It is clear from many recent studies that sphingolipids can be intimately involved in the onset and maintenance of inflammation. This indicates that the targeting of sphingolipid actions as part of an anti-inflammatory therapeutic strategy would be beneficial in a number of different clinical conditions. While this strategy is at an early stage of development, studies have begun to demonstrate the potential importance of these lipids as an effective area for pharmacological intervention. There are now several compounds that have been developed, which can pharmacologically manipulate different components of the sphingomyelin cycle, and in particular S1P synthesis and S1P receptors (recently reviewed in Huwiler and Pfeilschifter, 2008). Only a few of these compounds have been examined in the context of a therapeutic benefit in inflammation. The remainder of this review will concentrate on those areas where studies in animal models of specific disease states have used existing drugs or novel therapeutic agents that mediate an anti-inflammatory action via regulation of sphingolipids (summarized in Table 1).
The most high-profile drug that regulates sphingolipid effects on inflammation and has been assessed in vivo is the immunosuppressant FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol), a fungal metabolite. FTY720 is phosphorylated in vivo (Billich et al., 2003) and FTY720-P (structure shown in Figure 1) is an effective modulator of the immune system in transplant models and in renal transplant in humans (Mansoor & Melendez, 2008). Its mode of action is not via the typical immunosuppressant actions of existing drugs (inhibition of T-cell function), but from a sequestration of lymphocytes to the lymph nodes. This reduces the number of T-cells circulating between lymph nodes and the peripheral site of tissue inflammation (Pinschewer et al., 2000). Subsequent studies have now demonstrated that FTY720-P has high affinity for four S1P receptor subtypes: S1P1, S1P3, S1P4 and S1P5. Moreover, FTY720 is phosphorylated by SK in vivo (Billich et al., 2003). Using conditional knockout mice, it has been demonstrated that SK2 is the isoenzyme involved in this phosphorylation (Zemann et al., 2006). It is now clear that the mechanism of action of FTY720-P is via binding to S1P1 receptors on lymphocytes (Brinkmann et al., 2002; Mandala et al., 2002). Activation of this S1P receptor subtype is essential to allow egress of lymphocytes from the lymph nodes. Although FTY720-P is an agonist with nanomolar affinity for the S1P1 receptor, the mechanism of action is possibly not via its agonist properties. The important mode of action in this case may be via a down-regulation of the S1P1 receptor on lymphocytes (Matloubian et al., 2004). Therefore, by preventing recycling of the receptor to the plasma membrane, FTY720 effectively prevents activation of the S1P1 receptor and inhibits lymphocyte egress from lymph nodes. This subsequently decreases T-cell-induced activation of the inflammatory response. Although this drug is effective as an immunosuppressant in transplantation as well as having therapeutic potential in other areas, such as multiple sclerosis and cancer (Hiestand et al., 2008), its use as an anti-inflammatory drug is still at a preliminary stage of investigation. The possible roles of FTY720 as a drug in certain inflammatory diseases, together with other potential agents that modulate sphingolipids, are discussed below with reference to asthma and inflammatory bowel disease (IBD). Both these disease states have important inflammatory components. The remainder of this review will concentrate on drugs that directly block the anti-inflammatory actions of sphingolipid effects. Other potential therapeutic treatments for these diseases will not be covered and readers are referred to specialist reviews on asthma and IBD.
