Microglia: multifaceted cells

Microglial cells are, similar to macrophages in the periphery, highly versatile and adaptive cells. Their exact origin was a matter of debate for a long time. By now, there is accumulating evidence that microglia precursors originate from the embryonic yolk sac.

These precursors are detectable as early as day 7.5 - 8.5 of embryonic development and their generation requires the activity of Pu.1 and Irf8 transcription factors. How these precursors populate the brain is still under investigation, but a functional blood circulation and the activity of matrix metalloproteinases seem to be indispensable (Ginhoux et al.

2010; Greter & Merad 2013; Kierdorf et al. 2013).

Under healthy conditions, microglia display a quiescent, so-called “resting state”, which is characterized by a highly-ramified morphology. But the term “resting state” is misleading, as these cells are constantly active, scanning their environment by extending and retracting fine protrusions (Nimmerjahn et al. 2005). Thereby, microglia are able to detect biochemical alterations or invading pathogens. In addition, microglia form transient contacts with synapses and support remodeling of synaptic contacts (Hanisch &

Kettenmann 2007; Wake et al. 2009).

Microglia can be activated in response to environmental changes and adapt their phenotype accordingly. The concept of different activation states is well established for macrophages. A pro-inflammatory (often referred to as M1/ classical activation state) and an anti-inflammatory subtype (M2a/ alternative activation state) are clearly distinguishable

by a specific pattern of antigen presentation and cytokine release (Martinez et al. 2009).

Another activation state (designated M2c or acquired deactivation state) inducible by interleukin-10 (IL-10) or transforming growth factor (TGF displays anti-inflammatory properties, which are supposed to be distinct from M2a (Gordon 2003; Mantovani et al.

2004; Colton & Wilcock 2010).

The different activation states of microglia are currently in the focus of numerous research projects. Challenging microglia with pro-inflammatory substances, such as lipopolysaccharide (LPS) or interferon  (IFN leads to a rapid activation, which is accompanied by the production of pro-inflammatory cytokines as for example interleukin-6 (IL-6), tumor necrosis factor (TNF) or interleukin-1(IL-1). In addition, activated microglia can release large amounts of nitric oxide (NO) or reactive oxygen species (Lee et al. 1993; Colton et al. 1996; Hanisch 2002). This pro-inflammatory “M1-like” activation state (see Figure 3 a) is important during the induction of an inflammatory response to promote tissue defense and killing of pathogens by releasing cytotoxic molecules. In addition, microglia can act as antigen presenting cells. Therefore, they display major histocompatibility complex II (MHC II) peptide complexes and co-stimulatory molecules such as CD80 (B7-1), CD86 (B7-2) or even intercellular adhesion molecule-1 (ICAM-1) to invading T-cells, thus promoting the local adaptive immune response (Shrikant &

Benveniste 1996; Yang et al. 2010).

In order to prevent self-damage from an exacerbated immune response, the final phase of infection fades to wound healing processes, including tissue repair and phagocytosis of cellular debris. During this phase, the pattern of cytokine release changes. Microglia, as well as astrocytes and even neurons produce anti-inflammatory cytokines- mainly interleukin-4 (IL-4), interleukin-13 (IL-13), IL-10 or TGF (Colton &

Wilcock 2010). A shift in the cytokine environment leads to an alteration of microglial activity. Stimulation of microglia with IL-4 or IL-13 evokes a so-called “M2a-like”

phenotype (see Figure 3 b), which is characterized by reduced mRNA levels of the NO-synthesizing enzyme iNOS and a diminished release of pro-inflammatory cytokines (Ledeboer, et al. 2000; Colton et al. 2006). In contrast, surface expression of pattern recognition receptors as for example the macrophage mannose receptor (MMR) is increased (Colton 2009). Moreover, enhanced production was detected for molecules like chitinase-3-like-3 (Ym1) and found-in-inflammatory-zone-1 (FIZZ1), which are both involved in the generation of extracellular matrix (Raes et al. 2002; Colton et al. 2006).

Arginase 1 (Arg1), an enzyme involved in arginine metabolism, is also induced upon IL-4 stimulation. It competes with iNOS for arginine, thus reducing NO production in an indirect way (Colton & Wilcock 2010). Moreover, IL-4 treated microglia have been shown to generate insulin-like growth factor-1 (IGF-1), thus conveying a survival signal and

facilitating regenerative processes in oligodendrocyte-lineage cells or neurons, respectively (Neumann et al. 2009). The detection and subsequent phagocytosis of apoptotic cells by microglia enhances the production and release of TGF and IL-10.

Acting in an auto- and paracrine way, these molecules attenuate the immune response, promote neuronal survival and support the re-establishment of the blood brain barrier integrity (Colton 2009). The activity state induced by the encounter of apoptotic cells and accompanied with increased TGF and IL-10 production is here referred to as “M2c-like”

phenotype (see Figure 3 c). This phenotype is supposed to be distinct from the one induced by IL-4 and IL-13, although both phenotypes promote tissue repair and wound-healing processes (Colton & Wilcock 2010).

