Fig. 3: Triggering of CBRs inhibits inflammatory signalling pathways and promotes autophagy and metabolic rewiring in human DCs.

a Left, western blot of protein extracts from hmoDCs stimulated for 15 min in the indicated conditions. Right, quantification of the reactive phosphorylated bands by scanning densitometry. b TLR2L-induced NFκB/AP-1 activation and IL-8 production by THP1-XBlue^TM cells with different doses of WIN55212-2 (WIN) for 18 h (n = 5). c Left, western blot of hmoDCs stimulated for 30 min in the indicated conditions. Right, quantification of the reactive phosphorylated bands by scanning densitometry. d Left, western blot of hmoDCs stimulated for 18 h in the indicated conditions. Right, quantification of the reactive bands by scanning densitometry. e Representative confocal images of LC3 staining of hmoDCs stimulated for 18 h in the indicated conditions. LC3 (green), DAPI (blue). White bars, 5 µm. f Left, western blot of hmoDCs preincubated with chloroquine (CQ) or E64d plus pepstatin A (PA) and stimulated for 18 h with LPS plus WIN55212-2. Right, quantification of the reactive bands by scanning densitometry. g Heatmap of autophagy-related gene expression after stimulation of hmoDCs with LPS or LPS/WIN55212-2 for 4 h. h Quantification of the induced Warburg effect and lactate content in cell-free supernatants relative to unstimulated hmoDCs and glucose consumption determined as metabolic rate of the indicated conditions (n = 8). i Fluorescence intensity of stimulated hmoDCs stained with Mito Tracker Red (n = 8). j Intracellular ATP levels in hmoDCs after 18 h of stimulation with the indicated conditions (n = 6). k Kinetic study of mitochondrial OCR in LPS or LPS/WIN55212-2-stimulated hmoDCs by sequential addition of oligomycin (Olig), FCCP, and rotenone/antimycin A (Rot + AA). Quantification of basal respiration, ATP production coupled respiration and spare respiratory capacity of hmoDCs are included (n = 6 of two independent experiments). l Heatmap of metabolism-related gene expression after stimulation of hmoDCs with LPS or LPS/WIN55212-2 for 4 h. Values are mean ± SEM. Statistical significance was determined using One-way Anova. *P < 0.05, **P < 0.01. p-IKKα/β, phosphorylated-IκB kinase subunits alpha and beta; p-IκB, phosphorylated-inhibitor of NFκB; p-p38, phosphorylated-p38 kinase; p-JNK, phosphorylated-c-Jun N-terminal kinase; p-ERK, phosphorylated-extracellular signal regulated kinase; NFκB, nuclear factor κB; AP-1, activation protein 1; p-Akt, phosphorylated-protein kinase B; p-p70S6K, phosphorylated-p70S6 kinase; p-AMPK, phosphorylated-AMP-activated protein kinase; LC3-I or II, microtubule-associated protein 1 A/1B-light chain 3-I or II; 3-MA, 3-methyladenine; ULK1, Unc-51 like kinase; ATG5 12, 14 or 16 L, autophagy-related gene 5, 14 or 16 L; PIK3C3, phosphatidylinositol 3-kinase catalytic subunit type 3; BECN1, Beclin; RUBCN, Rubicon; GLUT1, glucose transporter 1; HK2, hexokinase 2; PFKFB3, phosphofructokinase 3; LDHA lactate dehydrogenase, HIF1A hypoxia-inducible factor 1 alpha, PDHA pyruvate dehydrogenase, IDH3A isocitrate dehydrogenase, SDHA succinate dehydrogenase, ATP5A1 ATP synthase subunit alpha, PINK1 PTEN-induced kinase 1, ACADM acyl-coenzyme A dehydrogenase, CPT1A carnitine O-palmitoyltransferase 1, GLS glutaminase, SLC1A3 amino acid transporter 1.