Sphingolipid therapeutic targets in asthma
Asthma is characterized by a bronchial hyperresponsiveness of airway smooth muscle cells and a proliferative remodelling of the airways (Halayko and Amrani, 2003). These effects are ultimately the result of an initial inflammatory cell infiltration of the airways following antigen challenge. This results in an increase in release of cytokines and chemokines from various cell types, such as macrophages and mast cells. Asthma is associated with increased TNF levels in bronchioalveolar lavage (Hallsworth et al., 1994). In addition, TNF has a prominent role in airway hyperresponsiveness (Hunter et al., 2003) and, within a pathophysiological concentration range found in asthmatic bronchioalveolar lavage, can induce DNA synthesis in ASM cells (Stewart et al., 1995). It should be noted that while the role of inflammation in asthmatic airway remodelling has been indicated in animal models, this remains to be established in humans (Tang et al., 2006). Both the hyperrresponsiveness (Roviezzo et al., 2007) and proliferation of airway smooth muscle (Waters et al., 2003) have been shown in some model systems to involve sphingolipids and in particular S1P (Ammit et al., 2001; Jolly et al., 2002). The individual mechanisms involved, however, can be secondary to the initial inflammation. For example, the proliferative effect may be via a transactivation of the PDGF receptor by the S1P1 receptor and subsequent MAPK activation (Waters et al., 2003). The hyperresponsiveness may involve S1P-induced activation of the RhoA/Rho-kinase pathway leading to an increased Ca2+ sensitivity of the contractile myofilaments (Kume et al., 2007). The source of sphingolipid, and more specifically in this case S1P, is likely to be either directly released from activated mast cells (Mitra et al., 2006), or the result of activation of the SMase pathway by TNF leading to de novo production. Indeed, S1P levels are increased in bronchioalveolar lavage from asthmatics following antigen challenge (Ammit et al., 2001), providing evidence that increased S1P release/production in asthma is directly linked to the inflammation. It is therefore not surprising that sphingolipids are potential anti-inflammatory targets in asthma.
A few studies have now examined the effectiveness of FTY720 in asthmatic models. Using an in vivo Th2 cell transfer mouse model, oral FTY720 treatment decreased infiltration of pro-inflammatory eosinophils and T-cells into the airway mucosa (Sawicka et al., 2003). FTY720 also had similar effects on a Th1 cell transfer mouse model. Both Th1 and Th2 cells express S1P1, S1P3, S1P4 and S1P5 receptor isoforms. Th2 cells are the T-cell subset that are considered to be predominantly responsible for initiating the inflammatory response in asthma via activation of oesinophils (Anderson and Coyle, 1994). Th2-mediated inflammation is causally related to several characteristics of asthma, such as airway hyperresponsiveness. Importantly, when the Th2 transfer mice were challenged with ovalbumin, FTY720 also decreased the airway hyperresponsiveness to agonist challenge. In this model, the mode of action of FTY720 is not clear and whether this occurs via down-regulation of the S1P1 receptor is not yet established. It appears unlikely to be an effect on mast cells as mast cell degranulation is via S1P2 receptor (Jolly et al., 2004), the S1P receptor isoform that does not bind FTY720P. Interestingly, in vitro FTY720 (unphosphorylated) inhibited cPLA2 activation independently of S1P receptor in RBL-2H3 mast cells (Payne et al., 2007). This effect was not observed with phosphorylated FTY720. Although it is not clear whether this occurs in vivo, it raises another possible mechanism for the FTY720-mediated anti-inflammatory effect in asthma.
Another study also examining the role of sphingolipids in mouse models of asthma has demonstrated that, while aerosol administration of FTY720 decreases Th2 cell-mediated inflammation and bronchial hyperresponsiveness, T-cell retention in the lymph nodes was not observed (Idzko et al., 2006). In this case the mode of action was an inhibition of dendritic cell migration. Dendritic cells, which express all five isoforms of the S1P receptor, present antigen to T-cells and initiate an immune response. Inhibition of dendritic cell migration subsequently decreased formation of Th2 cells in lymph nodes and thereby prevents airway inflammation. Therefore, in at least some asthmatic models, the mechanism of action of FTY720 may occur via mechanisms other than preventing T-cell egress from the lymph nodes. Whether this action is via direct agonist properties of FTY720 or is via down-regulation of S1P receptors remains to be determined.
Other potential therapeutic targets for asthma within the sphingomyelin cycle have also been examined. Results obtained using in vivo mouse models have indicated the possible benefits of SK inhibitors. In ovalbumin-challenged mice intraperitoneal administration of the selective SK inhibitor, N,N-dimethylsphingosine, decreased the infiltration of pro-inflammatory cells, such as eosinophils and macrophages and also decreased Th2 cell-mediated cytokine release (Lai et al., 2008). Importantly, the methacholine-induced airway hyperresponsiveness was also reduced by SK inhibition. To ensure that these effects were via SK inhibition, siRNA knockdown of SK1 in the asthmatic mice has similar effects. IgE levels were also lowered suggesting an inhibition of mast cell degranulation. A similar study has also shown that N,N-dimethylsphingosine and SK-I (2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole) administered via inhalation also decreased airway inflammation and hyperresponsiveness in ovalbumin-sensitized mice (Nishiuma et al., 2008). The increased S1P concentration in the bronchioalveolar lavage of these animals was decreased to basal levels by inhibition of SK, suggesting that the effects are directly linked to decreased S1P concentrations. These studies further indicate the potential of the SK/S1P pathway in providing a promising therapeutic target in asthma. Clarification on the potentially multiple mechanisms with different models will lead new directions for asthma therapy.
