Fingolimod ameliorates the development of experimental autoimmune encephalomyelitis by inhibiting Akt–mTOR axis in mice

doi:10.1016/j.intimp.2015.11.024

International Immunopharmacology

Volume 30, January 2016, Pages 171-178

https://doi.org/10.1016/j.intimp.2015.11.024 Get rights and content

Highlights

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Fingolimod treatment significantly reduces EAE development in mice.
•
Fingolimod treatment on EAE may inhibit the Akt-mTOR axis.
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Fingolimod treatment on EAE may affect Th1 and Treg function.

Abstract

Fingolimod is a new immunosuppressive agent approved by Food and Drug Administration (FDA) for treating multiple sclerosis (MS). It acts as a functional antagonist to downregulate the S1P1 receptor, which is known to signal through the Akt–mTOR pathway. We investigated the mechanism of fingolimod action in the classical animal model of MS: experimental autoimmune encephalomyelitis (EAE). Fingolimod treatment significantly reduced clinical scores and histopathology in this model, even when treatment was begun after the onset of pathology. The Akt–mTOR signaling pathway was shown to be activated in the EAE model, by measuring the abundance of downstream activation markers, pAkt and ps6k. And this pathway was inhibited when EAE mice were treated with fingolimod. Mice with EAE exhibited an increased frequency of Th1 cells in the spleen, with concomitant increases in the mRNA levels of Tbet and Ifng and increased IFN-γ production by activated splenocytes; the frequency of Treg cells, as well as mRNA levels of Foxp3 and Tgfb, was reduced, as was TGF-β production by activated splenocytes. After treatment with fingolimod, these parameters were reversed, suggesting that fingolimod treatment inhibits the Akt–mTOR axis in EAE, which affects the differentiation and function of Th1 and Treg cells. These results provide an insight into the mechanism of action of fingolimod treatment and may provide new ideas for treating EAE and MS.

Introduction

Multiple sclerosis (MS) is a globally distributed inflammatory demyelinating disease. In September 2010, fingolimod was approved as the first oral drug for treating MS by Food and Drug Administration (FDA) [1], [2]. Previous studies have confirmed that fingolimod prevents lymphocytes egress from peripheral lymphoid tissue and blocks lymphocytes in secondary lymphoid organs [3], [4], [5]. The number of lymphocytes in the blood is reduced, which inhibits pathogenic lymphocytes from attacking the central nervous system [6], [7], [8]. Recently, fingolimod was demonstrated to affect the differentiation of different T subsets [9], but the exact mechanism has remained unclear.

Fingolimod is a nonselective antagonist of sphingosine 1-phosphate (S1P) receptors, acting against the S1P1, S1P3, S1P4 and S1P5 receptors, but primarily targeting the S1P1 receptor [10]. It has been shown that S1P1 induces Akt–mTOR pathway activation to impede the function of regulatory T (Treg) cells while promoting T helper type 1 (Th1) cells, and that S1P1 deficiency can lead to the opposite changes [11], [12]. In experimental autoimmune encephalomyelitis (EAE), the classical mouse model for studying MS, therapeutic effects of fingolimod treatment have been observed [13], [14], [15]. We speculated that fingolimod, as an S1P1 receptor modulator, could block the Akt–mTOR pathway to modulate the differentiation and function of Th1 and Treg cells. The aim of this study was to determine the relationship between the Akt–mTOR pathway and the differentiation and function of Th1 and Treg cells in fingolimod treatment of EAE.

Section snippets

Animals

Female C57BL/6 mice at 8–10 weeks of age were purchased from Vital River (Beijing, China). Mice were kept on a 12 h dark/light cycle with free access to food and water, and a constant temperature of 22 ± 0.5 °C. All mouse experiments were approved by the Institutional Animal Care and Use Committee of Hebei Medical University.

Induction and evaluation of EAE

EAE was induced by immunization with 250-μg myelin oligodendrocyte glycoprotein (MOG)p35–55 (Lysine Bio-system, Xian, China) dissolved in complete Freund's adjuvant (Sigma, St.

