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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

Decrease in CD4+CD25+cells in MOG35-55-stimulated APC/TMOGco-cultures is not affected by CBD. Splenocytes (including accessory CD4+ T cells) were co-cultured with TMOG cells and stimulated with MOG35-55 at 5 μg/ml. CBD at 5 μM was added to the cells immediately before MOG35-55. (A) Bar graph showing the average percentage of CD4+ cells in APC/TMOG co-cultures (100% as equal to total number of cells). Neither treatment affected overall CD4+ T cell number; (B) dot plot histogram presenting CD4+ T cells in APC/TMOG co-cultures positive and negative for CD25 expression; (C) a representative dot plot showing the changes in CD4+CD25+ T cell population in control or MOG35-55-treated APC/TMOG co-cultures in the presence or absence of CBD; (D) bar graph showing the number of cells positive for CD4 and CD25 ± SEM. ANOVA F(3,8) = 7.8, P < 0.01. *P < 0.05 vs non-stimulated cells. n = 3 to 4.
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Fig1: Decrease in CD4+CD25+cells in MOG35-55-stimulated APC/TMOGco-cultures is not affected by CBD. Splenocytes (including accessory CD4+ T cells) were co-cultured with TMOG cells and stimulated with MOG35-55 at 5 μg/ml. CBD at 5 μM was added to the cells immediately before MOG35-55. (A) Bar graph showing the average percentage of CD4+ cells in APC/TMOG co-cultures (100% as equal to total number of cells). Neither treatment affected overall CD4+ T cell number; (B) dot plot histogram presenting CD4+ T cells in APC/TMOG co-cultures positive and negative for CD25 expression; (C) a representative dot plot showing the changes in CD4+CD25+ T cell population in control or MOG35-55-treated APC/TMOG co-cultures in the presence or absence of CBD; (D) bar graph showing the number of cells positive for CD4 and CD25 ± SEM. ANOVA F(3,8) = 7.8, P < 0.01. *P < 0.05 vs non-stimulated cells. n = 3 to 4.

Mentions: TMOG cells were co-cultured with splenocytes (APC/TMOG co-cultures) and stimulated with MOG35-55 at 5 μg/ml for 18 h in the presence or absence of 5 μM CBD, and the number of CD4+ cells was determined using flow cytometry. As presented in Figure 1A, neither MOG35-55-stimulation nor CBD addition affected the total number of CD4+ cells in these co-cultures. Next, we analyzed the frequency of CD4+CD25+ natural regulatory T cells in APC/TMOG co-cultures (Figure 1B). We observed that in control, non-stimulated co-cultures 11.0% ± 1.4% of CD4+ cells were CD25 positive (CD4+CD25+) and this frequency did not change following CBD treatment (10.4% ± 1.5%; Figure 1C,D). MOG35-55-stimulation of these APC/TMOG co-cultures led to a significant decrease in CD4+CD25+ cells (down to 5.5% ± 0.5% of all CD4+, P < 0.05), and this level was not affected by CBD co-incubation (5.6% ± 0.3%).Figure 1


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)

Decrease in CD4+CD25+cells in MOG35-55-stimulated APC/TMOGco-cultures is not affected by CBD. Splenocytes (including accessory CD4+ T cells) were co-cultured with TMOG cells and stimulated with MOG35-55 at 5 μg/ml. CBD at 5 μM was added to the cells immediately before MOG35-55. (A) Bar graph showing the average percentage of CD4+ cells in APC/TMOG co-cultures (100% as equal to total number of cells). Neither treatment affected overall CD4+ T cell number; (B) dot plot histogram presenting CD4+ T cells in APC/TMOG co-cultures positive and negative for CD25 expression; (C) a representative dot plot showing the changes in CD4+CD25+ T cell population in control or MOG35-55-treated APC/TMOG co-cultures in the presence or absence of CBD; (D) bar graph showing the number of cells positive for CD4 and CD25 ± SEM. ANOVA F(3,8) = 7.8, P < 0.01. *P < 0.05 vs non-stimulated cells. n = 3 to 4.
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Related In: Results  -  Collection

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Fig1: Decrease in CD4+CD25+cells in MOG35-55-stimulated APC/TMOGco-cultures is not affected by CBD. Splenocytes (including accessory CD4+ T cells) were co-cultured with TMOG cells and stimulated with MOG35-55 at 5 μg/ml. CBD at 5 μM was added to the cells immediately before MOG35-55. (A) Bar graph showing the average percentage of CD4+ cells in APC/TMOG co-cultures (100% as equal to total number of cells). Neither treatment affected overall CD4+ T cell number; (B) dot plot histogram presenting CD4+ T cells in APC/TMOG co-cultures positive and negative for CD25 expression; (C) a representative dot plot showing the changes in CD4+CD25+ T cell population in control or MOG35-55-treated APC/TMOG co-cultures in the presence or absence of CBD; (D) bar graph showing the number of cells positive for CD4 and CD25 ± SEM. ANOVA F(3,8) = 7.8, P < 0.01. *P < 0.05 vs non-stimulated cells. n = 3 to 4.
Mentions: TMOG cells were co-cultured with splenocytes (APC/TMOG co-cultures) and stimulated with MOG35-55 at 5 μg/ml for 18 h in the presence or absence of 5 μM CBD, and the number of CD4+ cells was determined using flow cytometry. As presented in Figure 1A, neither MOG35-55-stimulation nor CBD addition affected the total number of CD4+ cells in these co-cultures. Next, we analyzed the frequency of CD4+CD25+ natural regulatory T cells in APC/TMOG co-cultures (Figure 1B). We observed that in control, non-stimulated co-cultures 11.0% ± 1.4% of CD4+ cells were CD25 positive (CD4+CD25+) and this frequency did not change following CBD treatment (10.4% ± 1.5%; Figure 1C,D). MOG35-55-stimulation of these APC/TMOG co-cultures led to a significant decrease in CD4+CD25+ cells (down to 5.5% ± 0.5% of all CD4+, P < 0.05), and this level was not affected by CBD co-incubation (5.6% ± 0.3%).Figure 1

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