Anandamide modulation of monocyte-derived Langerhans cells: implications for immune homeostasis and skin inflammation

Abstract

Introduction: The endocannabinoid system (ECS), named after the chemical compounds found in the cannabis plant, is a regulatory network of neurotransmitters, receptors, and enzymes that plays crucial roles in skin health and disease. Endogenous ligands of the ECS, called endocannabinoids, have proven to be important regulators of immune responses. One of the most prevalent endocannabinoids, arachidonoylethanolamide (also known as anandamide), is known for its anti-inflammatory effects. Langerhans cells (LCs) are the sole antigen-presenting cells present in the human epidermis. They serve as the first line of defense against pathogens and are essential for the skin's specific immune responses and play a critical role in maintaining tissue homeostasis; however, little is known about the effect of endocannabinoids on these cells. Our research aimed to provide the connection between monocyte-derived Langerhans cells (moLCs) and the ECS, shedding light on their collaborative roles in immune homeostasis and inflammation.

Methods: Human monocytes were differentiated into moLCs using established protocols. Anandamide was applied during the differentiation process to test its effect on the viability, marker expression, and cytokine production of the cells, as well as in short term treatments for intracellular calcium measurement. TLR ligands applied after the differentiation protocol were used to activate moLCs. The impact of anandamide on the functionality of moLCs was further assessed using differential gene expression analysis of bulk RNA-Seq data, moLC-T cell cocultures, while ELISpot was employed to determine polarization of T cells activated in the aforementioned cocultures.

Results: Anandamide did not significantly affect the viability of moLCs up to 10 µM. When applied during the differentiation process it had only a negligible effect on CD207 expression, the prototypic marker of LCs; however, there was an observed reduction in CD1a expression by moLCs. Anandamide had no significant effects on the maturation status of moLCs, nor did it affect the maturation induced by TLR3 and TLR7/8 agonists. MoLCs differentiated in the presence of anandamide did however show decreased production of CXCL8, IL-6, IL-10 and IL-12 cytokines induced by TLR3 and TLR7/8 activation. Anandamide-treated moLCs showed an increased capability to activate naïve T cells; however, not to the level seen with combined TLR agonism. RNA sequencing analysis of moLCs differentiated with anandamide showed modest changes compared to control cells but did reveal an inhibitory effect on oxidative phosphorylation specifically in activated moLCs. Anandamide also promoted the polarization of naïve T cells towards a Th1 phenotype.

Discussion: Our results show that anandamide has nuanced effects on the differentiation, maturation, cytokine secretion, metabolism and function of activated moLCs. Among these changes the decrease in CD1a expression on moLCs holds promise to selectively dampen inflammation induced by CD1a restricted T cells, which have been implicated as drivers of inflammation in common inflammatory skin conditions such as psoriasis, atopic dermatitis and contact dermatitis.

Keywords: CD1a; Langerhans; anandamide; cells; epidermis; skin immunology.

Figure 1

Anandamide decreases CD1a expression, increases maturation markers on moLCs without affecting their viability. Monocytes were cultured in the presence of GM-CSF, TNFα, TGFβ and IL-4 (for 48 hrs) for 5 days to generate moLCs in the presence of 10 µM AEA, vehicle (0.1 v/v% absolute ethanol). Maturation was induced on day 4 by p(I:C) (20 µg/ml) and CL075 (0.5 µg/ml), both applied for 24 hours. N≥10 mean ± SD. Percentage of cells positive for 7-AAD (A), CD207 (B), CD1a (C), CD83 (D), CD86 (E), HLA-DQ (F), CCR7 (G) following the indicated treatments. *p < 0.05, ** p < 0.01, ***p < 0.001, **** p<0.0001 as indicated (determined by repeated measures one-way ANOVA). Individual donors are represented by symbols. 7-AAD, 7-aminoactinomycin; PCTR, Positive control; cells damaged by freezing (30 min), AEA, N-arachidonoylethanolamine, anandamide; CL075, TLR7/8 agonist; p(I:C), Polyinosinic:polycytidylic acid, TLR3 agonist.

Figure 2

AEA can decrease CXCL8, IL-6, IL-10 and IL-12 cytokine production induced by TLR3 and TLR7/8 activation on moLCs. Monocytes were cultured in the presence of GM-CSF, TNFα, TGFβ and IL-4 (for 48 hrs) for 5 days to generate moLCs in the presence of 10 µM AEA, vehicle (0.1 v/v% absolute ethanol). Maturation was induced on day 4 by p(I:C) (20 µg/ml) and CL075 (0.5 µg/ml), both applied for 24 hours. N≥3 mean ± SD. CXCL8 (A), IL-6 (B), IL-10 (C) and IL-12 (D) production was determined with ELISA from supernatants. Bar plots represent mean ± SD of representative results from N≥3 independent experiments, ** p < 0.01, ***p < 0.001, **** p<0.0001 compared to the marked groups as determined by repeated measures one-way ANOVA. ND, not determined. AEA, N-arachidonoylethanolamine, anandamide; CL075: TLR7/8 agonist, p(I:C), Polyinosinic:polycytidylic acid, TLR3 agonist.

