WO2013011135A1

WO2013011135A1 - Ceramide c16-cer and cers6 in the treatment and diagnosis of multiple sclerosis (ms)

Info

Publication number: WO2013011135A1
Application number: PCT/EP2012/064328
Authority: WO
Inventors: Gerd Geisslinger; Susanne SCHIFFMANN; Klaus Scholich; Ulf ZIEMANN
Original assignee: Goethe Universitaet Frankfurt am Main
Current assignee: Goethe Universitaet Frankfurt am Main
Priority date: 2011-07-20
Filing date: 2012-07-20
Publication date: 2013-01-24
Anticipated expiration: 2014-01-20
Also published as: GB201112455D0; GB2493142A

Abstract

The present invention relates to a method of diagnosis and/or monitoring the progression of multiple sclerosis, comprising the use of ceramide and/or ceramide synthase, specifically C₁₆-Cer and CerS6, as biomarkers for monitoring the onset and development of the disease. The invention discloses further a diagnostic kit for performing the inventive methods. Furthermore methods and compounds for modulating the activity of the ceramide and/or ceramide synthase are described which are useful for the treatment of multiple sclerosis. Also described is a method for screening compounds or combination of compounds capable of preventing and/or alleviating the clinical symptoms of an autoimmune disease based on the ceramide and/or ceramide synthase biomarkers disclosed herein, as well as compounds identified by the screening methods and their use in the treatment or prevention of multiple sclerosis.

Description

Ceramide Ci₆-Cer and CerS6 in the Treatment and Diagnosis of Multiple Sclerosis

(MS).

The present invention relates to a method of diagnosis and/or monitoring the progression of multiple sclerosis, comprising the use of ceramide and/or ceramide synthase, specifically C₁₆- Cer and CerS6, as biomarkers for monitoring the onset and development of the disease. The invention discloses further a diagnostic kit for performing the inventive methods. Furthermore methods and compounds for modulating the activity of the ceramide and/or ceramide synthase are described which are useful for the treatment of multiple sclerosis. Also described is a method for screening compounds or combination of compounds capable of preventing and/or alleviating the clinical symptoms of an autoimmune disease based on the ceramide and/or ceramide synthase biomarkers disclosed herein, as well as compounds identified by the screening methods and their use in the treatment or prevention of multiple sclerosis.

Background

Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease in which inflammatory processes play an important role in the onset of the disease. Although differing theories have implicated the involvement of various factors, dysfunction of the immune system, and alterations in the genome in the development of this disorder, the etiology of MS is still poorly unraveled. It is realistic to assume that any factor that results in an autoimmune reaction against the integrity and generation of myelin results in MS. In the European Union over 400 000 people have Multiple Sclerosis (MS). The onset of the disease develops in the prime of life of affected individuals and is usually diagnosed in the age 20 to 40. The disease significantly affects more women than men (3:2).

The pathological hallmark of MS is discrete and focal areas of myelin loss, known as plaques or lesions, which develop to scarred tissue areas. These plaques can consist of varying amounts of demyelination, gliosis, inflammation, edema and axonal degradation. Although the exact locations of the plaques vary among patients, a general anatomical pattern is evident. Plaques within the human brain are located periventricular, and more than half of MS patients have plaques within the cervical portion of the spinal cord. The physiological conse- quence of the plaques is the slowing or blocked transmission of nerve impulses which manifests itself as sensory and/or motor impairment which constitute the clinical manifestations of the disease.

Multiple sclerosis (MS) and its prototype animal model, the experimental autoimmune reactive encephalomyelitis (EAE), are induced by autoimmune responses against myelin components in the central nervous system (CNS). Activated autoreactive T-cells play an essential role in the development of the disease. These activated T-cells proliferate and secrete proinflammatory cytokines, which in turn stimulate microglia, macrophages and astrocytes, and recruit B cells, ultimately resulting in damage to myelin, the myelin forming oligodendrocytes and axons (McFarland and Martin, 2007). One hypothesis concerning the development of multiple sclerosis is that interferon-gamma (INF-γ) secreted from activated T-cell induces NO/TNF-a release in activated macrophages which in turn induces apoptosis of oligodendrocytes leading to the devastating demyelination process (Neuhaus et al, 2006). Oligodendrocyte apoptosis is one of the critical events in the development of multiple sclerosis (Kassmann et al., 2007) followed by glial activation and infiltration of leucocytes (lymphocytes and macrophages). TNF-a (Renno et al, 1995) and NO (Rejdak et al, 2004) are secreted by activated glia cells. Furthermore in cerebrospinal fluid NO metabolites are associated with the progression of MS (Rejdak et al., 2004). Interestingly, a partial knock-down of iNOS by pharmacological compounds is a feasible therapeutic approach for EAE (Hooper et al., 1997) and the treatment with soluble tumor necrosis factor receptor prevents EAE development (Selmaj et al, 1995).

