Advertisement

Cannabinoids Δ9-Tetrahydrocannabinol and Cannabidiol Differentially Inhibit the Lipopolysaccharide-activated NF-κB and Interferon-β/STAT Proinflammatory Pathways in BV-2 Microglial Cells*

  • Ewa Kozela
    Affiliations
    Neurobiology Department, Weizmann Institute of Science, 76100 Rehovot
    Search for articles by this author
  • Maciej Pietr
    Affiliations
    Neurobiology Department, Weizmann Institute of Science, 76100 Rehovot
    Search for articles by this author
  • Ana Juknat
    Footnotes
    Affiliations
    The Dr. Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel
    Search for articles by this author
  • Neta Rimmerman
    Footnotes
    Affiliations
    Neurobiology Department, Weizmann Institute of Science, 76100 Rehovot
    Search for articles by this author
  • Rivka Levy
    Affiliations
    Neurobiology Department, Weizmann Institute of Science, 76100 Rehovot
    Search for articles by this author
  • Zvi Vogel
    Correspondence
    To whom correspondence should be addressed. Fax: 972-8-9344131
    Affiliations
    Neurobiology Department, Weizmann Institute of Science, 76100 Rehovot

    The Dr. Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by the Dr. Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, by the Adelsons' Program for Nerve Regeneration and Repair, by the Nella and Leon Benoziyo Center for Neurosciences, and by the Israeli Ministry of Health (to Z. V.).
    1 Supported by the Center for Absorption in Science in Israel.
Open AccessPublished:November 12, 2009DOI:https://doi.org/10.1074/jbc.M109.069294
      Cannabinoids have been shown to exert anti-inflammatory activities in various in vivo and in vitro experimental models as well as ameliorate various inflammatory degenerative diseases. However, the mechanisms of these effects are not completely understood. Using the BV-2 mouse microglial cell line and lipopolysaccharide (LPS) to induce an inflammatory response, we studied the signaling pathways engaged in the anti-inflammatory effects of cannabinoids as well as their influence on the expression of several genes known to be involved in inflammation. We found that the two major cannabinoids present in marijuana, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), decrease the production and release of proinflammatory cytokines, including interleukin-1β, interleukin-6, and interferon (IFN)β, from LPS-activated microglial cells. The cannabinoid anti-inflammatory action does not seem to involve the CB1 and CB2 cannabinoid receptors or the abn-CBD-sensitive receptors. In addition, we found that THC and CBD act through different, although partially overlapping, mechanisms. CBD, but not THC, reduces the activity of the NF-κB pathway, a primary pathway regulating the expression of proinflammatory genes. Moreover, CBD, but not THC, up-regulates the activation of the STAT3 transcription factor, an element of homeostatic mechanism(s) inducing anti-inflammatory events. Following CBD treatment, but less so with THC, we observed a decreased level of mRNA for the Socs3 gene, a main negative regulator of STATs and particularly of STAT3. However, both CBD and THC decreased the activation of the LPS-induced STAT1 transcription factor, a key player in IFNβ-dependent proinflammatory processes. In summary, our observations show that CBD and THC vary in their effects on the anti-inflammatory pathways, including the NF-κB and IFNβ-dependent pathways.

      Introduction

      Δ9-Tetrahydrocannabinol (THC)
      The abbreviations used are: THC
      Δ9-tetrahydrocannabinol
      CBD
      cannabidiol
      abn-CBD
      abnormal cannabidiol
      STAT
      signal transducers and activators of transcription
      IL
      interleukin
      IFN
      interferon
      LPS
      lipopolysaccharide
      PI
      propidium iodide
      PBS
      phosphate-buffered saline
      ELISA
      enzyme-linked immunosorbent assay
      qPCR
      quantitative real time PCR
      ANOVA
      analysis of variance
      IRF3
      interferon-regulated factor 3
      ISRE
      interferon-stimulated response element.
      is a major constituent of Cannabis and serves as an agonist of the cannabinoid receptors CB1 (located mainly in neural cells) and CB2 (located mainly on immune cells). The second major constituent of Cannabis extract is cannabidiol (CBD), which is virtually inactive at the CB1 and CB2 receptors (
      • Thomas B.F.
      • Gilliam A.F.
      • Burch D.F.
      • Roche M.J.
      • Seltzman H.H.
      ). Thus, because of its negligible activity at the CB1 receptor, CBD lacks the psychoactive effects that accompany the use of THC. Moreover, CBD was demonstrated to antagonize some undesirable effects of THC, including intoxication, sedation, and tachycardia, while sharing neuroprotective, anti-oxidative, anti-emetic, and anti-carcinogenic properties (
      • Mechoulam R.
      • Peters M.
      • Murillo-Rodriguez E.
      • Hanus L.O.
      ,
      • Izzo A.A.
      • Borrelli F.
      • Capasso R.
      • Di Marzo V.
      • Mechoulam R.
      ,
      • Zuardi A.W.
      ). Both THC and CBD have been shown to exert anti-inflammatory properties and to modulate the function of immune cells, including suppression of humoral response, immune cell proliferation, maturation, and migration, and antigen presentation (
      • Malfait A.M.
      • Gallily R.
      • Sumariwalla P.F.
      • Malik A.S.
      • Andreakos E.
      • Mechoulam R.
      • Feldmann M.
      ,
      • Ben-Shabat S.
      • Hanus L.O.
      • Katzavian G.
      • Gallily R.
      ,
      • Klein T.W.
      • Cabral G.A.
      ,
      • Baker D.
      • Jackson S.J.
      • Pryce G.
      ,
      • Borrelli F.
      • Aviello G.
      • Romano B.
      • Orlando P.
      • Capasso R.
      • Maiello F.
      • Guadagno F.
      • Petrosino S.
      • Capasso F.
      • Di Marzo V.
      • Izzo A.A.
      ). Despite increasing amounts of such observations, the molecular mechanisms involved in these cannabinoid-mediated effects are not yet fully understood.
