Figure 1.

Analysis of the phagocytic activity of monocytes in peripheral blood mononuclear cells (PBMCs) of normal subjects (n=3) and gout patients (n=7) and its relationship to interleukin-1 beta (IL-1β) release in response to priming with Pam3CSK4, a toll-like receptor 2 ligand, and treatment with monosodium urate monohydrate (MSU) crystals. To assess phagocytic activation, PBMCs were treated with Pam3CSK4 (1μg/mL) for 24h followed by co-incubation with FITC-labeled rabbit IgG-coated latex beads and determination of percent bead-positive monocytes. Alternatively, PBMCs were incubated with MSU crystals (200μg/mL) and secreted IL-1β levels were determined by ELISA at 6h. Analysis of FITC-bead positive monocytes was performed using 2-way ANOVA followed by Tukey’s post-hoc test, and analysis of IL-1β levels was performed by multiple t-tests followed by a post-hoc false discovery rate analysis using two stage step-up approach. ns, non-significant; *p < 0.05; ***p < 0.001. (A) Flow cytometry gating strategy for monocytes in PBMCs. Identification of monocytes was conducted using the expected forward and side scatter (FSC-A and SSC-A) ranges and single cells were gated using FSC-A and FSC-H. Viable cells were identified using Zombie Violet viability dye and monocytes were confirmed using CD14 and CD45 surface markers. Subsequently, bead positive monocytes were gated based on thresholds established using fluorescence minus one control. (B) Representative flow cytometry histograms depicting a higher percentage of bead-positive monocytes in a gout patient compared to a normal subject. (C) Monocytes of gout patients had higher basal phagocytic activities compared to normal subjects. TLR2 ligand priming increased phagocytic activities of normal subjects’ monocytes but not gout patients and phagocytic activities in primed samples were not different between normal and gout subjects. (D) A combination of TLR2 ligand priming and MSU crystal challenge resulted in higher secreted IL-1β levels from gout patients’ PBMCs.