Unfortunately the limited diagnostic options for a clinical detection of MS and the lack of diagnostic laboratory tests are the main causes that impair an early diagnosis and hence of the disease. Furthermore, currently no possibilities exist to predict the prognosis and progression of the disease. New therapeutic and diagnostic options investigated include the use of anti- myelin antibodies, microarray gene expression and studies with serum and cerebrospinal fluid but none of them has yielded reliable positive results. Disease associated biomarkers investigated are interleukin-6, nitric oxide (NO) and nitric oxide synthase (iNOS), osteopontin, and fetuin-A. Since the disease progression is the result of a neurodegeneration process the roles of proteins indicative of neuronal, axonal, and glial loss such as neurofilaments, tau and N- acetylaspartate are under investigation. Recent findings indicate that sphingolipids may play a role in the disease process of multiple sclerosis (Jana and Pahan, 2010). The sphingo lipid transduction pathway induces apoptosis, differentiation, proliferation, growth arrest and inflammation depending upon cell and receptor types and downstream targets (Hannun and Obeid, 2008). As the backbone of several complex sphingolipids (sphingomyelins, glycosylceramides), ceramides are members of the rapidly expanding field of bioactive lipids.

Ceramides can be generated by de novo synthesis or by degradation of complex sphingolipids. The two rate limiting steps in the biosynthesis of ceramides are the synthesis of sphin- ganine from L-serine and palmitoyl-CoA by L-serine palmitoyl transferase (SPT) and the attachment of various acyl-CoA side chains by the ceramide synthases (CerS) to a sphingoide base. The ceramide synthases (CerS 1-6) act chain length specific and introduce side chains form C14-C26. Thus, CerSl synthesizes mainly C18-Cer, CerS4 synthesizes C18-/C20-Cer; CerS5 and CerS6 synthesizes mostly C16-Cer, CerS2 synthesizes mainly C22/C24-Cer and CerS3 synthesizes very long chain ceramides (Ben-David and Futerman, 2010). Additionally to the de novo synthesis the salvage pathway supplies also ceramides mainly via the activation of sphingomyelinases (SMase) (Zeidan and Hannun, 2010).

Ceramides were recently implicated to play an important role in inflammatory processes. Ceramides were linked to oligodendroglial injury in cerebral white matter disorder (Kim et al., 2011). Furthermore, in the inflammatory disease cystic fibrosis, ceramides induce the upregulation of pro-inflammatory mediators by a yet unknown mechanism (Becker et al., 2010). Cystic fibrosis patients treated with amitriptyline (inhibitor of SMase) significantly increased lung functions and ceramide levels determined in respiratory epithelial cells decreased significantly (Riethmuller et al., 2009). SMases and their product ceramide induce an increase in TNF-a as well as NO and its generating enzyme iNOS which play prominent roles during inflammatory processes (Perrotta et al., 2005; Sakata et al., 2007). Moreover, cell culture experiments reveal that pro-inflammatory stimuli such as lipopolysaccharide and cytokines activate macrophages and induce an increase in ceramides via the activation of sphingomyelinases (MacKichan and DeFranco, 1999).

Antagonizing the pro-apoptotic effects of ceramides in the field of medicine is described in US20100278907, wherein immunogenic compositions containing ceramide or ceramide analogs for treating or reducing the risk of developing one or more symptoms of a disease or dis- order associated with ceramide-induced cell death are provided. The immunogenic ceramide compositions were intended to induce a humoral immune response including the production of anti-ceramide antibodies effective to bind to and reduce levels of extracellular ceramide and/or to inhibit one or more biological activities of extracellular ceramide.

For the treatment of MS based on the modulation of the sphingolipid metabolism DEI 02008029734 relates to novel thiazolyl piperidine derivatives which are inhibitors of spingosine kinase and can be used in inflammatory and/or proliferative diseases. It was found that the compounds according to DEI 02008029734 cause specific inhibition of sphingosine kinase 1 , but not of sphingosine kinase 2, and thereby reduce degenerative changes of car- diomyocytes and myocardial fibrosis, as shown in an animal model.

