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. Recent studies have suggested that PRG4 may have anti-inflammatory properties in MSU-crystal induced arthritis. Inflammasomes are proteins platforms linking recognition of danger-associated molecular patterns such as MSU by cytosolic sensory proteins to caspase-1 activation. The objective of this study was to further evaluate the anti-inflammatory mechanisms of rhPRG4 in the inflammasome in MSU in acute gout inflammation.

Methods: We evaluated the impact of recombinant human PRG4(rhPRG4) on MSU-induced release, secretion and expression of interleukin-1 beta (IL-1b), tumor necrosis factor alpha (TNF-a), interleukin-8 (IL-8), by ELISA, immunohistochemistry, and western-blot analysis in MSU-stimulated THP-1 cell. Cytosolic and nuclear levels of nuclear factor kappa B (NFkB) p50 and p65 subunits, IkB were studied using western blot. We also evaluated the role of rhPRG4 in the expression of the NLRP3 inflammasome components using qPCR, immunohistochemistry and western-blot analysis. Immunoprecipitation experiments were performed to evaluate if rhPRG4 inhibited NALP3 inflammasome assembly by binding to ASC and prevent caspase-1 activation and cytokine release.

Results: In THP-1 cell line, rhPRG4 significantly inhibited IL-1b, TNF-a and IL-8 production to MSU-crystals using ELISA, western blot analysis and immunohistochemistry in a dose dependent manner. rhPRG4 inhibited the expression of NALP3, PYCARD, Caspase-1 and IL-18 using immunohistochemistry, western blot analysis and qPCR in a dose dependent manner compared to colchicine. rhPRG4 inhibited nuclear factor kappa B (NFkB) and IkB. Considering the activation of the NALP3 inflammasome requires oligomerization of the proteins NALP3, ASC and procaspase, we explored changes in the association of these proteins in response to rhPRG4 in MSU-stimulated THP-1 cells. After immunoprecipitation of ACS, there was a decrease in association of NALP3, activated (cleaved) caspase-1 in rhPRG4 treated THP-1 cells exposed to MSU-crystals.

Conclusion:

Our findings advance our understanding of the complex anti-inflammatory mechanisms of PRG4 in joint homeostasis. rhPRG4 retards the progression MSU-crystal induced inflammatory arthritis by inhibiting the expression and assembly of the NALP3 inflammasome. Our findings provide a better understanding of the molecular mechanism(s) of PRG4 in MSU-crystal induced arthritis with important implications in the development of novel biological strategies in gout.

Acknowledgements

This work is support from a grant from the Arthritis Foundation and COBRE grant P20GM104937 from the National Institutes of Health.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-inflammatory-mechanism-of-lubricinproteoglycan-4-prg4-in-monosodium-urate-msu-crystal-induced-arthritis-in-thp-1-macrophages-is-mediated-by-nalp3-inflammasome/