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Comparative Study
. 2019 Apr 15:369:82-89.
doi: 10.1016/j.taap.2019.02.013. Epub 2019 Feb 23.

Suppression of CpG-ODN-mediated IFNα and TNFα response in human plasmacytoid dendritic cells (pDC) by cannabinoid receptor 2 (CB2)-specific agonists

Joseph E Henriquez  1 , Robert B Crawford  2 , Norbert E Kaminski  3
Affiliations
Comparative Study

Suppression of CpG-ODN-mediated IFNα and TNFα response in human plasmacytoid dendritic cells (pDC) by cannabinoid receptor 2 (CB2)-specific agonists

Joseph E Henriquez et al. Toxicol Appl Pharmacol. .

Abstract

Plasmacytoid dendritic cells (pDC) compose 0.2-0.5% of circulating leukocytes but play a significant role in mounting host immune responses. Elevated and chronic activation of pDC are implicated in autoimmune disease like systemic lupus erythematosus and rheumatoid arthritis. Δ9-tetrahydrocannabinol (THC) is a well characterized cannabinoid with potent anti-inflammatory activity, but acceptance of THC as a treatment for autoimmune disorders has been hindered due to psychotropic activity. The psychotropic effects of THC are mediated through cannabinoid receptor 1 (CB1) expressed in the central nervous system while the immunomodulatory effects of THC result from THC binding to CB1 and CB2 on immune cells. Synthetic CB2-selective agonists have been developed to explore immune modulation by cannabinoids in the absence of psychotropic effects. The goal of these studies was to determine if the CB2-selective agonists, JWH-015 and JWH-133, have comparable efficacy to THC in modulating IFNα and TNFα responses by primary human pDC. Treatment with JWH-133 and JWH-015 inhibited CpG-induced IFNα and TNFα responses by pDC. Further, the phosphorylation of IRF7, TBK1, NFκB, and IKKγ, key events in pDC activation, were suppressed by THC, JWH-133, and JWH-015. Likewise, the phosphorylation of AKT at the S473 and T308 residues were differentially modulated by treatment with THC and both JWH compounds. Collectively, these results demonstrate the potential for CB2 targeted therapeutics for treatment of inflammatory conditions involving aberrant pDC activity.

Keywords: Cannabinoids; Interferon α; Plasmacytoid dendritic cells; Toll-like receptors; Tumor necrosis factor α; Δ9-Tetrahydrocannabinol.

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Conflict of interest statement

Conflict of interest

The authors report no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Treatment with THC, JWH-015 and JWH-133 inhibited CpG-induced IFNα and TNFα responses in pDC. Isolated human PBMCs were treated with either Vehicle (VH; 0.03% Ethanol), CBD (10 μM), THC (0.1, 1, or 10 μM), JWH-015 (10−2, 10−1, 100 μM) or JWH-133 at (10−3, 10−2, 10−1 μM) for 30 min, stimulated with CpG-ODN at 15 μg/ml for 6 h, and intracellularly stained for either IFNα or TNFα (N = 5 donors). pDC > 90% viability within 6 h of treatment. (A) pDC were identified by first gating on singlets, then focusing on the interface of leukocytes and monocytes based on forward and side scatter area, and then on CD303+ CD123+ cells. (B) Example of gating for IFNα and TNFα positive pDC in resting cells cultured in the presence of VH and following CpG stimulation. (C) CpG induced intracellular expression of IFNα which was suppressed by THC, JWH-015, and JWH-133. (D) CpG induced intracellular expression of TNFα which was suppressed by THC, JWH-015, and JWH-133. Asterisks indicate statistically significant differences in IFNα or TNFα expression compared to 0 THC + CpG (*** = P < .001, ** = P < .01, * = P < .05) using a 1-Way ANOVA with Dunnett’s posttest.
Fig. 2.
Fig. 2.
Treatment with THC, JWH-015, and JWH-133 inhibited the CpG-induced phosphorylation of IRF7 and TBK1 in pDC. Isolated human PBMCs were treated with either Vehicle (VH; 0.03% Ethanol), CBD (10 μM), THC (0.1, 1, or 10 μM), JWH-015 (10−2, 10−1, 100 μM) or JWH-133 at (10−3, 10−2, 10−1 μM) for 30 min, stimulated with CpG-ODN at 15 μg/ml for 5 h and intracellularly stained for pIRF7 or pTBK1 (N = 5 donors). pDC > 90% viability within 5 h of treatment (A) Example of gating for pIRF7 and pTBK1 in the presence of VH, with and without CpG stimulation. (B) CpG induced intracellular expression of pIRF7 was suppressed by THC, JWH-015, and JWH-133. (C) CpG induced intracellular expression of pTBK1 was suppressed by THC, JWH-015, and JWH-133. Asterisks indicate statistically significant differences in IRF7 and TBK1 phosphorylation compared to VC + CpG (*** = P < .001, ** = P < .01, * = P < .05) using a 1-Way ANOVA with Dunnett’s posttest.
Fig. 3.
Fig. 3.
Treatment with THC, JWH-015, and JWH-133 inhibited the CpG-induced phosphorylation of the p65 subunit of NFκB and IKKγ (NEMO) in pDC. Isolated human PBMCs were treated with either Vehicle (VH; 0.03% Ethanol), CBD (10 μM), THC (0.1, 1, or 10 μM), JWH-015 (10−2, 10−1, 100 μM) or JWH-133 at (10−3, 10−2, 10−1 μM) for 30 min, stimulated with CpG-ODN at 15 μg/ml for 5 h and intracellularly stained for p65 (pNFκB) or IKKγ (N = 5 donors). pDC > 90% viability within 5 h of treatment. (A) Example of gating for p65 (pNFκB) and pIKKγ with VH treated resting and CpG stimulated pDC. (B) CpG induced intracellular expression of pNFκB was suppressed by THC, JWH-015, and JWH-133. (C) CpG induced intracellular expression of IKKγ was suppressed by THC and both CB2-selective agonists. Asterisks indicate statistically significant differences in p65 and IKKγ phosphorylation compared to VC + CpG (*** = P < .001, ** = P < .01, * = P < .05) using a 1-Way ANOVA with Dunnett’s posttest.
Fig. 4.
Fig. 4.
Treatment with THC, JWH-015, and JWH-133 differentially affected the phosphorylation of the AKT at the T308 and S473 residues. Isolated human PBMCs were treated with either Vehicle (VC; 0.03% Ethanol), CBD (10 μM), THC (0.1, 1, or 10 μM), JWH-015 (10−2, 10−1, 100 μM) or JWH-133 at (10−3, 10−2, 10−1 μM) for 30 min, stimulated with CpG-ODN at 15 μg/ml for 5 h and intracellularly stained for either pT308 or pS473 AKT (N = 5 donors). pDC > 90% viability within 5 h of treatment. (A) Example of gating for pT308 and pS473 with VH treated resting and CpG stimulated pDC. (B) CpG induced intracellular expression of pT308 which was suppressed by THC, JWH-015, and JWH-133. (C) CpG had no significant effect on pS478 expression and treatment with THC reduced pS478 while neither JWH-015 or JWH-133 had a significant effect. Asterisks indicate statistically significant differences in p65 and IKKγ phosphorylation compared to VC + CpG (*** = P < .001, ** = P < .01, * = P < .05) using a 1-Way ANOVA with Dunnett’s posttest.
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