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

Abstract

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.

Targeting Tristetraprolin Expression or Functional Activity Regulates Inflammatory Response Induced by MSU Crystals

“…To date, the best known PP2A activator is the sphingosine analog FTY720, FTY720 has other roles, most notably as a functional antagonist of the S1P pathway. Previous study has shown that the active metabolites of FTY720 can activate PP2A and inhibit the production of IL-1b induced by MSU crystals (37), but the molecular mechanism of its inhibition of IL-1b production has not been thoroughly explored. Arctigenin is a natural product, so we chose to study the effect of ATG on the inflammatory response induced by MSU crystals and its molecular mechanism.…”

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

“…To further characterize the contribution of PP2A to rhPRG4’s mechanism of action, we evaluated the effect of co-incubating rhPRG4 with okadaic acid, a potent PP2A inhibitor ( 30 ). Normal and gout PBMCs and THP-1 monocytes were primed with a TLR2 ligand, Pam3CSK4, for 24h prior to adding urate crystals, as PBMCs and monocytes fail to secrete significant IL-1β in response to crystals alone ( 22 , 26 ). Across all experiments, assays were performed with two technical replicates per experimental group, with 3-4 independent experiments for THP-1 monocytes.…”

“…THP-1 monocytes were treated with Pam3CSK4 (1μg/mL) for 24h followed by MSU crystals (200μg/mL) ± IL-1RA (250ng/mL) or rhPRG4 (200μg/mL) for 6h. IL-1β gene expression was performed as previously described ( 26 ), using commercially available primers and probes for IL-1β (Hs01555410_m1) and β-actin (Hs00194899_m1) (ThermoFisher Scientific, USA), and the cycle threshold (Ct) value of IL-1β was normalized to the Ct value of β-actin in the same sample, and the relative expression in the different experimental groups compared to untreated controls was computed using the 2 -ΔΔCt method ( 26 ). In another set of experiments, cells were treated and subsequently collected and lysed using RIPA buffer + 1% protease inhibitor (ThermoFisher Scientific), and cell lysate total protein was determined using the micro-BCA assay kit (Sigma Aldrich).…”

“…PP2A was immunoprecipitated from THP-1 monocyte cell lysates following Pam3CSK4 priming for 24h and MSU crystals (200μg/mL) ± IL-1RA (250ng/mL) or rhPRG4 (200μg/mL) for 6h. PP2A activity was determined as previously described ( 26 ), and normalized to cell lysate total protein. To further investigate the role of PP2A in mediating rhPRG4’s effect, THP-1 monocytes were stimulated with Pam3CSK4 for 24h followed by MSU crystals (200μg/mL) ± rhPRG4 (200μg/mL) ± okadaic acid (5nM) (Cayman Chemicals) for 6h.…”

“…The efficacy of rhPRG4 was likely due to its binding CD44 receptor, as CD44 was shown to mediate phagocytic uptake of urate crystals by murine and human macrophages ( 24 , 25 ). The significance of the PRG4/CD44/PP2A axis was further highlighted by our recent observations that uptake of urate crystals by human monocytes was associated with a reduction in PP2A activity and that a small molecule PP2A activator reduced IL-1β secretion in human monocytes via attenuating pro-IL-1β production ( 26 ). While available in vitro and in vivo data suggest that rhPRG4 may be of considerable utility as a novel therapeutic in acute gout, the efficacy of rhPRG4 in gout monocytes remains unclear.…”