Role of Protein Phosphatase-2A in Regulating Monocyte Activation by Soluble and Crystalline Uric Acid in Gout

Gout is a chronic inflammatory disease caused by the phagocytosis of monosodium urate monohydrate (MSU) crystals by monocytes/macrophages resulting in downstream expression and production of interleukin-1 beta (IL-1β) and chemokines. The activation of monocytes by MSU crystals involves the priming of monocytes with danger signals e.g. lipopolysaccharide (LPS) or soluble uric acid (UA), crystal phagocytosis and subsequent NLRP3 inflammasome activation and conversion of pro-IL-1β to active IL-1β. Protein-phosphatase-2A (PP2A) is a serine/threonine phosphatase that plays an important role in cell growth and inflammation. The prodrug Fingolimod (FTY720) and its phosphorylated active metabolite (p-FTY720) activate intracellular PP2A. We hypothesized that monocyte activation by MSU crystals is mediated by a reduction in intracellular PP2A activity and restoring PP2A activity reduces MSU-induced inflammation in monocytes. We aimed to investigate the role of PP2A in regulating monocyte priming and activation by MSU crystals and evaluate whether intracellular PP2A activation exerts an anti-inflammatory effect in MSU-stimulated monocytes. Human THP-1 monocytes were primed with a combination of UA and LPS. MSU stimulation was performed for 4-6 hours and MSU crystal phagocytosis, PP2A activity, IL-1β expression and production were studied in primed and unprimed monocytes. We performed PP2A knockdown in THP-1 monocytes and evaluated the impact of PP2A attenuation on IL-1β expression and production in unprimed THP-1 monocytes. Time-dependent intracellular PP2A activation in response to FTY720 or p-FTY720 treatments was studied and we evaluated the impact of p-FTY720 treatment on IL-1β expression and production in MSU stimulated human monocytes. Priming with UA+LPS increased MSU phagocytosis and IL-1β expression and production in monocytes. This effect was associated with a reduction in intracellular PP2A activity. PP2A knockdown increased IL-1β expression and production. FTY720 and p-FTY720 increased intracellular PP2A activity in monocytes. p-FTY720 treatment reduced IL-1β expression and production in UA+LPS pre-treated monocytes following MSU stimulation mediated by an increase in PP2A activity with no alteration in PP2A gene expression. In summary, UA and LPS enhanced MSU phagocytosis, expression and production of IL-1β via a reduction in PP2A activity. Pharmacological restoration of PP2A activity exerted an antiinflammatory effect. We conclude that PP2A is a novel therapeutic target for gout treatment

PRG4/CD44/PP2A Signaling Axis: A Significant Regulator of Gout Arthritis

Introduction: Gout is a common chronic arthritis caused by monosodium urate (MSU) crystal deposition in joints leading to activation of synovial resident macrophages and downstream production of interleukin-1b (IL-1b). Xanthine oxidase (XO) catalyzes purines into uric acid (UA) and reactive oxygen species (ROS) that activate NLRP3 inflammasome. Protein phosphatase-2A (PP2A) may be involved in regulating inflammatory pathways in macrophages. Proteoglycan 4 (PRG4) and its receptor CD44 and its transducer PP2A signaling axis play a biologically significant role in regulating urate crystal inflammation. Methods: Monocytes and macrophages were primed with TLR2 agonist and stimulated with MSU crystals. Fingolimod (PP2A activating drug) and febuxostat (XO inhibitor) were assessed for their ability to regulate PP2A and XO activity, UA production, ROS generation and IL-1b secretion. Anti-inflammatory effect of fingolimod was assessed in vivo. Phagocytic activation of gout and normal monocytes was compared along with intracellular ROS generation and IL-1b production. rhPRG4 and IL-1RA (IL-1b receptor antagonist) were examined for their ability to regulate phagocytosis, PP2A activity, ROS generation and IL-1b secretion. Peritoneal influx of classical monocytes (CMs) and non-classical monocytes (NCMs) in Prg4 deficient mice was assessed. Results: Fingolimod and febuxostat enhanced PP2A activity and suppressed XO activity, UA and ROS generation and IL-1b secretion. Fingolimod reduced influx of CMs and neutrophils and increased influx of NCMs in an in vivo model of acute gout. Phagocytic activation, ROS generation and IL-1b were higher in gout monocytes. rhPRG4 showed superior efficacy in reduction of phagocytosis, ROS generation and increasing PP2A activity. rhPRG4 showed faster resolution of inflammation in Prg4 deficient mice. 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. The anti-inflammatory effect of rhPRG4 in monocytes is mediated by PP2A and PRG4 plays a role in regulation of influx of immune cells in site of gout flare

