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. 2023 Sep 26;14:1178172. doi: 10.3389/fimmu.2023.1178172

Figure 1.

Figure 1

CB inhibited TLR-7/8-mediated TNF-α, IL-1β and IL-6 production by human monocytes. (A, B) THP1 NF-κB reporter cells were treated with increasing concentration of Histamine (A) or CB (B) ranging from 1 to 100 µM then stimulated with R848 (5 μg/ml) for 24 hours. NF-κB reporter activity was measured using QUANTI-Blue, a SEAP detection reagent. (C) mRNA levels of TNF-α, IL-1β and IL-6 from THP1-dual pre-incubated with increased doses of CB and stimulated overnight with R848 (5 μg/ml), were measured by RT-qPCR and normalized to RPL13A. Kruskal-Wallis with Dunn’s multiple comparisons test. (D-G) PBMCs from healthy donors (HD) were preincubated with CB (20 μM) then stimulated with R848 (5 μg/mL) overnight. (D, E) Cytokine production was measured in the supernatant using a bead-based multiplexed immunoassay system Luminex. (D) Heatmap representation of statistically different cytokines (P<0.05) between the different conditions (Null, R848, R848+CB), ordered by hierarchical clustering. Up-regulated cytokines are shown in orange, and down-regulated in blue. P values were determined with the Kruskal-Wallis test. (E) Individual cytokines from the Luminex are represented. Mann-Whitney test. (F) PBMCs were analyzed by mass cytometry (CyTOF) and tSNE analysis were performed using CD56, CD3, CD11c, BDCA4, CD14, HLADR, CD123, CD4, CD8, and CD19 markers. Intracellular levels of TNF-α, IL-1β, IL-6, IFNγ, and IL-17were evaluated. (G) Mean Metal Intensity (MMI) of TNF-α, IL-1β, and IL-6 was evaluated on gated monocytes from four different donors. (H) Cytokine production was measured in the supernatants of purified monocytes from nine HD using the multiplex bead-based immunoassay LEGENDplex. Mann-Whitney test. (I) Purified monocytes from five HD were preincubated with increased doses of CB then stimulated with R848 during 5 h. Intracellular levels of TNF-α and IL-1β as well as viability were evaluated by flow cytometry. SSC-A, side scatter. FSC-A, forward scatter. 2-way ANOVA. All data are presented as median ± range. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.