In view of the above, an ongoing demand exists for the development of new and effective methods to allow an accurate and early diagnosis of multiple sclerosis. Furthermore, the limited treatment options to date cause a constant need for the development of new alternative therapeutic starting points, specifically for the screening and development of a novel therapeutically active substances which may find their way into clinic application to tackle multiple sclerosis.

The above objective is solved in a first aspect of the present invention by method of diagnosis and/or monitoring the progression of an autoimmune disease, comprising a step of determining the level of ceramide and/or ceramide synthase in a test sample.

The inventors surprisingly found that Ci6_:o-Cer is involved in the induction of an animal model of MS (EAE) by activation of NO and TNF-a synthesis. Ci₆:o-Cer and CerS6 are upregu- lated in the lesion site in activated microglia, astroglia and migrated leucocytes in EAE mice. The inhibition of Ci₆:o-Cer synthesis caused a reduced iNOS and TNF-a expression and a remission of the disabilities of EAE mice. Moreover, it was demonstrated that Ci6_:o-Cer plays a critical role in the INF-γ induced iNOS/ TNF-a expression and NO/ TNF-a release Accordingly, in INF- γ KO EAE mice the CerS6, iNOS/TNF-α expression were reduced. The antiinflammatory substance methylprednisolone prevents the increase in Ci6_:o-Cer in vivo and in vitro. Additionally, in the cerebrospinal fluid (CSF) of MS patients the Ci₆:o-Cer levels were increased as compared to control patients. Moreover, the Ci₆:o-Cer levels were also increased in the cerebrospinal fluid of EAE mice. Since a significant upregulation of ceramide and ceramide synthase was surprisingly observed in MS, the present invention relates in one embodiment of the above aspect to a method of diagnosis and/or monitoring the progression of an autoimmune disease, wherein an increased level of ceramide and/or ceramide synthase in said test sample compared to a control sample and/or reference value is indicative for an autoimmune disease.

Autoimmune diseases originate from an over reactivity of the immune system which eventually leads to an immune reaction directed to the hosts own proteins and structures. In the context of the present invention an autoimmune disease is preferably an autoimmune disease of the central nervous system, more preferably a demyelinating disease, such as and most preferred, multiple sclerosis.

A further embodiment of the invention relates to the above method, wherein the increased level of ceramide and/or ceramide synthase is indicative for the onset of multiple sclerosis. The inventors found that the elevation of the expression of ceramide and of ceramide synthase is present shortly after the induction of EAE in mice, which speaks for the use of ceramide and of ceramide synthase as biomarkers for MS in the early phase of the onset of the disease.

Yet further preferred for the methods of the present invention is that the ceramide is Ci₆:o-Cer and/or the ceramide synthase is CerS6. The present invention surprisingly found that Ci6_:o-Cer and/or CerS6 can be used as biomarkers in the diagnosis or in monitoring the progression of an autoimmune disease, specifically of multiple sclerosis, for example to diagnose the disease in the onset phase.

A next embodiment of the invention is directed to the method as described above, wherein the test sample is a biological sample from a subject to be diagnosed. A biological sample contains preferably tissue, cells or cerebrospinal fluid (CSF) of the patient to be diagnosed. The biological sample is preferably of mammalian origin, preferably of human origin. Typically, the sample is derived from a human patient suspected to suffer from multiple sclerosis. Such a test sample is in one embodiment a fluid from the subject to be diagnosed, preferably a blood, serum or CSF sample; or is a tissue biopsy, such as a biopsy of the central nervous system, preferably a biopsy of the white matter, preferably derived from the patient's spinal cord. In another preferred embodiment the present test and/or control sample of the methods of the invention contain at least one cell type selected from leucocytes (macrophages, lymphocytes), microglia and astroglia.

In the context of this invention a control sample corresponds to the test sample with the difference that it is derived from a subject that is known not to have the autoimmune disease to be diagnosed and/or monitored. In this respect the control sample is preferably a sample from a subject not having said autoimmune disease. Alternatively the level of ceramide and/or ceramide synthase observed in the test sample may be compared with a reference value. The reference value represents the level of ceramide and/or ceramide synthase in a subject not having said autoimmune disease.

