Gout is a chronic inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystal deposits in joints of lower limbs. Phagocytic uptake of MSU crystals by joint-resident macrophages and recruited circulating monocytes results in IL-1 expression and production. Current acute gout treatments have serious toxicities and suffer suboptimal clinical outcomes. Protein phosphatase 2A (PP2A) plays an important role in regulating signaling pathways relevant to inflammation. We hypothesized that innate immune danger signals e.g. lipopolysaccharide (LPS) and soluble uric acid (sUA) prime human monocytes towards MSU crystal phagocytosis, and increased IL-1production mediated by a reduction in PP2A activity and restoring PP2A activity exerts an anti-inflammatory effect in this setting. Priming monocytes with LPS + sUA increased cytosolic pro-IL-1 and mature IL-1 and enhanced MSU crystal phagocytosis and its downstream IL-1 expression (p<0.001). A combination of LPS + sUA priming and MSU crystals reduced PP2A activity in monocytes by 60% (p=0.013). PP2A catalytic subunit gene knockdown reduced PP2A activity and exacerbated MSU crystal induced IL-1 expression and secretion (p<0.0001). Fingolimod (FTY720) and its active metabolite, fingolimod phosphate (FTY720-P) were evaluated for their ability to activate PP2A in human monocytes over 24 h. FTY720 and FTY720-P activated PP2A to a similar extent, and maximal enzyme activity occurred at 24 h for FTY720 and at 6 h for FTY720-P. FTY720-P (2.5M) reduced pro-IL-1 production and IL-1 secretion in primed and MSU crystal stimulated monocytes (p<0.0001) without changing the magnitude of crystal phagocytosis. We conclude that PP2A is a promising new target in acute gout.

ObjectivesTo 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.MethodsAcute 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.ResultsEnhanced phagocytic activation of gout monocytes under basal conditions (p&lt;0.001) was associated with ROS generation and MSU stimulated IL-1β secretion (p&lt;0.05). rhPRG4 reduced bead phagocytosis by normal and gout monocytes compared to IL-1RA and both treatments were efficacious in reducing IL-1β secretion (p&lt;0.05). rhPRG4 reduced pro-IL-1β content, caspase-1 activity, conversion of pro-IL-1β to mature IL-1β and restored PP2A activity in monocytes (p&lt;0.05). 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 (p&lt;0.01) and rhPRG4 treatment reduced neutrophil accumulation and enhanced anti-inflammatory monocyte influx (p&lt;0.05).ConclusionsMSU 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 in vivo, PRG4 plays a role in regulating the trafficking of immune cells into the site of a gout flare.

In gout, hyperuricemia promotes urate crystal deposition, which stimulates the NLRP3 inflammasome and interleukin-1β (IL-1β)-mediated arthritis. Incident gout without background hyperuricemia is rarely reported. To identify hyperuricemia-independent mechanisms driving gout incidence and progression, we characterized erosive urate crystalline inflammatory arthritis in a young female patient with normouricemia diagnosed as having sufficient and weighted classification criteria for gout according to the American College of Rheumatology (ACR)/EULAR gout classification criteria (the proband).Methods. We conducted whole-genome sequencing, quantitative proteomics, whole-blood RNA-sequencing analysis using serum samples from the proband. We used a mouse model of IL-1β-induced knee synovitis to characterize proband candidate genes, biomarkers, and pathogenic mechanisms of gout.Results. Lubricin level was attenuated in human proband serum and associated with elevated acute-phase reactants and inflammatory whole-blood transcripts and transcriptional pathways. The proband had predicted damaging gene variants of NLRP3 and of inter-α trypsin inhibitor heavy chain 3, an inhibitor of lubricin-degrading cathepsin G. Changes in the proband's serum protein interactome network supported enhanced lubricin degradation, with cathepsin G activity increased relative to its inhibitors, SERPINB6 and thrombospondin 1. Activation of Toll-like receptor 2 (TLR-2) suppressed levels of lubricin mRNA and lubricin release in cultured human synovial fibroblasts (P < 0.01). Lubricin blunted urate crystal precipitation and IL-1β induction of xanthine oxidase and urate in cultured macrophages (P < 0.001). In lubricin-deficient mice, injection of IL-1β in knees increased xanthine oxidase-positive synovial resident M1 macrophages (P < 0.05).Conclusion. Our findings linked normouricemic erosive gout to attenuated lubricin, with impaired control of cathepsin G activity, compounded by deleterious NLRP3 variants. Lubricin suppressed monosodium urate crystallization and blunted IL-1β-induced increases in xanthine oxidase and urate in macrophages. The collective activities of articular lubricin that could limit incident and erosive gouty arthritis independently of hyperuricemia are subject to disruption by inflammation, activated cathepsin G, and synovial fibroblast TLR-2 signaling.

Background: Gout is a common arthritis, due to deposition of monosodium urate (MSU) crystals which results in IL-1β secretion by tissue-resident macrophages. Xanthine oxidase (XO) catalyzes uric acid (UA) production and in the process, reactive oxygen species (ROS) are generated which contributes to NLRP3 inflammasome activation. Protein phosphatase 2A (PP2A) may be involved in regulating inflammatory pathways in macrophages. The objective of this study was to investigate whether PP2A regulates gout inflammation, mediated by XO activity modulation. We studied UA and ROS generations in MSU stimulated murine bone marrow derived macrophages (BMDMs) in response to fingolimod phosphate, a PP2A activator, and compared its anti-inflammatory efficacy to that of an XO inhibitor, febuxostat.Methods: BMDMs were stimulated with MSU, GM-CSF/IL-1β or nigericin ± fingolimod (2.5 μM) or febuxostat (200 μM) and UA levels, ROS, XO, and PP2A activities, Xdh (XO) expression and secreted IL-1β levels were determined. PP2A activity and IL-1β in MSU stimulated BMDMs ± N-acetylcysteine (NAC) (10 μM) ± okadaic acid (a PP2A inhibitor) were also determined. M1 polarization of BMDMs in response to MSU ± fingolimod treatment was assessed by a combination of iNOS expression and multiplex cytokine assay. The in vivo efficacy of fingolimod was assessed in a murine peritoneal model of acute gout where peritoneal lavages were studied for pro-inflammatory classical monocytes (CMs), anti-inflammatory nonclassical monocytes (NCMs) and neutrophils by flow cytometry and IL-1β by ELISA.Results: Fingolimod reduced intracellular and secreted UA levels (p &lt; 0.05), Xdh expression (p &lt; 0.001), XO activity (p &lt; 0.001), ROS generation (p &lt; 0.0001) and IL-1β secretion (p &lt; 0.0001), whereas febuxostat enhanced PP2A activity (p &lt; 0.05). NAC treatment enhanced PP2A activity and reduced XO activity and PP2A restoration mediated NAC’s efficacy as co-treatment with okadaic acid increased IL-1β secretion (p &lt; 0.05). Nigericin activated caspase-1 and reduced PP2A activity (p &lt; 0.001) and fingolimod reduced caspase-1 activity in BMDMs (p &lt; 0.001). Fingolimod reduced iNOS expression (p &lt; 0.0001) and secretion of IL-6 and TNF-α (p &lt; 0.05). Fingolimod reduced CMs (p &lt; 0.0001), neutrophil (p &lt; 0.001) and IL-1β (p &lt; 0.05) lavage levels while increasing NCMs (p &lt; 0.001).Conclusion: Macrophage PP2A is inactivated in acute gout by ROS and a PP2A activator exhibited a broad anti-inflammatory effect in acute gout in vitro and in vivo.