Interestingly, the mechanisms that give rise to either the protective or the damaging microglial phenotypes are not fully elucidated. Enhancing the microglial-mediated innate immunity in the CNS and/or preventing the harmful effects associated with their chronic activation may offer new therapeutic approaches for the treatment of brain injury and neurodegenerative diseases. One of the most potent stimuli for microglia activation is the bacterial endotoxin LPS, that mimics infection by Gram-negative bacteria. LPS activates intracellular signaling pathways in a complex way leading to secretion of cytokines and to overexpression of several markers of the immune response. Previous studies reported that LPS stimulation induces gene expression of TNFa, IL-1b, IL-6, iNOS and COX-2 as well as the production of NO and PGE2 in primary and BV-2 microglial cell cultures. We reported that CBD reduces the activity of the NF-kB pathway and upregulates the activation of STAT3 transcription factor in LPSstimulated BV-2 cells, and that both CBD and THC decrease the activation of the LPS-induced STAT1 transcription factor, a key player in IFNb-dependent pro-inflammatory processes. Moreover, performing comparative microarray analysis of genome-wide mRNA levels in the BV-2 cells, we reported that CBD, but less so THC, shows a specific gene expression profile associated with oxidative stress and glutathione depletion involving the GCN2/eIF2a/p8/ATF4/Chop-Trib3 pathway. Furthermore, the CBD-stimulated genes were shown to be controlled by nuclear factors known to be involved in regulation of stress response and inflammation, mainly via the -Nrf2/ Atf4 system and the Nrf2/Hmox axis.
We reported that CBD, but less so THC, affects the expression of genes involved in zinc homeostasis,procona valencia buckets suggesting that the regulation of zinc levels could have an important role through which CBD may exert its antioxidant and anti-inflammatory effects. Although the inhibitory functions of cannabinoids on LPSactivated NF-kB and IFN-b/STAT proinflammatory pathways and on the secretion of selected cytokines in BV-2 microglial cells has been studied, a genome-wide search for cannabinoid molecular targets in LPS-activated BV-2 cells has not yet been performed. We have, therefore, performed gene array studies and comparative gene profiling analysis of BV-2 cells treated with LPS, CBD+LPS or THC+LPS. This approach allowed us to analyze the changes induced by CBD and THC on gene expression patterns in LPS-treated BV-2 cells and to explore the genomewide interaction network affected by these treatments. In this regard, a structured network knowledge-based approach to analyze genome-wide transcriptional responses in the context of known functional interactions among proteins, small molecules and phenotypes has been established. We applied this analysis to show the interactions and signaling networks elicited by the cannabinoids in LPS-stimulated BV-2 cells. Our results show that CBD is a potent modulator of microglial activation. Identification of the CBD- and THC-regulated genes and related networks provides a molecular basis for understanding the effects of these cannabinoids on LPS-activated microglia.BV-2 microglial cells were pretreated for 2 h with either THC or CBD followed by the addition of LPS to the incubation medium for another 4 h. The control treatment with cannabinoids alone lasted for 6 h and with LPS alone for 4 h.
The choice of these time points for transcriptional profiling was guided by our previous studies as well as by other reports which investigated the general temporal pattern of microglial activation by LPS. Moreover, according to our previous results, neither THC nor CBD treatments significantly affected the viability of the BV-2 microglial cells during this 6 h period. The RNA prepared from these samples was analyzed for changes in transcriptional levels using the MouseRef- 8 v1.1 Expression BeadChip Illumina Arrays. Each of these arrays has .24,000 mouse targets based on the NCBI mouse Reference Sequence Database, including 16,287 constitutive exons/islands based on the splice variants in the mouse transcriptome and NCBI LocusLink databases. The results of the analyses of the arrays showed that 32% of the transcripts were consistently ‘‘present’’ in the BV-2 RNA samples across all arrays. Moreover, clustering based on inter-array Pearson correlation coefficient indicated no batch effects. Microarray analysis based on a threshold of p# 0.005, revealed that a total of 22% of the Illumina gene set was differentially regulated across treatments. Of these, 1319 gene probe sets were up regulated and 1829 transcripts were down regulated by the LPS treatment ; and from these numbers of genes, 400 transcripts were found to be up regulated and 145 down regulated by LPS by 2-fold or more. When the fold change was set on $3-fold , we found that 226 gene products were up regulated and 33 were down regulated by LPS . The vast majority of the LPS-affected transcripts represented genes that were exclusively responsive to LPS stimulation, and not to treatment with CBD alone or THC alone . the CBD+LPS treatment was analyzed, 1381 gene probe sets were up regulated and 1666 transcripts were down regulated by CBD+LPS . From these numbers of genes, 379 gene products were found to be up regulated and 489 down regulated exclusively as a response to the combination of CBD+LPS and not affected by LPS or CBD alone . When the fold change was set on $2-fold, 502 transcripts were up regulated and 297 gene probe sets were down regulated by the CBD+LPS treatment .