For determining the level of ceramide and/or ceramide synthase in the test and/or control sample any method can be used that allows the quantification of sphingolipid concentrations, the determination of protein content in said sample and/or the quantification of expressed m NA of the analyzed ceramide synthase. In a preferred embodiment the content of sphin- golipids in a sample to be analyzed is determined by tandem liquid chromatography mass spectrometry (LC-MS/MS), by quantitative ELISA using a Ci6:o-Cer-specific antibody or by thin layer chromatography.

The protein content and specifically the expression of one or multiple ceramide synthases in context of the present invention can be determined both on the nucleic acid and the protein level. Typical methods include quantitative PCR techniques, like quantitative real-time PCR, probe hybridization-based techniques, like the micro array technology, or classical approaches like northern blots. Any method known in the art to quantify the amount of mRNA in a sample is usable and preferred in context of the methods described herein. On the protein level the expression of one or multiple ceramide synthases is preferably quantified using antibody based techniques, like for example quantitative western blots, ELISA, FACS analysis or im- munohistochemistry. Preferably an antibody for use in the methods of the invention is an antibody which is specifically directed to ceramide, preferably Ci₆:o-Cer and/or to a ceramide synthase, preferably to CerS6.

In a preferred embodiment, the inventive methods described herein are performed in-vitro. It is specifically preferred that the methods described herein do not comprise methods for treat- ment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body.

In a second aspect the problems of the state of the art are solved by providing a diagnostic kit, comprising means to perform a method of diagnosis and/or monitoring the progression of an autoimmune disease as described herein, and instructions for their use. The means comprised by the preferred diagnostic kits are usable for performing specifically the ceramide and/or ceramide synthase quantification methods mentioned herein.

Another aspect of the present invention relates to an inhibitor of the activity and/or the expression of a ceramide synthase for use in the treatment of an inflammatory disease, preferably multiple sclerosis. In a one embodiment of the aspect the inhibitor is an inhibitor of CerS6.

Yet further preferred is that the described inhibitor of the invention is selected from an inhibitory nucleic acid, such as a siRNA, an inhibitory CerS6 antibody, or a small molecule capable of binding and inhibiting the enzymatic activity of CerS6. A nucleic acid based inhibitor is preferably an RNA molecule comprising a sequence which is complementary and specific to the gene sequence of CerS6. In a preferred embodiment of this aspect, the RNA sequence of the RNAi molecule should be selected to allow a specific targeting of CerS6 expression and not of any other ceramide synthase.

In a preferred embodiment the inhibitory nucleic acid comprises the sequence of SEQ ID No. l , or a variant thereof having preferably 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity to the sequence shown in SEQ ID No. 1. Another preferred embodiment relates to a RNAi construct, such as an siRNA, comprising the sequence of SEQ ID No. l, or a variant thereof having preferably 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity to the sequence shown in SEQ ID No. In this embodiment the thymin bases in SEQ ID No. 1 are exchanged with Uracil in the RNA sequence. Preferably the siRNA molecule for use in the context of the present invention is modified at the 5' or 3' ends. Most preferred is the siRNA molecule provided by Applied Biosystems, Ambion, under catalogue number #sl09529, having the sequence 5'-GAC CUU CAC UAC UAU UAC Att3'. The object of the present invention is further solved by an antagonist of a ceramide for use in the treatment of multiple sclerosis. Such an antagonist is a molecule that specifically binds to ceramide, preferably Ci₆-Cer, and impairs it's function as a second messenger. Such antagonists are preferably inhibitory antibodies which bind specifically to ceramide resulting in the formation of an inactive ceramide-antibody complex. Preferably the ceramide is Ci₆-Cer.

Another aspect of the invention relates to a method for screening compounds or combination of compounds capable of preventing and/or alleviating the clinical symptoms of an autoimmune disease, the method comprising the steps of

a) contacting a cell with a candidate compound or candidate combination of compounds, b) inducing in said cell the expression of ceramide or ceramide synthase,

c) monitoring the level of ceramide or ceramide synthase in said cell,

wherein an reduced level of ceramide or ceramide synthase compared to a control is indicative for the capability of the candidate compound or candidate combination of compounds to prevent and/or alleviate the clinical symptoms of said autoimmune disease.

In another embodiment of the above aspect, the steps of the method, specifically steps a) and b) may be performed in reverse order.

