Ana Juknat

Cannabinoids Δ9-Tetrahydrocannabinol and Cannabidiol Differentially Inhibit the Lipopolysaccharide-activated NF-κB and Interferon-β/STAT Proinflammatory Pathways in BV-2 Microglial Cells

Cannabinoids have been shown to exert anti-inflammatory activities in various in vivo and in vitro experimental models as well as ameliorate various inflammatory degenerative diseases. However, the mechanisms of these effects are not completely understood. Using the BV-2 mouse microglial cell line and lipopolysaccharide (LPS) to induce an inflammatory response, we studied the signaling pathways engaged in the anti-inflammatory effects of cannabinoids as well as their influence on the expression of several genes known to be involved in inflammation. We found that the two major cannabinoids present in marijuana, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), decrease the production and release of proinflammatory cytokines, including interleukin-1β, interleukin-6, and interferon (IFN)β, from LPS-activated microglial cells. The cannabinoid anti-inflammatory action does not seem to involve the CB1 and CB2 cannabinoid receptors or the abn-CBD-sensitive receptors. In addition, we found that THC and CBD act through different, although partially overlapping, mechanisms. CBD, but not THC, reduces the activity of the NF-κB pathway, a primary pathway regulating the expression of proinflammatory genes. Moreover, CBD, but not THC, up-regulates the activation of the STAT3 transcription factor, an element of homeostatic mechanism(s) inducing anti-inflammatory events. Following CBD treatment, but less so with THC, we observed a decreased level of mRNA for the Socs3 gene, a main negative regulator of STATs and particularly of STAT3. However, both CBD and THC decreased the activation of the LPS-induced STAT1 transcription factor, a key player in IFNβ-dependent proinflammatory processes. In summary, our observations show that CBD and THC vary in their effects on the anti-inflammatory pathways, including the NF-κB and IFNβ-dependent pathways.

Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis-like disease in C57BL/6 mice

BACKGROUND AND PURPOSE Cannabis extracts and several cannabinoids have been shown to exert broad anti‐inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration.

Differential changes in GPR55 during microglial cell activation

We examined how lipopolysaccharide (LPS) and interferon gamma (IFN‐γ), known to differentially activate microglia, affect the expression of G protein‐coupled receptor 55 (GPR55), a novel cannabinoid receptor. We found that GPR55 mRNA is significantly expressed in both primary mouse microglia and the BV‐2 mouse microglial cell line, and that LPS down‐regulates this message. Conversely, IFN‐γ slightly decreases GPR55 mRNA in primary microglia, while it upregulates this message in BV‐2 cells. Moreover, the GPR55 agonist, lysophosphatidylinositol, increases ERK phosphorylation in BV‐2 stimulated with IFN‐γ, in correlation with the increased amount of GPR55 mRNA. Remarkably, these stimuli‐induced changes in GPR55 expression are similar to those observed with CB2‐R, suggesting that both receptors might be involved in neuroinflammation and that their expression is concomitantly controlled by the state of microglial activation.

In vivo up‐regulation of brain‐derived neurotrophic factor in specific brain areas by chronic exposure to Δ9‐tetrahydrocannabinol

Cannabinoids are widely abused drugs. Here we show that chronic administration of Δ9‐tetrahydrocannabinol (Δ9‐THC), the active psychotropic agent in marijuana and hashish, at 1.5 mg per kg per day intraperitoneally for 7 days, increases the expression, at both mRNA and protein levels, of brain‐derived neurotrophic factor (BDNF), in specific rat brain areas, notably in those involved in reward and addiction. Real‐time PCR revealed a 10‐fold up‐regulation of BDNF mRNA in the nucleus accumbens (NAc) upon chronic Δ9‐THC treatment, but there was no change at 3 or 24 h after a single injection. Smaller increases in mRNA levels were found in the ventral tegmental area (VTA), medial prefrontal cortex and paraventricular nucleus (PVN). Immunohistochemistry showed large increases in BDNF‐stained cells in the NAc (5.5‐fold), posterior VTA (4‐fold) and PVN (1.7‐fold), but no change was observed in the anterior VTA, hippocampus or dorsal striatum. Altogether, our study indicates that chronic exposure to Δ9‐THC up‐regulates BDNF in specific brain areas involved with reward, and provides evidence for different BDNF expression in the anterior and posterior VTA. Moreover, BDNF is known to modulate synaptic plasticity and adaptive processes underlying learning and memory, leading to long‐term functional and structural modification of synaptic connections. We suggest that Δ9‐THC up‐regulation of BDNF expression has an important role in inducing the neuroadaptive processes taking place upon exposure to cannabinoids.