      Microglial cells are resident macrophages of the central nervous system and serve as early host defense against pathogens. Activation of microglial cells leads to the release of proinflammatory and neurotoxic factors and serves as part of the neuroinflammatory process (
      • Dheen S.T.
      • Kaur C.
      • Ling E.A.
      ). The BV-2 murine microglial cell line is known to retain morphological, phenotypic, and functional properties associated with freshly isolated microglia such as expression of nonspecific esterase activity, phagocytic ability, and the absence of peroxidase activity (
      • Blasi E.
      • Barluzzi R.
      • Bocchini V.
      • Mazzolla R.
      • Bistoni F.
      ,
      • Bocchini V.
      • Mazzolla R.
      • Barluzzi R.
      • Blasi E.
      • Sick P.
      • Kettenmann H.
      ). Furthermore, these cells release lysozyme and, when stimulated, interleukin (IL)-1 and tumor necrosis factor α (
      • Blasi E.
      • Barluzzi R.
      • Bocchini V.
      • Mazzolla R.
      • Bistoni F.
      ,
      • Bocchini V.
      • Mazzolla R.
      • Barluzzi R.
      • Blasi E.
      • Sick P.
      • Kettenmann H.
      ). Close similarities between BV-2 and primary microglia in mechanisms mediating microglial stimulations, e.g. by lipopolysaccharide (LPS), S100B, or β-amyloid, were reported (
      • Kim S.H.
      • Smith C.J.
      • Van Eldik L.J.
      ). These properties make BV-2 cells an appropriate model for studying the activation of microglia in vitro. It has recently been shown that BV-2 cells express elements of the cannabinoid signaling systems, including the presence of endocannabinoids, i.e. anandamide and 2-arachidonoylglycerol, and cannabinoid or cannabinoid-like receptors such as CB2, GPR55, and abnormal cannabidiol (abn-CBD)-sensitive receptors but very little CB1 cannabinoid receptor (
      • Walter L.
      • Franklin A.
      • Witting A.
      • Wade C.
      • Xie Y.
      • Kunos G.
      • Mackie K.
      • Stella N.
      ,
      • Carrier E.J.
      • Kearn C.S.
      • Barkmeier A.J.
      • Breese N.M.
      • Yang W.
      • Nithipatikom K.
      • Pfister S.L.
      • Campbell W.B.
      • Hillard C.J.
      ,
      • Pietr M.
      • Kozela E.
      • Levy R.
      • Rimmerman N.
      • Lin Y.H.
      • Stella N.
      • Vogel Z.
      • Juknat A.
      ).
      In this study, we used the BV-2 microglial cell line and assessed the effects of THC and CBD on the LPS-activated microglial secretion of proinflammatory cytokines such as interleukin IL-1β, IL-6, and of interferon β (IFNβ). LPS signaling through TLR4 (toll-like receptor 4) is known to activate several intracellular pathways and to induce broad changes in gene expression, eventually inducing the release of various proinflammatory cytokines and neurotoxic factors (
      • Gay N.J.
      • Gangloff M.
      ). LPS activates two basic intracellular pathways via specific adaptor proteins. The first is the myeloid differentiation factor 88 (MyD88)-adaptor protein-dependent pathway that leads to activation of NF-κB-dependent transcription. The second pathway (the MyD88-independent pathway) is dependent on the toll-interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon-β (TRIF) protein. Its activation turns on the interferon-regulated factor 3 (IRF3)-dependent pathway that enhances the production of IFNβ (
      • Kawai T.
      • Takeuchi O.
      • Fujita T.
      • Inoue J.
      • Mühlradt P.F.
      • Sato S.
      • Hoshino K.
      • Akira S.
      ). IFNβ, in an autocrine way, acts via the type I interferon receptor and via signal transducers and activators of transcription (STAT)-dependent pathways and activates a second wave of gene expression including chemokines such as chemokine 2 (CCL2 (C-C motif ligand 2)). We studied the effects of THC and CBD on these two pathways. In addition, we studied the effect of these materials on the expression of several genes, belonging to suppressors of cytokine signaling (SOCS) family, that are involved in the negative regulation of proinflammatory events.
      We found that although both THC and CBD exert inhibitory effects on the production of inflammatory cytokines in activated microglial cells in culture, their activities seem to involve both different and overlapping intracellular pathways. These effects are not mediated via CB1, CB2, nor abn-CBD-sensitive receptors.

      DISCUSSION

      In this study, we activated BV-2 microglial cells with LPS and observed vast release of IL-1β, IL-6, and IFNβ cytokines, all well recognized as key mediators of inflammatory responses (
      • Suzumura A.
      • Takeuchi H.
      • Zhang G.
      • Kuno R.
      • Mizuno T.
      ). Cannabinoid treatment by either THC or CBD strongly reduced the LPS-induced release of IL-1β, IL-6, and IFNβ. The inhibitory effects of these two cannabinoids on the release of IL-1β and IFNβ were similar. On the other hand, the release of IL-6 was inhibited to a much stronger extent by CBD than by THC.
      It is well known that THC and CBD differ in their pharmacology toward the currently known cannabinoid receptors. THC is a CB1 and CB2 receptor partial agonist, whereas CBD exhibits a very low affinity toward both receptors (
      • Showalter V.M.
      • Compton D.R.
      • Martin B.R.
      • Abood M.E.
      ,
      • Pertwee R.G.
      ). Because CB2 receptors are expressed on various immune cells (
      • Mackie K.
      ), including primary microglia and the BV-2 microglial cell line (
      • Walter L.
      • Franklin A.
      • Witting A.
      • Wade C.
      • Xie Y.
      • Kunos G.
      • Mackie K.
      • Stella N.
      ,
      • Pietr M.
      • Kozela E.
      • Levy R.
      • Rimmerman N.
      • Lin Y.H.
      • Stella N.
      • Vogel Z.
      • Juknat A.
      ), they seem to be primary candidates to mediate cannabinoid immunomodulation. Immunosuppressive effects of THC were shown to be predominantly CB2 mediated as revealed by using knock-out systems (
      • Buckley N.E.
