AUTHOR=ElSayed Sandy , Jay Gregory D. , Cabezas Ralph , Qadri Marwa , Schmidt Tannin A. , Elsaid Khaled A. 

TITLE=Recombinant Human Proteoglycan 4 Regulates Phagocytic Activation of Monocytes and Reduces IL-1β Secretion by Urate Crystal Stimulated Gout PBMCs

JOURNAL=Frontiers in Immunology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.771677

DOI=10.3389/fimmu.2021.771677

ISSN=1664-3224

ABSTRACT=<sec><title>Objectives</title><p>To compare phagocytic activities of monocytes in peripheral blood mononuclear cells (PBMCs) from acute gout patients and normal subjects, examine monosodium urate monohydrate (MSU) crystal-induced IL-1β secretion ± recombinant human proteoglycan 4 (rhPRG4) or interleukin-1 receptor antagonist (IL-1RA), and study the anti-inflammatory mechanism of rhPRG4 in MSU stimulated monocytes.</p></sec><sec><title>Methods</title><p>Acute gout PBMCs were collected from patients in the Emergency Department and normal PBMCs were obtained from a commercial source. Monocytes in PBMCs were identified by flow cytometry. PBMCs were primed with Pam3CSK4 (1μg/mL) for 24h and phagocytic activation of monocytes was determined using fluorescently labeled latex beads. MSU (200μg/mL) stimulated IL-1β secretion was determined by ELISA. Reactive oxygen species (ROS) generation in monocytes was determined fluorometrically. PBMCs were incubated with IL-1RA (250ng/mL) or rhPRG4 (200μg/mL) and bead phagocytosis by monocytes was determined. THP-1 monocytes were treated with MSU crystals ± rhPRG4 and cellular levels of NLRP3 protein, pro-IL-1β, secreted IL-1β, and activities of caspase-1 and protein phosphatase-2A (PP2A) were quantified. The peritoneal influx of inflammatory and anti-inflammatory monocytes and neutrophils in Prg4 deficient mice was studied and the impact of rhPRG4 on immune cell trafficking was assessed.</p></sec><sec><title>Results</title><p>Enhanced phagocytic activation of gout monocytes under basal conditions (<italic>p&lt;0.001</italic>) was associated with ROS generation and MSU stimulated IL-1β secretion (<italic>p&lt;0.05</italic>). rhPRG4 reduced bead phagocytosis by normal and gout monocytes compared to IL-1RA and both treatments were efficacious in reducing IL-1β secretion (<italic>p&lt;0.05</italic>). rhPRG4 reduced pro-IL-1β content, caspase-1 activity, conversion of pro-IL-1β to mature IL-1β and restored PP2A activity in monocytes (<italic>p&lt;0.05</italic>). PP2A inhibition reversed rhPRG4’s effects on pro-IL-1β and mature IL-1β in MSU stimulated monocytes. Neutrophils accumulated in peritoneal cavities of Prg4 deficient mice (<italic>p&lt;0.01</italic>) and rhPRG4 treatment reduced neutrophil accumulation and enhanced anti-inflammatory monocyte influx (<italic>p&lt;0.05</italic>).</p></sec><sec><title>Conclusions</title><p>MSU phagocytosis was higher in gout monocytes resulting in higher ROS and IL-1β secretion. rhPRG4 reduced monocyte phagocytic activation to a greater extent than IL-1RA and reduced IL-1β secretion. The anti-inflammatory activity of rhPRG4 in monocytes is partially mediated by PP2A, and <italic>in vivo</italic>, PRG4 plays a role in regulating the trafficking of immune cells into the site of a gout flare.</p></sec>