When LPS was applied in the presence of THC, 1216 gene probe sets were up regulated and 1638 transcripts were down regulated; and from these, 424 transcripts were found to be up regulated and 149 down regulated by 2-fold or more . From the 1216 up regulated genes, 157 transcripts were exclusively responsive to THC+LPS treatment alone and from the 1638 down regulated transcripts, 285 gene probes were affected only when THC and LPS were together . When the fold change was set on $3-fold, we found that 230 gene products were up regulated by CBD+LPS and 236 transcripts by THC+LPS whereas 51 gene products were down regulated by CBD+LPS and 31 transcripts by THC+LPS . Our results also reveal that from the 5338 transcripts found to be differentially regulated by the various treatments , 680 gene probe sets were found to be up regulated by CBD alone and 58 gene products by THC alone. CBD had also a much larger effect compared to THC on the number of down regulated genes, 524 gene products were down regulated by CBD and 36 by THC . The groups of LPS-up regulated and down regulated genes that showed a change in expression of $2-fold in either direction were subjected to gene ontology analysis allowing functional annotation using the DAVID Bio-informatics Resources and the KEGG pathways . All major Biological Processes, Cellular Components and Molecular Functions within GO for the LPS-up regulated transcripts, were for the most part, genes associated with the immune and defense responses as well as apoptosis and cell death. KEGG database analysis included genes related to Toll-like receptor pathways and antigen processing as well as to the MAPK pathways. IPA global functional analysis of the LPS-up regulated and down regulated genes confirmed the DAVID representation of genes within IPA specific GO categories . IPA global functional analysis of the LPS-up regulated transcripts show gene products mainly implicated in cell-to-cell signaling, cellular movements, growth and proliferation, as well as cell death,procona buckets immune response and signaling, including genes involved in the NF-kB pathway and in the activation of the liver X receptor/retinoic X receptor . The largest subsets of down regulated transcripts included genes known to be involved in the regulation of macromolecules and nucleotide metabolism, differentiation and development as well as regulation of gene expression and transcription . A list of 183 LPS-up regulated genes are presented in Table S2. This list includes LPS-up regulated genes that were significantly affected by CBD+LPS or by THC+LPS. They appear in the Table according to their categories and specific annotations based on their GO and IPA. The highly up regulated transcripts were related to inflammation, host defense and adaptive response. Inflammatory cytokines and chemokines together with their cognate receptors comprised a large group of more than 20 genes. Interferon related transcripts formed a significant population comprised of 12 genes. Based on GO and relevant literature references, genes were also classified into other functional categories including metabolic enzymes, membrane transport and secretion, kinases, phosphatases as well as transmembrane Gprotein coupled receptors. The remainder of the LPS-up regulated genes was grouped according to their involvement in several other cellular processes such as apoptosis, proliferation and cell cycle progression, transcriptional and translational control as well as stress response. Additionally, we have identified a cluster of genes associated with regulation of extracellular matrix that are known to be involved in molecular recognition between cells, cell adhesion and migration. Genes implicated in cytoskeleton remodeling and control of cell motility and morphogenesis form a separate category.This study addresses the effects of the two major cannabinoids present in cannabis, CBD and THC, on mRNA expression in LPS-stimulated BV-2 microglial cells.
Our results show that pretreatments with CBD or THC differentially affect LPSregulated gene transcription. This result is in line with the more profound effects of CBD on mRNA regulation in surveillant microglial cells, as previously described by our group. Here we show that CBD, more than THC, suppresses the LPSinduction of many proinflammatory genes in selected functional categories including cytokines and chemokines . Moreover, CBD also repressed the basal expression of Ccl2, Ccl7 and Ccl9. Interestingly, Ccl2, Ccl7 and Ccl12 bind to the same receptor , thus, showing binding promiscuity while they activate different signal transduction pathways in different cell populations. From these chemokines, Ccl2 plays a critical role in multiple sclerosis and in its murine model, experimental autoimmune encephalomyelitis . Several reports show that increased expression of Ccl2 in immune cells is closely associated with the clinical activity of EAE . Thus, the CBD down regulation of Ccl2 mRNA is in line with the finding that CBD is ameliorating the EAE disease symptoms. Another hallmark of inflammation is the increased expression of proinflammatory mediators like matrix metalloproteinases . Here, we show that CBD down regulates the expression of the LPS-up regulated matrix metalloproteinase13 . MMP13 level and activity are enhanced in correlation with the degenerative changes in osteoarthritis cartilage and this molecule co-localizes with its specific type II collagen cleavage products. In agreement with this result, Malfait et al., reported that in the murine collagen-induced arthritis CBD treatment blocks the progression of the disease. CD69 antigen is another mediator found to be involved in the CIA model of rheumatoid arthritis. This antigen is highly expressed in the leukocyte infiltrates of various chronic inflammatory diseases. In this regard, CIA disease severity is increased by antibody-induced blockade of the transforming growth factor b in wild type but not in CD69-/- mice, suggesting that CD69 is a negative modulator of autoimmune response and inflammatory reaction. Our current results show that CBD down regulates the mRNA expressions of the LPS-up regulated Cd69 antigen and of Mmp13 suggesting a possible mechanism for the therapeutic activity of CBD in the murine CIA model. The expression of several genes in the inflammatory cytokine functional category was found to be enhanced by CBD . These include the LPS-up regulated expression of Gdf15/MIC-1 and Il-15. From these genes, Gdf15 mRNA expression is up regulated in surveillant/ resting BV-2 cells by CBD but synergistically up regulated by CBD+LPS . Gdf15/ MIC-1 is a member of the TGF-b super family that plays key roles in the regulation of cellular responses to stress signals, inflammation and tissue repair. Gdf15 mRNA expression was found to be up regulated in activated macrophages by secreted proinflammatory cytokines suggesting that Gdf15 may act through an autocrine loop as an inhibitory factor in the late phases of inflammation by suppressing inflammation through the inhibition of macrophage activation. Other genes highly up regulated by CBD are Lcn2 and Aqp9. Lcn2 has been implicated in many cellular processes, such as cell death/survival, cell migration/invasion, cell differentiation, inflammation and iron sequestration, including a role in the acute phase response . With regard to Aqp9, we have previously shown that CBD up regulates Aqp9 mRNA in BV-2 cells . Moreover, CBD synergistically increases the LPS-up regulated Aqp9 . Aqp9 participates in the transport of small solutes and takes part in osmotic swelling induced by apoptotic stimuli.