Anandamide, Acting via CB2 Receptors, Alleviates Neuroinflammation

Anandamide, Acting via CB2 Receptors, Alleviates LPS-Induced Neuroinflammation in Rat Primary Microglial Cultures | Neural Plast. 2015| Free Full Text article

Microglial activation is a polarized process divided into potentially neuroprotective phenotype M2 and neurotoxic phenotype M1, predominant during chronic neuroinflammation.

The endocannabinoid system provides an attractive target to control the balance between microglial phenotypes. Anandamide as an immune modulator in the central nervous system acts via not only cannabinoid receptors (CB1 and CB2) but also other targets (e.g., GPR18/GPR55).  

We studied the effect of anandamide on lipopolysaccharide-induced changes in rat primary microglial cultures. Microglial activation was assessed based on nitric oxide (NO) production.

Analysis of mRNA was conducted for M1 and M2 phenotype markers possibly affected by the treatment. Our results showed that lipopolysaccharide-induced NO release in microglia was significantly attenuated, with concomitant downregulation of M1 phenotypic markers, after pretreatment with anandamide.

This effect was not sensitive to CB1 or GPR18/GPR55 antagonism. Administration of CB2 antagonist partially abolished the effects of anandamide on microglia.

Interestingly, administration of a GPR18/GPR55 antagonist by itself suppressed NO release. In summary, we showed that the endocannabinoid system plays a crucial role in the management of neuroinflammation by dampening the activation of an M1 phenotype. This effect was primarily controlled by the CB2 receptor, although functional cross talk with GPR18/GPR55 may occur.

1. Introduction

Neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, stroke, and chronic pain, are associated with ongoing inflammation in the central nervous system (CNS). One of the striking hallmarks of these neurodegenerative disorders is chronic microglial activation.

Recent studies have shown that activated microglia can be divided into two phenotypic profiles.

  1. The classical M1 state, characterized by proinflammatory factors for example, interleukins (IL-1Β, IL-18, and IL-6) and inducible nitric oxide synthase (NOS2), is neurotoxic and therefore contributes to secondary neuronal damage, cell death, and demyelination, which lead to neurodegeneration.
  2. The neuroprotective M2 state, known as “alternative activation,” is associated with the release of anti-inflammatory factors, such as IL-10, IL-4, and NGF.

Several studies indicate that the endocannabinoid system provides an attractive target for managing microglial-derived neuroinflammation and may regulate many aspects of the brain’s inflammatory response, including the release of M1 phenotype specific cytokines. The endocannabinoid system modulates both neuronal and immune functions through two protein-coupled cannabinoid receptors (CB1 and CB2), although endocannabinoids, especially anandamide (AEA), can activate numerous other receptors like PPARS, TRPV1, and GPR18/GPR55. The latter, involved in immunological responses, represents an interesting molecular target for the control of neuroinflammation

Both cannabinoid receptors are expressed in microglia and may act as immune modulators in the CNS

Moreover, it has been proposed that microglia are the main population of cells responsible for the production of AEA in the CNS, as primary microglial cultures produce approximately 20-fold more of this compound than neuronal or astrocyte cultures. Therefore, it is important to investigate the effect of AEA on microglia,

Many of the neurodegenerative conditions of the CNS result in increased levels of endogenous AEA; therefore, the endocannabinoid system may provide an attractive target to influence microglial phenotype during chronic inflammation.

Therefore, to increase our understanding of the role of the endocannabinoid system in the modulation of microglial polarization, we explored the possible therapeutic action of AEA and AM-251, AM-630, and CID-16020046 at cannabinoid and GPR18/GPR55 receptors in the in vitro model of LPS-induced microglial activation.

3. Results

3.1. The Concentrations of Compounds Used in the Biochemical and Molecular Analyses Did Not Show Signs of Toxicity

3.2. Involvement of Cannabinoid and GPR Receptors in the AEA-Mediated Alleviation of NO Production

3.3. Alteration of Cb1, Cb2, Gpr18, and Gpr55 Expression in LPS-Stimulated Primary Microglial Cells after Treatment with the Tested Compounds

3.5. Changes in M2 Phenotype-Related Molecules in LPS-Stimulated Rat Primary Microglia after Treatment with the Tested Compounds

Expression of anti-inflammatory IL-10 mRNA was significantly elevated after AEA treatment in LPS-stimulated cells (Figure 5(a)). Moreover, administration of AM-251 also showed similar results.

4. Discussion

In the present study, we have demonstrated that the alleviating effect of AEA on NO production in primary microglial cultures is mediated mainly through CB2 receptors. Activated upon CNS damage, microglia initiate and play a critical role in the development of CNS inflammation.

Various stimuli can activate microglia, causing proinflammatory or anti-inflammatory functions depending on the duration, nature, and scale of the stimulus

It has been shown that the inflammatory response of LPS-stimulated microglia, which leads to increased secretion of NO, contributes to events underlying brain inflammation and neuronal degeneration

Some studies have indicated that short-term cannabinoid exposure can have a neuroprotective effect at the time of the sudden failure of CNS tissues.

Bursts of AEA, which is synthesized “on demand” in areas of cellular stress (e.g., in damaged tissue or at the site of inflammation), have been suggested as the mechanism that inhibits the immune response in both normal and injured tissues, where it is involved in the migration of immune cells to the site of inflammation

Our study demonstrated a reduction in NO release after pretreatment with AEA in LPS-stimulated primary microglial cultures, which suggests it has a neuroprotective action during CNS tissue damage

studies have shown that CB2 receptor activation reduces the immune response during CNS inflammation, brain edema, and the death of neurons, alleviating the symptoms of neurodegenerative diseases in animal models. CB2 receptor stimulation inhibits the activation of microglia, slowing down the development of Alzheimer’s disease

Similarly, CB2 receptor activation in microglial cells in the spinal cord can reduce inflammatory reactions and pain after peripheral nerve injury

Microglial activation is a polarized process that can be divided into M1 and M2 phenotypes. During the short-term activation of microglia, the presence of both the M1 and M2 phenotypes is balanced, allowing the restoration of CNS homeostasis; however, chronic inflammation causes a shift toward the proinflammatory M1 phenotype.

One of the actions of activated microglia is the promotion of inflammation, which causes an influx of immune cells to the site of injury. To this end, the M1 phenotype of microglial cells initiates neuroinflammation by producing cytotoxic factors such as cytokines and enzymes (NOS2 and COX2), which, in addition to acting as chemoattractants, may lead to neuronal damage upon chronic activation

Moreover, cannabinoids can modulate cytokine production, which in turn contributes to a reduction of the immune response and can be beneficial in autoimmune diseases.

Our studies showed that AEA causes a reduction in microglial cell activation, especially by dampening activation of the M1 phenotype. We demonstrated the involvement of the CB2 receptor in the cytoprotective effect of AEA.

Summing up, the use of pharmacological tools to control the phenotype of microglia through the endocannabinoid system may be useful in the treatment of neurodegenerative conditions.  


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