Endocannabinoids drive the acquisition of an alternative phenotype in microglia
Introduction
The innate primary immune response in the brain is mediated by an inherent and characteristic program that involves the dynamic activation of microglia. Like other tissue macrophages, microglia participate in injury, repair and resolution processes to restore normal tissue homeostasis. It is well known that macrophages adopt different activation states in response to diverse signals and that these states can be identified by upregulation of specific markers (MacMicking et al., 1997, Gordon, 2003). The main activation states are described as the classically-activated or M1 phenotype, and the alternatively-activated or M2 phenotype. Stimuli like LPS or IFN-γ induce the M1 phenotype which is mainly identified by enhanced iNOS expression, while the alternatively-activated or M2a phenotype was first used to describe a macrophage which adopted a phenotype distinct from that induced by IFN-γ and LPS (Stein et al., 1992). This phenotype is induced by the Th2 cell derived cytokines, IL-4 and IL-13 and it is identified by increased expression of Arginase-1 (Arg-1), IGF-1, the mannose receptor, chitinase 3-like 3 and FIZZ-1, etc. The acquired deactivated (M2c) phenotype is induced by the immunosuppressive cytokines, TGFβ and IL-10, and it is mainly identified by the expression of SOCS3. Significantly, apoptotic cells are capable of enhancing microglial TGFβ expression and release thereby shifting the microglia phenotype towards alternative activation M2c (Spittau et al., 2015). Indeed, it was recently reported that these phenotypes can be identified in microglia cultures (Chhor et al., 2013).
Rather than discrete and stable states, both macrophages and microglia adopt a spectrum of activated phenotypes, retaining their plasticity after polarization to adapt to changing environments. Thus, deciphering the factors that regulate the physiological behavior of macrophages and microglia is essential to understand pathogenic and neuroprotective pathways, and will aid the development of new therapies. The microglial phenotypes are distinguished by cell surface receptor markers expression and the release of soluble factors with recognized functions. These elements in turn drive distinct cell responses as well as the production of cytokines and chemokines, thereby promoting the multiple responses to microglia associated with neuroinflammation (Chhor et al., 2013, Hu et al., 2015). Endocannabinoids (eCBs) are lipid transmitters that are released from their membrane precursors by specific lipases, allowing them to activate cannabinoid (CB) receptors, and their inactivation involves their uptake and ensuing hydrolysis (Pacher et al., 2008, Mechoulam and Parker, 2013). For example, various lipases and hydrolases including NAPE-PLD can provoke the release of anandamide (AEA) from its membrane precursor (Liu et al., 2006, Simon and Cravatt, 2006, Di Marzo, 2011), and AEA activates both CB1 and CB2 receptors as a partial agonist. Ultimately, AEA is inactivated by uptake followed by fatty acid amide hydrolase (FAAH)-mediated hydrolysis (Cravatt and Lichtman, 2003, Fowler, 2102). The other main eCB, 2-arachidonoylglycerol (2-AG) is released from its membrane precursor by phospholipase C (PLC) and diacylglycerol lipases (DAGLs), isoforms α and β (Stella et al., 1997, Bisogno et al., 2003, Murataeva et al., 2014). 2-AG activates CB1 and CB2 receptors as a full agonist; after uptake, it is hydrolyzed by monoacylglycerol lipase (MAGL), and to a lesser extent by α,β-hydrolase-6 (ABDH6) and α,β-hydrolase-12 (ABDH12) (Freund et al., 2003, Litchman et al., 2010, Pertwee, 2010). In the CNS, AEA and 2-AG are produced and inactivated by neurons and glia. The production of eCBs is increased in response to specific stimuli, like receptor activation, ion channel opening and calcium influx. The levels of extracellular eCBs and the ensuing activation of CB1 and CB2 receptors is a reflection of the balance between their production and inactivation. In vivo and in vitro models of inflammation have shown that exogenous and endogenous CBs have immunomodulatory properties that may mediate some of their beneficial effects in diverse pathological states (Arévalo-Martín et al., 2008, Maroof et al., 2013). Microglia express a functional eCB signaling system (eCBSS) and the activation of CB receptors modulates microglia behaviors such as migration, proliferation, cytokines and free radicals release, or phagocytosis (Stella, 2009). Indeed, the expression of CB2 receptors has been related to the activation state of microglia, as observed in macrophages (Carlisle et al., 2002, Walter et al., 2003, Martín-Couce et al., 2012). The upregulation of CB2 receptors in pathological neuroinflammatory conditions has been associated to a restoration of tissue homeostasis (Miller and Devi, 2011). However, the direct implication of the eCBSS in the acquisition of the different microglia phenotypes has not been addressed to date.
Here, by studying primary cultures of microglia, we show that the eCBSS machinery in microglia is altered as a function of their activation state. Alternatively activated microglia synthesized different types of eCBs, depending on the stimuli they are exposed to, and these eCBs influenced the acquisition of the M2 activation state. Moreover, the presence of functional CB2 receptors was necessary for the upregulation of Arg-1 in the M2 state, and for the phagocytic function of microglia. Our results support the role of eCBs as novel drivers of the alternative state of rodent and human microglia.
Section snippets
Animals
Animal handling and care was performed in compliance with European Union guidelines 2010/63/EU and Spanish regulations (BOE67/8509-12; BOE 1201/2005) regarding the use and care of laboratory animals, and all the protocols were approved by the local Animal Care and Ethics Committee of the CSIC. Wistar rats and C57BL/6 WT mouse pups were obtained from our in-house colony (Instituto Cajal, CSIC) and from Jackson Lab (USA). The B6.129P2-Cnr2tml1Dgen/J mouse used is a null mutant knockout for the
M2a and M2c activated microglia selectively produce 2-AG or AEA, respectively
The M1–M2 paradigm, traditionally distinguished by the expression of Arg-1 in M2 cells, has been useful to characterize the functional phenotypes of macrophages, and more recently that of microglia in neuroinflammatory disorders. Nevertheless, both these cell types show remarkable plasticity and they can probably adopt a spectrum of polarized states in response to environmental cues. The morphological changes that distinguish the different phenotypes are one of the principal hallmarks of
Discussion
We suggest here a new role for eCBs as important messengers for microglial function that drive the acquisition of an alternative (or reparative) phenotype. ECBs are produced by both immune cells and neural cells (Salzet et al., 2000, Freund et al., 2003) and they appear to fulfill a key role in neuroimmune networks (García-Ovejero et al., 2013, Hernangómez et al., 2014). The first goal of this study was to examine whether the synthesis of eCBs is altered in M2a or M2c alternative activated
Acknowledgments
We thank to Laura Ramos for excellent technical support. This work was supported by Grants from the Ministry of the Economy and Competition (MINECO SAF2013-42784-R and SAF2013-48271, the Comunidad de Madrid (S2010/BMD-2308 and S2010/BMD-2353) and the Red Española de Esclerosis Múltiple (REEM) RD12/0032/0008 (CG) sponsored by the Fondo de Investigación Sanitaria (FIS). A.R.Z. is a predoctoral fellow from UCM. M.M. is a senior postdoctoral supported by the Comunidad de Madrid (S2010/BMD-2308).
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