Differential transcriptional profiles mediated by exposure to the cannabinoids cannabidiol and Δ9-tetrahydrocannabinol in BV-2 microglial cells.

BACKGROUND AND PURPOSE Apart from their effects on mood and reward, cannabinoids exert beneficial actions such as neuroprotection and attenuation of inflammation. The immunosuppressive activity of cannabinoids has been well established. However, the underlying mechanisms are largely unknown. We previously showed that the psychoactive cannabinoid Δ9‐tetrahydrocannabinol (THC) and the non‐psychoactive cannabidiol (CBD) differ in their anti‐inflammatory signalling pathways.

Microarray and pathway analysis reveal distinct mechanisms underlying cannabinoid-mediated modulation of LPS-induced activation of BV-2 microglial cells.

Cannabinoids are known to exert immunosuppressive activities. However, the mechanisms which contribute to these effects are unknown. Using lipopolysaccharide (LPS) to activate BV-2 microglial cells, we examined how Δ9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, and cannabidiol (CBD) the non-psychoactive component, modulate the inflammatory response. Microarray analysis of genome-wide mRNA levels was performed using Illumina platform and the resulting expression patterns analyzed using the Ingenuity Pathway Analysis to identify functional subsets of genes, and the Ingenuity System Database to denote the gene networks regulated by CBD and THC. From the 5338 transcripts that were differentially expressed across treatments, 400 transcripts were found to be upregulated by LPS, 502 by CBD+LPS and 424 by THC+LPS, while 145 were downregulated by LPS, 297 by CBD+LPS and 149 by THC+LPS, by 2-fold or more (p≤0.005). Results clearly link the effects of CBD and THC to inflammatory signaling pathways and identify new cannabinoid targets in the MAPK pathway (Dusp1, Dusp8, Dusp2), cell cycle related (Cdkn2b, Gadd45a) as well as JAK/STAT regulatory molecules (Socs3, Cish, Stat1). The impact of CBD on LPS-stimulated gene expression was greater than that of THC. We attribute this difference to the fact that CBD highly upregulated several genes encoding negative regulators of both NFκB and AP-1 transcriptional activities, such as Trib3 and Dusp1 known to be modulated through Nrf2 activation. The CBD-specific expression profile reflected changes associated with oxidative stress and glutathione depletion via Trib3 and expression of ATF4 target genes. Furthermore, the CBD affected genes were shown to be controlled by nuclear factors usually involved in regulation of stress response and inflammation, mainly via Nrf2/Hmox1 axis and the Nrf2/ATF4-Trib3 pathway. These observations indicate that CBD, and less so THC, induce a cellular stress response and that this response underlies their high immunosuppressant activities.

Anandamide Protects from Low Serum-induced Apoptosis via Its Degradation to Ethanolamine

Anandamide (AEA) is a lipid molecule belonging to the family of endocannabinoids. Various studies report neuroprotective activity of AEA against toxic insults, such as ischemic conditions and excitotoxicity, whereas some show that AEA has pro-apoptotic effects. Here we have shown that AEA confers a protective activity in N18TG2 murine neuroblastoma cells subjected to low serum-induced apoptosis. We have demonstrated that the protection from apoptosis by AEA is not mediated via the CB1 receptor, the CB2 receptor, or the vanilloid receptor 1. Interestingly, breakdown of AEA by fatty acid amide hydrolase is required for the protective effect of AEA. Furthermore, the ethanolamine (EA) generated in this reaction is the metabolite responsible for the protective response. The elevation in the levels of reactive oxygen species during low serum-induced apoptosis is not affected by AEA or EA. On the other hand, AEA and EA reduce caspase 3/7 activity, and AEA attenuates the cleavage of PARP-1. Taken together, our results demonstrate a role for AEA and EA in the protection against low serum-induced apoptosis.