Fingolimod treatment reduces the severity of EAE

We examined the effect of fingolimod on the development and severity of mice with EAE. C57BL/6 mice were immunized with MOG35-55, and then randomly divided into an EAE group and fingolimod treatment group. Healthy control mice were treated with normal saline. We monitored the development and severity of EAE in each group over time (Fig. 1A). In the EAE group, the onset of clinical signs began on post-immunization day 10, and the mean onset time was 12.4 days. In the fingolimod treatment group,

Discussion

Fingolimod inhibits the S1P1 receptor through a mechanism involving activation of the receptor, which leads to its degradation [20], [21]. Because of its effectiveness and availability, fingolimod has found a clinical application in treating MS. In addition, Chinese researchers found that fingolimod was well tolerated and effective in treating patients with intracerebral hemorrhage [22] or with acute ischemic stroke [23], [24]. Although it is known to prevent lymphocyte egress from peripheral

Acknowledgments

We would like to thank Chunyan Li, Jingci Yang, Yansu Guo, Dongxia Wu and Hongran Wu for expert technical assistances.

Financial support was obtained from the National Natural Science Foundation of China (No. 81100884) and the key project of Medical Science Research in Hebei Province (No. 20150213).

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Two studies employing mice with S1P1 deletion or overexpression in CD4+ T cells demonstrate that S1P1 signalling inhibits Treg development and promotes Th1 differentiation via activation of the Akt-mTOR pathway (Liu et al., 2009; Liu et al., 2010). Importantly, FTY720 administration to mice with experimental autoimmune encephalomyelitis (EAE, the animal model of MS) dampens Akt-mTOR signalling and Th1 cell frequency while enhancing Treg counts (Hou et al., 2016). Moreover, TCF-1, a transcription factor inhibiting Th1/Th17 differentiation and downregulated in MS CD4+ cells (Mazzola et al., 2015), can be induced by in vitro exposure to FTY720 in T lymphocytes where it binds to regulatory regions of inflammatory genes, thus reducing their expression (Mazzola et al., 2015).
Sphingosine 1-phosphate (S1P) is a potent bioactive sphingolipid binding to specific G protein-coupled receptors expressed in several organs. The relevance of S1P-S1P receptor axis in the pathophysiology of immune and nervous systems has encouraged the development of S1P receptor modulators for the treatment of neurological, autoimmune and/or inflammatory disorders. Currently, four S1P receptor modulators are approved drugs for multiple sclerosis (MS), an inflammatory disorder of the central nervous system. As main pharmacologic effect, these treatments induce lymphopenia due to the loss of responsiveness to S1P gradients guiding lymphocyte egress from lymphoid organs into the bloodstream. Recent data point to immunological effects of the S1P modulators beyond the inhibition of lymphocyte trafficking. Further, these drugs may cross the blood-brain barrier and directly target CNS resident cells expressing S1P receptors. Here we review the role of S1P signalling in neuroimmunology at the light of the evidences generated from the study of the mechanism of action of S1P receptor modulators in MS and integrate this information with findings derived from neuroinflammatory animal models and in vitro observations. These insights can direct the application of therapeutic approaches targeting S1P receptors in other disease areas.
Fingolimod (FTY720) prevents chronic rejection of rodent cardiac allografts through inhibition of the RhoA pathway
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We tested many commercially available inhibitors of RhoA/ROCK and although they all were very effective, none of them was approved for the clinical use [1,3,4]. In our search for clinically applicable RhoA pathway inhibitors, we found very limited data indicating that FTY720 besides downmodulating S1P1 on lymphocytes also inhibits RhoA and mTOR/mTORC2 [13,14,21]. We confirmed these data in our own research on the effect of FTY720 and Siponimod, on the mouse macrophages in vitro [12,15].
The Fingolimod (FTY720, Gilenya) is clinically approved for the treatment of multiple sclerosis (MS). Its therapeutic effect on MS is based on the ability to bind sphingosine 1-phosphate (S1P) receptors and block the exit of immune cells from the lymphoid organs, thus preventing immune cell-dependent injury to the central nervous system (CNS). We showed recently that, besides the S1P-related activity, the FTY720 also down-regulates RhoA, which is a master regulator of the actin cytoskeleton. Our previous studies showed that FTY720 also down-regulates Rictor, which is a signature molecule of mTORC2 complex, which regulates RhoA and dictates actin cytoskeleton specificity. Because, our previous studies showed that chronic rejection correlates with the upregulation of RhoA and mTORC2 components and that the inhibition of RhoA pathway prevents chronic rejection, here we studied the effect of FTY720 on the chronic rejection of rat and mouse cardiac allografts. We show that FTY720 in conjunction with the inhibitors of early T cell response, (CTA4-Ig in mice and Everolimus in rats) blocks macrophage infiltration into the grafts and prevents chronic rejection of rat and mouse cardiac transplants. This indicates that FTY720 may be repurposed from the MS application to the clinical transplantation as an anti-chronic rejection drug.
A new lipophilic amino alcohol, chemically similar to compound FTY720, attenuates the pathogenesis of experimental autoimmune encephalomyelitis by PI3K/Akt pathway inhibition
2020, International Immunopharmacology
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The phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (PKB/Akt)/ mechanistic target of rapamycin (mTOR) signaling pathway has been linked to the pathogenesis of EAE since it acts in important biological processes, such as survival, proliferation, and migration of immune cells. A previous study showed that splenocytes derived from EAE animals presented an increase in the activation of this signaling pathway, which was inhibited by treatment with the compound FTY720, the first oral drug approved by the FDA for the treatment of MS [12]. In previous studies, we have investigated the role of long-chain lipophilic amino alcohols in reducing inflammatory mediators in carrageenan-induced mice paw edema and delayed-type hypersensitivity mice paw edema models, by their immunosuppressive activities [13].
