydroxy-5,7,8-trimethyl-3-(2-methylbenzyl)-2H-chromen-2-one KIT-17 KIT-21 Fig. 1 Molecular structure of the synthesized compounds a KIT 3: 8-isopropyl-3-(2-methoxybenzyl)-5-methyl-2H-chromen-2-one, MW = 336,1725 g/mol. b KIT 17: 3-benzyl-6-hydroxy-7,8-dimethyl-2H-chromen-2-one, MW = 294,1256 g/mol. c KIT 21: 6-hydroxy-7,8-dimethyl-3-(2-methylbenzyl)- 2H-chromen-2-one, MW = 308,1412 g/mol Saliba et al. Journal of Neuroinflammation (2018) 15:322 Page 3 of 13 Primary microglia cultures after 24 h of incubation by adding 100 μL of reconsti- As described in our previous studies [19–24], primary tuted substrate and incubating for 10 min. Lumines- mixed glial cell cultures were prepared from cerebral cence was measured using a Modulus™ II Microplate cortices of 1-day neonatal Sprague-Dawley rats. Under Multimode Reader (Turner BioSystems, USA). sterile conditions, the brains were carefully taken, and the cerebral cortices were isolated and the meninges re- RNA isolation and quantitative PCR moved. Then, the cortices were gently dissociated and Quantitative real-time PCR (qPCR) was performed