Cannabidiol exerts multitarget immunomodulatory effects on PBMCs from individuals with psoriasis vulgaris

Abstract

Introduction: The involvement of endocannabinoid system (ECS) in the inflammatory cascade, and the ability of phytocannabinoids, endocannabinoids and their synthetic analogues to modulate it has become an interesting research area for new therapeutic approaches in inflammatory skin diseases. Cannabidiol (CBD) appears to be the most promising among phytocannabinoids, due to the lack of psychotropic effects and low toxicity profile. Its anti-inflammatory action has been highlighted in different preclinical models, ranging from experimental colitis to arthritis and neuroinflammation. Our aim was to evaluate CBD immune-modulatory effects in peripheral blood mononuclear cells (PBMC) of psoriasis individuals with particular attention to both innate and adaptative immune arms.

Methods: We performed in vitro immune functional experiments to analyze CBD action on various immune cells active in psoriatic lesions.

Results: The results showed that CBD produced a shift from Th1 to Th2 response, while boosting cytotoxic activity of Natural Killer (NK) cells. Furthermore, it also exerted a potent action on monocyte differentiation as, after CBD treatment, monocytes from psoriatic individuals were unable to migrate in response to inflammatory stimuli and to fully differentiate into mature dendritic cells. Finally, a M2 skewing of monocyte-derived macrophages by CBD also contributed to the fine tuning of the magnitude of immune responses.

Conclusions: These data uncover new potential immunomodulatory properties of this cannabinoid suggesting a possible therapeutic action in the treatment of multiple inflammatory skin diseases.

Keywords: CBD; dendritic cells; macrophage; monocyte; natural killer; psoriasis.

Figure 1

Cytokine profile analysis of PBMCs from psoriatic individuals. PBMCs not treated (CBD-, DMSO 0,1%) or treated with CBD+ (10μM) for 18 hours. The intensity of the positive control (reference spot) was considered 100% and the protein expression was expressed as a percentage of the intensity of the reference spots on the array. Spots related to TARC, MIP, IL-24, IL-19, IL-1RA, IFNγ, GM-CSF, ENA-78 are bordered in the representative array blots on the left and their expression is indicated in the histogram graph on the right.

Figure 2

Effect of CBD on PBMCs’ release of TNFα and IFNγ from psoriatic individuals. Histograms report the levels of IFNγ (A) and TNFα (B) secretion quantified by ELISA assay. Psoriatic individuals’ PBMCs were treated with CBD (0-10μM) or with vehicle alone (DMSO 0,1%) for 18h hours in presence or absence of the activation stimulus PHA. Multiple comparisons statistically significant are indicated (ANOVA; ****p< 0,0001).

Figure 3

CBD affects the degranulating and inhibitory activity of Natural killer cells. Natural killer cells were isolated as indicated in Materials and methods section. Healthy donors’ and psoriatic individuals’ NKs (2*10⁶/ml in complete RPMI) were treated with CBD (10μM) or with vehicle alone (DMSO 0,1%) for 18 hours. (A) The panel shows a representative flow cytometry dot plot reporting the CD69 (early activation marker of NKs) expression on gated CD56 positive cells. Below, bar graph shows the flow cytometry histogram profile of CD69 protein levels at the cell surface of CD56+ NKs. (B-D) The flow cytometry histograms show the mean fluorescence intensity (MFI) of CD107a (marker of NKs degranulation), NKG2D (NK activator cell receptor) and KIR3DL1 (NK inhibitory cell receptor), respectively, in CD56 positive NK cells, after exposing NKs to CBD (10μM) for 18h hours in presence or absence of IL-2 (200IU/ml). We gated on low side scatter lymphocytes and then used CD3 and CD56 to identify NK cells (CD56+ CD3−). (ANOVA; *p< 0,05; **p< 0,01; ***p< 0,001).

Figure 4

CBD impacts the expression of chemokine receptors on NK cells. The flow cytometry bar charts show respectively the percentage of (A) CXCR3 positive cells and (B) CCR5 positive cells on gated CD56 positive NK cells, after exposing healthy donors’ and psoriatic individuals’ NK cells, stimulated for 18h hours in presence or absence of IL-2 (200IU/ml) at a concentration of 2*10⁶/ml in complete RPMI medium. (ANOVA; *p< 0,05; **p<0,01; ***p< 0,001).

Figure 5

CBD alters monocytes’ migration to MCP-1. Monocytes were isolated as indicated in Materials and methods section. Psoriatic individuals’ monocytes were treated with CBD (10μM) or with vehicle alone (DMSO 0,1%) for 18 hours. (A) The bar graph shows the flow cytometry histogram profile of CD16, CD68, CD69, CCR7, CXCR3, CD62L, CX3CR1, CCR5 and CCR2 expression at the cell surface of CD14+ monocytes. Pairwise comparisons statistically significant are indicated. (B) The histogram shows the percentage of CD14+ monocytes migrating in response to chemotactic stimuli (monocytes’ migration was observed in free medium with addition of MCP-1 0,05 μg/mL or free medium +10% FBS). (ANOVA; **p<0,01; ***p< 0,001).

Figure 6

CBD interferes with LPS-induced maturation of DC and activation of T cell dependent responses. (A) Monocyte-derived dendritic cells from psoriatic individuals were generated in presence of CBD (10μM) or with vehicle alone (DMSO 0,1%) for 7 days and then exposed to LPS maturation for additional 24h. The bar graph shows the flow cytometry analysis of the maturation surface marker CD86 on CD1a positive monocyte-derived dendritic cells from psoriatic individuals. (B) Monocyte-derived dendritic cells from psoriatic individuals were co-cultured with T cells in presence of CBD (10μM) or with vehicle alone (DMSO 0,1%). The histogram highlights the release levels of IFNγ and IL-4 following 7 days-co-culture. (ANOVA; *p<0,05, ***p<0,001).

Figure 7

CBD skews monocytes toward M2 pro-resolutive macrophage differentiation. Macrophages were generated from PBMCs of healthy donors and psoriatic individuals upon 7 days in vitro culture in presence or absence of CBD (10μM). (A) Panel shows the M1 phenotype through the flow cytometry histogram profiles of CD86 (on the left) and CD14 (on the right) protein levels at the cell surface of recovered macrophages of healthy donors and psoriatic individuals. (B) Panel shows the M2 phenotype through the flow cytometry histogram profiles of CD163 (on the left) and CCR5 (on the right) protein levels at the cell surface of recovered macrophages of healthy donors and psoriatic individuals. Both bars graph in panel’s reports the mean fluorescence intensity (MFI) values. Data are reported as mean ± SD. (ANOVA; *p<0,05).