Using gene array analysis, we have observed that the relative levels of CB1, CB2, GPR18 and TRPV2 as well as of the fatty acid amide hydrolase gene transcripts were not significantly affected by the cannabinoid treatments and their levels did not exceed the 2-fold induction or 50% reduction by either CBD or THC treatment. On the other hand, we show here that LPS markedly down regulates CB2 and GPR55 and that this down regulation is not affected by either CBD or THC pretreatment. This result is in agreement with our previous report showing that LPS markedly down regulates CB2 and GPR55 mRNAs in BV- 2 microglial cells and in microglial primary cultures. Network analysis and signaling pathways A relationship between CBD-mediated oxidative stress response and glutathione depletion was previously reported . More recently, we showed that CBD-specific gene expression profile in BV-2 cells displays changes normally occurring under either nutrient limiting conditions or proteasome inhibition, and that are attributed to activation of GCN2/eIF2a/ p8/ATF4/CHOP-Trib3 pathway leading to autophagy as well as to apoptotic cell death. The Trib3 gene product seem to be of high importance to the CBD effect due to its ability to serve as a master regulator of an array of pathways including AP-1, ER stress, Akt/PKB and NF-kB . Trib3 expression is significantly upregulated by CBD as well as by THC and as observed here, remains upregulated after LPS treatment . According to these gene array studies and the qPCR results, LPS by itself does not significantly affect the expression of Trib3 mRNA. IPA interactome analysis of the micro-arrays data reveals an interaction between the CBD-upregulated Trib3 and the NF-kB transcription factor pathway . This interaction seems to be responsible for the attenuation by CBD of the transcription of many pro-inflammatory genes. There are several indications suggesting interaction between these two pathways. First, a direct interaction between p65/RelA and Trib3 protein which induces inhibition of PKA dependent p65 phosphorylation, was described . Second, Trib3 protein can negatively regulate the serinethreonine kinase Akt/PKB, a downstream effector of PI3K that has been implicated in the potentiation of NF-kB-induced transcription of pro-inflammatory mediators.
This negative regulation of Akt activity by the highly induced Trib3 gene product could point to the mechanism for the CBD-mediated regulation of LPS-stimulated gene expression. Indeed,cannabis grow set up the effect of CBD treatment on a number of LPS-stimulated genes as reported here is reminiscent of the effects described for the PI3K inhibitor and for the NFkB inhibitor in the murine macrophage cell line RAW264.7 activated with LPS. Both PI3K and NFkB signaling pathways exert important roles in gene expression in response to LPS, but they are not overlapping. Specifically, treatment with CBD repressed a number of typical pro-inflammatory genes stimulated by LPS, which are known to be NFkB dependent and of other genes including Csf3, Il-1b, Il-1a and Cox2/Ptgs2, which are under the control of both PI3K and NFkB pathways. Finally, Trib3 was documented to interfere with the inflammatory MAPK signaling via direct interaction with MEK-1 and MKK7 leading to attenuation of AP-1 mediated transcriptional activity in cancer HeLa cells. AP-1 is a transcription factor involved in the regulation of inflammation-mediated cellular functions and has been shown to be inhibited by Nrf2-activating agents. Indeed, our IPA network analysis indicates that the observed decrease in mRNA levels for a number of genes is probably related to a reduction in AP-1 dependent transcription. Additionally, according to these IPA results, this repression is reinforced by combined treatments of CBD and LPS as observed by the induction of FosL1 gene product, another negative regulator of AP-1 . Trib3 has been shown to down regulate PPARc transcription and serve as a potent negative regulator of adipocyte differentiation and PPARc is a molecular target for CBD that could be involved in mediating transcriptional effects in BV-2 microglial cells. Indeed, CBD has been shown to bind to PPARc in vitro as well as to activate its transcriptional activity in 3T3L1 fibroblast and in HEK293 transfected cells. In addition, Necela et al., described a regulatory feedback loop in which PPARc represses NF-kB-mediated inflammatory signaling in unstimulated macrophages. Moreover, they show that upon activation of TLR4 in LPS-stimulated macrophages, NF-kB drives down PPARc expression.
These results are in agreement with our results showing that LPS highly down regulates the expression of Pparg1 and Pparg2 in BV-2 cells. The profiles of CBD-induced gene expression with either resting or LPS-activated BV-2 cells, show that CBD stimulates the transcription of several anabolic genes encoding amino acid bio-synthetic enzymes, amino acid transporters and aminoacyltRNA synthetases known to be activated by ATF4, a basic leucine zipper transcription factor, that is increased when cultured cells are deprived of amino acids or subjected to endoplasmic reticulum stress . The divergent types of stress converge on a single event—phosphorylation of the translation initiation factor eIF2a, resulting in a general translational pause followed by selective increase in ATF4 mRNA translation and subsequent stimulation of expression of ATF4 target genes. Many of the CBD-affected transcripts are indeed classified as Nrf2-mediated oxidative stress response genes, including enzymes involved in the biosynthesis of glutathione. Thus, the observed CBD-mediated induction of ATF4-dependent anabolic genes may serve to replenish the amino acids reduced during the elevated turnover of GSH . The mechanism underlying CBD action presumably engages generation of ROS which in turn depletes intracellular GSH. Perturbations in redox tone and GSH levels activate the ‘‘phase 2 response’’, a mechanism used by cells to mitigate oxidative stress . As we have previously shown, many of the ‘‘phase 2’’ gene products are significantly upregulated by CBD. Our present results show that CBD, and less so THC, have immuno suppressive and protective activities that are reminiscent of other clinically applied drugs such as glucocorticoids , rexinoids and synthetic triterpenoids. GCs are immunomodulatory agents known to act as suppressive and protective mediators against inflammation. GCs are known to clear antigens by stimulating cell trafficking as well as scavenger systems and matrix metallo proteinases while they stop cellular immune responses by inhibiting antigen presentation and T cell activation.
