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. 2021 Dec 21;12:771677. doi: 10.3389/fimmu.2021.771677

Figure 2.

Figure 2

Dose-response of interleukin-1 receptor antagonist (IL-1RA) and recombinant human proteoglycan 4 (rhPRG4) in Pam3CSK4 (P), a toll-like receptor 2 ligand, and monosodium urate monohydrate (M) crystal-stimulated peripheral blood mononuclear cells (PBMCs) of normal subjects and efficacy of IL-1RA and rhPRG4 in modulating phagocytic activation of monocytes of normal subjects (n=3) and gout patients (n=7 to 12) and secretion of mature interleukin-1 beta (IL-1β) by urate crystal-stimulated gout PBMCs. 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, using the gating strategy shown in Figure 1A . Alternatively, PBMCs were incubated with urate crystals (200μg/mL) and secreted IL-1β levels were determined by ELISA. Gout PBMCs were categorized, based on urate crystal-induced IL-1β secretion, as either high IL-1β secreting (IL-1β concentrations > 500pg/mL) (n=6) or low IL-1β secreting (IL-1β concentrations < 500pg/mL) (n=6) and the differential efficacies of IL-1RA and rhPRG4 on both populations was investigated. Statistical analyses included one and two-way ANOVAs followed by Tukey’s post-hoc test. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (A) IL-1RA (250ng/mL and higher) reduced IL-1β secretion by PBMCs of normal subjects. (B) rhPRG4 (100&200μg/mL) reduced IL-1β secretion by PBMCs of normal subjects. (C) IL-1RA (250ng/mL) and rhPRG4 (200μg/mL) reduced phagocytic activation of monocytes in PBMCs of normal subjects. rhPRG4 treatment reduced bead phagocytosis by normal monocytes compared to IL-1RA. (D) rhPRG4 (200μg/mL) reduced bead phagocytosis by gout monocytes, while IL-1RA (250ng/mL) did not alter fluorescent bead uptake by the same monocytes. (E) IL-1RA (250ng/mL) and rhPRG4 (200μg/mL) reduced IL-1β secretion by gout PBMCs by similar magnitudes. PBMCs from patients receiving colchicine (n=5) are highlighted in red. 4 out of 5 samples are classified as low-IL-1β secreting gout PBMCs. (F) IL-1RA (250ng/mL) and rhPRG4 (200μg/mL) reduced IL-1β secretion from high-IL-1β secreting gout PBMCs but not from low-IL-1β secreting PBMCs.