Journal: bioRxiv

Article Title: Targeting the chemokine receptor CXCR4 with histamine analogue to reduce inflammation in juvenile arthritis: a proof of concept for COVID-19 therapeutic approach

doi: 10.1101/2021.10.24.465080

Figure Lengend Snippet: ( 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-17 were 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.

Article Snippet: Human monocytes were purified by positive selection with Human CD14 microbeads (Miltenyi).

Techniques: Concentration Assay, Activity Assay, Incubation, Quantitative RT-PCR, Luminex, MANN-WHITNEY, Mass Cytometry, Purification, Multiplex Assay, Bead-based Assay, Flow Cytometry