title: Modulation of IL-33/ST2-TIR and TLR signalling pathway by fingolimod and analogues in immune cells.
abstract: For the immune modulatory drug fingolimod (FTY720), lymphocyte sequestration has been extensively studied and accepted as mode of action. Further, direct effects on immune cell signalling are incompletely understood. Herein, we used the parent drug and newly synthesized analogues to investigate their effects on dendritic cell (DC) calcium signalling and on Th1, Th2 and Th17 responses. DC calcium signalling was determined with a single cell-based confocal assay and IL-33/ST2-TIR Th2-like response with ST2-transduced EL4-6.1 thymoma cells. The Th1/Th17 responses were examined with a LPS/TLR-enhanced antigen presentation assay with OVA-TCRtg CD4 and CD8 spleen cells. Our results revealed a comparable influence of fingolimod and S1P on intracellular calcium level in DC, while an oxy-derivative of fingolimod exhibited an EC50 of 3.3 nm, being 14 times more potent than FTY720-P. The IL-33/ST2-TIR Th2-like response in ST2-EL4 cells was inhibited by fingolimod and analogues at varying degrees. Using the OVA-TCRtg LPS/TLR-enhanced spleen cell assay, we found that fingolimod inhibited both IL-17 and IFN-γ production. In contrast, fingolimod phosphate failed to decrease Th1 cytokines. Interestingly, the effects of the parent compound fingolimod were modulated by the PP2A inhibitor okadaic acid, thus suggesting PP2A as relevant intracellular target. These studies describe detailed immune-modulating properties of fingolimod, including interference with a prototypical Th2 response via IL-33/ST2-TIR. Moreover, differential effects of fingolimod versus its phosphorylated derivative on TLR-activated and antigen-dependent Th1 activation suggest PP2A as an additional target of fingolimod immune therapy. Together with the analogues tested, these data may guide the development of more specific fingolimod derivatives.
authors.affiliation: pharmazentrum frankfurt/ZAFES, Clinic of the J.W. Goethe University, Frankfurt am Main, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Rüger
authors.forename: F
authors.initials: F
authors.lastname: Ottenlinger
authors.forename: M
authors.initials: M
authors.lastname: Schröder
authors.forename: A
authors.initials: A
authors.lastname: Zivković
authors.forename: H
authors.initials: H
authors.lastname: Stark
authors.forename: J M
authors.initials: JM
authors.lastname: Pfeilschifter
authors.forename: H H
authors.initials: HH
authors.lastname: Radeke
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte; D000068876:Fingolimod Hydrochloride; D006706:Homeostasis; D007166:Immunosuppressive Agents / Q000494:pharmacology; D000072179:Interleukin-1 Receptor-Like 1 Protein; D000067596:Interleukin-33; D007378:Interleukins / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D051379:Mice; D011409:Propylene Glycols / Q000494:pharmacology*; D018123:Receptors, Interleukin / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016176:T-Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051193:Toll-Like Receptors / Q000378:metabolism*
chemical_list: D016207:Cytokines; D018984:Epitopes, T-Lymphocyte; C486189:Il1rl1 protein, mouse; C506535:Il33 protein, mouse; D007166:Immunosuppressive Agents; D000072179:Interleukin-1 Receptor-Like 1 Protein; D000067596:Interleukin-33; D007378:Interleukins; D011409:Propylene Glycols; D018123:Receptors, Interleukin; D051193:Toll-Like Receptors; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine; D002118:Calcium