Background/Purpose: Lubricin/proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblast and superficial zone chondrocytes. PRG4 has a multifaceted homeostatic role in the joint including boundary lubrication, friction lowering of apposed cartilage surfaces and prevention of synovial overgrowth. PRG4 is abundant in the synovial fluid (SF) and its levels are reduced in SF from patients with inflammatory arthropathies. Therapeutically, the recombinant and native form of PRG4 has been shown to exhibit a disease a disease-modifying effect in pre-clinical osteoarthritis (OA) models. We recently showed that rhPRG4 reduces phagocytosis of MSU-crystals, activation of chemokines and cytokines, NLRP3 expression, generation of mature IL-1ß, and NFkB nuclear translocation. The objective of this study was to further evaluate role of Lubricin/PRG4 in NLRP3 inflammasome activation, ASC recruitment and speck formation in MSU-crystal stimulation in THP-1 macrophages and wild-type and Prg4 knock-out macrophages.

Methods: We evaluated the impact of recombinant human PRG4 (rhPRG4) on MSU-induced release, secretion and expression of interleukin-1 beta (IL-1ß), NLRP3 induction, caspase-1 activation by ELISA, immunohistochemistry, and Western-blot analysis in MSU-stimulated THP-1 cell and MSU-stimulated peritoneal macrophages (PEM) and bone marrow derived macrophages (BDMPs) from wildtype and Prg4 knock-out (KO) mice. We also evaluated the role of rhPRG4in the expression of the NLRP3 inflammasome components using qPCR, immunohistochemistry and western-blot analysis. Immunohistochemistry experiments were performed to evaluate if rhPRG4 inhibited ASC speck formation and oligomerization. Colocalization of rhodamine rhPRG4 and NLRP3, ASC was evaluated in MSU-stimulated THP-1 cells.

Results: rhPRG4 inhibited the expression of NALP3, PYCARD, Caspase-1 and IL-18 in MSU-stimulated THP-1 cell using immunohistochemistry, western blot analysis and qPCR in a dose dependent manner. rhPRG4 inhibited the formation of ASC speck, a critical marker of NLRP3 inflammasome assembly, in MSU-stimulated cells. PEM from Prg4 -/- mice showed increased NLRP3 induction, caspase-1 activation and conversion of pro-IL-1ß to mature IL-1ß compared to wild-type mice. Colocalization of rhodamine rhPRG4 in MSU-stimulated THP-1 cells showed colocalization with NLRP3 but not ASC.

Conclusion: Our findings advance our current understanding of the complex anti-inflammatory mechanisms of PRG4in gout. One of the rhPRG4’s anti-inflammatory mechanism may be due to targeting the NALP3 inflammasome assembly.

This work is supported by P20GM104937 (AMR and GDT) and Arthritis Foundation Grant (AMR).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/lubricin-proteoglycan-4-prg4-inhibits-nlrp3-inflammasome-assembly-in-monosodium-urate-msu-crystal-induced-arthritis/