      • McCoy K.L.
      • Mezey E.
      • Bonner T.
      • Zimmer A.
      • Felder C.C.
      • Glass M.
      • Zimmer A.
      ). Accordingly, CB2 agonists were shown to be immunosuppressive in rat primary microglial cultures (
      • Romero-Sandoval E.A.
      • Horvath R.
      • Landry R.P.
      • DeLeo J.A.
      ). Rat primary microglial cells were also shown to express CB1 receptors whose activation leads to induction of NO production (
      • Cabral G.A.
      • Harmon K.N.
      • Carlisle S.J.
      ). To determine whether CB1 or CB2 receptors mediate the THC immunosuppression in our system, we applied the respective antagonists before THC treatment. We found that neither CB1 nor CB2 receptor antagonists interfered with the THC inhibitory effect on LPS-induced IL-1β release, suggesting that other receptor(s) or nonreceptor targets seem to be involved. The exact reasons for these different results are not clear. One possibility is that THC is a very weak agonist of the CB2 (
      • Bayewitch M.
      • Rhee M.H.
      • Avidor-Reiss T.
      • Breuer A.
      • Mechoulam R.
      • Vogel Z.
      ) and that the CB1 is almost absent in BV-2 cells (
      • Pietr M.
      • Kozela E.
      • Levy R.
      • Rimmerman N.
      • Lin Y.H.
      • Stella N.
      • Vogel Z.
      • Juknat A.
      ). However, we cannot rule out that different inflammatory models may involve somewhat different mechanisms.
      As for CBD, whereas binding assays show negligible affinity of CBD toward CB1 and CB2 receptors (
      • Showalter V.M.
      • Compton D.R.
      • Martin B.R.
      • Abood M.E.
      ), several functional studies showed that some of the CBD effects may involve cannabinoid receptors. Thus, the CB1 antagonist AM251 reversed the slowing of gastrointestinal motility by CBD in LPS-treated septic mice (
      • de Filippis D.
      • Iuvone T.
      • d'amico A.
      • Esposito G.
      • Steardo L.
      • Herman A.G.
      • Pelckmans P.A.
      • de Winter B.Y.
      • de Man J.G.
      ). Moreover, Sacerdote et al. (
      • Sacerdote P.
      • Martucci C.
      • Vaccani A.
      • Bariselli F.
      • Panerai A.E.
      • Colombo A.
      • Parolaro D.
      • Massi P.
      ) showed that CB1 and CB2 receptor antagonists reversed the CBD modulation of IL-12 and IL-10 release in mouse peritoneal macrophages in vitro and showed that CBD decreased the formyl-methionyl-leucyl-phenylalanine-induced chemotaxis of macrophages in a CB2-dependent manner. However, using our model of microglial activation and applying CB1 and CB2 antagonists prior to CBD treatment, we found that neither of these antagonists affected CBD diminution of IL-1β release suggesting that this CBD activity is not CB1/CB2-mediated.
      CBD may act as a partial agonist toward abn-CBD-sensitive receptors. These are putative new members of the cannabinoid receptor family, not yet cloned, but pinpointed pharmacologically in mice lacking CB1 and CB2 receptors (
      • Járai Z.
      • Wagner J.A.
      • Varga K.
      • Lake K.D.
      • Compton D.R.
      • Martin B.R.
      • Zimmer A.M.
      • Bonner T.I.
      • Buckley N.E.
      • Mezey E.
      • Razdan R.K.
      • Zimmer A.
      • Kunos G.
      ). abn-CBD-sensitive receptors were shown to be present in microglial cells and regulate their migration (
      • Walter L.
      • Franklin A.
      • Witting A.
      • Wade C.
      • Xie Y.
      • Kunos G.
      • Mackie K.
      • Stella N.
      ,
      • Franklin A.
      • Stella N.
      ). However, in our hands abn-CBD pretreatment did not have any effect by itself and did not interfere with the CBD suppression of LPS-induced IL-1β release, thus excluding the involvement of abn-CBD-sensitive receptors in these CBD mediated effects.
      Several studies pointed out that cannabinoids could have CB1/CB2 receptor-independent mechanisms of action (
      • Makriyannis A.
      • Yang D.P.
      • Griffin R.G.
      • Das Gupta S.K.
      ,
      • Felder C.C.
      • Veluz J.S.
      • Williams H.L.
      • Briley E.M.
      • Matsuda L.A.
      ,
      • Puffenbarger R.A.
      • Boothe A.C.
      • Cabral G.A.
      ,
      • Berdyshev E.V.
      • Schmid P.C.
      • Krebsbach R.J.
      • Hillard C.J.
      • Huang C.
      • Chen N.
      • Dong Z.
      • Schmid H.H.
      ,
      • Price T.J.
      • Patwardhan A.
      • Akopian A.N.
      • Hargreaves K.M.
      • Flores C.M.
      ). This observation is in agreement with our current findings and with the results of Kaplan et al. (
      • Kaplan B.L.
      • Rockwell C.E.
      • Kaminski N.E.
      ) showing that CBD and WIN 55212-3 exhibit immunosuppressive effects via non-CB1 and non-CB2 mechanisms. Moreover, Kaplan et al. (
      • Kaplan B.L.
      • Rockwell C.E.
      • Kaminski N.E.
      ) showed that both cannabinol (a weak CB2 agonist) and CBD inhibit IL-2 release from T lymphocytes, although for cannabinol the effect is CB2-dependent but not for CBD. Altogether, these results indicate that the immunoregulating effects of cannabinoids can be both CB2 receptor- and nonreceptor-mediated even in the same system.
      Despite the increasing data on the immune regulatory effects of cannabinoids in vitro (
      • Puffenbarger R.A.
      • Boothe A.C.
      • Cabral G.A.
      ,
      • Watzl B.
      • Scuderi P.
      • Watson R.R.
      ,
      • Srivastava M.D.
      • Srivastava B.I.
      • Brouhard B.
      ) and in vivo (
      • Gallily R.
      • Yamin A.