Protein Phosphatase 2A Regulates Xanthine Oxidase-derived ROS Production in Macrophages and Influx of Inflammatory Monocytes in a Murine Gout Model

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 \u3c 0.05), Xdh expression (p \u3c 0.001), XO activity (p \u3c 0.001), ROS generation (p \u3c 0.0001) and IL-1β secretion (p \u3c 0.0001), whereas febuxostat enhanced PP2A activity (p \u3c 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 \u3c 0.05). Nigericin activated caspase-1 and reduced PP2A activity (p \u3c 0.001) and fingolimod reduced caspase-1 activity in BMDMs (p \u3c 0.001). Fingolimod reduced iNOS expression (p \u3c 0.0001) and secretion of IL-6 and TNF-α (p \u3c 0.05). Fingolimod reduced CMs (p \u3c 0.0001), neutrophil (p \u3c 0.001) and IL-1β (p \u3c 0.05) lavage levels while increasing NCMs (p \u3c 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

Fingolimod Phosphate (FTY720-P) Activates Protein Phosphatase 2A in Human Monocytes and Inhibits Monosodium Urate Crystal-Induced Interleukin-1 β Production

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-1β 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 toward MSU crystal phagocytosis and that increased IL-1β 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-1β and mature IL-1β and enhanced MSU crystal phagocytosis and its downstream IL-1β expression (P \u3c 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-1β expression and secretion (P \u3c 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 hours. FTY720 and FTY720-P activated PP2A to a similar extent, and maximal enzyme activity occurred at 24 hours for FTY720 and at 6 hours for FTY720-P. FTY720-P (2.5 μM) reduced pro-IL-1β production and IL-1β secretion in primed and MSU crystal–stimulated monocytes (P \u3c 0.0001) without changing the magnitude of crystal phagocytosis. We conclude that PP2A is a promising new target in acute gout

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

Objectives 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. Methods 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. Results Enhanced phagocytic activation of gout monocytes under basal conditions (p\u3c0.001) was associated with ROS generation and MSU stimulated IL-1β secretion (p\u3c0.05). rhPRG4 reduced bead phagocytosis by normal and gout monocytes compared to IL-1RA and both treatments were efficacious in reducing IL-1β secretion (p\u3c0.05). 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\u3c0.05). 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\u3c0.01) and rhPRG4 treatment reduced neutrophil accumulation and enhanced anti-inflammatory monocyte influx (p\u3c0.05). Conclusions 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 in vivo, PRG4 plays a role in regulating the trafficking of immune cells into the site of a gout flare

Needs assessment to strengthen capacity in water and sanitation research in Africa:experiences of the African SNOWS consortium

Despite its contribution to global disease burden, diarrhoeal disease is still a relatively neglected area for research funding, especially in low-income country settings. The SNOWS consortium (Scientists Networked for Outcomes from Water and Sanitation) is funded by the Wellcome Trust under an initiative to build the necessary research skills in Africa. This paper focuses on the research training needs of the consortium as identified during the first three years of the project

Amplification of Inflammation by Lubricin Deficiency Implicated in Incident, Erosive Gout Independent of Hyperuricemia

Objective In gout, hyperuricemia promotes urate crystal deposition that stimulates the NLRP3 inflammasome and 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 meeting ACR/EULAR gout classification criteria in a normouricemic young adult female. Methods Whole genome sequencing, quantitative proteomics, whole blood RNA-seq, and IL-1β-induced murine knee synovitis characterized proband candidate genes, biomarkers, and pathogenic mechanisms. Results Lubricin was attenuated in proband serum, 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-Alpha-Trypsin Inhibitor Heavy Chain 3, an inhibitor of lubricin-degrading Cathepsin G. Proband serum protein interactome network changes supported enhanced lubricin degradation, with Cathepsin G activity increased relative to its inhibitors SERPINB6 and Thrombospondin1. TLR2 activation suppressed cultured human synovial fibroblast lubricin mRNA and release (p\u3c0.01). Lubricin blunted urate crystal precipitation, and IL-1β induction of xanthine oxidase and urate in cultured macrophages (p\u3c0.001). In lubricin-deficient mice, IL-1β knee injection increased xanthine oxidase positive synovial resident M1 macrophages (p\u3c0.05). Conclusion We 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 macrophage xanthine oxidase and urate. 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 TLR2 signaling

