Differential expression of ECS-related genes in microglia

Comprehensive gene expression profiling performed within this work revealed that the combined application of LPS and IFN achieved the broadest impact on gene regulation in primary microglia. The subsequent phenotypic changes that were induced by these substances were well detectable, as described before. In comparison, the effect of IL-4 and TGF on gene transcription was by far less pronounced. Nevertheless, only a small proportion of regulated genes were commonly affected by IL-4 and TGF, pointing out distinct modulatory activities and supporting the idea of distinct activation states. The obtained lists of genes that were responsive to the respective stimulation represent an important source to analyze involved signaling pathways in detail in the near future.

As mentioned earlier, the regulation of genes that are related to the ECS was in the focus of this thesis. Gene expression profiling revealed a distinct expression pattern for several ECS-related molecules in differentially activated microglia. An overview of the

corresponding ECS components and of identified marker moelcules for the respective microglial activation state is given in Table 3.

Table 3: Regulation of ECS-related genes in differentially activated microglia. The subtypes

“M1-like”, “M2a-like” and “M2c-like” were induced by LPS/IFN IL-4 or TGFstimulation, respectively. The sets of confirmed marker molecules and the responsive components of the ECS are listed below. LPS: Lipopolysaccharide, IFN interferon IL-4: interleukin-4, TGF:

transforming growth factor ICAM-1: intercellular adhesion molecule-1, CD80/CD40:

costimulatory molecules, TNF: tumor necrosis factor , IL-6: interleukin-6, iNOS: inducible nitric oxide synthase, MMR: macrophage mannose receptor, YM1: chitinase-3-like-3, FIZZ1: found-in-inflammatory-zone 1, Arg1: arginase 1, HGF: hepatocyte growth factor, CB2: cannabinoid receptor 2, DAGLβ: diacylglycerol lipase , ABHD12: -hydrolase 12, NAPE-PLD: N-arachidonoyl-phosphatidylethanolamine specific phospholipase D, GPR18: G-protein coupled receptor 18;

upregulated compared to unstimulated control, downregulated compared to unstimulated control, no change compared to unstimulated control synthesis of the two main endocannabinoids AEA and 2-AG. Moreover, the expression of 2 cannabinoid receptors, CB₂ and GPR18, and ABHD12, a 2-AG degrading enzyme, were altered.

LPS/IFN stimulation had a stronger impact on gene expression levels than other stimuli in microglia. On closer consideration it becomes obvious that the genes encoding DAGLβ, the CB_ receptor and ABHD12 were regulated in a similar way. Their respective expression levels were diminished in response to LPS/IFN. This is an interesting aspect, as all these components are closely related to 2-AG signaling. CB2 expression was down regulated under pro-inflammatory conditions, while 2-AG production might have remained homeostatic with less synthesis and less degradation. In contrast, the gene encoding NAPE-PLD was upregulated in response to LPS/IFN, just as the newly identified

cannabinoid receptor GPR18, which can be activated by a metabolite of NAPE-PLD-produced AEA.

IL-4 stimulation did not result in significant changes in genes that encode components of the ECS. In contrast, TGF stimulation enhanced CB2 receptor expression in primary microglia. The regulation of the CB2 receptor in immune cells is definitely the aspect, which has been most intensively studied by others. Nevertheless, it is still not completely understood. It is generally assumed that CB₂ is not or only marginally expressed in healthy brain tissue (Munro et al. 1993; Galiègue et al. 1995a; Carlisle, et al.

2002). Cultured primary microglial cells are in a “primed”, responsive state and express detectable amounts of CB2 (Becher & Antel 1996; Carlisle, et al. 2002; Walter et al. 2003).

It has also been reported that fully activated microglia occurring in brain tissue of patients suffering from multiple sclerosis or Alzheimer’s disease express CB2 (Benito et al. 2003;

Yiangou et al. 2006). Therefore, CB₂ receptor upregulation is thought to be associated with the activation process of microglia under inflammatory conditions. In contrast, in vitro studies revealed that pro-inflammtory stimuli such as IFN alone did not affect CB2 levels or, in the case of LPS, even reduced its expression in microglia (Carlisle, et al. 2002;

Maresz et al. 2005). This observation is in line with the results reported here and supports the idea that CB₂ activation promotes rather immunosuppressive activities. However, it might also be possible, that CB₂ is upregulated in a short time frame during the process of activation to promote proliferation and migration to lesion sites, as this has been shown to occur in a 2-AG dependent manner (Walter et al. 2003; Carrier et al. 2004). Arranging these reports with the obtained data in this work, the following scenario might be possible:

Downregulation of the CB2 receptor would occur during the reactive pro-inflammatory phase of an immune response. A second wave of CB₂ expression would be induced in a later phase of inflammation coinciding with the switch to an immunosuppressive “M2a- or M2c-like” subtype. However, discrepancies of in vivo and in vitro data as well as differences in stimulation protocols complicate the generation of a consistent picture.

A completely different induction pattern was observed here for the newly identified cannabinoid receptor GPR18. The expression level of the corresponding gene was strongly elevated in response to LPS/IFNin primary microglia. Similar results were reported for macrophages. Takenouchi and colleagues observed an upregulation of Gpr18 mRNA levels in pertioneal macrophages and RAW264.7 macrophages upon LPS/IFN stimulation, while IL-4 treatment had no effect (Takenouchi et al. 2012). GPR18 can be activated by NaGly, which is a metabolite of AEA and the most efficacious agonist (McHugh et al. 2010). But also AEA and - to a lesser extent - 2-AG are able to activate GPR18 (McHugh et al. 2012). Addition of NaGly effectively induced apoptosis in macrophages (Takenouchi et al. 2012). It was proposed that NaGly might serve as an

immunosuppressive mediator, which reduces the number of pro-inflammatory M1 macrophages by inducing apoptosis (Takenouchi et al. 2012). Up to now, these observations were not verified in microglia and this study is the first to report enhanced GPR18 expression in LPS/IFN treated primary microglia. However, the interpretation of Takenouchi and colleagues supports the general view that the ECS exerts beneficial functions during the course of an immune response by balancing pro-and anti-inflammatory reactions.

In conclusion, gene expression profiling of differentially activated microglia revealed that each substance induced a distinct pattern of gene regulation. The obtained data will provide helpful insights into the function of different microglia subtypes on the level of gene expression. Some genes encoding components of the ECS were shown to be responsive to different stimuli and underline an important role of the ECS in immune regulation. Nevertheless, further research has to be performed. Comprehensive analysis of the gene structure and corresponding promotor elements of CB2 and GPR18, for example, could provide important data, which might reveal the link between the signaling pathways elicited by different stimulants and the phenotypical outcome.