      • Waksmann Y.
      • Ovadia H.
      • Weidenfeld J.
      • Bar-Joseph A.
      • Biegon A.
      • Mechoulam R.
      • Shohami E.
      ,
      • Smith S.R.
      • Terminelli C.
      • Denhardt G.
      ,
      • Roche M.
      • Diamond M.
      • Kelly J.P.
      • Finn D.P.
      ), the signaling pathways responsible for these effects are for the most part unknown. Moreover, the modulation of IFNβ-related processes by cannabinoids has been only poorly addressed.
      The expression of IL-1β, IL-6, and IFNβ gene products are tightly regulated, and their promoter regions possess binding sites for specific inducible transcription factors. NF-κB is a primary (although not the only one) regulator of IL-1β and IL-6 cytokines, whereas IFNβ is expressed in response to IRF-3 factor activation (
      • Covert M.W.
      • Leung T.H.
      • Gaston J.E.
      • Baltimore D.
      ).
      The NF-κB pathway is a primary intracellular pathway controlling the transcription of many inflammatory genes (
      • Rothwarf D.M.
      • Karin M.
      ). The NF-κB p65-p50 protein complex is present in the cytoplasm through association with the inhibitor protein, IκB, which masks the nuclear localization signal present within the NF-κB p65 subunit. The activation of NF-κB by extracellular inducers (including LPS) depends on the rapid phosphorylation of IκB by upstream IRAK-1 followed by ubiquitination and targeting for proteosome degradation of both proteins, IRAK-1 and IκB. The NF-κB p65 subunit then undergoes phosphorylation, followed by translocation to the nucleus where it regulates the expression of various inflammatory genes, including IL-1β and IL-6 (
      • Covert M.W.
      • Leung T.H.
      • Gaston J.E.
      • Baltimore D.
      ).
      Our data provide several indications for the effects of CBD in decreasing the activity of the NF-κB signaling pathway. The first includes the partial reversal of the LPS-induced degradation of IRAK-1 intermediate kinase; the second is the reversal of IκB degradation, and the third is the lowering of the NF-κB p65 subunit phosphorylation. Interestingly, treatment with THC did not significantly affect this cascade at any of these steps, thus questioning the involvement of the NF-κB pathway in the THC-mediated effects in BV-2 microglia. Several previous studies suggested the involvement of the NF-κB pathway in cannabinoid-induced immunosuppression in macrophages (
      • Jeon Y.J.
      • Yang K.H.
      • Pulaski J.T.
      • Kaminski N.E.
      ), thymocytes (
      • Herring A.C.
      • Kaminski N.E.
      ), monocytes (
      • Rajesh M.
      • Mukhopadhyay P.
      • Haskó G.
      • Huffman J.W.
      • Mackie K.
      • Pacher P.
      ), and granulomatous tissue (
      • De Filippis D.
      • Russo A.
      • De Stefano D.
      • Maiuri M.C.
      • Esposito G.
      • Cinelli M.P.
      • Pietropaolo C.
      • Carnuccio R.
      • Russo G.
      • Iuvone T.
      ). In all of these cases, the effects were shown to be CB2 receptor-mediated. It is interesting to note that 2-arachidonoylglycerol endocannabinoid was shown to decrease NF-κB activation in injured murine brain via CB1 receptors (
      • Panikashvili D.
      • Mechoulam R.
      • Beni S.M.
      • Alexandrovich A.
      • Shohami E.
      ). However, in our microglial system neither the CB1 (which is present in a low concentration if at all (
      • Pietr M.
      • Kozela E.
      • Levy R.
      • Rimmerman N.
      • Lin Y.H.
      • Stella N.
      • Vogel Z.
      • Juknat A.
      )) nor the CB2 cannabinoid receptors seem to be involved. Interestingly, the non-CB1/CB2-mediated anti-inflammatory effects of cannabinoids mediated via NF-κB and other pathways were also observed in several nonimmune cells, including astrocytes and neuronal PC12 cells (
      • Esposito G.
      • De Filippis D.
      • Maiuri M.C.
      • De Stefano D.
      • Carnuccio R.
      • Iuvone T.
      ,
      • Curran N.M.
      • Griffin B.D.
      • O'Toole D.
      • Brady K.J.
      • Fitzgerald S.N.
      • Moynagh P.N.
      ).
      Other pathways could be affected by cannabinoids promoting anti-inflammatory activities in microglial cells. For example, the released IL-1β could promote activation of the NF-κB pathway, whereas IFNβ promotes the interferon-stimulated response element (ISRE) pathway, and IL-6 induces the NF-κB as well as several other pathways (e.g. STAT- and ISRE-dependent) (
      • Gay N.J.
      • Gangloff M.
      ). Both THC and CBD decrease LPS-induced IFNβ production and release. These cannabinoids exert their inhibitory activity upstream of IFNβ synthesis, e.g. at the level of the MyD88-independent pathway that is leading to the activation of IRF-3. The IRF-3 pathway is activated following its phosphorylation by TBK1 (TANK-binding kinase 1) associated with the TRIF adaptor protein of TLR4 receptors. The activated IRF-3 binds the ISRE DNA sequence inducing the production of the IFNβ cytokine (
      • Gay N.J.
      • Gangloff M.
      ). IFNβ expression activates a second wave of gene expression (including chemokines such as CXCL10, CCL5, and CCL2) via the IFN receptor and the Janus tyrosine kinase/STAT pathways. Briefly, the released IFNβ binds to IFN receptor and induces phosphorylation of the Janus tyrosine kinase family members leading to the activation of STAT multifamily proteins. Upon activation, the members of the STAT family induce the expression of pro- as well as of anti-inflammatory genes through binding to the various ISRE as well as to some IFN-γ-activated sequence promoter sites to induce expression of interferon-stimulated genes (
      • Wesoly J.
      • Szweykowska-Kulinska Z.
      • Bluyssen H.A.
      ). The major mediators of IFNβ signaling are STAT1 and STAT3 (
      • Wesoly J.
