Cannabinoid WIN55,212-2 reprograms monocytes and macrophages to inhibit LPS-induced inflammation

Pérez-Diego | Martín-Cruz | de la Rocha-Muñoz | Sevilla-Ortega

The IL-10/TNFα, IL-10/IL-1β and IL-10/IL-6 cytokine ratios were significantly higher in LPS-stimulated WIN-hmoDCs than conventional hmoDCs (Figure 1C), indicating that WIN55,212-2 reprograms the differentiation of monocytes towards DCs displaying anti-inflammatory features upon LPS stimulation. FIGURE 1 WIN55,212-2 inhibits the activation of pro-inflammatory M1 human macrophages To investigate the effects of WIN55,212-2 in human macrophages under inflammatory conditions, we first generated human macrophages from the THP-1 monocytic cell line (THP-1 MΦs) and stimulated them with medium (control), LPS/IFNγ alone (M1) or in the presence of WIN55,212-2 (M1+WIN) (Figure 2A). LPS/IFNγ stimulation significantly enhanced the mRNA expression levels of all the assayed molecules, which were in all the cases significantly impaired by WIN55,212-2 (Figure 2J), confirming its potential capacity to rescue human macrophages from this inflammatory cell death pathway. In addition, WIN55,212-2 impaired the pro-inflammatory polarization of human macrophages by inhibiting cytokine production, inflammasome activation and rescuing macrophages from pyroptotic cell death. Abbreviations 2-AG, 2-arachidonoylglycerol; 2-DG, 2-deoxy-D-glucose; A488, Alexa Fluor 488; AA, antimycin A; AEA, anandamide; APC, allophycocyanin; BSA, bovine serum albumin; CBD, cannabidiol; CBRs, cannabinoid receptors; ChIP, chromatin immunoprecipitation; DCs dendritic cells; ECS, endocannabinoid system; FBS, fetal bovine serum; FITC, fluorescein-5-isothiocyanate; glycoPER, glycolytic proton efflux rate; GM-CSF, granulocyte-macrophage colony-stimulating factor; H3K27ac, histone 3 acetylation of lysine 27; hmoDCs, human monocyte-derived dendritic cells; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MAS, macrophage activation syndrome; mTORC1, mammalian target of rapamycin complex 1; PBMCs, peripheral blood mononuclear cells; PE, phycoerythrin; PerCP, peridinin chlorophyll protein complex; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; PMΦs, peritoneal macrophages; PPARα, peroxisome proliferator-activated receptor α; PRRs, pattern-recognition receptors; qPCR, quantitative PCR; ROI, region of interest; Rot, rotenone; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SEAP, secreted embryonic alkaline phosphatase; THC, Δ9-tetrahydrocannabinol; THP-1 MΦs, THP-1 Macrophages; Tregs, regulatory T cells; WIN, WIN55,212-2.

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