AUY954, a selective S1P1 modulator, prevents experimental autoimmune neuritis

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Abstract

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune inflammatory demyelinating disease of the peripheral nervous system and an animal model of human inflammatory demyelinating polyradiculoneuropathy. AUY954, which targets selectively the sphingosine-1-phosphate receptor 1 (S1P1), is known to sequester lymphocytes into secondary lymphoid tissues. In EAN rats, AUY954 greatly prevented paraparesis if administrated from the day of immunization. T cell, B cell, and macrophage infiltration, inflammatory demyelination, and local expression of interleukine-17 and matrix metalloproteinase-9 in sciatic nerves of EAN rats were significantly decreased by AUY954 treatment. Therefore, S1P1 modulation might be a potential treatment option for inflammatory neuropathies.

Introduction

Experimental autoimmune neuritis (EAN) is a T cell-mediated inflammatory demyelinating disease of the peripheral nervous system (PNS) that shares many clinical, electrophysiological and immunological features with human acute and chronic inflammatory demyelinating polyradiculoneuropathies. Therefore, the EAN model is widely applied to investigate therapeutic principles and disease mechanisms of inflammatory polyneuropathies (Zhang et al., 2009a).

Acute inflammatory demyelinating polyneuropathy (AIDP) is the most common subtype of Guillain–Barré Syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) is considered the chronic counterpart of GBS among peripheral nerve abnormalities. GBS is the world's leading cause of acute autoimmune neuromuscular paralysis and caused by an autoimmune attack on the peripheral nervous system (Winer, 2001). Existing treatments of these polyneuropathies can be divided into supportive management, such as good intensive care and respiratory assistance, as well as active treatment including plasma exchange (PEX) and intravenous immunoglobulin (IVIG) (Hughes, 2008). Nevertheless, only around 65% GBS patients respond to plasma exchange or intravenous immunoglobulin (Meyer zu Hörste et al., 2007). About 8% of GBS patients die, and up to 20% remain disabled after a year despite modern treatment. Even in those who recover well, residual weakness and loss of motor units can usually be detected and could explain the fatigue that is a common problem. Differently from GBS and AIDP, steroids are first line therapy for CIDP, up to 65%–95% of CIDP patients respond to steroids, but steroids have very limited effect and the side effects are noticeable (Lunn and Willison, 2009). Plasma exchange and intravenous immunoglobulins represent second stage therapies of CIDP, but they are expensive and cumbersome to apply. Therefore, more efficacious and even orally applicable therapeutic options represent an unmet medical need.

EAN can be induced in rats by active immunization with autoantigens of the PNS, like myelin, myelin proteins P0 and P2, or peptides. Following immunization, injected peripheral myelin autoantigen is processed by local dendritic cells or macrophages and brought to the secondary lymphoid organs (SLOs) where naive peripheral myelin autoantigen-specific lymphocytes encounter antigen, begin to divide vigorously and remain sequestered in the SLOs for several days during which the peripheral myelin autoantigen-specific T lymphocytes are activated to get the ability to attack the source of the antigen and the peripheral myelin autoantigen-specific B lymphocytes become plasma cells producing high-affinity and class-switched auto-antibodies (Cyster, 2003, Cyster, 2005). Thereafter, activated peripheral myelin autoantigen-specific T cells enter the blood and penetrate the blood nerve barrier (BNB) by the use of matrix metalloproteinases (MMP), mainly MMP-9 (Leppert et al., 1999, Renaud and Leppert, 2007), to reach peripheral nerves where these T cells further proliferate and produce cytokines in response to local antigen to amplify inflammation in the PNS. This initial inflammatory response recruits monocytes and induces their differentiation into reactive macrophages that act as effectors destroying myelin. The increase of macrophages at the local inflammatory sites is accompanied by an increase of monocytes in the circulating blood, which are either from the intravascular pool of monocytes or from precursors in the bone marrow (Goto et al., 2003).