      • Szweykowska-Kulinska Z.
      • Bluyssen H.A.
      ,
      • Regis G.
      • Pensa S.
      • Boselli D.
      • Novelli F.
      • Poli V.
      ). Indeed, we observed profound activation of STAT1 and STAT3 following LPS stimulation.
      STAT1 and STAT3 have similar structures; both are phosphorylated on tyrosine residues upon cytokine stimulation, and both form homo- or heterodimers through the reciprocal Src homology 2 domain/phosphotyrosine interactions, move to the nucleus, bind to respective sequences on promoter sites, and activate transcription of a large number of genes. Various STAT1 and STAT3 dimers bind selectively to very similar but not identical elements and thus activate different but to some extent overlapping genes. This is likely to account for their different biological effects. For example, STAT1 homodimers exert proinflammatory effects via binding to ISRE and IFN-γ-activated sequence elements, inducing the expression of many chemokines (e.g. CCL2, ICAM1, and CXCL10), which regulate the migration or adhesion of immune cells (
      • Wesoly J.
      • Szweykowska-Kulinska Z.
      • Bluyssen H.A.
      ). In contrast, STAT3 exerts anti-inflammatory effects via the increased synthesis of IL-10 (an anti-inflammatory interleukin) or via direct binding to consensus elements of various IL-10-inducible genes (
      • Murray P.J.
      ). Several reports have revealed mechanisms responsible for STAT3-mediated attenuation of immune responses. For example, activated STAT3 was shown to suppress LPS-induced IL-6, tumor necrosis factor α, and IL-12 gene expression in macrophages and in dendritic cells (
      • Butcher B.A.
      • Kim L.
      • Panopoulos A.D.
      • Watowich S.S.
      • Murray P.J.
      • Denkers E.Y.
      ,
      • Barton B.E.
      ). STAT3 deficiency (or inactivation) makes the mutant mice highly susceptible to LPS shock and results in increased production of inflammatory cytokines such as tumor necrosis factor α, IL-1, and IFNγ from macrophages or neutrophils (
      • Takeda K.
      • Clausen B.E.
      • Kaisho T.
      • Tsujimura T.
      • Terada N.
      • Förster I.
      • Akira S.
      ,
      • Akira S.
      ). In addition, studies on STAT3-deficient cells revealed the existence of reciprocal STAT1/STAT3 regulatory mechanisms and explained the increase in proinflammatory STAT1 activity in the absence/inactivation of STAT3 (
      • Costa-Pereira A.P.
      • Tininini S.
      • Strobl B.
      • Alonzi T.
      • Schlaak J.F.
      • Is'harc H.
      • Gesualdo I.
      • Newman S.J.
      • Kerr I.M.
      • Poli V.
      ,
      • Qing Y.
      • Stark G.R.
      ,
      • Tanabe Y.
      • Nishibori T.
      • Su L.
      • Arduini R.M.
      • Baker D.P.
      • David M.
      ). Indeed, the balance between the proinflammatory STAT1 and the anti-inflammatory STAT3 seems to determine the final outcome of cell activation, i.e. immune tolerance versus chronic inflammatory state (
      • Wesoly J.
      • Szweykowska-Kulinska Z.
      • Bluyssen H.A.
      ,
      • Regis G.
      • Pensa S.
      • Boselli D.
      • Novelli F.
      • Poli V.
      ). Thus, STAT3 forms a feedback loop that is switched on by LPS and serves as a counterbalance mechanism to reduce the risk of chronic inflammation.
      In our experiments, we observed that although both cannabinoids reduce the activation of the proinflammatory STAT1, CBD (but not THC) strengthens the activation of STAT3. Thus, CBD seems to decrease the ongoing pro-inflammatory processes as well as intensify events counteracting inflammation. Moreover, we observed that LPS-induced STAT1-dependent expression of CCL2 mRNA was down-regulated following CBD (but not THC) pretreatment. CCL2 was shown to be up-regulated in STAT3 knock-out macrophages (
      • Matsukawa A.
      • Kudo S.
      • Maeda T.
      • Numata K.
      • Watanabe H.
      • Takeda K.
      • Akira S.
      • Ito T.
      ) pointing to a tight regulation of CCL2 levels by STAT3. Indeed, we found that the effect of CBD on the down-regulation of CCL2 mRNA parallels the up-regulation of STAT3 activation and STAT1 down-regulation and further indicates that these STAT molecules have a role in CBD-induced anti-inflammatory effects.
      The IFNβ-inducible Janus tyrosine kinase/STAT pathway is under the control of feedback inhibitors belonging to the SOCS family that include SOCS1–SOCS7 and CISH (
      • Jin H.J.
      • Shao J.Z.
      • Xiang L.X.
      • Wang H.
      • Sun L.L.
      ,
      • Croker B.A.
      • Kiu H.
      • Nicholson S.E.
      ). The transcription of several of these inhibitors is up-regulated as a feedback response to immune activation by a variety of immune cytokines and by LPS (
      • Wormald S.
      • Hilton D.J.
      ). Indeed, our qPCR measurement of mRNAs for SOCS3 and CISH shows that LPS profoundly up-regulates the expression of SOCS3 and of CISH in BV-2 microglial cells. CBD, and to a lesser extent THC, significantly suppressed the expression of SOCS3, but not of CISH, suggesting the involvement of particular negative regulators in the anti-inflammatory activity of the cannabinoids. Because SOCS3 has been shown to decrease STAT3 activity (
      • Hong F.
      • Jaruga B.
      • Kim W.H.
      • Radaeva S.
      • El-Assal O.N.
      • Tian Z.
      • Nguyen V.A.
      • Gao B.
      ,
      • Yasukawa H.
      • Ohishi M.
      • Mori H.
      • Murakami M.
      • Chinen T.
      • Aki D.
      • Hanada T.
      • Takeda K.
      • Akira S.
      • Hoshijima M.
      • Hirano T.
      • Chien K.R.
      • Yoshimura A.
