Modulation of IL‐33/ST2‐TIR and TLR Signalling Pathway by Fingolimod and Analogues in Immune Cells

Abstract

a These authors contributed equally to this work. AbstractFor 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 EC 50 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-c 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 TLRactivated 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.

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

“…This cell line expresses S1PR2, S1PR4, and S1PR5 on mRNA level (Fig. 1B), which has been previously confirmed by our group with another set of primers [13]. The expression pattern of S1P receptors is in line with the expression of NK-and B-cell markers of EL4 cells, with S1PR4 expressed by B cells and S1PR5 highly expressed by NK cells [8,43].…”

“…Therefore, we hypothesize that the agonistic effect of S1P on IL‐33 induced IL‐2 formation may be mediated by agonistic effects on S1P receptors, while the inhibitory effects from the FTY720‐P is mediated by its partial antagonistic activities. FTY720 was not inhibiting IL‐2 in both experimental conditions, although we determined an inhibition of IL‐33 induced IL‐2 in the same cells stimulated with 5 ng/mL IL‐33 in one of our previous publications . Thus, FTY720 can inhibit IL‐2 formation exclusively, when the cells are stimulated with very low concentrations of IL‐33, indicating a low turnover rate from FTY720 to FTY720‐P in EL4‐ST2 cells.…”

“…(B) Representative PAA‐gel showing S1P receptor expression in EL4‐ST2 cells on mRNA level. Data shown are representative of two independent experiments, confirmed by qRT‐PCR with a different set of primer . (C or D) FTY720, FTY720‐P, or S1P was incubated for 24 h on EL4‐ST2 cells stimulated with (C) 20 ng/mL IL‐33 or (D) 5 ng/mL IL‐33 + 250 nM ionomycin.…”

“…E and F). Our group already investigated that FTY720 but not FTY720‐P was specifically able to inhibit the OVA/LPS‐induced IFN‐ γ formation in transgenic OVA CD8 + spleen cells (OT‐II), which was reversible by okadaic acid . While preparing this manuscript Ntranos et al.…”

“…4E and F). Our group already investigated that FTY720 but not FTY720-P was specifically able to inhibit the OVA/LPS-induced IFN-γ formation in transgenic OVA CD8 + spleen cells (OT-II), which was reversible by okadaic acid [13]. While preparing this manuscript Ntranos et al [46] showed that FTY720 also inhibited the αCD3/αCD28-induced production of IFN-γ and cytotoxicity of CD8 + T cells in vitro and in vivo.…”