The Cannabinoid Receptor Agonist, WIN-55212-2, Suppresses the Activation of Proinflammatory Genes Induced by Interleukin 1 Beta in Human Astrocytes

Abstract

Background: Alterations of astrocyte function play a crucial role in neuroinflammatory diseases due to either the loss of their neuroprotective role or the gain of their toxic inflammatory properties. Accumulating evidence highlights that cannabinoids and cannabinoid receptor agonists, such as WIN55,212-2 (WIN), reduce inflammation in cellular and animal models. Thus, the endocannabinoid system has become an attractive target to attenuate chronic inflammation in neurodegenerative diseases. However, the mechanism of action of WIN in astrocytes remains poorly understood. Objective: We studied the immunosuppressive property of WIN by examining gene expression patterns that were modulated by WIN in reactive astrocytes. Materials and Methods: Transcriptomic analysis by RNA-seq was carried out using primary human astrocyte cultures stimulated by the proinflammatory cytokine interleukin 1 beta (IL1β) in the presence or absence of WIN. Real-time quantitative polymerase chain reaction analysis was conducted on selected transcripts to characterize the dose-response effects of WIN, and to test the effect of selective antagonists of cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptors (PPAR). Results: Transcriptomic analysis showed that the IL1β-induced inflammatory response is robustly inhibited by WIN pretreatment. WIN treatment alone also induced substantial gene expression changes. Pathway analysis revealed that the anti-inflammatory properties of WIN were linked to the regulation of kinase pathways and gene targets of neuroprotective transcription factors, including PPAR and SMAD (mothers against decapentaplegic homolog). The inhibitory effect of WIN was dose-dependent, but it was not affected by selective antagonists of CB1 or PPAR. Conclusions: This study suggests that targeting the endocannabinoid system may be a promising strategy to disrupt inflammatory pathways in reactive astrocytes. The anti-inflammatory activity of WIN is independent of CB1, suggesting that alternative receptors mediate the effects of WIN. These results provide mechanistic insights into the anti-inflammatory activity of WIN and highlight that astrocytes are a potential therapeutic target to ameliorate neuroinflammation in the brain.

Keywords: immunosuppression; inflammation; neurobiology; synthetic cannabinoids.

FIG. 1.

Exploratory data analysis of transcriptomic data. (A) Hierarchical clustering of top 2000 genes showing that the variation between replicates was minimal and that substantial changes were induced by IL1β and WIN treatments. (B) PCA plot showing that the majority of variance is explained by PC1 (68%) and PC2 (21%). IL, interleukin; PCA, principal component analysis. Color images are available online.

FIG. 2.

IL1β and WIN treatments induced substantial changes in gene expression. Volcano plots visualizing the up- and downregulated genes by plotting the statistical significance (−log 10 FDR) versus the log2-fold change for pairwise comparisons between IL1β versus vehicle (A), WIN versus vehicle (B) and WIN+IL1β versus vehicle (C). (D–F) Enrichment KEGG pathways analysis of DEGs is shown for each pairwise comparison. The relationship among enriched KEGG pathways is visualized as a tree. DEGs, differentially expressed genes; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes. Color images are available online.

FIG. 3.

WIN diminished the IL1β-induced inflammatory response. (A) k-Means clustering visualizing 4 groups using the top 2000 differentially expressed genes. (B) The top GO biological process terms from each cluster is shown with associated p values. (C) For each cluster, the normalized expression values for three representative genes are plotted and error bars represent standard deviation values. GO, Gene Ontology. Color images are available online.

FIG. 4.

TFs associated with inflammatory responses in astrocytes. Pathway analysis of DEGS was performed using the TRED database. Enriched TF are ranked by p-values (FDR <0.05 cutoff). Heatmaps are shown for pairwise comparisons between IL1β versus vehicle (A), WIN versus vehicle (B) and WIN+IL1β versus vehicle (C). TF, transcription factor; TRED, Transcriptional Regulatory Element Database. Color images are available online.

FIG. 5.

WIN reduced IL1β-induced inflammatory gene expression in astrocytes in a dose-dependent manner. Fold-change of IL6 (A) or C3 (B) mRNA levels normalized to ACTB mRNA levels in total RNA isolated from human astrocytes. One-way ANOVA was conducted for the effect of treatment on IL6 [F (9, 10)=24.83, p<0.0001] and C3 [F (9, 10)=107.3, p<0.0001]. A post hoc Tukey's test was conducted; corrected p-values are shown (***p<0.001; ****p<0.0001 vs. vehicle; ^{^^}p<0.01, ^{^^^}p<0.001, ^{^^^^}p<0.0001 vs. IL1β-treated cells). ANOVA, analysis of variance.

FIG. 6.

WIN treatment before, at the same time, and after IL1β treatment robustly blocked IL1β-induced inflammatory gene expression. (A–C) Fold-change of IL6 mRNA transcript levels normalized to ACTB mRNA levels in total RNA isolated from human astrocytes. One-way ANOVA was conducted for WIN 24 h before IL1β [F (3, 4)=377.6, p<0.0001], WIN at the same time as IL1β [F (3, 4)=91.26, p=0.0004], WIN after IL1β [F (3, 4)=90.3, p=0.0004]. (D–F) Fold-change of C3 mRNA transcript levels normalized to ACTB mRNA levels in total RNA isolated from human astrocytes. One-way ANOVA was conducted for WIN 24 h before IL1β [F (3, 4)=233.8, p<0.0001], WIN at the same time as IL1β [F (3, 4)=148.4, p=0.0001], WIN after IL1β [F (3, 4)=270.0, p<0.0001]. A post hoc Tukey's test was conducted; corrected p-values are shown (**p<0.01; ***p<0.001; ****p<0.0001 vs. vehicle, ^{^^}p<0.01, ^{^^^}p<0.001, ^{^^^^}p<0.0001 vs. IL1β-treated cells).

FIG. 7.

PPARα, PPARγ, and CB1 antagonists did not block the inhibitory effect of WIN on the IL1β-induced inflammatory gene transcription. (A) Fold-change of IL6 mRNA transcript levels normalized to ACTB mRNA levels in total RNA isolated from human astrocytes. One-way ANOVA was conducted [F (12, 13)=55.96, p<0.0001]. (B) Fold-change of C3 mRNA transcript levels normalized to ACTB mRNA levels in total RNA isolated from human astrocytes. One-way ANOVA was conducted [F (12, 13)=90.84, p<0.0001]. A post hoc Tukey's test was conducted; corrected p-values are shown (***p<0.001, ****p<0.0001 vs. vehicle; n.s. vs. WIN+IL1β). CB1, cannabinoid receptor 1; PPAR, peroxisome proliferator-activated receptors.