In the present study, we characterized the CBD effects on the transcriptional activity of mouse MOG35-55-activated encephalitogenic T_MOG cells, the latter known to induce autoimmune pathologies. We previously showed that CBD decreases IL-17 and IL-6 mRNA production and protein secretion in MOG35-55-stimulated T_MOG co-cultured with APC while increasing anti-inflammatory IL-10 mRNA synthesis [24, 26]. In parallel, we showed that CBD negatively regulates STAT3 phosphorylation while enhancing STAT5 activity, main positive and negative regulators of Th17 differentiation, respectively [36], further confirming the regulatory effect of CBD on Th17 function of memory T cells [26]. The present gene profiling reveals that CBD targets a relatively small number of genes (163) within a large group of those significantly affected by MOG35-55 stimulation (reaching 1936 transcripts). Bioinformatics analysis performed on these CBD affected transcripts assembled them into various pathways and gene networks mediating the effects of CBD on the MOG35-55-activated memory T cells. In line with our previous studies, this gene profiling confirms that CBD affects various processes controlling “Th17 differentiation”, including “IL-6 signaling” (a pathway promoting memory Th17 generation) and anti-inflammatory “IL10 signaling” (known to restrain Th17 function and Th17-mediated autoimmunity [25, 37]).

CBD upregulated the transcription of 81 genes and downregulated 82 genes in MOG35-55-stimulated T_MOG. For example, CBD was found to decrease the MOG35-55-upregulated mRNA levels of various proinflammatory mediators including interleukins (Il3, Il1b, and Il12a), chemokines (Xcl1 and Cxcl9) and receptors (Cxcr1, Ifngr1, Gpr83), molecules known to be involved in Th17 generation and function [38–40]. In addition, CBD downregulated elements of various signaling cascades, including members of the TNF superfamily (e.g., Tnfsf11, Tnfrsf18), molecules mediating cell adhesion (e.g., Amica1) growth, structure, and metabolism (e.g., Cdk5r1, Tubb2d, Dgat1). The effect of CBD on the TNF superfamily members was found to be very diverse. This is not surprising since the TNF superfamily consists of a large number of molecules involved in multiple cellular processes including inflammation, cell growth, cell cycle, differentiation, metabolism, and cytokine production [41]. The mRNA of the cAMP-responsive element modulator (CREM) transcription factor was also significantly suppressed by CBD in activated encephalitogenic T_MOG cells. CREM activity was shown to control IL-2 and IL-17 expression during CD4 lineage commitment and distribution [42]. Indeed, human autoimmune T cells were shown to display increased expression of CREM which accounted for their increased IL-17 release and autoimmunity (e.g., in systemic lupus erythematosus and MS-like pathologies [43, 44]). Thus, the decrease in Crem mRNA found here may serve as an important contribution to the CBD-induced suppression of the Th17-like activity of the T_MOG cells. This downregulation of Crem mRNA by CBD does not seem to be mediated via the cannabinoid receptors CB1 and CB2, both known to affect adenylyl cyclase activity and cAMP formation [45, 46]. This is due to the fact that CBD was shown to have negligible affinity toward these receptors [47]. Moreover, we showed previously that CBD effects on T_MOG do not involve these receptors [12, 24].

Several of the MOG35-55-induced transcripts were preserved at high levels or even further upregulated in the presence of CBD. Egr2 transcription factor was one of the highest upregulated representative of this group along with a number of cytokines (Csf2, Ifng, Tnf), chemokines (Ccl3, Ccl4, Cxcl10) and regulatory glycoproteins (Sema7a). This gene expression pattern may indicate a promotion by CBD of tolerogenic conditions driving exhaustion of T cells (anergy) and thus leading to the functional deactivation of T cells [48]. Indeed, EGR2 is an acknowledged T cell anergy marker which induction turns on a genetic program driving cell cycle arrest, eventually dampening immune responses [49, 50] including IL-17 production [51, 52]. Indeed, we have recently shown that CBD induces Egr2-dependent anergy in encephalitogenic T cells and that CBD enhances the expression of several other anergy signature genes including Il10 and Lag3 [26].

MOG35-55-upregulated Ifng mRNA production and secretion were not affected by CBD in stimulated T_MOG cells [24]. This result was reproduced by the current microarray analysis showing no effect of CBD on the IFNγ upstream transcription factor Tbx21 mRNA. On the other hand, the present results show that CBD has a potent enhancing effect on several IFN-dependent transcripts including Irf4, Ifit3, Rsad2/Viperin, and Mx2. Type I (IFN-α and -β) and type II (IFN-γ) interferons are known to mediate innate immune responses and inhibit the proliferation of virus-infected cells [53]. IFNs exert antiproliferative and immunomodulatory effects and IFNγ expressing cells were shown to counteract Th17 activity [54]. Alterations in IFN-stimulated gene patterns (including in Ifit, Mx2, Cxcl10) and/or in Jak/STAT activity, a main IFN-dependent pathway, cause a failure in immunological tolerance resulting in T cell-mediated autoimmune pathologies (e.g., type I diabetes) [55]. Thus, we assume that CBD-enhanced IFN-controlled pathways may contribute to CBD anti-proliferative effects in activated memory T cells, as already observed by us previously [12].

Interferon regulatory factor 4 (IRF4, a lymphocyte-specific nuclear factor) was shown to be a core promoter of other IFN-dependent genes such as Rsad2 and Ifit3 [56]. IRF4 was reported to mediate the differentiation of naive CD4+ T cells into various T helper lineages [57]. IRF4 potently synergizes with NFAT signaling to enhance the promoter activity of anti-inflammatory IL-4 cytokine [58]. Indeed, in our hands Nfatc1 mRNA, an element of the NFAT signaling axis, was upregulated by CBD.

