Fingolimod (FTY720) Activates Protein Phosphatase‐2A in Human Monocytes and Inhibits Uric Acid Crystal‐Induced Interleukin‐1 Beta Production

Abstract

IntroductionGout is a chronic inflammatory disease due to monosodium urate (MSU) crystal deposits in articular joints. Phagocytosis of MSU crystals by monocytes/macrophages results in downstream expression and production of interleukin‐1 beta (IL‐1β). The two‐step process of gout inflammation involves 1) priming of monocytes with danger signals e.g. lipopolysaccharide (LPS) or soluble uric acid (UA) and 2) MSU phagocytosis and activation of the NLRP3 inflammasome in monocytes. Protein‐phosphatase‐2A (PP2A) is a serine/threonine phosphatase, and a reduction in intracellular macrophage PP2A activity was shown to aggravate LPS‐induced proinflammatory cytokine production. The prodrug Fingolimod (FTY720) and its phosphorylated active metabolite (p‐FTY720) are efficacious in multiple sclerosis and their mechanism of action involves activation of intracellular PP2A. The objective of this study is to investigate the role of PP2A in regulating monocyte priming and activation by MSU crystals and whether intracellular PP2A activation exerts an anti‐inflammatory effect in MSU‐stimulated monocytes.MethodsHuman THP‐1 monocytes (ATCC; 500,000 cells per well) were treated with UA (50 mg/dL) and LPS (10ng/mL) for 24 hours. Subsequently, cells were treated with MSU crystals (100ug/mL) and MSU phagocytosis was determined at 4 hours using flow cytometry and the percentage of cells displaying side‐scattering above a threshold value, indicative of crystal phagocytosis, were determined. Intracellular PP2A activity was determined at 6 hours following MSU stimulation using the PP2A Immunoprecipitation Phosphatase Assay Kit (Fisher Scientific). IL‐1β expression and protein levels at 6 hours were determined using quantitative polymerase chain reaction and ELISA, respectively. THP‐1 monocytes were treated with FTY720 or p‐FTY720 (2.5mM for both treatments; Cayman Chemicals) and PP2A activity was determined at 1, 3, 6 and 24 hours. THP‐1 monocytes were treated with UA+LPS followed by MSU ± p‐FTY720 (2.5mM for 6 hours) and IL‐1β expression and protein levels were determined. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey’s post‐hoc test.ResultsUA+LPS pre‐treatment increased MSU phagocytosis by THP‐1 monocytes (fig. 1A and 1B; p<0.001) and IL‐1β expression (fig. 1C; p<0.001) and production (fig. 1D; p<0.001). This effect was associated with a reduction in intracellular PP2A activity (fig. 1E; p<0.001). FTY720 and p‐FTY720 increased intracellular PP2A activity in monocytes (fig. 2A; p<0.001) with a higher peak effect achieved at 1 hour for p‐FTY720 (p<0.01). p‐FTY720 treatment reduced IL‐1β expression (fig. 2B; p<0.001) and mature IL‐1β production (fig. 2C; p<0.001) in UA+LPS pre‐treated human monocytes following MSU stimulation.ConclusionUA and LPS enhanced MSU phagocytosis, expression and production of IL‐1β, potentially via a reduction in intracellular PP2A activity. FTY720 and p‐FTY720 activated PP2A in monocytes with an onset of effect of 1 hour and correspondingly reduced IL‐1β expression and production. PP2A is a novel therapeutic target for gout treatment.Impact of soluble uric acid (UA) and lipopolysaccharide (LPS) pre‐treatments on THP‐1 monocyte phagocytic activity towards monosodium urate (MSU) crystals, interleukin‐1 beta (IL‐1β) expression and production and association with intracellular protein phosphatase‐2A (PP2A) activity. A) Representative flow cytometry scatter plots showing enhanced MSU crystal phagocytosis (as shown by more cells with increased side‐scatter in P2 region of interest) by THP‐1 monocytes following UA+LPS pre‐treatment. B) UA and UA+LPS pre‐treatment increased the percentage of THP‐1 monocytes positive for MSU phagocytosis. C) UA+LPS pre‐treatment increased IL‐1β expression by THP‐1 monocytes following MSU challenge. D) UA+LPS pre‐treatment increased IL‐1β production by THP‐1 monocytes following MSU challenge. E) Intracellular PP2A activity was reduced in UA or UA+LPS pre‐treated THP‐1 monocytes. *p<0.001; **p<0.01; n.s.: non significant.Figure 1Impact of FTY720 (Fingolimod; prodrug) and phosphorylated FTY720 (p‐FTY‐720; active metabolite) treatments on protein phosphatase‐2A (PP2A) activity in human THP‐1 monocytes and impact of p‐FTY720 treatment on interleukin‐1 beta (IL‐1β) expression and production by monosodium urate (MSU) crystal stimulated THP‐1 monocytes ± soluble uric acid (UA) and lipopolysaccharide (LPS) pre‐treatment. Treatments were conducted using 2.5μM concentrations. A) FTY720 and p‐FTY720 increased intracellular PP2A activity in THP‐1 monocytes. B) p‐ FTY720 reduced IL‐1β expression in THP‐1 monocytes. C) p‐FTY720 reduced IL‐1β production in THP‐1 monocytes. *p<0.001; **p<0.01; n.s.: non significant.Figure 2