Pathways and gene networks mediating the regulatory effects of cannabidiol, a nonpsychoactive cannabinoid, in autoimmune T cells.

BackgroundOur previous studies showed that the non-psychoactive cannabinoid, cannabidiol (CBD), ameliorates the clinical symptoms in mouse myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis model of multiple sclerosis (MS) as well as decreases the memory MOG35-55-specific T cell (TMOG) proliferation and cytokine secretion including IL-17, a key autoimmune factor. The mechanisms of these activities are currently poorly understood.MethodsHerein, using microarray-based gene expression profiling, we describe gene networks and intracellular pathways involved in CBD-induced suppression of these activated memory TMOG cells. Encephalitogenic TMOG cells were stimulated with MOG35-55 in the presence of spleen-derived antigen presenting cells (APC) with or without CBD. mRNA of purified TMOG was then subjected to Illumina microarray analysis followed by ingenuity pathway analysis (IPA), weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) elucidation of gene interactions. Results were validated using qPCR and ELISA assays.ResultsGene profiling showed that the CBD treatment suppresses the transcription of a large number of proinflammatory genes in activated TMOG. These include cytokines (Xcl1, Il3, Il12a, Il1b), cytokine receptors (Cxcr1, Ifngr1), transcription factors (Ier3, Atf3, Nr4a3, Crem), and TNF superfamily signaling molecules (Tnfsf11, Tnfsf14, Tnfrsf9, Tnfrsf18). "IL-17 differentiation" and "IL-6 and IL-10-signaling" were identified among the top processes affected by CBD. CBD increases a number of IFN-dependent transcripts (Rgs16, Mx2, Rsad2, Irf4, Ifit2, Ephx1, Ets2) known to execute anti-proliferative activities in T cells. Interestingly, certain MOG35-55 up-regulated transcripts were maintained at high levels in the presence of CBD, including transcription factors (Egr2, Egr1, Tbx21), cytokines (Csf2, Tnf, Ifng), and chemokines (Ccl3, Ccl4, Cxcl10) suggesting that CBD may promote exhaustion of memory TMOG cells. In addition, CBD enhanced the transcription of T cell co-inhibitory molecules (Btla, Lag3, Trat1, and CD69) known to interfere with T/APC interactions. Furthermore, CBD enhanced the transcription of oxidative stress modulators with potent anti-inflammatory activity that are controlled by Nfe2l2/Nrf2 (Mt1, Mt2a, Slc30a1, Hmox1).ConclusionsMicroarray-based gene expression profiling demonstrated that CBD exerts its immunoregulatory effects in activated memory TMOG cells via (a) suppressing proinflammatory Th17-related transcription, (b) by promoting T cell exhaustion/tolerance, (c) enhancing IFN-dependent anti-proliferative program, (d) hampering antigen presentation, and (d) inducing antioxidant milieu resolving inflammation. These findings put forward mechanism by which CBD exerts its anti-inflammatory effects as well as explain the beneficial role of CBD in pathological memory T cells and in autoimmune diseases

miRNA expression profiles and molecular networks in resting and LPS-activated BV-2 microglia-Effect of cannabinoids.

