Cannabinoids Delta(9)-tetrahydrocannabinol and cannabidiol differentially inhibit the lipopolysaccharide-activated NF-kappaB and interferon-beta/STAT proinflammatory pathways in BV-2 microglial cells

Abstract

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, Delta(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD), decrease the production and release of proinflammatory cytokines, including interleukin-1beta, interleukin-6, and interferon (IFN)beta, 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-kappaB 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 IFNbeta-dependent proinflammatory processes. In summary, our observations show that CBD and THC vary in their effects on the anti-inflammatory pathways, including the NF-kappaB and IFNbeta-dependent pathways.

FIGURE 1.

THC and CBD inhibit the LPS-induced release of IL-1β and IL-6 from BV-2 cells. Cells were preincubated for 2 h with the indicated concentrations of THC or CBD and then activated for 4 h with 100 ng/ml LPS. Cell-free media were then collected, and the release of IL-1β (A) and IL-6 (B) was measured using ELISA. The percentage compared with LPS applied alone (marked as 100%) is expressed as the mean ± S.E. of three independent experiments. One-way ANOVA was used as follows: IL-1β, F(7,16) = 10.21, p < 0.001 and IL-6 F(7,16) = 81.8, p < 0.0001. Bonferroni post hoc test: *, p < 0.05; **, p < 0.01; ***, p < 0.001 show significant differences from LPS-treated cells.

FIGURE 2.

THC and CBD decrease the LPS-induced release of IFNβ from BV-2 cells. Cells were pretreated for 2 h with THC or CBD (both at 10 μm) and then activated for 4 h with 100 ng/ml LPS. Cell-free media were then collected and subjected to ELISA for IFNβ. Each bar represents the mean (in pg/ml) ± S.E. from three independent experiments. One-way ANOVA was used as follows: F(3,8) = 35.4, p < 0.001; Bonferroni post hoc test: *, p < 0.05; ***, p < 0.001 versus LPS-treated BV-2 cells.

FIGURE 3.

CBD and THC decrease the mRNA levels of LPS-up-regulated IL-1β and IFNβ. Cells were treated for 2 h with 10 μm THC or CBD. LPS (100 ng/ml) was then added, and 4 h later the cells were harvested, and RNA was extracted for qPCR analysis. The bar graphs present the percent of mRNA expression (average ± S.E. from three independent experiments) versus LPS-only treated samples (taken as 100%). One-way ANOVA was used as follows: IL-1β F(5,12) = 57.2, p < 0.001; IFNβ F(5,10) = 25.16, p < 0.001; Dunnett's post hoc tests: *, p < 0.05, ***, p < 0.001 versus LPS.

FIGURE 4.

CB1 and CB2 receptor antagonists as well as abn-CBD do not affect the THC- and CBD-induced inhibition of IL-1β release from LPS-stimulated BV-2 cells. Cells were pretreated for 30 min with SR141716 or SR144528 (both at 0.5 μm) (A) or abn-CBD (1 μm) (B), followed by the addition of 10 μm THC or CBD and 2 h later of LPS (100 ng/ml). Cell-free media were collected 4 h later and assayed for released IL-1β by ELISA. The data are expressed as percentage of released IL-1β ±S.E. from three to four independent experiments. The amount released with LPS alone is represented as 100%. A, one-way ANOVA was used as follows: F(6,14) = 6.58, p < 0.01. Bonferroni post hoc analysis showed that neither SR141716 nor SR144528 affected THC or CBD inhibition of IL-1β release. B, one-way ANOVA was used as follows: F(3,8) = 14.34, p < 0.01. Bonferroni post hoc analysis showed that pretreatment with abn-CBD did not change the effect of CBD on LPS stimulated IL-1β release. ns, not significant.

FIGURE 5.

CBD, but less so THC, partially reverses the LPS-induced degradation of IRAK-1 and of IκB in LPS-stimulated BV-2 cells. Cells were pretreated for 2 h with THC or CBD at the indicated concentrations followed by 15 min of incubation with LPS (100 ng/ml) and lysed in RIPA buffer, and 20 μg of protein aliquots were subjected to Western blot analysis for IRAK-1 (A) and IκB (B). β-Actin served as a loading control. Bars show the average results of three repetitions with 100% representing the amounts of proteins in control cells. One-way ANOVA was used as follows for IRAK-1 expression: THC-treated cells F(6,14) = 16.79, p < 0.0001, and for CBD-treated cells F(6,14) = 6.00, p < 0.01. One-way ANOVA was used as follows for IκB protein expression: THC-treated samples F(6,14) = 36.59, p < 0.0001, and for CBD-treated samples F(6,14) = 6.39, p = 0.01, followed by Bonferroni post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control. Cannabinoid vehicle (0.1% ethanol; Et) did not affect the LPS-induced IRAK-1 or IκB degradation.

FIGURE 6.

CBD, but not THC, reduces LPS-induced NF-κB p65 subunit phosphorylation. Cells were pretreated for 2 h with CBD or THC at the indicated concentrations followed by LPS (100 ng/ml) for 15 min. Cell homogenates (20 μg of protein aliquots) were subjected to Western blot analysis using antibodies against the phospho-form of NF-κB p65 (Ser-536) subunit. The amount of total p65 served as a loading control. Et, ethanol.

FIGURE 7.

THC and CBD decrease LPS-induced STAT1 phosphorylation. Cells were pretreated for 2 h with various concentrations of either THC or CBD prior to the addition of LPS for an additional 2 h. A shows representative Western blots for pSTAT1 (Tyr-701) and the general form of STAT1; B, bar graph shows the level of phosphorylation of STAT1 from three independent experiments. One-way ANOVA was used s follows: F(8,18) = 8.1, p < 0.001, followed by Bonferroni post hoc test. *, p < 0.05, ***, p < 0.001 versus ethanol (Et) + LPS.

FIGURE 8.

CBD increases LPS-induced STAT3 phosphorylation. A, cells were pretreated for 2 h with various concentrations of either THC or CBD prior to the addition of LPS for additional 2 h. Representative Western blots for pSTAT3 (Tyr-705) and for the general form of STAT3 are shown. B and C show the phosphorylation of STAT3 in bar graphs presenting the results from three independent experiments on cells stimulated with LPS for 2 h (B) or 4 h (C). B, one-way ANOVA was used as follows: F(3,11) = 8.32, p < 0.01, followed by Dunnett's post hoc test. *, p < 0.05 versus LPS. C, ANOVA was used as follows: F(3,8) = 27.09, p < 0.001; *, p < 0.05; ***, p < 0.001 versus LPS alone. Et, ethanol.

FIGURE 9.

LPS up-regulated SOCS3, CISH, and CCL2 mRNAs are differently modulated by CBD and THC. Cells were treated for 2 h with 10 μm THC or CBD. LPS (100 ng/ml) was then added, and 4 h later the cells were harvested, and RNA was extracted for qPCR analysis. The bar graphs present the percent of mRNA expression (average ± S.E. from three to four independent experiments) versus LPS-only treated samples (taken as 100%). One-way ANOVA was used as follows: for SOCS3 F(5,12) = 100.5, p < 0.001; for CISH F(5,12) = 20.7, p < 0.001; for CCL2 F(5,12) = 32.81, p < 0.0001. Dunnett's post hoc test: ***, p < 0.001 versus LPS.

SCHEME 1.

Schematic figure showing possible sites for the anti-inflammatory activities of THC and CBD in LPS-activated BV-2 microglia. Pointed arrows indicate activation, and blunt arrows indicate inhibition.