Experimental autoimmune encephalomyelitis (EAE) is one of the main animal models used for the study of Multiple Sclerosis (MS). Long-chain lipophilic amino alcohols with immunoregulatory activities have already been studied in some models of inflammatory diseases, but the action of these compounds in EAE and MS is still unknown. In this study, we investigated whether the lipophilic amino alcohol 4b would act to improve the clinical signs of EAE and reduce the demyelination process and the neuroinflammatory parameters in the spinal cord, as well as the inflammatory process in the inguinal lymph nodes, of C57Bl/6 mice induced with EAE after stimulation with MOG_35-55 and pertussis toxin. The 4b treatment (1.0 mg/kg/day) was orally administered, starting on the day of onset of clinical signs of the disease (10th) and ending on the 20th day after immunization. This treatment was able to reduce the cell count on the inguinal lymph nodes, the migration of inflammatory cells into the central nervous system (CNS), as well as the processes of microgliosis, astrogliosis, and the production of chemokines and pro-inflammatory cytokines, thus increasing the IL-10 anti-inflammatory cytokine levels in EAE mice. The inhibition of Akt phosphorylation in the CNS of EAE mice after treatment with 4b indicates that the immunoregulatory action of 4b is related to the PI3K/Akt signaling pathway. Our results indicate the immunoregulatory efficacy of the new compound 4b in the control of some inflammatory parameters and in the glial proliferation. In addition, 4b was able to reduce the demyelination of neurons and the worsening of clinical signs of EAE as effectively as the compound FTY720, the first oral drug approved by the FDA for the treatment of MS.
Chronic mechanical hypersensitivity in experimental autoimmune encephalomyelitis is regulated by disease severity and neuroinflammation
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Chronic pain severely affects quality of life in more than half of people living with multiple sclerosis (MS). A commonly-used model of MS, experimental autoimmune encephalomyelitis (EAE), typically presents with hindlimb paralysis, neuroinflammation and neurodegeneration. However, this paralysis may hinder the use of pain behavior tests, with no apparent hypersensitivity observed post-peak disease. We sought to adapt the classic actively-induced EAE model to optimize its pain phenotype. EAE was induced with MOG_35-55/CFA and 100–600 ng pertussis toxin (PTX), and mice were assessed for mechanical, cold and thermal sensitivity over a 28-day period. Spinal cord tissue was collected at 14 and 28 days post-injection to assess demyelination and neuroinflammation. Only mice treated with 100 ng PTX exhibited mechanical hypersensitivity. Hallmarks of disease pathology, including demyelination, immune cell recruitment, cytokine expression, glial activation, and neuronal damage were higher in EAE mice induced with moderate (200 ng) doses of pertussis toxin, compared to those treated with low (100 ng) levels. Immunostaining demonstrated activated astrocytes and myeloid/microglial cells in both EAE groups. These results indicate that a lower severity of EAE disease may allow for the study of pain behaviors while still presenting with disease pathology. By using this modified model, researchers may better study the mechanisms underlying pain.
Fingolimod modulates T cell phenotype and regulatory T cell plasticity in vivo
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Citation Excerpt :
We thus examined the direct effects of Fingolimod on T cells populations in vivo, with a focus on cytokine secretion and expression of exhaustion markers. We first confirmed published data demonstrating the decrease in T cell numbers in the circulation of patients under Fingolimod treatment [38,55–59] and the lack of effects on monocyte and NK cell counts [60]. These differential results on the effect of Fingolimod on T cells as compared to NK cells and monocytes are in agreement with recent reports [46,54], and could be due to differential levels of expression of the five S1P receptors in these cell populations [61,62].
Fingolimod is an approved therapeutic option for patients with relapsing-remitting multiple sclerosis that primarily functions by sequestering T cells in lymph nodes inhibiting their egress to the central nervous system. However, recent data suggests that Fingolimod may also directly affect the immune cell function. Here we examined the in vivo effects of Fingolimod in modulating the phenotype and function of T cell and Foxp3 regulatory T cell populations in patients with multiple sclerosis under Fingolimod treatment. Besides decreasing the cell numbers in peripheral blood and sera levels of pro-inflammatory cytokines, Fingolimod inhibited the expression of Th1 and Th17 cytokines on CD4⁺ T cells and increased the expression of exhaustion markers. Furthermore, treatment increased the frequency of regulatory T cells in blood and inhibited the Th1-like phenotype that is characteristic of patients with multiple sclerosis, augmenting the expression of markers associated with increased suppressive function. Overall, our data suggest that Fingolimod performs other important immunomodulatory functions besides altering T cell migratory capacities, with consequences for other autoimmune pathologies characterized by excessive Th1/Th17 responses and Th1-like regulatory T cell effector phenotypes.
Rapamycin and fingolimod modulate Treg/Th17 cells in experimental autoimmune encephalomyelitis by regulating the Akt-mTOR and MAPK/ERK pathways
2018, Journal of Neuroimmunology
Citation Excerpt :
At present, fingolimod (FTY720) has been approved for MS by the U.S. Food and Drug Administration (FDA) (Cohen et al. 2010; Kappos et al. 2010) and shown to ameliorate development of EAE (de Bruin et al. 2016; Kataoka et al. 2005; Rothhammer et al. 2017; Wang et al. 2013). Fingolimod, is an antagonist of the sphingosine 1-phosphate (S1P) receptor, inhibiting the mTOR signaling pathway (Hou et al. 2016; Liu et al. 2010) and inducing downregulation of pERK in breast cancer cells (Nagaoka et al. 2008). In addition, fingolimod behaves as an S1P receptor agonist to alleviate pain in EAE by reducing cellular markers of central sensitization of neurons in the dorsal horn of the spinal cord via stimulus-evoked pERK expression (Doolen et al. 2017).
Rapamycin prevents experimental autoimmune encephalomyelitis (EAE) and activates the MAPK/ERK pathway in EAE. Thus, we hypothesized combining rapamycin and fingolimod treatments would have synergistic effects in EAE. We show that combination therapy ameliorated EAE and regulated spinal cord IL-17 and TGF-β levels in EAE mice. Combination therapy also modulated IL-17 and TGF-β concentration, RoRγt and Foxp3 mRNA levels, and Th17 cell and Treg frequencies in the spleen. Moreover, rapamycin decreased ps6k and increased pAkt and pERK, while combination therapy downregulated pAkt, ps6 k and pERK in EAE mice. Our findings provide insight into using this drug combination to treat EAE.