The term "capable of preventing and/or alleviating the clinical symptoms of an autoimmune disease" shall be understood as referring to the capability of a compound or combination of compounds to be useful in the treatment of said autoimmune disease. Furthermore the term is to be understood as a compound that is capable of modifying the level ceramide or ceramide synthase in said cell.

Yet a further embodiment of the above method of the invention comprises the optional, alternative to step c), or additional step d), monitoring the level of NO and/or TNF-a release in said cell.

In this embodiment of the above inventive method a reduced level of NO and/or TNF-a release compared to a control is indicative for the capability of the candidate compound or candidate combination of compounds to prevent and/or alleviate the clinical symptoms of said autoimmune disease. In another embodiment of the present invention a reduced level of NO and/or TNF-a release compared to a control and a reduced level of ceramide or ceramide synthase compared to a control is indicative for the capability of the candidate compound or candidate combination of compounds to prevent and/or alleviate the clinical symptoms of said autoimmune disease.

In a preferred embodiment of the invention the cell for use in the method is a cell capable of expressing ceramide or ceramide synthase, more preferably a cell selected from the group comprising oligodendrocytes, migrated leucocytes (macrophages, lymphocytes), microglia, preferably activated microglia and astroglia. Preferred for the methods of the present invention is that said cell is a macrophage.

In another embodiment said autoimmune disease is preferably a demyelinating disease, such as multiple sclerosis.

In macrophages, one of the key inducers of the formation of NO and/or TNF-a is Interferon-γ. Hence, it is further preferred that in one embodiment step b) of the inventive method comprises the use of Interferon-γ (INF-γ). INF-γ acts according to the invention as an upstream factor of ceramide dependent NO/ TNF-a release, the latter being causal to the apoptotic cell death of oligodendrocytes and demeylination.

In a further embodiment it is preferred that the ceramide is C16-Cer and/or the ceramide synthase is CerS6.

A further aspect of the invention relates to a compound or combination of compounds capable of preventing and/or alleviating the clinical symptoms of a demyelinating disease identified with the screening method described above, for use in the treatment and/or prevention of said demyelinating disease. In one embodiment said demyelinating disease is preferably multiple sclerosis.

A next embodiment of the invention is directed at a compound or combination of compounds capable of preventing and/or alleviating the clinical symptoms of a demyelinating disease identified with the screening method described above, wherein the compound is selected from fumonisin Bl, a fumonisin Bl derivative, L-cycloserine, L-cycloserine derivatives, myriocin, myriocin derivatives, FTY720 (fingolimod) and its derivatives or a pharmaceutically acceptable salt of these compounds.

The present invention will now be explained in the fol lowing examples with reference to the accompanying figures, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures,

Figure 1: C16:0-Cer level is altered in EAE mice. A) Clinical score of EAE mice. Mice were immunized with M OG 5-55-pept ide dissolved in complete Freud^'s adjuvant on day 0 and received pertussis toxin on day 0 and day 1. Onset of disease comprises mice with clinical scO.5 - scl .5. The acute phase included mice with a clinical sc2 - sc3. B) Ceramide levels of unt eated mice, of CFA-treated mice alone or EAE mice were determined by LC-MS/MS. The ceramide level of the EAE mice and the CFA-treated mice were related to the ceramide levels of untreated mice. The ceramide levels of untreated mice were for C16:0-Cer 0.47 ± 0.13 iimol 'mg tissue, for C18:0-Cer 10.05 ± 3.76 iimo!/mg tissue, for C20:0-Cer 14.41 ± 6.44 iimol mg tissue and for C24:0-Cer 19.06 ± 8.91 iimol mg tissue. The ceramide levels of untreated mice were set as 100%. * (p < 0.05 ) ** (p < 0.01 ) indicate significant difference between EAE and CFA-treated mice.

Figure 2: C16:0-Cer level is upregulated in cerebrospinal fluid of MS patients (n=18) as compared to control patients (n=13). C16:0-Cer levels were determined by LC-MS/MS. * (p < 0.05 ) indicate significant difference between MS patients and control patients.

Figure 3: CerS6 is elevated in the onset of the disease. A) The relative mRNA expression of CerSl, CerS2, CerS4, CerS5, CerS6 and GAPDH (control) was normalized to 18SRNA and was calculated using the mRNA level of untreated mice at the same age as basal level. The unchanged level of GAPDH indicates that in all experiments a comparable amount of mRNA was used. Data are mean ± s. e. m. of number of mice as indicated; each measurement achieved in triplicate. B) Densitometric analysis of the CerS6 protein expression. The CerS6 expression was related to the expression level of the loading control GAPDH. The data are mean ± s. e. m. of three independent experiments. C) Western blot analysis of CerS6. As loading control GAPDH was used (one of three independent experiments is shown). * (p < 0.05), ** (p < 0.01), *** (p < 0.001) indicate significant difference between EAE and CFA-treated mice.