Synthetic oleanane triterpenoids were shown to be highly effective in many in vivo models in the prevention and treatment of cancer and other diseases with an inflammatory component. Molecular targets of SO include KEAP1 , PPARc, IkB kinase, TGF-b signaling and STAT signaling. SO are among the most potent known inducers of the phase 2 response both in vivo and in vitro and affect the expression of several key cell cycle proteins . In some cancer cells, SO signal through PPARc to inhibit proliferation. The rexinoids bind almost exclusively to the RXRs and are involved in regulation of development, cell proliferation, differentiation and apoptosis. Because RXRs heterodimerize with other receptors , rexinoids modulate the actions of many steroid-like molecules that control metabolism and cellular energetics. In view of these results, triterpenoids and rexinoids are defined as multifunctional drugs. Their targets are either regulatory proteins that control the activity of transcription factors or transcription factors themselves . These complex modulatory activities exerted by GCs,grow rack systems rexinoids and SO display a panorama of effects that closely resembles the complex actions of CBD.Humans support the growth and maintenance of diverse sets of microbes in niches in contact with the environment including skin, lungs, mouth and gut. Studies of these microbes in the gut and oral cavity have uncovered key interactions between bacteria and human hosts in a wide variety of normal and pathological states. Many of these interactions are inferred from correlations between the composition of the microbial populations and changes in health status. For example, in gingivitis, an increase in Gram negative and anaerobic bacteria causes inflammation in the mouth.Our understanding of the basis for changes in microbial composition, and of how these changes influence human phenotypes, is still a work in progress. Clearly environmental factors and host genetic factors have important influences, perhaps best demonstrated to date by studies in the gut.By this approach, informed hypotheses about human genes that may conceivably influence a particular microbiological phenotype are tested with family or population-based studies to identify human variants that are statistically consistent with the hypothesis.
Examples include MHC genes,SLC11A1, the MEFV gene,FUT2 gene, and loci linked to susceptibility to infectious disease. While often successful, the candidate gene approach is limited by the ability to formulate hypotheses given current knowledge. They are neither comprehensive nor sufficient to identify the entire range of human genes involved in population changes associated with complex phenotypes or with maintenance of the composition of the “normal” micro-biome. In addition the significant inter-individual variation in micro-biome composition often masks specific effects of human genes if insufficient numbers of individuals are studied. Moreover, the micro-biome of a niche includes complex mixtures of organisms and is in part defined by interactions among its members making the identification of a “microbial phenotype” complicated. The oral micro-biome is one of the most diverse microbial niches in the human body, including over 600 different microorganisms . It is in continual contact with the environment, and has been shown to be susceptible to many environmental effects. These environmental factors include tobacco use , romantic partners, and cohabitation. The microbes reside in sub-niches along the oral cavity including on the tongue, cheek, and teeth. The salivary micro-biome has been shown to be representative of many the oral micro-biome niches, which is thought to be due to the fact that microorganisms from the oral cavity surfaces shed into the saliva. Previous salivary micro-biome studies have identified specific micro-biota that are present in almost all individuals, referred to as the core micro-biome. Saliva is also accessible, making it ideal for surveys of populations for micro-biome studies. In this paper, we describe an unbiased approach to studying the effects of human genes on the oral micro-biome with a two-step strategy. The first step utilizes twin information to establish heritable phenotypes related to the micro-biome; and the second identifies DNA sequence variation associated with the identified highly heritable traits. From 16S rRNA sequence information, a large number of potential phenotypes can be explored with the twin studies to allow identification of the most heritable and therefore the phenotypes most likely to be mapped in the association study. A key strength of this approach lies in the independence of the data underlying the two steps reducing multiple testing and type 1 effects on the power to carry out the test for association. The ability to refine a phenotype prior to carrying out an association study can lead to greater likelihood of detecting specific SNPs that influence it. We show, with the largest oral micro-biome twin study to date, that multiple phenotypes of the salivary micro-biome are heritable. Using these phenotypes, we identify promising host gene candidates in a genome wide association study of an separate sample that may play a role in establishing the oral micro-biome.Twin samples were obtained from the Colorado Twin Registry . The twin sample included 366 monozygotic pairs , 263 same sex, and 123 opposite sex dizygotic pairs . Unrelated individuals were ascertained from community and clinical samples participating in the Colorado Center for Antisocial Drug Dependence and isolation of DNA from saliva and characterization of their genotypes was as previously described.Pooled DNA from triplicate PCR with the 16S V4 hyper variable primers 515F/806R was done according to the Earth Microbiome Project 16S rRNA amplicon Protocol, with unique barcode indices for multiplex sequencing on the forward primer. Concentration of pooled products was determined by picogreen. 240 ng from each sample was pooled for multiplex paired-end sequence determination on the Illumina MiSeq platform.Samples from 1504 twins of whom 111 within-twin longitudinal samples with at least 3500 reads and DNA samples from 1481 unrelated individuals with at least 3000 reads produced 2664 and 2679 OTUs respectively. All samples were rarefied to 2500 sequences to retain as many samples as possible to improve power with little effect to results.To avoid analyses of OTUs that were the result of sequencing or PCR error, OTUs that were not present in at least 2 subjects and observed at least 10 times were removed, resulting in 895 OTUs in the twins and 931 OTUs in the unrelated individuals. One of the unrelated individuals was later removed during analysis due to cryptic relatedness leaving 1480 people in the unrelated sample.β-diversity was analyzed via Bray Curtis and UniFrac using QIIME and R.