      ) and the absence of SOCS3 (e.g. in SOCS3−/− macrophages) led to increased STAT3-mediated anti-inflammatory effects (
      • Yasukawa H.
      • Ohishi M.
      • Mori H.
      • Murakami M.
      • Chinen T.
      • Aki D.
      • Hanada T.
      • Takeda K.
      • Akira S.
      • Hoshijima M.
      • Hirano T.
      • Chien K.R.
      • Yoshimura A.
      ), the inducible effect of CBD on the activation of STAT3 could be mediated via its effect on SOCS3 expression. This mode of regulation is in line with the CBD anti-inflammatory activity in LPS-activated microglial cells.
      The NF-κB pathway can also be regulated by STAT-dependent molecules. Nishinakamura et al. (
      • Nishinakamura H.
      • Minoda Y.
      • Saeki K.
      • Koga K.
      • Takaesu G.
      • Onodera M.
      • Yoshimura A.
      • Kobayashi T.
      ) showed that activated STAT3 (STAT3C, a modified form of STAT3) reduced LPS-induced NF-κB transcription through αCP-1 (an RNA-binding protein that contains a K-homology domain with specificity for C-rich pyrimidine tracts) without affecting the TLR4 signal transduction, meaning without affecting phosphorylation of IκB and without affecting the DNA binding activity of NF-κB. We hypothesize that this regulation may be responsible at least in part for the diminution of IL-6 release by CBD.
      As for THC, it did not affect STAT3 phosphorylation and had a reduced effect on NF-κB. This could explain its reduced effect on the LPS-induced release of IL-6, in comparison with the effects of CBD. As for its effects on IL-1β, this might be due to the effect of THC on the release of IFNβ and the concomitant reduction in STAT1 phosphorylation. Although we did not observe a direct effect of THC on the NF-κB pathway, an increasing number of genome-wide analyses indicate that modulation of IFNβ pathway activity results in diminished transcription of NF-κB-dependent genes (
      • Ogawa S.
      • Lozach J.
      • Benner C.
      • Pascual G.
      • Tangirala R.K.
      • Westin S.
      • Hoffmann A.
      • Subramaniam S.
      • David M.
      • Rosenfeld M.G.
      • Glass C.K.
      ,
      • Lacaze P.
      • Raza S.
      • Sing G.
      • Page D.
      • Forster T.
      • Storm P.
      • Craigon M.
      • Awad T.
      • Ghazal P.
      • Freeman T.C.
      ). This reciprocal regulation may be involved in THC-exerted anti-inflammatory effects.
      In summary, our results show that although both THC and CBD exert anti-inflammatory effects, the two compounds engage different, although to some extent overlapping, intracellular pathways. Both THC and CBD decrease the activation of proinflammatory signaling by interfering with the TRIF/IFNβ/STAT pathway (see Scheme 1). CBD additionally suppresses the activity of the NF-κB pathway and potentiates an anti-inflammatory negative feedback process via STAT3. It is well known that NF-κB, IRF-3, and the STAT factors are induced by a broad spectrum of endogenous signals whose level is increased in response to cytotoxic changes. These include mitogens, cytokines, and neurotoxic factors (
      • Ock J.
      • Jeong J.
      • Choi W.S.
      • Lee W.H.
      • Kim S.H.
      • Kim I.K.
      • Suk K.
      ,
      • Pais T.F.
      • Figueiredo C.
      • Peixoto R.
      • Braz M.H.
      • Chatterjee S.
      ). The cannabinoids by moderating or disrupting these signaling networks may show promise as anti-inflammatory agents.
      Figure thumbnail grs1
      SCHEME 1Schematic figure showing possible sites for the anti-inflammatory activities of THC and CBD in LPS-activated BV-2 microglia. Pointed arrows indicate activation, and blunt arrows indicate inhibition.

      REFERENCES

        • Thomas B.F.
        • Gilliam A.F.
        • Burch D.F.
        • Roche M.J.
        • Seltzman H.H.
        J. Pharmacol. Exp. Ther. 1998; 285: 285-292
        • Mechoulam R.
        • Peters M.
        • Murillo-Rodriguez E.
        • Hanus L.O.
        Chem. Biodivers. 2007; 4: 1678-1692
        • Izzo A.A.
        • Borrelli F.
        • Capasso R.
        • Di Marzo V.
        • Mechoulam R.
        Trends Pharmacol. Sci. 2009; 30: 515-527
        • Zuardi A.W.
        Rev. Bras. Psiquiatr. 2008; 30: 271-280
        • Malfait A.M.
        • Gallily R.
        • Sumariwalla P.F.
        • Malik A.S.
        • Andreakos E.
        • Mechoulam R.
        • Feldmann M.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9561-9566
        • Ben-Shabat S.
        • Hanus L.O.
        • Katzavian G.
        • Gallily R.
        J. Med. Chem. 2006; 49: 1113-1117
        • Klein T.W.
        • Cabral G.A.
        J. Neuroimmune Pharmacol. 2006; 1: 50-64
        • Baker D.
        • Jackson S.J.
        • Pryce G.
        Br. J. Pharmacol. 2007; 152: 649-654
        • Borrelli F.
        • Aviello G.
        • Romano B.
        • Orlando P.
        • Capasso R.
        • Maiello F.
        • Guadagno F.
        • Petrosino S.
        • Capasso F.
        • Di Marzo V.
        • Izzo A.A.
        J. Mol. Med. 2009; 87: 1111-1121
        • Dheen S.T.
        • Kaur C.
        • Ling E.A.
        Curr. Med. Chem. 2007; 14: 1189-1197
        • Blasi E.
        • Barluzzi R.
        • Bocchini V.
        • Mazzolla R.
        • Bistoni F.
        J. Neuroimmunol. 1990; 27: 229-237
        • Bocchini V.
        • Mazzolla R.
        • Barluzzi R.
        • Blasi E.
        • Sick P.
        • Kettenmann H.
        J. Neurosci. Res. 1992; 31: 616-621
        • Kim S.H.
        • Smith C.J.
        • Van Eldik L.J.