Zerumbone reduces TLR2 stimulation-induced M1 macrophage polarization pattern via upregulation of Nrf-2 expression in murine macrophages

Hyperuricemia contributes significantly to gout arthritis pathogenesis, which promotes urate crystal deposition in the joints and activates joint-resident macrophages and circulating monocytes to initiate a state of inflammatory arthritis. In the joint, macrophages have an immune defense role where the presence of urate crystals results in the inflammatory mediators secretion, inflammatory cells recruitment to the joint, and shift macrophage population toward M1 pro-inflammatory phenotypes. Current treatment modalities of gout arthritis have side effects that limit their use in the elderly. A novel treatment that targets macrophage polarization to re-establish homeostasis may initiate a drug discovery program of novel disease-modifying agents for gout. Zerumbone (Zer) is a sesquiterpenoid bioactive compound found in the rhizome of Zingiberaceae family and possesses anti-inflammatory, antioxidant, and anti-proliferative activity. Our study hypothesized that soluble uric acid (sUA) and Pam3CSK4 (TLR2 agonist) reduce the anti-inflammatory function of murine M2 bone marrow-derived macrophages and change the expression of M2 genetic markers toward M1 phenotypes. We observed that priming of M2 macrophages with sUA and Pam3CSK4 significantly decreased M2 specific markers expression, e.g., Arg-1, Ym-1, and Fizz-1, enhanced mRNA expression of IL-1β, TNF-α, CXCL2, and iNOS and increased oxidative stress in M2 macrophages, as exhibited by a reduction in Nrf2 expression. We also aimed to study the impact of Zer on reducing the pro-inflammatory effect of sUA in TLR2-stimulated M2 macrophages. We noticed that Zer treatment significantly reduced L-1β and TNF-α production following Pam3CSK4 + sUA treatment on M2 macrophages. Furthermore, Zer reduced the caspase-1 activity without altering cytosolic NLRP3 content in challenged M2 BMDMs. We also observed that Zer significantly enhanced M2-associated marker’s expression, e.g., Arg-1, Ym-1, and Fizz-1, and augmented Nrf-2 and other antioxidant proteins, including HMOX1 and srxn1expression following Pam3CSK4 + sUA treatment. We draw the conclusion that Zer is a potentially effective anti-inflammatory treatment for gout arthritis linked to hyperuricemia

North American Neuromodulation Society Educational Curriculum for Intrathecal Drug Delivery Systems Implantation and Management

OBJECTIVES: Intrathecal drug delivery systems (IDDSs) are used for the treatment of pain and spasticity. A wide range of educational criteria exist for these devices. The North American Neuromodulation Society (NANS) Education Committee developed a comprehensive IDDS curriculum to function as a standard for physician graduate education and assessment through training and into practice. MATERIAL AND METHODS: A multidisciplinary and diverse task force gathered by the NANS Education Committee met in person and virtually over several sessions and developed an IDDS curriculum modeling their previous work on spinal cord stimulation and following the Accreditation Council for Graduate Medical Education (ACGME) Milestones. There were iterative revisions and adaptations to the curriculum, and the final version was approved by the NANS Board of Directors. RESULTS: The curriculum was developed with distinction between implanting physicians and managing physician and physicians who perform both tasks. There is a lateral temporal progression from early learner to practitioner, with advanced learner in the middle. In addition, there is a modular vertical organization that divides the curriculum into the six educational competencies outlined by the ACGME. CONCLUSION: A comprehensive, modular, graduated, and segmented educational curriculum for IDDSs was developed by NANS. We propose the curriculum to be the standard for guidance and assessment of trainees and physicians pursuing training in implanting or managing IDDSs