Based on these observations, Th1 cell-mediated cellular immune responses were considered dominant in EAN (Maurer et al., 2002). Recently, a new linage of Th cells was identified, called Th17 cells, which are characterized by their production of proinflammatory cytokines Interleukin-17 (IL-17) and IL-17F (Kurts, 2008). Accumulating evidence suggest an important role for this new linage of Th cells in the pathology of various autoimmune diseases (Bettelli et al., 2008), and this pathological role might largely be fulfilled through the secretion of IL-17 and related cytokines (Kurts, 2008, Liao et al., 2007). In experimental autoimmune encephalomyelitis (EAE), the central nervous system analogue of EAN, besides Th1 cells, Th17 cells are considered to play important pathological roles in the progression of disease (Aranami and Yamamura, 2008).

IL-17, also called IL-17A, is a member of IL-17 family (IL-17A–F), and has no direct effects on T cells, but directly stimulates epithelial cells, endothelial cells and fibroblasts to produce proinflammatory cytokines and chemokines to recruit neutrophils. Physiologically, IL-17 has been associated with host defence against extracellular infectious agents. However, an increased level of IL-17 expression was observed in different autoimmune and inflammatory diseases, including rheumatoid arthritis, multiple sclerosis, asthma, lupus, allograft rejection and psoriasis (Bettelli et al., 2008, Dong, 2008). Thus, the IL-17 and Th17 axes might play an important role in EAN pathogenesis. Therefore, prevention of activated T cells from homing to the peripheral nerves could suppress local inflammation in EAN.

Sphingosine-1-phosphate (S1P) is an evolutionarily conserved bioactive sphingolipid and regulates diverse physiological processes such as cell migration, angiogenesis, vascular maturation, and immunity by serving as a ligand to cell-surface G-protein-coupled S1P receptors (Brinkmann, 2007, Zhang and Schluesener, 2007). We recently reported that administration of FTY720 (fingolimod), an effective S1P receptor modulator, in EAN rats resulted in reversible reduction of blood lymphocyte counts, significantly reduced lymphocyte and macrophage infiltration into the peripheral nervous system, prevented the onset of diseases and reduced established neurological deficits (Zhang et al., 2008). FTY720 induces the internalization of S1P receptor subtype 1 which leads to a sequestration of lymphocytes into secondary lymphoid tissues, resulting in reduction of blood lymphocyte counts (Yopp et al., 2006, Brinkmann, 2007, Huwiler and Pfeilschifter, 2008). Phosphorylated FTY720 shares structural homology with S1P and targets four of the five S1P receptors, namely S1P1, S1P3, S1P4 and S1P5. However, only interaction with S1P1 has been proven to be essential for lymphocyte egress from peripheral lymphoid tissues. A newly developed mono-selective S1P1 modulator, AUY954, demonstrates comparable activity to FTY720 in mediating reduction of blood lymphocyte counts in rats and is reported to prevent allograft rejection in a stringent rat heart transplantation model (Pan et al., 2006). AUY954 has an attractive pharmacokinetic profile in rats as well (Pan et al., 2006). Our aim was to investigate whether AUY954 might prevent EAN in rats.

Section snippets

Animals

Male Lewis rats (8–10 weeks, 180–200 g, Elevage-Janvier, France) were housed under a 12 h light–12 h dark cycle and with free access to food and water. All animal procedures were in accordance with a protocol approved by the local Administration District Official Committee. All efforts were made to minimize the number of animals and their suffering.

EAN induction

For EAN induction, rats were immunized by subcutaneous injection into both hind footpads with 100 μL of an inoculum containing 100 μg of synthetic

Suppressive treatment of EAN by AUY954

EAN was induced by subcutaneous injection of neuritogenic synthetic P2 peptide. For suppressive treatment, 1% CMC in water (the control group) or AUY954 was orally administrated immediately after immunization and then once daily until Day 22. The first neurologic signs (reduced tail tonus) of control EAN rats were observed at Day 9 (mean clinical score: 0.2 ± 0.1). The neurologic severity of EAN increased fast in the control group with a maximal score at Day 13 (mean neurologic score: 4.8 ± 0.5).

Discussion

EAN is the prime animal model for inflammatory demyelinating polyneuropathies and proven to be useful in investigating new therapeutic approaches. Here we have studied the effects of AUY954, a mono-selective S1P1 modulator, on EAN. Our results demonstrate that AUY954 almost prevented EAN and inhibited paraparesis through substantial reduction of infiltration of lymphocytes and macrophages into the peripheral nerves, decreased local demyelination and suppressed local expression of

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