Fingolimod targeting protein phosphatase 2A differently affects IL‐33 induced IL‐2 and IFN‐γ production in CD8⁺ lymphocytes

Abstract

Multiple sclerosis patients are treated with fingolimod (FTY720), a prodrug that acts as an immune modulator. FTY720 is first phosphorylated to FTY720-P and then internalizes sphingosine-1-phosphate receptors, preventing lymphocyte sequestration. IL-33 is released from necrotic endothelial cells and contributes to MS severity by coactivating T cells. Herein we analyzed the influence of FTY720, FTY720-P, and S1P on IL-33 induced formation of IL-2 and IFN-γ, by using IL-33 receptor overexpressing EL4 cells, primary CD8 + T cells, and splenocytes. EL4-ST2 cells released IL-2 after IL-33 stimulation that was inhibited dose-dependently by FTY720-P but not FTY720. In this system, S1P increased IL-2, and accordingly, inhibition of S1P producing sphingosine kinases diminished IL-2 release. In primary CD8 + T cells and splenocytes IL-33/IL-12 stimulation induced IFN-γ, which was prevented by FTY720 but not FTY720-P, independently from intracellular phosphorylation. The inhibition of IFN-γ by nonphosphorylated FTY720 was mediated via the SET/protein phosphatase 2A (PP2A) pathway, since a SET peptide antagonist also prevented IFN-γ formation and the inhibition of IFN-γ by FTY720 was reversible by a PP2A inhibitor. While our findings directly improve the understanding of FTY720 therapy in MS, they could also contribute to side effects of FTY720 treatment, like progressive multifocal leukoencephalopathy, caused by an insufficient immune response to a viral infection.Keywords: CD8 + T cells r Fingolimod (FTY720) r IL-33 r Multiple sclerosis r Protein phosphatase 2a (PP2A)Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionMultiple sclerosis is an autoimmune disorder, characterized by the destruction of the myelin sheath by autoreactive immune cells, leading to astrogliosis, demyelination, and increasing disability. Most patients are diagnosed between 20 and 40 years, Correspondence: Dr. Heinfried H. Radeke e-mail: radeke@em.uni-frankfurt.de with a 2:1 female to male ratio [1]. Fingolimod (2-amino-2-[2-(4-octylphenyl)ethyl]-propane-1,3-diol; FTY720) is an immunemodulating prodrug derived from the natural compound myriocin and was approved in 2010 as the first oral treatment for relapsing-remitting MS. Its phosphorylated derivative is an analog of the bioactive sphingolipid sphingosine-1-phosphate (S1P), which is involved in cellular processes such as cell cycle, apoptosis, or immune modulatory processes such as migration or the regulation of cytokine expression [2,3]. Extracellular lipophilic C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 942Florian Ottenlinger et al. Eur. J. Immunol. 2016. 46: 941-951 FTY720 or sphingosine easily passes the membrane of immune cells to the intracellular space. Here, sphingosine is phosphorylated by the sphingosine kinases (SphKs) SphK1 and SphK2 to S1P, whereas FTY720 is phosphorylated by SphK2 to FTY720-phosphate (FTY720-P) [4]. Once phosphorylated, the amphiphilic nature of FTY720-P or S1P prev...

FTY720 ameliorates renal fibrosis by simultaneously affecting leucocyte recruitment and TGF-β signalling in fibroblasts

“…Regarding its detailed mechanisms, other studies have discovered that FTY720 can promote PP2A phosphatase activities in many types of cells [30][31][32] and dephosphorylate activated signal transducer and activator of transcription-1 (STAT-3) [33,34] and AKT [35,36]. In our study, FTY720 reduced a-SMA and collagen expression, suggesting that FTY720 prevents the genesis of myofibroblasts and reduces the synthesis of ECM protein in vivo.…”

“…This effective interference with multiple signalling pathways in TGF-b-induced fibrogenesis would constitute the second advantage of this drug in the treatment of renal fibrosis. Regarding its detailed mechanisms, other studies have discovered that FTY720 can promote PP2A phosphatase activities in many types of cells [30][31][32] and dephosphorylate activated signal transducer and activator of transcription-1 (STAT-3) [33,34] and AKT [35,36]. In addition, it is now clear that FTY720 targets G protein-coupled S1P receptors as a potent agonist [18], thereby regulating a variety of intracellular signalling pathways.…”

Interferon-Beta Increases Plasma Ceramides of Specific Chain Length in Multiple Sclerosis Patients, Unlike Fingolimod or Natalizumab

“…Furthermore, injection of C 16:0 ceramide into the hippocampus is sufficient to induce depression-like behavior in WT mice (Gulbins et al, 2013). Downstream of ceramides, S1P is another very important signaling molecule, especially in MS. By activating five known S1PRs on the plasma membrane of various cells, S1P influences cellular processes such as the cell cycle, apoptosis and the regulation of cytokine expression (Schröder et al, 2011; Arlt et al, 2014; Ottenlinger et al, 2016). Furthermore, a steep S1P gradient between blood and secondary lymphoid compartments regulates the egress of lymphocytes out of secondary lymphoid organs.…”

“…Its additional effects include activation of astrocytic or neuronal S1PRs by FTY720-P or the inhibition of IFN-γ formation by non-phosphorylated FTY720 (Groves et al, 2013; Ottenlinger et al, 2016). The humanized anti-α4-integrin antibody natalizumab (Tysabri ® , Biogen, Cambridge, MA, USA) blocks the migration of T cells across intracerebral vessel walls, resulting in an ARR of 68% (Polman et al, 2006).…”

Activation-Induced Cell Death of Dendritic Cells Is Dependent on Sphingosine Kinase 1

“…Thus, Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis by inhibiting central memory T cell egress from secondary lymphoid organs (Brinkmann et al, 2010 ). However, Fingolimod has also direct influences on immune cell function (Ottenlinger et al, 2016 ) and is able to accumulate intracellularly (Schroder et al, 2015 ).…”