Anti-Inflammatory Activity of a CB2 Selective Cannabinoid Receptor Agonist: Signaling and Cytokines Release in Blood Mononuclear Cells

Abstract

The endocannabinoid system (ECS) exerts immunosuppressive effects, which are mostly mediated by cannabinoid receptor 2 (CBR2), whose expression on leukocytes is higher than CBR1, mainly localized in the brain. Targeted CBR2 activation could limit inflammation, avoiding CBR1-related psychoactive effects. Herein, we evaluated in vitro the biological activity of a novel, selective and high-affinity CBR2 agonist, called JT11, studying its potential CBR2-mediated anti-inflammatory effect. Trypan Blue and MTT assays were used to test the cytotoxic and anti-proliferative effect of JT11 in Jurkat cells. Its pro-apoptotic activity was investigated analyzing both cell cycle and poly PARP cleavage. Finally, we evaluated its impact on LPS-induced ERK1/2 and NF-kB-p65 activation, TNF-α, IL-1β, IL-6 and IL-8 release in peripheral blood mononuclear cells (PBMCs) from healthy donors. Selective CB2R antagonist SR144528 and CBR2 knockdown were used to further verify the selectivity of JT11. We confirmed selective CBR2 activation by JT11. JT11 regulated cell viability and proliferation through a CBR2-dependent mechanism in Jurkat cells, exhibiting a mild pro-apoptotic activity. Finally, it reduced LPS-induced ERK1/2 and NF-kB-p65 phosphorylation and pro-inflammatory cytokines release in human PBMCs, proving to possess in vitro anti-inflammatory properties. JT11 as CBR2 ligands could enhance ECS immunoregulatory activity and our results support the view that therapeutic strategies targeting CBR2 signaling could be promising for the treatment of chronic inflammatory diseases.

Keywords: CB2R; CB2R agonist; anti-inflammatory activity; immunomodulation.

Figure 1

Radioligand Binding Data of 1,8-Naphthyridin-2(1H)-one-3-carboxamide Derivative JT11 ^a. ^a Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM) for CB1R and CB2R binding assays. ^b Affinity of compounds for CBR1 was evaluated using membranes from HEK-293 cells transfected with CB1R and [3H]CP-55,940. ^c Affinity of compounds for CB2R was evaluated using membranes from HEK-293 cells transfected with CB1R and [3H]CP-55,940 [26].

Figure 2

JT11 regulates cell viability and growth through a CB2R-dependent mechanism in Jurkat cells. ((A), left panel) Jurkat cells were treated with 0.1, 1 or 2 μM dose of JT11 for 24, 48 and 72 h. The number of viable cells was determined by Trypan Blue assay. Data are reported as the mean ± SD among ten independent experiments. *** p < 0.0001 vs. vehicle; ((A), right panel) To verify that JT11 acted via CB2R, Jurkat cells were pretreated with CB2R selective antagonist, SR144528 (1 μM), exposed to JT11 for 72 h and then analyzed for cell viability. *** p < 0.0001 vs. vehicle; §§§ p < 0.0001 vs. SR144528 + JT11; ((B), left panel) Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in Jurkat cells. The results represent the mean ± SD of five independent experiments performed in triplicate and represent cell viability as a percentage of untreated control cells. * p < 0.01 vs. vehicle; ((B), right panel) Jurkat cells were pretreated with selective CB2R antagonist (SR144528, 1 μM), exposed to JT11 for 72 h and then analyzed for cell proliferation. * p < 0.01 vs. vehicle; §§ p < 0.001 vs. SR144528 + JT11.