Mammalian microRNAs (miRNAs) play a critical role in modulating the response of immune cells to stimuli. Cannabinoids are known to exert beneficial actions such as neuroprotection and immunosuppressive activities. However, the underlying mechanisms which contribute to these effects are not fully understood. We previously reported that the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signaling pathways. Using lipopolysaccharide (LPS) to stimulate BV-2 microglial cells, we examined the role of cannabinoids on the expression of miRNAs. Expression was analyzed by performing deep sequencing, followed by Ingenuity Pathway Analysis to describe networks and intracellular pathways. miRNA sequencing analysis revealed that 31 miRNAs were differentially modulated by LPS and by cannabinoids treatments. In addition, we found that at the concentration tested, CBD has a greater effect than THC on the expression of most of the studied miRNAs. The results clearly link the effects of both LPS and cannabinoids to inflammatory signaling pathways. LPS upregulated the expression of pro-inflammatory miRNAs associated to Toll-like receptor (TLR) and NF-κB signaling, including miR-21, miR-146a and miR-155, whereas CBD inhibited LPS-stimulated expression of miR-146a and miR-155. In addition, CBD upregulated miR-34a, known to be involved in several pathways including Rb/E2f cell cycle and Notch-Dll1 signaling. Our results show that both CBD and THC reduced the LPS-upregulated Notch ligand Dll1 expression. MiR-155 and miR-34a are considered to be redox sensitive miRNAs, which regulate Nrf2-driven gene expression. Accordingly, we found that Nrf2-mediated expression of redox-dependent genes defines a Mox-like phenotype in CBD treated BV-2 cells. In summary, we have identified a specific repertoire of miRNAs that are regulated by cannabinoids, in resting (surveillant) and in LPS-activated microglia. The modulated miRNAs and their target genes are controlled by TLR, Nrf2 and Notch cross-talk signaling and are involved in immune response, cell cycle regulation as well as cellular stress and redox homeostasis

Biosíntesis enzimática de porfirinógenos

La Porfobilinogenasa (PBG-asa) es un complejo enzimático que cataliza la ciclotetramerización del monopirrol Porfobilinógeno (PBG) en el Uroporfirinógeno III (Urogen III), intermediario fisiológico en la sintesis de hemos,clorofilas y corrinas. Está constituido por dos enzimas, la Deaminasa o Urogen I Sintetasa y la Isomerasa o Urogen III Cosintetasa. En ausencia o deficiencia de la segunda, la Deaminasa forma el Urogen I, que sólo se detecta naturalmente en condiciones anormales. Por fallas en el camino metabólico de las porfirinas, se producen una familia de enfermedades conocidas como Porfirias. En ellas, una deficiencia enzimática especifica y primaria, acompañada generalmente de un aumento secundario en la actividad de la enzima limitante ALA-Sintetasa, conduce a una sintesis anormal de precursores y/o porfirinas, responsables de los cuadros clinicos que caracterizan a las distintas porfirias. En algunas de estas porfirias, la falla metabólica se localiza precisamente a nivel de la conversión del PBG en Uroporfirinógenos, como en la Porfiria Aguda Intermitente (PAI), Porfiria Congénita Eritropoyética (PCE) y en algunos casos de intoxicación por plomo. Este sistema enzimático se ha venido estudiando en nuestro laboratorio durante más de 20 años, desde múltiples puntos de vista y en los más variados sistemas. Entre ellos, uno de los más fascinantes ha sido el alga protozoo Euglena gracilis que en virtud de sus peculiares caracteristicas ha constituido uno de los modelos experimentales más atractivos para la investigación de ciertos aspectos del metabolismo de los tetrapirroles. Empleando justamente Euglena gracilis, hemos logrado pruebas acerca de la existencia de un factor regulador de la biosintesis de porfirinas en este organismo, y el estudio de algunasde sus propiedades nos ha permitido identificarlo con un compuesto de naturaleza pteridinica, al tiempo que desarrollar una nueva terapia de la PAI, administrando ácido fólico a pacientes en fase aguda, luego de lo cual se ha logrado una positiva recuperación clinica y bioquimica. En consecuencia resultó importante continuar nuestros estudios acerca de este factor regulador. De manera que, entre los objetivos del presente trabajo nos habíamos propuesto: Emplear como fuentes, además de Euglena gracilis por las razones conocidas, una bacteria fotosíntética, que aparte de poder compartir algunas de las propiedades de la Euglena nos permitiera ampliar nuestros conocimientos acerca de la PBG-asa, poco investigada en estos organismos. Entre ellos, los datos existentes sobre las enzimas involucradas en la sintesis de porfirinas en Rhodopseudomonas palustris se refieren solo al ALA-Sintetasa y también provienen de nuestro laboratorio; de manera que la elección de la segunda fuente fue simple y lógica. Este trabajo comprendía asi, el uso de dos organismos diferentes para el logro de una serie de objetivos. Como etapa previa se planteó entonces un estudio de las condiciones necesarias para la medición de la actividad de PBG-asa EN Rp. Palustris es decir, curvas de crecimiento y actividad en función de los días de desarrollo de las células, asi como condiciones óptimas de extracción y medición de la enzima, en cuanto a atmósfera, tiempo, temperatura y pH de incubación. Se desarrollaría luego un método para la obtención de preparaciones altamente purificadas sobre las cuales se planeaban estudiar algunas propiedades generales como peso molecular,cinética de la reacción y efecto de ciertos cationes y aniones. Pero entre los fines de este trabajo se encontraba además, tratar de identificar y determinar en otros tejidos, la presencia de un factor regulador, análogo al encontrado en Euglena gracilis de manera, que se planearon una serie de experiencias en RP. palustris tendientes al logro de este propósito. Los estudios que se enfocarían en Euglena gracilis estában principalmente relacionados con la obtención de mayores datos experimentales acerca del factor regulador. Se trataba deconocer sus efectos sobre la cinética de la reacción de diferentes preparaciones de la enzima. De datos previos, nos interesó estab1ecer su relación con ciertos compuestos como sulfas y derivados sulfurados. Y dado el conocido efecto activante del factor de Euglena sobre la enzima de igual fuente y del ácido fólico en pacientes con PAI, se pretendía determinar el comportamiento de amboscompuestos frente a distintas preparaciones de la enzima proveniente de los origenes más diversos, con el objeto de aclarar el posible mecanismo de acción de este factor regulador.Fil: Juknat, Adela Ana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Porphyrin biosynthesis in Rhodopseudomonas palustris-IX. PBG-deaminase. Kinetic studies