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Fingolimod ameliorates the development of experimental autoimmune encephalomyelitis by inhibiting Akt–mTOR axis in mice

Highlights

Abstract

Introduction

Section snippets

Animals

Induction and evaluation of EAE

Fingolimod treatment reduces the severity of EAE

Discussion

Acknowledgments

Trends Pharmacol. Sci.

Immunity

Neurobiol. Dis.

J. Biol. Chem.

FEBS Lett.

Neurobiol. Dis.

Cell

J. Neuroimmunol.

J. Neuroimmunol.

Exp. Neurol.

Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis

N. Engl. J. Med.

A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis

N. Engl. J. Med.

FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats. I. FTY720 selectively decreases the number of circulating mature lymphocytes by acceleration of lymphocyte homing

J. Immunol.

Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors

Nat. Chem. Biol.

Promotion of lymphocyte egress into blood and lymph by distinct sources of sphingosine-1-phosphate

Science

Finding a way out: lymphocyte egress from lymphoid organs

Nat. Immunol.

Multiple sclerosis: closing in on an oral treatment

Nature

FTY720 ameliorates Th1-mediated colitis in mice by directly affecting the functional activity of CD4 + CD25 + regulatory T cells

J. Immunol.

The receptor S1P1 overrides regulatory T cell-mediated immune suppression through Akt–mTOR

Nat. Immunol.