Figure 4: Expression of CerS6 in the lumbar spinal cord. A) Expression of oligodendrocyte specific protein (OSP) (green channel) and Cers6 (red channel) in untreated mice. B) Expression of CerS6 (red channel) in the lesion site in the white matter of the ventral horn in two magnifications as indicated. C) Expression of CerS6 (red channel) and specific cell type markers (green channel). Activated astroglia (GFAP)) and migrated lymphocytes and macrophages (CD45), in scl .5 mice express CerS6. D) Expression of CerS6 (red channel), specific cell type marker F4/80 (green channel) (macrophage and activated microglia) and nuclei staining (Dapi) in scl .5 EAE mice. Expression of CerS6 (red channel) and specific cell type marker CD l ib (green channel) and Ibal (blue channel) (CD l ib and Ibal are expressed on macrophage and activated microglia) in scl .5 mice. E) Expression of CerS6 (red channel) and the death receptor 5 (green channel) in scl .5 mice. Merge = overlay of CerS6 (red channel) and cell type specific proteins (green channel). All data are representative images of three independent experiments. Scale bar, 10 μιη (A/C, 60x), Scale bar, 100 μΜ (B, 5x) Scale bar, 25 μΜ (B, 20x).

Figure 5: INF-γ induced in RAW macrophages an alteration of the ceramide levels and an increase in CerS6 A) RAW macrophages were treated for 16 h with 10 ng/ml INF-γ, 5 ng/ml TNF-alpha and 1 ng/ml IL-1B. The ceramide levels were determined by LC-MS/MS and normalized to the number of treated cells. The relative increase in the specific ceramides was calculated using the ceramide level of untreated cells as 100% value. Data are mean ± s. e. m. of three independent experiments. * (p < 0.05), ** (p <0.01) indicate significant difference between cytokine-treated and untreated RAW macrophages. B/C/D) The time dependent effect of INF-γ treated RAW macrophages on the ceramide levels (B), on the mRNA levels (C) and on the protein levels (D) of CerSs. RAW macrophages were treated for the time points as indicated with 10 ng/ml INF-γ or were not treated (control). B) The ceramide levels were determined by LC-MS/MS and normalized to the number of treated cells. The relative increase in the specific ceramides was calculated using the ceramide level of untreated cells as 100% value. Data are mean ± s. e. m. of three independent experiments each achieved in duplicate. ** (p < 0.01) indicates significant difference between INF-γ treated and untreated RAW macrophages. C) The relative mRNA expression of CerSs was normalized to GAPDH and was calculated using the mRNA level of untreated cells at the same time point as basal level. Data are mean ± s. e. m. of three independent experiments, each achieved in triplicate. * (p < 0.05), * * (p < 0.01 ), * * * (p < 0.001) indicate significant difference between INF-γ treated and untreated cells. D) Densitometric analysis of the CerS6 expression. The CerS6 expression was related to the expression level of GAPDH. * (p < 0.05) indicate significant difference between INF-γ treated and untreated cells. Western blot analysis of CerS6 at several time points as indicated. As loading control GAPDH was used (one of two independent experiments is shown).

Figure 6: The INF-γ expression correlates with disease progress. A) The relative mRNA levels were normalized to 18SRNA and were calculated using untreated mice at the same age as basal level. * (p < 0.05), ** (p < 0.01) indicate significant difference between EAE and CFA-treated mice. B) INF-γ expression was determined by mouse INF-γ ELISA Kit (Bioleg- end, Uithoorn, Netherlands). * (p < 0.05) indicate significant difference between EAE and untreated mice (control).

