title: Cannabidiol, a non-psychoactive cannabinoid, leads to EGR2-dependent anergy in activated encephalitogenic T cells.
abstract: BACKGROUND
  Cannabidiol (CBD), the main non-psychoactive cannabinoid, has been previously shown by us to ameliorate clinical symptoms and to decrease inflammation in myelin oligodendrocyte glycoprotein (MOG)35-55-induced mouse experimental autoimmune encephalomyelitis model of multiple sclerosis as well as to decrease MOG35-55-induced T cell proliferation and IL-17 secretion. However, the mechanisms of CBD anti-inflammatory activities are unclear.
  
  
  METHODS
  Here we analyzed the effects of CBD on splenocytes (source of accessory T cells and antigen presenting cells (APC)) co-cultured with MOG35-55-specific T cells (TMOG) and stimulated with MOG35-55. Using flow cytometry, we evaluated the expression of surface activation markers and inhibitory molecules on T cells and B cells. TMOG cells were purified using CD4 positive microbead selection and submitted for quantitative PCR and microarray of mRNA transcript analyzes. Cell signaling studies in purified TMOG were carried out using immunoblotting.
  
  
  RESULTS
  We found that CBD leads to upregulation of CD69 and lymphocyte-activation gene 3 (LAG3) regulatory molecules on CD4(+)CD25(-) accessory T cells. This subtype of CD4(+)CD25(-)CD69(+)LAG3(+) T cells has been recognized as induced regulatory phenotype promoting anergy in activated T cells. Indeed, we observed that CBD treatment results in upregulation of EGR2 (a key T cell anergy inducer) mRNA transcription in stimulated TMOG cells. This was accompanied by elevated levels of anergy promoting genes such as IL-10 (anti-inflammatory cytokine), STAT5 (regulatory factor), and LAG3 mRNAs, as well as of several enhancers of cell cycle arrest (such as Nfatc1, Casp4, Cdkn1a, and Icos). Moreover, CBD exposure leads to a decrease in STAT3 and to an increase in STAT5 phosphorylation in TMOG cells, positive and negative regulators of Th17 activity, respectively. In parallel, we observed decreased levels of major histocompatibility complex class II (MHCII), CD25, and CD69 on CD19(+) B cells following CBD treatment, showing diminished antigen presenting capabilities of B cells and reduction in their pro-inflammatory functions.
  
  
  CONCLUSIONS
  Our data suggests that CBD exerts its immunoregulatory effects via induction of CD4(+)CD25(-)CD69(+)LAG3(+) cells in MOG35-55-activated APC/TMOG co-cultures. This is accompanied by EGR2-dependent anergy of stimulated TMOG cells as well as a switch in their intracellular STAT3/STAT5 activation balance leading to the previously observed decrease in Th17 activity.
authors.affiliation: The Dr Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. ewa.kozela@weizmann.ac.il.
authors.forename: Ewa
authors.initials: E
authors.lastname: Kozela
authors.affiliation: The Dr Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Ana.juknat@weizmann.ac.il.
authors.forename: Ana
authors.initials: A
authors.lastname: Juknat
authors.affiliation: Neurobiology Department, Weizmann Institute of Science, Rehovot, Israel. nathali.kaushansky@weizmann.ac.il.
authors.forename: Nathali
authors.initials: N
authors.lastname: Kaushansky
authors.affiliation: Immunology Department, Weizmann Institute of Science, Rehovot, Israel. avi.ben-nun@weizmann.ac.il.
authors.forename: Avraham
authors.initials: A
authors.lastname: Ben-Nun
authors.affiliation: Neurology Department, UCLA, Los Angeles, CA, USA. gcoppola@ucla.edu.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Coppola
authors.affiliation: The Dr Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. vogelz@post.tau.ac.il.|Neurobiology Department, Weizmann Institute of Science, Rehovot, Israel. vogelz@post.tau.ac.il.
authors.forename: Zvi
authors.initials: Z
authors.lastname: Vogel
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000187:drug effects; D015703:Antigens, CD / Q000378:metabolism; D002185:Cannabidiol / Q000494:pharmacology*; D002460:Cell Line; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism*; D051767:Early Growth Response Protein 2 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D008213:Lymphocyte Activation / Q000187:drug effects*; D051379:Mice; D008810:Mice, Inbred C57BL; D063308:Myelin-Oligodendrocyte Glycoprotein / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011619:Psychotropic Drugs / Q000494:pharmacology*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects*; D000093322:Lymphocyte Activation Gene 3 Protein
chemical_list: D015703:Antigens, CD; D016207:Cytokines; D051767:Early Growth Response Protein 2; C497327:Egr2 protein, mouse; D063308:Myelin-Oligodendrocyte Glycoprotein; D010446:Peptide Fragments; D011619:Psychotropic Drugs; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; C494087:Stat3 protein, mouse; C445081:myelin oligodendrocyte glycoprotein (35-55); D002185:Cannabidiol; D000093322:Lymphocyte Activation Gene 3 Protein; C000720327:Lag3 protein, mouse