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Cannabidiol, a non-psychoactive cannabinoid, leads to EGR2-dependent anergy in activated encephalitogenic T cells.

Kozela E, Juknat A, Kaushansky N, Ben-Nun A, Coppola G, Vogel Z - J Neuroinflammation (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

CBD treatment results in upregulation of CD69 and of LAG3 regulatory molecules on CD4+CD25−T cells in MOG35-55-stimulated APC/TMOGco-cultures. APC/TMOG co-cultures were treated with MOG35-55 and CBD. (A) Percentage of CD4+CD25− T cells expressing CD69 (ANOVA F(3,12) = 68.9; P < 0.001); (B) percentage of CD4+CD25− T cells expressing LAG3 (ANOVA F(3,12) = 13.0, P < 0.001); (C) the representative contour plot density graphs showing the co-expression of CD69 and LAG3 in the CD4+CD25− subpopulation; (D) percentage of CD4+CD25− cells expressing both CD69 and LAG3 ± SEM. ANOVA F(3,7) = 7.3, P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001 vs non-stimulated cells; #P < 0.05, ##P < 0.01, ###P < 0.001 vs MOG35-55-stimulated cells. n = 3 to 4.
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Fig2: CBD treatment results in upregulation of CD69 and of LAG3 regulatory molecules on CD4+CD25−T cells in MOG35-55-stimulated APC/TMOGco-cultures. APC/TMOG co-cultures were treated with MOG35-55 and CBD. (A) Percentage of CD4+CD25− T cells expressing CD69 (ANOVA F(3,12) = 68.9; P < 0.001); (B) percentage of CD4+CD25− T cells expressing LAG3 (ANOVA F(3,12) = 13.0, P < 0.001); (C) the representative contour plot density graphs showing the co-expression of CD69 and LAG3 in the CD4+CD25− subpopulation; (D) percentage of CD4+CD25− cells expressing both CD69 and LAG3 ± SEM. ANOVA F(3,7) = 7.3, P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001 vs non-stimulated cells; #P < 0.05, ##P < 0.01, ###P < 0.001 vs MOG35-55-stimulated cells. n = 3 to 4.

Mentions: At the second step, we analyzed the CD4+CD25− population for the expression of CD69 molecule. In control, non-stimulated APC/TMOG co-cultures 9.3% ± 0.4% of the CD4+CD25− cells were found to be positive for CD69 (Figure 2A). Interestingly, 18 h co-incubation with CBD resulted in a very significant increase in CD4+CD25−CD69+ cells reaching 22.5% ± 1.3% (P < 0.001 vs 9.3% ± 0.4% observed in the non-stimulated APC/TMOG co-cultures). MOG35-55-stimulation of APC/TMOG co-cultures resulted in doubling the CD69 expression on CD4+CD25− cells up to 18.0% ± 1.5% (P < 0.001 vs non-stimulated cells), and CBD addition together with MOG35-55 further increased the frequency of CD4+CD25−CD69+ cells up to 36.6% ± 1.9% (P < 0.001 vs MOG-treated cells).Figure 2


Cannabidiol, a non-psychoactive cannabinoid, leads to EGR2-dependent anergy in activated encephalitogenic T cells.

Kozela E, Juknat A, Kaushansky N, Ben-Nun A, Coppola G, Vogel Z - J Neuroinflammation (2015)

CBD treatment results in upregulation of CD69 and of LAG3 regulatory molecules on CD4+CD25−T cells in MOG35-55-stimulated APC/TMOGco-cultures. APC/TMOG co-cultures were treated with MOG35-55 and CBD. (A) Percentage of CD4+CD25− T cells expressing CD69 (ANOVA F(3,12) = 68.9; P < 0.001); (B) percentage of CD4+CD25− T cells expressing LAG3 (ANOVA F(3,12) = 13.0, P < 0.001); (C) the representative contour plot density graphs showing the co-expression of CD69 and LAG3 in the CD4+CD25− subpopulation; (D) percentage of CD4+CD25− cells expressing both CD69 and LAG3 ± SEM. ANOVA F(3,7) = 7.3, P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001 vs non-stimulated cells; #P < 0.05, ##P < 0.01, ###P < 0.001 vs MOG35-55-stimulated cells. n = 3 to 4.
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Fig2: CBD treatment results in upregulation of CD69 and of LAG3 regulatory molecules on CD4+CD25−T cells in MOG35-55-stimulated APC/TMOGco-cultures. APC/TMOG co-cultures were treated with MOG35-55 and CBD. (A) Percentage of CD4+CD25− T cells expressing CD69 (ANOVA F(3,12) = 68.9; P < 0.001); (B) percentage of CD4+CD25− T cells expressing LAG3 (ANOVA F(3,12) = 13.0, P < 0.001); (C) the representative contour plot density graphs showing the co-expression of CD69 and LAG3 in the CD4+CD25− subpopulation; (D) percentage of CD4+CD25− cells expressing both CD69 and LAG3 ± SEM. ANOVA F(3,7) = 7.3, P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001 vs non-stimulated cells; #P < 0.05, ##P < 0.01, ###P < 0.001 vs MOG35-55-stimulated cells. n = 3 to 4.
Mentions: At the second step, we analyzed the CD4+CD25− population for the expression of CD69 molecule. In control, non-stimulated APC/TMOG co-cultures 9.3% ± 0.4% of the CD4+CD25− cells were found to be positive for CD69 (Figure 2A). Interestingly, 18 h co-incubation with CBD resulted in a very significant increase in CD4+CD25−CD69+ cells reaching 22.5% ± 1.3% (P < 0.001 vs 9.3% ± 0.4% observed in the non-stimulated APC/TMOG co-cultures). MOG35-55-stimulation of APC/TMOG co-cultures resulted in doubling the CD69 expression on CD4+CD25− cells up to 18.0% ± 1.5% (P < 0.001 vs non-stimulated cells), and CBD addition together with MOG35-55 further increased the frequency of CD4+CD25−CD69+ cells up to 36.6% ± 1.9% (P < 0.001 vs MOG-treated cells).Figure 2

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus