Fingolimod Attenuates Splenocyte-Induced Demyelination in Cerebellar Slice Cultures

Abstract

The family of sphingosine-1-phosphate receptors (S1PRs) is G-protein-coupled, comprised of subtypes S1PR1-S1PR5 and activated by the endogenous ligand S1P. The phosphorylated version of Fingolimod (pFTY720), an oral therapy for multiple sclerosis (MS), induces S1PR1 internalisation in T cells, subsequent insensitivity to S1P gradients and sequestering of these cells within lymphoid organs, thus limiting immune response. S1PRs are also expressed in neuronal and glial cells where pFTY720 is suggested to directly protect against lysolecithin-induced deficits in myelination state in organotypic cerebellar slices. Of note, the effect of pFTY720 on immune cells already migrated into the CNS, prior to treatment, has not been well established. We have previously found that organotypic slice cultures do contain immune cells, which, in principle, could also be regulated by pFTY720 to maintain levels of myelin. Here, a mouse organotypic cerebellar slice and splenocyte co-culture model was thus used to investigate the effects of pFTY720 on splenocyte-induced demyelination. Spleen cells isolated from myelin oligodendrocyte glycoprotein immunised mice (MOG-splenocytes) or from 2D2 transgenic mice (2D2-splenocytes) both induced demyelination when co-cultured with mouse organotypic cerebellar slices, to a similar extent as lysolecithin. As expected, in vivo treatment of MOG-immunised mice with FTY720 inhibited demyelination induced by MOG-splenocytes. Importantly, in vitro treatment of MOG- and 2D2-splenocytes with pFTY720 also attenuated demyelination caused by these cells. In addition, while in vitro treatment of 2D2-splenocytes with pFTY720 did not alter cell phenotype, pFTY720 inhibited the release of the pro-inflammatory cytokines such as interferon gamma (IFNγ) and interleukin 6 (IL6) from these cells. This work suggests that treatment of splenocytes by pFTY720 attenuates demyelination and reduces pro-inflammatory cytokine release, which likely contributes to enhanced myelination state induced by pFTY720 in organotypic cerebellar slices.

Galactosylsphingosine (psychosine)-induced demyelination is attenuated by sphingosine 1-phosphate signalling

“…5A). In agreement with our previous studies (Pritchard et al, 2014;Sheridan and Dev, 2012), pFTY720 attenuated LPC-induced demyelination compared with control, as observed by expression of myelin oligodendrocyte glycoprotein (MOG) (Fig. 5B) and myelin basic protein (MBP) (Fig.…”

“…Specific treatment details are indicated in the figure legends. Organotypic cerebellar slice cultures were prepared exactly as we have described previously (Pritchard et al, 2014;Sheridan and Dev, 2012). In brief, tissue isolated from postnatal day 10 (P10) C57BL/6 mice and 400 µm parasagittal slices of cerebellum were grown on cell culture inserts (5-6 slices each) (Millicell PICMORG50 Millipore).…”

“…For cytokine analysis supernatants from cell culture were removed and examined for their cytokine content using ELISA kits for IL6 (DY406), TNFα (DY410) and IL-1β (DY401) according to the manufacturer's instructions (R&D Systems), and exactly as we have described before (Pritchard et al, 2014). For western blotting, cerebellar slices were scraped from the culture membrane and suspended in PTxE buffer (PBS, 1% Tritonx, 1 mM EDTA) using mechanical homogenisation and sonication.…”

“…For western blotting, cerebellar slices were scraped from the culture membrane and suspended in PTxE buffer (PBS, 1% Tritonx, 1 mM EDTA) using mechanical homogenisation and sonication. Samples were denatured and electrophoresis carried out on 10% SDS-polyacrylamide gels exactly as we have previously reported (Pritchard et al, 2014). Primary antibodies used were: anti-MOG (1:2000; Millipore: MAB5680) and antitubulinβ (1:5000; Millipore MAB3408).…”

Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair

“…28,[46][47][48] Furthermore, it has also been shown that FTY720 can enhance remyelination in organotypic slice cultures. 27,49,50 In vivo studies demonstrated that FTY720 is effective at treating acute EAE by promoting OPC proliferation and differentiation and by stimulating the sonic hedgehog (Shh) signaling pathway. 51 These results, together with our findings, indicate that FTY720 has both anti-inflammatory and neuroregenerative effects.…”