Figure 3

Anandamide causes delayed calcium transients in moLCs. Monocytes were cultured in the presence of GM-CSF, TNFα, TGFβ and IL-4 (for 48 hrs) for 5 days to generate moLCs. Cells were seeded at a density of 5 × 10⁴ cells/ml in Ibidi μ-Slide chambers on day 5. Representative graphs showing changes in intracellular Ca²⁺ concentration in response to vehicle (0.1 v/v% absolute ethanol) (A), 10 µM AEA (B), p(I:C) (20 µg/ml) and CL075 (0.5 µg/ml) (C) and the three compounds together (AEA + p(I:C) + CL075) (D) as determined by measuring changes in Fluo-4 fluorescence in real time by Olympus IX-81 Microscope. Arrows show the approximate time of application of treating compounds. Representative images above the line graphs show typical changes in fluorescence intensity at the marked time points. AEA, N-arachidonoylethanolamine, anandamide; CL075: TLR7/8 agonist, p(I:C), Polyinosinic:polycytidylic acid, TLR3 agonist.

Figure 4

Anandamide treatment enhances T cell proliferation-inducing capability of moLCs. Monocytes were cultured in the presence of GM-CSF, TNFα, TGFβ and IL-4 (for 48 hrs) for 5 days to generate moLCs in the presence of 10 µM AEA, vehicle (0.1 v/v% absolute ethanol). Maturation was induced on day 4 by p(I:C) (20 µg/ml) and CL075 (0.5 µg/ml), both applied for 24 hours. Percentage of proliferating T cells after 5 days of coculture of naïve T cells and moLCs at a ratio of 6:1 (A). Representative histograms after 5 days co-culture of naïve T cells and moLCs at a ratio of 6:1 (B). N=8, mean ± SD, individual donors are represented by symbols. * p<0.05, ** p < 0.01 as determined by repeated measures one-way ANOVA compared to the marked groups. AEA, N-arachidonoylethanolamine, anandamide; CL075: TLR7/8 agonist, p(I:C), Polyinosinic:polycytidylic acid, TLR3 agonist.

Figure 5

Anandamide treatment alone results in modest changes to transcriptome as determined by RNASeq. Monocytes were cultured in the presence of GM-CSF, TNFα, TGFβ and IL-4 (for 48 hrs) for 5 days to generate moLCs in the presence of 10 µM AEA, vehicle (0.1 v/v% absolute ethanol). Maturation was induced on day 4 by p(I:C) (20 µg/ml) and CL075 (0.5 µg/ml), both applied for 24 hours. The gene expression of vehicle-treated (blue) and AEA-treated moLCs (red) cluster together in PCA, as does that of mature moLCs with, or without AEA co-treatment (green and purple, respectively) (A). A confidence ellipse is drawn for each group with a confidence interval level of 0.95. Volcano plots of RNASeq data with the top 15 most highly changed genes highlighted for each plot. Red dots indicate genes with log₁₀ P>2 and fold change>2, while blue dots indicate genes with -log₁₀ P>2 and fold change<-2, when compared to vehicle-treated control cells (B-D), or to matured cells that did not receive AEA treatment (E). Lollipop graphs of upregulated pathways as determined by Gene Ontology analysis in AEA treated cells compared to vehicle-treated control (F), p(I:C) and CL075, and p(I:C), CL075 and AEA treated cells compared to vehicle-treated control (G and H, respectively). The size of the circle corresponds to the number of induced genes, while the color of the line corresponds to the -log10 of the False Discovery Rate (FDR). AEA, N-arachidonoylethanolamine, anandamide; CL075: TLR7/8 agonist, p(I:C), Polyinosinic:polycytidylic acid, TLR3 agonist.

Figure 6

Maturation-induced switch to oxidative phosphorylation is absent from anandamide-treated moLCs. Monocytes were cultured in the presence of GM-CSF, TNFα, TGFβ and IL-4 (for 48 hrs) for 5 days to generate moLCs in the presence of 10 µM AEA, vehicle (0.1 v/v% absolute ethanol). Maturation was induced on day 4 by p(I:C) (20 µg/ml) and CL075 (0.5 µg/ml) both applied for 24 hours. Venn diagram showing number of genes upregulated by p(I:C), CL075 and AEA (left side) and p(I:C) and CL075 (right side) when compared to vehicle-treated control cells. Arrows point to upregulated pathways as determined by Gene Ontology analysis of genes that only appear in p(I:C), CL075 and AEA treated cells (marked in red on the Venn diagram, left side) or in p(I:C) and CL075 treated cells (marked in green on the Venn diagram, left side). The size of the circle corresponds to the number of induced genes, while the color of the line corresponds to the -log10 of the False Discovery Rate (FDR).

Figure 7

Anandamide slightly increases Th1 polarization induced by moLCs as determined by IFNγ ELISpot. Monocytes were cultured in the presence of GM-CSF, TNFα, TGFβ and IL-4 (for 48 hrs) for 5 days to generate moLCs in the presence of 10 µM AEA, vehicle (0.1 v/v% absolute ethanol). Maturation was induced by on day 4 p(I:C) (20 µg/ml) and CL075 (0.5 µg/ml), applied both for 24 hours. Representative ELISpot image showing the quantity of IFNγ producing T cells in three parallel technical repeats in brown (A). The total intensity of all foreground objects per well (B) in panel (A). The mean values and the converted well area of spot numbers were counted based on 4 independent donors (C). Bar plots represent mean ± SD of representative results from N≥3 independent experiments, * p < 0.05, **** p<0.0001 compared to the marked groups as determined by repeated measures one-way ANOVA. ND, not determined. AEA, N-arachidonoylethanolamine, anandamide; CL075: TLR7/8 agonist, p(I:C), Polyinosinic:polycytidylic acid, TLR3 agonist.