Protein phosphatase 2A regulates xanthine oxidase-derived ROS production in macrophages and influx of inflammatory monocytes in a murine gout model

Abstract

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 < 0.05), Xdh expression (p < 0.001), XO activity (p < 0.001), ROS generation (p < 0.0001) and IL-1β secretion (p < 0.0001), whereas febuxostat enhanced PP2A activity (p < 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 < 0.05). Nigericin activated caspase-1 and reduced PP2A activity (p < 0.001) and fingolimod reduced caspase-1 activity in BMDMs (p < 0.001). Fingolimod reduced iNOS expression (p < 0.0001) and secretion of IL-6 and TNF-α (p < 0.05). Fingolimod reduced CMs (p < 0.0001), neutrophil (p < 0.001) and IL-1β (p < 0.05) lavage levels while increasing NCMs (p < 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.

Proteoglycan 4 (PRG4)/Lubricin and the Extracellular Matrix in Gout

“…This is in line with a previous study, in which ROS generated from XOR were identified as a significant driver of NLRP3 inflammasome activation and IL-1β production [91]. XOR-derived ROS accumulation inactivates PP2A in macrophages [90]. In addition, a PP2A-activating drug treatment inhibits XOR expression, ROS and urate production and stimulates the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcriptional activator of antioxidant response in macrophages [90,92].…”

“…The ROS, in turn, inactivate the enzyme via mechanisms that include tyrosine phosphorylation, leucine carboxy methylation, and the dissociation of the enzyme's catalytic subunit [87][88][89]. We identified XOR in macrophages as a major source of ROS production in MSU crystal-stimulated BMDMs [90]. This is in line with a previous study, in which ROS generated from XOR were identified as a significant driver of NLRP3 inflammasome activation and IL-1β production [91].…”

The potential relationship of coronary artery disease and hyperuricemia: A cardiometabolic risk factor

“…Uric acid is produced by the breakdown of nucleotides in the body through a variety of uricase enzymes and the consumption of adenosine triphosphate. During uric acid production, the activity of xanthine oxidase (XO) increases, prompting the elevation of ROS [ 58 ]. Elevated ROS can induce the migration and proliferation of small arterial smooth muscle cells and the production of monocyte chemotactic protein (MCP)-1, participating in the development of atherosclerosis [ 58 ].…”

“…During uric acid production, the activity of xanthine oxidase (XO) increases, prompting the elevation of ROS [ 58 ]. Elevated ROS can induce the migration and proliferation of small arterial smooth muscle cells and the production of monocyte chemotactic protein (MCP)-1, participating in the development of atherosclerosis [ 58 ]. ROS causes oxidation of low-density lipoprotein to OX-LDL, inducing an inflammatory response in vascular endothelial cells.…”

Atherogenic Activation of Human Vascular Smooth Muscle Cells by Monosodium Urate Crystals

“…Moreover, small, tophus-like intimal and adventitial coronary artery lesions have also been reported [10]. Notably, inflammatory macrophages express xanthine oxidase (XO) [11], and XO has been reported to be significantly upregulated in the macrophage-rich atherosclerotic plaques of patients with symptomatic central nervous system (CNS) ischemia, compared to plaques of those without CNS symptoms [12,13]. XO and urate are particularly enriched in the shoulder and subendothelial regions of such carotid atherosclerotic plaques, and increased XO and urate aggregate in plaque [13].…”