Sphingolipid therapeutic targets in IBD
IBD is a collective definition for several different diseases, principally ulcerative colitis and Crohn's disease, which are the result of damage to the intestinal mucosa. This damage is caused by cellular inflammation due to an imbalanced cytokine production from dysfunctional T-cells (Braegger and MacDonald, 1994). Specifically, regulatory T-cells do not respond to normal (effector) T-cell stimulation, but they prevent inflammation by the production of suppressor cytokines, including IL-10 and transforming growth factor-β (Maloy and Powrie, 2001). In a recent study the therapeutic potential of FTY720 in a chemically induced mouse model of colitis was examined (Daniel et al., 2007). Treatment with FTY720 significantly reduced all clinical and pathological indications of intestinal inflammation in this model. This was correlated with a decrease in inflammatory cytokines, for example, TNF, released from effector T-cells with a concomitant increase in the release of IL-10 and transforming growth factor-β from regulatory T-cells. The exact signalling mechanisms of these effects are not clear, and the S1P receptor subtypes involved on T-cells are not yet defined. However, it does indicate further the therapeutic potential for FTY720.
Another study has also examined regulation of S1P receptors in models of colitis. The effects of a novel S1P agonist KRP-203, which has structural homology to FTY-720 and is similarly phosphorylated in vivo, was examined in an IL-10−/− mouse model of colitis (Song et al., 2008). KRP-203 has similar agonist efficacy to FTY720 at S1P1 and S1P4 receptors but has much lower efficacy at S1P3. Oral administration of KRP-203 resulted in a decrease in the pathological symptoms of colitis, including decreased weight loss and normal intestinal wall thickness. This was attributed to a sequestration of lymphocytes to lymph nodes. KRP-203 therefore produces its action by preventing infiltration of lymphocytes into the intestinal mucosa and results in a decreased release of cytokines, including TNF. Although it is not known if KRP-203 acts to down-regulate S1P1 receptor, similar to FTY720, the results indicate that this may be the case. Certainly, its low efficacy at S1P3 receptors suggests that S1P1 and/or possibly S1P4 are involved.
In addition to S1P receptors, the de novo production of S1P via SK activation has also been examined as a potential therapeutic target in IBD. This has been verified by a recent study using SK1−/− mice (Snider et al., 2009). SK−/− mice treated with dextran sulphate sodium (DSS) to induce colitis had significantly less intestinal damage compared with controls. Also, unlike DSS-treated controls, SK1−/− mice did not display a systemic inflammatory response and did not have any colonic COX-2 induction. Pharmacological evidence has further validated SK1 inhibition as a potential therapeutic target in IBD. In the DSS-induced colitis mouse model, two orally active novel SK inhibitors (ABC294640 and ABC747080), which were effective in vitro, were examined (Maines et al., 2008). Both inhibitors decreased the development and progression of colitis, including less colon shortening and colonic inflammation. Inflammatory cytokines, such as TNF IL-1β and IL-6, were reduced. SK inhibitors may therefore represent another potential target in IBD.
Evidence that other points on the sphingomyelin cycle may also be therapeutic targets in colitis has been demonstrated by a study using a novel acid SMase inhibitor. SMA-7 inhibited LPS-induced activation of NF-κB and release of the pro-inflammatory cytokines TNF, Il-1β and IL-6 in macrophages (Sakata et al., 2007). This was correlated with decreased ceramide production. In an in vivo chemically induced mouse model of colitis, oral administration of SMA-7 resulted in decreased cytokine levels in the colon and lower severity of colonic injury. Whether the inhibition of ceramide is the key mechanism in this effect, or a decreased ceramide production prevents further downstream conversion to sphingosine and S1P, is not known.