1. 1. PBG-Deaminase obtained from Rp. palustris exhibited classical Michaelis-Menten kinetics in the absence or presence of different ions. 2. 2. Detailed kinetic studies were carried out in the presence of ammonium, phosphate and magnesium ions. 3. 3. It has been found that the different effects observed are dependent on both the substrate and the ion concentration. © 1987.Fil:Kotler, M.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Fumagalli, S.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Juknat, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Batlle, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Porphyrin biosynthesis in rhodopseudomonas palustris-V. Purification of porphyrinogen decarboxylase and some unusual properties

1. 1. Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified 16-fold from Rp. palustris to a specific activity of 210 nmol of total decarboxylated porphyrinogens III formed/hr per mg of protein and about 50% yield. The Rp. palustris enzyme exhibits some unusual properties as compared with URO-D from other sources. 2. 2. The purified enzyme is a monomer with a molecular weight of ∼46,000, an isoelectric point of 4.6 and an optimum pH of 6.9 and 6.8 with urogen III and I substrate. Neither GSH nor EDTA seem to be necessary for activity, and the decarboxylation rate and the distribution of the reaction products was not affected either by the presence or absence of oxygen. 3. 3. The Rp. palustris enzyme is a thermo-stable protein, heating at 60°C for 15 min enhanced several times activity. This is the first time that the heat treatment is included as one of the steps to purify URO-D. 4. 4. Thermal activation followed an identical profile using either substrate. The ratios of specific activity for the type III and I isomer of urogen remained constant throughout the purification. These findings are indicating that a single enzyme catalyzes the four decarboxylations occurring from urogen to coprogen. 5. 5. Kinetic data employing urogen III and I as substrate showed that the pattern of accumulated intermediates was rather different depending on whether type III or I isomer was used. 6. 6. While decarboxylation of urogen III responds to the usual scheme: {A figure is presented} where v1≫v2 and decarboxylation of heptagen III is the rate-controlling step. 7. 7. Decarboxylation of urogen I revealed a completely different and characteristic picture fitting the scheme: {A figure is presented} where again v′1≫v′2 and the removal of the final carboxyl group from pentagen I becomes the rate-limiting step. © 1986.Fil:Juknat de Geralnik, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:del C. Batlle, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