The transcription of several genes was downregulated by MOG35-55 stimulation. This group of genes included IL-17 receptors (Il17re variant 1 and 2, Il17rc). Interestingly, CBD did not reverse this Il17r mRNA downregulation suggesting that CBD does not interfere with intrinsic negative feedback mechanisms restraining the activity of memory T cells.

Antigen-induced T cell activation occurs following peptide presentation by major histocompatibility complex class II (MHCII) molecules on APC cells. A number of molecules negatively regulate these APC/T interactions [59]. Our study shows that CBD is a powerful inducer of negative regulators of APC/T interactions as CBD increased the mRNA levels of: B and T lymphocyte attenuator (Btla/Cd272); lymphocyte-activation gene 3 (Lag3/Cd223); cluster of differentiation 69 (Cd69) and of T cell receptor associated transmembrane adaptor 1/T-cell receptor interacting molecule (Trat1/TRIM). These molecules are members of a broad, structurally related group of immunoglobulin receptors that were found to serve as markers of various inducible T cell regulatory phenotypes involved in Th17 suppression and amelioration of MS-like pathologies [59–62]. For example, LAG3 is a CD4 receptor homologue that by interfering with MHCII on APC upon antigen exposure inhibits the function and expansion of memory T cells [63] decreasing IL-17 and increasing IL-10 secretions, this way preventing autoimmunity in mice [64, 65]. Mice lacking the CD69 transmembrane receptor develop aggravated forms of autoimmune pathologies including arthritis, contact dermatitis, allergic asthma, and autoimmune myocarditis [66]. Indeed, CD69-deficient T cells demonstrate increased differentiation into Th17 cells, higher IL-17 release as well as Jak3/Stat5 and RORγt pathways’ increased activities [67] (Martin 2010). Similarly, BTLA ligation with its ligand, herpes virus entry mediator (HVEM/TNFRSF14), was shown to attenuate T cell proliferation [68, 69] and to promote anergy [70]. Functional BTLA/HVEM pathway maintains immune tolerance and prevents autoimmunity (for review see [71] and BTLA-deficiency in mice leads to T cell-mediated rheumatoid arthritis, lymphocytic infiltration, autoimmune hepatitis-like diseases, and EAE [72–74]. CTLA-4 (CD152) is an inhibitory receptor which in its inactive state is located intracellularly and upon demand is trafficked by TRAT1/TRIM protein (that is upregulated by CBD) to the membrane [75]. CTLA-4 negatively competes on T cells with CD28, a co-stimulatory T cell molecule, in binding to the co-activators CD80 and CD86 on APC cells [76, 77]. TRAT1/TRIM-dependent increase in CTLA4 expression led to suppression of encephalitogenic T cells expansion and of EAE development [78]. In agreement with this, impaired CTLA-4 expression on T cells was found to correlate with increased IL-17 release and more severe EAE [79]. Moreover, CTLA-4-deficient mice show an autoimmune phenotype with T cell-mediated organ destruction [80–82]. Thus, CBD upregulation of these powerful inhibitors of APC/T communication should exert robust immunoregulatory effect in MOG35-55 activated T_MOG resulting in their suppressed activity. Indeed, complement WGCNA analysis confirmed the inhibitory role of CBD in processes related to antigen presentation and processing and APC/T_MOG interactions. Moreover, these observations are in agreement with our previous report showing that CBD decreases the activation of B cells (the main APC in the periphery) incubated with T_MOG as well as MHCII expression on these cells [26].

Reactive oxygen species generated primarily by macrophages and damaged neurons, increase immune response and T cell activation, propagate demyelination and neurodegeneration [83]. The present gene profiling links the CBD effects to nuclear factor-erythroid 2-related factor 2 (Nfe2l2/Nrf2)-driven antioxidant genetic program, known to exert immunoregulatory effects [84, 85]. Our results show that CBD enhances the expression of Nrf2 target genes including Heme oxygenase-1 (Hmox-1 encoding HO-1 protein) and metallothioneins (Mt1 and Mt2). Indeed, Mt(−/−) mice show increased macrophage and T-lymphocyte infiltration into the CNS as well as increased cytokine release, oxidative stress and axon damage as compared to their wildtype EAE littermates [86]. HO-1 was shown to diminish inflammatory reactions, e.g., via induction of the anti-inflammatory IL10 cytokine and modulation of Jak/STAT pathway [87]. Indeed, pharmacological or genetic induction of Nrf2-dependent anti-oxidant genes (i.e., Hmox1, Mt1, and Mt2) in EAE mice led to reduction in IL-17 levels and decreased EAE severity [88]. Moreover, Hmox1−/− C57BL/6 mice develop enhanced EAE-like paralysis and CNS demyelination while HO-1 restoration diminished EAE progression and relapses [89]. Interestingly, this protective effect of HO-1 has been associated with inhibition of MHCII expression by APCs via mechanisms involving carbon monoxide, an antioxidant product of HO-1 activity. Thus, CBD induction of Nfe2l2/Nrf2-dependent antioxidant genes may be one of the key mechanisms driving the anti-inflammatory effects of this cannabinoid in T_MOG cells and EAE. Similar CBD regulation of anti-oxidant genes was reported by us in quiescent and LPS-stimulated microglial cells [30, 90]. These findings suggest that Nrf2-dependent immunoregulation may be an important feature of this cannabinoid contributing to its anti-inflammatory effects. However, we cannot rule out that CBD is also affecting several other pathways, e.g., receptors for A2A as well as dimers of CB2/5HT1A [91]. In addition, CBD was shown to increase the endocannabinoid levels, known to modulate immune functions [92], thus it may indirectly exert immunoregulatory actions on T cells through CB2 receptors, previously suggested to modulate T cell-mediated autoimmunity [93].