        Neurobiol. Aging. 2004; 25: 431-439
        • Walter L.
        • Franklin A.
        • Witting A.
        • Wade C.
        • Xie Y.
        • Kunos G.
        • Mackie K.
        • Stella N.
        J. Neurosci. 2003; 23: 1398-1405
        • Carrier E.J.
        • Kearn C.S.
        • Barkmeier A.J.
        • Breese N.M.
        • Yang W.
        • Nithipatikom K.
        • Pfister S.L.
        • Campbell W.B.
        • Hillard C.J.
        Mol. Pharmacol. 2004; 65: 999-1007
        • Pietr M.
        • Kozela E.
        • Levy R.
        • Rimmerman N.
        • Lin Y.H.
        • Stella N.
        • Vogel Z.
        • Juknat A.
        FEBS Lett. 2009; 583: 2071-2076
        • Gay N.J.
        • Gangloff M.
        Annu. Rev. Biochem. 2007; 76: 141-165
        • Kawai T.
        • Takeuchi O.
        • Fujita T.
        • Inoue J.
        • Mühlradt P.F.
        • Sato S.
        • Hoshino K.
        • Akira S.
        J. Immunol. 2001; 167: 5887-5894
        • Nicoletti I.
        • Migliorati G.
        • Pagliacci M.C.
        • Grignani F.
        • Riccardi C.
        J. Immunol. Methods. 1991; 139: 271-279
        • Butovsky E.
        • Juknat A.
        • Elbaz J.
        • Shabat-Simon M.
        • Eilam R.
        • Zangen A.
        • Altstein M.
        • Vogel Z.
        Mol. Cell. Neurosci. 2006; 31: 795-804
        • Pfaffl M.W.
        Nucleic Acids Res. 2001; 29: e45
        • Franklin A.
        • Stella N.
        Eur. J. Pharmacol. 2003; 474: 195-198
        • Rothwarf D.M.
        • Karin M.
        Sci. STKE. 1999; 1999: RE1
        • Wesoly J.
        • Szweykowska-Kulinska Z.
        • Bluyssen H.A.
        Acta Biochim. Pol. 2007; 54: 27-38
        • Regis G.
        • Pensa S.
        • Boselli D.
        • Novelli F.
        • Poli V.
        Semin. Cell Dev. Biol. 2008; 19: 351-359
        • Suzumura A.
        • Takeuchi H.
        • Zhang G.
        • Kuno R.
        • Mizuno T.
        Ann. N.Y. Acad. Sci. 2006; 1088: 219-229
        • Showalter V.M.
        • Compton D.R.
        • Martin B.R.
        • Abood M.E.
        J. Pharmacol. Exp. Ther. 1996; 278: 989-999
        • Pertwee R.G.
        Br. J. Pharmacol. 2008; 153: 199-215
        • Mackie K.
        J. Neuroendocrinol. 2008; 20: 10-14
        • Buckley N.E.
        • McCoy K.L.
        • Mezey E.
        • Bonner T.
        • Zimmer A.
        • Felder C.C.
        • Glass M.
        • Zimmer A.
        Eur. J. Pharmacol. 2000; 396: 141-149
        • Romero-Sandoval E.A.
        • Horvath R.
        • Landry R.P.
        • DeLeo J.A.
        Mol. Pain. 2009; 5: 25
        • Cabral G.A.
        • Harmon K.N.
        • Carlisle S.J.
        Adv. Exp. Med. Biol. 2001; 493: 207-214
        • Bayewitch M.
        • Rhee M.H.
        • Avidor-Reiss T.
        • Breuer A.
        • Mechoulam R.
        • Vogel Z.
        J. Biol. Chem. 1996; 271: 9902-9905
        • de Filippis D.
        • Iuvone T.
        • d'amico A.
        • Esposito G.
        • Steardo L.
        • Herman A.G.
        • Pelckmans P.A.
        • de Winter B.Y.
        • de Man J.G.
        Neurogastroenterol. Motil. 2008; 20: 919-927
        • Sacerdote P.
        • Martucci C.
        • Vaccani A.
        • Bariselli F.
        • Panerai A.E.
        • Colombo A.
        • Parolaro D.
        • Massi P.
        J. Neuroimmunol. 2005; 159: 97-105
        • Járai Z.
        • Wagner J.A.
        • Varga K.
        • Lake K.D.
        • Compton D.R.
        • Martin B.R.
        • Zimmer A.M.
        • Bonner T.I.
        • Buckley N.E.
        • Mezey E.
        • Razdan R.K.
        • Zimmer A.
        • Kunos G.
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 14136-14141
        • Makriyannis A.
        • Yang D.P.
        • Griffin R.G.
        • Das Gupta S.K.
        Biochim. Biophys. Acta. 1990; 1028: 31-42
        • Felder C.C.
        • Veluz J.S.
        • Williams H.L.
        • Briley E.M.
        • Matsuda L.A.
        Mol. Pharmacol. 1992; 42: 838-845
        • Puffenbarger R.A.
        • Boothe A.C.
        • Cabral G.A.
        Glia. 2000; 29: 58-69
        • Berdyshev E.V.
        • Schmid P.C.
        • Krebsbach R.J.
        • Hillard C.J.
        • Huang C.
        • Chen N.
        • Dong Z.
        • Schmid H.H.
        Biochem. J. 2001; 360: 67-75
        • Price T.J.
        • Patwardhan A.
        • Akopian A.N.
        • Hargreaves K.M.
        • Flores C.M.
        Br. J. Pharmacol. 2004; 142: 257-266
        • Kaplan B.L.
        • Rockwell C.E.
        • Kaminski N.E.
        J. Pharmacol. Exp. Ther. 2003; 306: 1077-1085
        • Watzl B.
        • Scuderi P.
        • Watson R.R.
        Int. J. Immunopharmacol. 1991; 13: 1091-1097
        • Srivastava M.D.
        • Srivastava B.I.
        • Brouhard B.
        Immunopharmacology. 1998; 40: 179-185
        • Gallily R.
        • Yamin A.
