Recombinant Human Proteoglycan-4 Regulates Phagocytic Activation Of Monocytes And Reduces Il-1b Secretion By Urate Crystal Stimulated Pbmcs

Introduction Gout is a chronic inflammatory disease due to monosodium urate (MSU) crystal deposits in articular joints. MSU phagocytosis by monocytes/macrophages results in interleukin-1 beta (IL-1β) secretion. Pharmacologic treatments of gout are inadequate and are associated with significant side effects. Interleukin-1 receptor antagonist (IL-1RA) is an endogenous inhibitor of IL-1b signaling and is efficacious in acute gout management where other treatments are contraindicated. Proteoglycan-4 (PRG4) is an anti-inflammatory glycoprotein that has shown efficacy in reducing MSU induced IL-1b release from macrophages. Our objective was to compare the activation of monocytes, of normal subjects and gout patients, by MSU crystals and examine the comparative efficacy of rhPRG4 and rhIL-1RA in regulating phagocytic activation of monocytes and IL-1b secretion. Methods Collection of peripheral blood mononuclear cells (PBMCs) and sera from patients with gout was approved by the IRB at RIH. PBMCs and sera of normal subjects were obtained from ATCC. IL-1b and IL-1RA levels were determined by ELISA. PBMCs were seeded (1 million cells/well) in 6 well plates and treated with Pam3CSK4 (1mg/mL; TLR2 agonist) ± rhIL-1RA or rhPRG4 for 24 h followed by MSU crystals (200mg/mL) for 6 h. Monocytes in PBMCs were identified by flow cytometry using anti-CD14-APC-Cy7 and anti-CD45-PE-A antibodies (BioLegend). Monocytes’ phagocytic activity was determined by co-incubation of PBMCs with FITC-labeled rabbit IgG-coated latex beads (Cayman Chemicals) for 2 h followed by determination of percent bead-positive monocytes. Statistical analyses included Student's t-test and one-way ANOVA followed by post-hoc Tukey's test. Results Sera from patients with gout had lower IL-1RA levels compared to sera from normal subjects (p<0.01; fig. 1A). The gating of monocytes and corresponding bead phagocytosis is shown in fig. 1B. Representative plots showing increased bead phagocytosis by gout PBMCs compared to normal PBMCs are shown in fig. 1C. Basal phagocytic activities of gout PBMCs were higher than corresponding activities of normal PBMCs (p<0.0001; fig. 1D). IL-1b secretion in MSU crystal stimulated gout PBMCs was higher than corresponding secretion in normal PBMCs (p<0.01; fig. 1E). rhIL-1A and rhPRG4 dose-dependently reduced IL-1b secretion by normal PBMCs (p<0.001; fig. 2A & 2B). rhPRG4 was efficacious in reducing phagocytic activities of monocytes from normal and goutsubjects (p<0.001; fig. 2C & 2E). rhPRG4 exhibited a non-significant trend towards reducing IL-1b secretion by gout PBMCs (p=0.13) and the efficacy of rhPRG4 was not different from IL-1RA (p>0.05; fig. 2D). Conclusion Gout PBMCs secreted more IL-1b associated with higher phagocytic activation of monocytes. Uniquely, rhPRG4 reduced phagocytic activation of monocytes, from normal and gout patients. rhIL-1RA and rhPRG4 showed comparable anti-inflammatory efficacy. rhPRG4 is a promising new systemic therapy in gout.