The Non-Psychoactive Plant Cannabinoid, Cannabidiol Affects Cholesterol Metabolism-Related Genes in Microglial Cells

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that is clinically used in a 1:1 mixture with the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) for the treatment of neuropathic pain and spasticity in multiple sclerosis. Our group previously reported that CBD exerts anti-inflammatory effects on microglial cells. In addition, we found that CBD treatment increases the accumulation of the endocannabinoid N-arachidonoyl ethanolamine (AEA), thus enhancing endocannabinoid signaling. Here we proceeded to investigate the effects of CBD on the modulation of lipid-related genes in microglial cells. Cell viability was tested using FACS analysis, AEA levels were measured using LC/MS/MS, gene array analysis was validated with real-time qPCR, and cytokine release was measured using ELISA. We report that CBD significantly upregulated the mRNAs of the enzymes sterol-O-acyl transferase (Soat2), which synthesizes cholesteryl esters, and of sterol 27-hydroxylase (Cyp27a1). In addition, CBD increased the mRNA of the lipid droplet-associated protein, perilipin2 (Plin2). Moreover, we found that pretreatment of the cells with the cholesterol chelating agent, methyl-β-cyclodextrin (MBCD), reversed the CBD-induced increase in Soat2 mRNA but not in Plin2 mRNA. Incubation with AEA increased the level of Plin2, but not of Soat2 mRNA. Furthermore, MBCD treatment did not affect the reduction by CBD of the LPS-induced release of the proinflammatory cytokine IL-1β. CBD treatment modulates cholesterol homeostasis in microglial cells, and pretreatment with MBCD reverses this effect without interfering with CBD’s anti-inflammatory effects. The effects of the CBD-induced increase in AEA accumulation on lipid-gene expression are discussed.

Cannabidiol, a non-psychoactive cannabinoid, leads to EGR2-dependent anergy in activated encephalitogenic T cells

BackgroundCannabidiol (CBD), the main non-psychoactive cannabinoid, has been previously shown by us to ameliorate clinical symptoms and to decrease inflammation in myelin oligodendrocyte glycoprotein (MOG)35-55-induced mouse experimental autoimmune encephalomyelitis model of multiple sclerosis as well as to decrease MOG35-55-induced T cell proliferation and IL-17 secretion. However, the mechanisms of CBD anti-inflammatory activities are unclear.MethodsHere we analyzed the effects of CBD on splenocytes (source of accessory T cells and antigen presenting cells (APC)) co-cultured with MOG35-55-specific T cells (TMOG) and stimulated with MOG35-55. Using flow cytometry, we evaluated the expression of surface activation markers and inhibitory molecules on T cells and B cells. TMOG cells were purified using CD4 positive microbead selection and submitted for quantitative PCR and microarray of mRNA transcript analyzes. Cell signaling studies in purified TMOG were carried out using immunoblotting.ResultsWe found that CBD leads to upregulation of CD69 and lymphocyte-activation gene 3 (LAG3) regulatory molecules on CD4+CD25− accessory T cells. This subtype of CD4+CD25−CD69+LAG3+ T cells has been recognized as induced regulatory phenotype promoting anergy in activated T cells. Indeed, we observed that CBD treatment results in upregulation of EGR2 (a key T cell anergy inducer) mRNA transcription in stimulated TMOG cells. This was accompanied by elevated levels of anergy promoting genes such as IL-10 (anti-inflammatory cytokine), STAT5 (regulatory factor), and LAG3 mRNAs, as well as of several enhancers of cell cycle arrest (such as Nfatc1, Casp4, Cdkn1a, and Icos). Moreover, CBD exposure leads to a decrease in STAT3 and to an increase in STAT5 phosphorylation in TMOG cells, positive and negative regulators of Th17 activity, respectively. In parallel, we observed decreased levels of major histocompatibility complex class II (MHCII), CD25, and CD69 on CD19+ B cells following CBD treatment, showing diminished antigen presenting capabilities of B cells and reduction in their pro-inflammatory functions.ConclusionsOur data suggests that CBD exerts its immunoregulatory effects via induction of CD4+CD25−CD69+LAG3+ cells in MOG35-55-activated APC/TMOG co-cultures. This is accompanied by EGR2-dependent anergy of stimulated TMOG cells as well as a switch in their intracellular STAT3/STAT5 activation balance leading to the previously observed decrease in Th17 activity.

Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveolin-1

BACKGROUND AND PURPOSE N‐acyl ethanolamines (NAEs) and 2‐arachidonoyl glycerol (2‐AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2‐AG into lipid raft fractions isolated from the caveolin‐1‐lacking microglial cell line BV‐2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin‐1‐positive F‐11 cell line.