        • Waksmann Y.
        • Ovadia H.
        • Weidenfeld J.
        • Bar-Joseph A.
        • Biegon A.
        • Mechoulam R.
        • Shohami E.
        J. Pharmacol. Exp. Ther. 1997; 283: 918-924
        • Smith S.R.
        • Terminelli C.
        • Denhardt G.
        J. Pharmacol. Exp. Ther. 2000; 293: 136-150
        • Roche M.
        • Diamond M.
        • Kelly J.P.
        • Finn D.P.
        J. Neuroimmunol. 2006; 181: 57-67
        • Covert M.W.
        • Leung T.H.
        • Gaston J.E.
        • Baltimore D.
        Science. 2005; 309: 1854-1857
        • Jeon Y.J.
        • Yang K.H.
        • Pulaski J.T.
        • Kaminski N.E.
        Mol. Pharmacol. 1996; 50: 334-341
        • Herring A.C.
        • Kaminski N.E.
        J. Pharmacol. Exp. Ther. 1999; 291: 1156-1163
        • Rajesh M.
        • Mukhopadhyay P.
        • Haskó G.
        • Huffman J.W.
        • Mackie K.
        • Pacher P.
        Br. J. Pharmacol. 2008; 153: 347-357
        • De Filippis D.
        • Russo A.
        • De Stefano D.
        • Maiuri M.C.
        • Esposito G.
        • Cinelli M.P.
        • Pietropaolo C.
        • Carnuccio R.
        • Russo G.
        • Iuvone T.
        J. Mol. Med. 2007; 85: 635-645
        • Panikashvili D.
        • Mechoulam R.
        • Beni S.M.
        • Alexandrovich A.
        • Shohami E.
        J. Cereb. Blood Flow Metab. 2005; 25: 477-484
        • Esposito G.
        • De Filippis D.
        • Maiuri M.C.
        • De Stefano D.
        • Carnuccio R.
        • Iuvone T.
        Neurosci. Lett. 2006; 399: 91-95
        • Curran N.M.
        • Griffin B.D.
        • O'Toole D.
        • Brady K.J.
        • Fitzgerald S.N.
        • Moynagh P.N.
        J. Biol. Chem. 2005; 280: 35797-35806
        • Murray P.J.
        Biochem. Soc. Trans. 2006; 34: 1028-1031
        • Butcher B.A.
        • Kim L.
        • Panopoulos A.D.
        • Watowich S.S.
        • Murray P.J.
        • Denkers E.Y.
        J. Immunol. 2005; 174: 3148-3152
        • Barton B.E.
        Expert Opin. Ther. Targets. 2006; 10: 459-470
        • Takeda K.
        • Clausen B.E.
        • Kaisho T.
        • Tsujimura T.
        • Terada N.
        • Förster I.
        • Akira S.
        Immunity. 1999; 10: 39-49
        • Akira S.
        Oncogene. 2000; 19: 2607-2611
        • Costa-Pereira A.P.
        • Tininini S.
        • Strobl B.
        • Alonzi T.
        • Schlaak J.F.
        • Is'harc H.
        • Gesualdo I.
        • Newman S.J.
        • Kerr I.M.
        • Poli V.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 8043-8047
        • Qing Y.
        • Stark G.R.
        J. Biol. Chem. 2004; 279: 41679-41685
        • Tanabe Y.
        • Nishibori T.
        • Su L.
        • Arduini R.M.
        • Baker D.P.
        • David M.
        J. Immunol. 2005; 174: 609-613
        • Matsukawa A.
        • Kudo S.
        • Maeda T.
        • Numata K.
        • Watanabe H.
        • Takeda K.
        • Akira S.
        • Ito T.
        J. Immunol. 2005; 175: 3354-3359
        • Jin H.J.
        • Shao J.Z.
        • Xiang L.X.
        • Wang H.
        • Sun L.L.
        Mol. Immunol. 2008; 45: 1258-1268
        • Croker B.A.
        • Kiu H.
        • Nicholson S.E.
        Semin. Cell Dev. Biol. 2008; 19: 414-422
        • Wormald S.
        • Hilton D.J.
        Curr. Opin. Hematol. 2007; 14: 9-15
        • Hong F.
        • Jaruga B.
        • Kim W.H.
        • Radaeva S.
        • El-Assal O.N.
        • Tian Z.
        • Nguyen V.A.
        • Gao B.
        J. Clin. Invest. 2002; 110: 1503-1513
        • Yasukawa H.
        • Ohishi M.
        • Mori H.
        • Murakami M.
        • Chinen T.
        • Aki D.
        • Hanada T.
        • Takeda K.
        • Akira S.
        • Hoshijima M.
        • Hirano T.
        • Chien K.R.
        • Yoshimura A.
        Nat. Immunol. 2003; 4: 551-556
        • Nishinakamura H.
        • Minoda Y.
        • Saeki K.
        • Koga K.
        • Takaesu G.
        • Onodera M.
        • Yoshimura A.
        • Kobayashi T.
        Int. Immunol. 2007; 19: 609-619
        • Ogawa S.
        • Lozach J.
        • Benner C.
        • Pascual G.
        • Tangirala R.K.
        • Westin S.
        • Hoffmann A.
        • Subramaniam S.
        • David M.
        • Rosenfeld M.G.
        • Glass C.K.
        Cell. 2005; 122: 707-721
        • Lacaze P.
        • Raza S.
        • Sing G.
        • Page D.
        • Forster T.
        • Storm P.
        • Craigon M.
        • Awad T.
        • Ghazal P.
        • Freeman T.C.
        BMC Genomics. 2009; 10: 372
        • Ock J.
        • Jeong J.
        • Choi W.S.
        • Lee W.H.
        • Kim S.H.
        • Kim I.K.
        • Suk K.
        J. Neurosci. Res. 2007; 85: 1989-1995
        • Pais T.F.
        • Figueiredo C.
        • Peixoto R.
        • Braz M.H.
        • Chatterjee S.
        J. Neuroinflammation. 2008; 5: 43