N-arachidonoyl glycine, an abundant endogenous lipid, potently drives directed cellular migration through GPR18, the putative abnormal cannabidiol receptor

Background: Microglia provide continuous immune surveillance of the CNS and upon activation rapidly change phenotype to express receptors that respond to chemoattractants during CNS damage or infection. These activated microglia undergo directed migration towards affected tissue. Importantly, the molecular species of chemoattractant encountered determines if microglia respond with pro- or anti-inflammatory behaviour, yet the signaling molecules that trigger migration remain poorly understood. The endogenous cannabinoid system regulates microglial migration via CB2 receptors and an as yet unidentified GPCR termed the 'abnormal cannabidiol' (Abn-CBD) receptor. Abn-CBD is a synthetic isomer of the phytocannabinoid cannabidiol (CBD) and is inactive at CB1 or CB2 receptors, but functions as a selective agonist at this Gi/o-coupled GPCR. N-arachidonoyl glycine (NAGly) is an endogenous metabolite of the endocannabinoid anandamide and acts as an efficacious agonist at GPR18. Here, we investigate the relationship between NAGly, Abn-CBD, the unidentified 'Abn-CBD' receptor, GPR18, and BV-2 microglial migration. Results: Using Boyden chamber migration experiments, yellow tetrazolium (MTT) conversion, In-cell Western, qPCR and immunocytochemistry we show that NAGly, at sub-nanomolar concentrations, and Abn-CBD potently drive cellular migration in both BV-2 microglia and HEK293-GPR18 transfected cells, but neither induce migration in HEKGPR55 or non-transfected HEK293 wildtype cells. Migration effects are blocked or attenuated in both systems by the 'Abn-CBD' receptor antagonist O-1918, and low efficacy agonists N-arachidonoyl-serine and cannabidiol. NAGly promotes proliferation and activation of MAP kinases in BV-2 microglia and HEK293-GPR18 cells at low nanomolar concentrations - cellular responses correlated with microglial migration. Additionally, BV-2 cells show GPR18 immunocytochemical staining and abundant GPR18 mRNA. qPCR demonstrates that primary microglia, likewise, express abundant amounts of GPR18 mRNA. Conclusions: NAGly is the most effective lipid recruiter of BV-2 microglia currently reported and its effects mimic those of Abn-CBD. The data generated from this study supports the hypothesis that GPR18 is the previously unidentified 'Abn-CBD' receptor. The marked potency of NAGly acting on GPR18 to elicit directed migration, proliferation and perhaps other MAPK-dependent phenomena advances our understanding of the lipid-based signaling mechanisms employed by the CNS to actively recruit microglia to sites of interest. It offers a novel research avenue for developing therapeutics to elicit a self-renewing population of neuroregenerative microglia, or alternatively, to prevent the accumulation of misdirected, pro-inflammatory microglia which contribute to and exacerbate neurodegenerative disease

Clinical studies and anti-inflammatory mechanisms of treatments

In this exciting era, we are coming closer and closer to bringing an anti‐inflammatory therapy to the clinic for the purpose of seizure prevention, modification, and/or suppression. At present, it is unclear what this approach might entail, and what form it will take. Irrespective of the therapy that ultimately reaches the clinic, there will be some commonalities with regard to clinical trials. A number of animal models have now been used to identify inflammation as a major underlying mechanism of both chronic seizures and the epileptogenic process. These models have demonstrated that specific anti‐inflammatory treatments can be effective at both suppressing chronic seizures and interfering with the process of epileptogenesis. Some of these have already been evaluated in early phase clinical trials. It can be expected that there will soon be more clinical trials of both “conventional, broad spectrum” anti‐inflammatory agents and novel new approaches to utilizing specific anti‐inflammatory therapies with drugs or other therapeutic interventions. A summary of some of those approaches appears below, as well as a discussion of the issues facing clinical trials in this new domain