Taken together, there is increasing evidence that microglia play an important role in different phases of a CNS immune response. They act as sensitive surveillants of the CNS and can adjust their activity in response to environmental stimuli. However, an immune response is complex, involving a variety of different cell types and a sophisticated system of intercellular communication, one of which is the ECS.

Figure 3: Different activation states in microglia. a) Challenging microglia with pro-inflammatory stimuli results in a reactive “M1-like” phenotype. b) Stimulation of microglia with IL-4 and/or Il-13 evokes an anti-inflammatory response and shifts microglia towards an “M2a-like” phenotype. c) A phenotype distinct from M2a, but also with anti-inflammatory properties is induced by apoptotic cells and/or stimulation with IL-10 and TGFand is referred to as "M2c-like" activation state. LPS:

lipopolysaccharide, IFN: interferon , ICAM-1 intercellular adhesion molecule-1, CD80/CD86.

costimulatory molecules, NO: nitric oxide, MHC II: major histocompatibility complex II, IL-6:

interleukin-6, TNF: tumor necrosis factor , IL-1: interleukin-1, MMR: macrophage mannose receptor, IGF-1: insulin-like growth factor-1, IL-4: interleukin-4, IL-13: interleukin-13, iNOS:

inducible nitric oxide synthase, Arg1: arginase 1, Ym1: chitinase-3-like-3, FIZZ1: found-in-inflammatory-zone 1, IL-10: interleukin-10, TGF : transforming growth factor , BBB: blood brain barrier

1.3.1 ECS signaling in microglia

The effect of cannabis consumption on immune function has been addressed in numerous in vivo and in vitro studies (for review see Klein et al. 1998). By now, an immunomodulatory function of the ECS is well accepted. The pivotal point, which creates the connection of the ECS with the immune system, is the CB₂ receptor. Careful expression analysis revealed its prevailing appearance in immune cells of the myeloid, and lymphoid lineage (Munro et al. 1993; Galiègue et al. 1995).

Microglia in the CNS are capable of expressing CB1 and CB2 receptors. However, while CB₂ levels are variable with respect to changes in phenotype and activation state, the CB1 receptor expression appears to be rather constant (Carlisle, et al. 2002).

Compared to resting microglia, the expression level of the CB₂ receptor is elevated, when these cells become activated. Various pro-inflammatory stimuli, such as LPS or IFNin combination with GM-CSF (granulocyte macrophage-colony stimulating factor) have been shown to modulate CB₂ expression (Carlisle et al. 2002; Maresz et al. 2005; Stella 2011).

However, the extend of CB₂ production by microglia in response to a neuroinflammatory process is dependent on the molecular environment generated by pathogens, toxins or cytokines (Stella 2009). More recent findings indicate that the putative cannabinoid receptor GPR18 is expressed in microglia. Activation of this receptor by N-arachidonoyl glycine (NAGly), a metabolite of anandamide, influences migration of microglia derived from the cell line BV-2 (McHugh et al. 2012).

In addition to cannabinoid receptors, microglia are able to produce 2-AG as well as anandamide (Carrier et al. 2004). Another study addressing the production of endocannabinoids in microglial cells revealed that exogenous ATP triggers the production of 2-AG by activation of purinergic P2X₇ receptors (Witting, et al. 2004). Degradation of endocannabinoids in this cell type occurs through the activation of FAAH and MAGL (Witting, et al. 2004; Muccioli et al. 2007). More recently, the -hydrolase ABHD12 has been shown to be abundantly expressed in microglia. Up to now, 2-AG is the only known substrate for this hydrolase and it is tempting to speculate that ABHD12 activity accounts for the degradation of the main proportion of microglia-derived 2-AG (Fiskerstrand et al.

2010).

Taken together, microglia express all important components of the ECS and signaling appears disengaged from the classical pre-and postsynaptic arrangement of the ECS in neurons. How endocannabinoid signaling is orchestrated between different cell types under pathological conditions is still under investigation. Neuronal damage is accompanied by extensive release of glutamate and ATP. Both molecules can stimulate endocannabinoid production and release in adjacent neurons and surrounding microglia (Stella 2009; Pandey et al. 2009). 2-AG stimulates proliferation of microglia and induces

site-specific migration, an effect, which was shown to be sensitive to CB₁- and CB₂ antagonists (Walter et al. 2003; Carrier et al. 2004; Eljaschewitsch et al. 2006). Acting on LPS-stimulated microglia, anandamide effectively reduced the production and the release of pro-inflammatory mediators such as NO, IL-6, IL-1 and IL-1(Puffenbarger et al.

2000; Eljaschewitsch et al. 2006). In contrast, anandamide was shown to enhance the production of the anti-inflammatory cytokine IL-10 in a CB₂-mediated manner (Correa et al. 2010). Across the plethora of studies that were performed in this field, the major findings suggest that the ECS functions as a communication and regulation system among different cell types. However, neuroinflammation can be triggered by a multitude of events. Thus, careful investigation is required to dissect the signaling events which involve the ECS and influence the respective activation state of microglia during different phases of the inflammatory response of the CNS.