Figure 3

JT11 exhibits a CB2R-dependent pro-apoptotic effect in Jurkat cells. (A) Jurkat cells were incubated with a 0.1, 1, or 2 μM JT11 for 24, 48 and 72 h, and their DNA content was measured by flow cytometry after propidium iodide (PI) staining. The histograms show the distribution of cells in the phases of cell cycle based on their DNA content, and the amplitude of the subG1 peak, i.e., the percentage of hypodiploid peak, indicates the percentage of dead cells; Percentage of cells with hypodiploid DNA content. Data are reported as mean ± SD from three independent experiments. * p < 0.01 vs. vehicle; ** p < 0.001 vs. vehicle; PI staining was performed also in the presence of 1 μM SR144528. Data are reported as mean ± SD from three independent experiments. ** p < 0.001 vs. vehicle; §§ p < 0.001 vs. SR144528 + JT11; (B) Analysis of poly (ADP ribose) polymerase (PARP) cleavage by Western blot. Jurkat cells were treated with 2 μM JT11, or alternatively pretreated with 1 μM SR144528 and then incubated with 2 μM JT11 for 12, 24 or 48 h. The protein extracts were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed using anti-PARP antibody (Ab). The loading control was evaluated using anti-β-actin Ab; Densitometric cleaved PARP/full length PARP ratio. Data are reported as mean ± SD from three independent experiments. * p < 0.01 vs. vehicle; ** p < 0.001 vs. vehicle; §§§ p < 0.0001 vs. SR144528 + JT11.

Figure 4

JT11 induces ERK1/2 phosphorylation through a CB2R-dependent mechanism in Jurkat cells. Jurkat cells were transfected with CB2R siRNA or with Scrambled siRNA, and after they were treated with 2 μM JT11 for 10 min. Evaluation of CB2R expression after 72 h siRNA transfection, where a scrambled siRNA was used as control, by flow cytometric analysis ((A), left panel) and Western blot analysis. Densitometric CB2R/β-tubulin ratio is shown. Data are reported as mean ± SD from three independent experiments. * p < 0.01 vs. scrambled siRNA ((A), right panel). ((B), left panel) Western blot analysis of the activation (phosphorylation) of extracellular signal-regulated kinase (ERK)1/2. The protein extracts were separated by SDS-PAGE and analyzed using anti-phospho-ERK1/2 and anti-ERK1/2 Abs. The loading control was evaluated using anti-β-tubulin Ab. ((B), right panel) Densitometric phosphorylated ERK1/2/total ERK1/2 ratio is shown. Data are reported as mean ± SD from three independent experiments. ** p < 0.001 vs. vehicle; §§ p < 0.001 vs. treated, transfected, cells.

Figure 5

JT11 modulates the pro-inflammatory signal activated by bacterial lipopolysaccharide (LPS) in human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy donors were pretreated for 1 h with JT11 (2 μM) and then stimulated with LPS (100 ng/mL) for 1 h. Alternatively, PBMCs were analyzed in presence of selective CB2R antagonist SR144528 (1 μM). Analysis of the phosphorylated ERK1/2 (A) and p65 subunit of nuclear factor-κB (NF-κB-p65) (B) by Western blot. Protein extracts were separated by SDS-PAGE (10% acrylamide) and analyzed using anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-NF-κB-p65 and anti-NF-κB-p65 Abs. Densitometric phosphor-ERK1/2/total ERK1/2 and phospho-NF-κB-p65/total NF-κB-p65 ratios are shown in the right panels of the figure. Data are reported as mean ± SD from three independent experiments. Statistical analysis indicated: ** p < 0.001 vs. vehicle; §§§ p < 0.0001 vs. JT11 + LPS; °° p < 0.001 vs. SR144528 + JT11 + LPS.

Figure 6

JT11 reduces LPS-induced release of pro-inflammatory cytokines in human PBMCs. PBMCs from healthy donors were pretreated for one hour with 2 μM JT11 and stimulated for 24 h with 100 ng/mL LPS. Alternatively, PBMCs were analyzed in the presence of selective CB2R antagonist SR144528 (1 μM). The culture medium was separated by centrifugation, and the concentration of cytokines interleukin (IL)-1β (A), tumor necrosis factor (TNF)-α (B), IL-6 (C) and IL-8 (D) was measured with an immunoassay using Luminex^® xMAP^® technology. Data are reported as mean ± SD of three independent experiments performed in duplicate. *** p < 0.0001 vs. vehicle; § p < 0.01 vs. JT11 + LPS; §§ p < 0.001 vs. JT11 + LPS; ° p < 0.01 vs. SR144528 + JT11 + LPS.