Figure 7: FBI and L-cycloserine prevent the INF-γ induced increase in NO synthesis. A) The mRNA level of iNOS is transiently increased in EAE mice and in INF-γ (10 ng/ml) time dependently stimulated RAW macrophages. The relative mRNA expression of iNOS was normalized to 18SRNA (EAE) and to GAPDH (RAW macrophages) and was calculated using the mRNA level of untreated mice at the same age or untreated cells as basal level. EAE data are mean ± s. e. m. of number of mice as indicated and each measurement was achieved in triplicate. RAW macrophage data are mean ± s. e. m. of three independent experiments each achieved in duplicate. B) The protein level of iNOS is increased in EAE mice and in INF-γ treated RAW macrophages. Western blot analysis of iNOS in EAE mice (in vivo) and in RAW macrophages (in vitro). As loading control GAPDH was used (one of two independent experiments is shown). C) The NO level is time dependent increased in INF-γ (10 ng/ml) induced RAW macrophages. The NO amount was determined from the supernatant and related to the number of cells. D/E)70 μΜ fumonisin Bl (FBI), 500 μΜ L-cycloserine (Cyclo) or 1 μΜ methylprednisolone (MP) were preincubated for 30 min or 90 min, respectively, subsequently 10 ng/ml INF-γ were added for 16 h. From the supernatant the NO release (D) and from the cell pellet the mRNA level of iNOS (E) were measured. The NO amount was related to the number of treated cells. The relative mRNA expression of iNOS was normalized to GAPDH and was calculated using untreated cells at the same time point as basal level. Data are mean ± s. e. m. of three comparable independent experiments, each achieved in duplicate. * (p < 0.05), ** (p < 0.01), *** (p < 0.001) indicate significant difference between INF-γ treated and untreated RAW macrophages as well as between EAE mice and untreated mice.

Figure 8: L-Cycloserine and Fumonisin Bl prevent the INF-γ induced increase in cera- mides. 500 μΜ L-cycloserine (Cyclo) or 70 μΜ fumonisin Bl (FBI) or DMSO (control) were preincubated for 2 h, subsequently 10 ng/ml INF-γ were added for 16 h. From the cell pellet the ceramide level were determined by LC-MS/MS. The ceramide levels were related to the number of treated cells. The ceramide amount of the control cells was set as 100% value. * p<0.05, ** p<0.01 , *** p<0.001 indicate significant difference between INF-γ treated cells and cells co-treated with INF-γ and an inhibitor of the sphingolipid synthesis.

Figure 9: Co-stimulation of INF-γ and L-cycloserine (Cyclo) or fumonisin Bl (FBI) or methylprednisolone (MP) didn^'t reduce macrophage viability. RAW macrophages were treated for 16 h with INF-γ (10 ng/ml) alone or co-treated with L-cycloserine (500 μΜ), fumonisin Bl (70 μΜ) or methylprednisolone (1 μΜ). The cell viability was determined with the WST proliferation assay (Roche Diagnostics). The cell viability was determined by using INF-γ treated cells as 100 % value.

Figure 10: Down-regulation of CerS6 prevents at least partly the INF-γ induced NO release and exogenously added palmitic acid amplified the INF-γ induced NO synthesis. A-C) mRNA levels of CerS5, CerS6 and iNOS (A), ceramide levels (B) and NO release (C) in scrambled (scr.) siRNA and siCerS6 treated RAW macrophages, which were co-incubated with 10 ng/ml INF-γ for 16 h. The relative mRNA levels were normalized to GAPDH and were calculated using untreated cells at the same time point as basal level. The ceramide levels were determined by LC-MS/MS and normalized to the number of treated cells. The NO amount were related to the number of treated cells. * (p < 0.05), *** (p < 0.001) indicate significant difference between siCerS6 treated and scrambled siRNA treated RAW macrophages. D/E) NO release (D) and C16:0-Cer level (E) of RAW macrophages stimulated simultaneously with 25 μΜ palmitic acid and 0.5 ng/ml INF-γ for 16 h. Ceramide levels were detected by LC-MS/MS. The relative increase in the C 16:0-Cer was calculated using the ceramide level of untreated cells as 100% value. NO amounts were related to the number of treated cells. Data are mean ± s. e. m. of one of three comparable independent experiments. ** (p < 0.01), *** (p < 0.001) indicate significant difference between INF-γ stimulated and INF- γ/palmitic acid co-stimulated RAW macrophages. Figure 11: Palmitic acid don't increase C14:0-Cer, C24: l-Cer and C24:0-Cer neither in control nor in INF-γ treated RAW macrophages. DMSO (control), 25 μΜ palmitic acid, 0.5 ng/ml INF-γ or 25 μΜ palmitic acid and 0.5 ng/ml INF-γ were incubated for 20 h. From the cell pellet the ceramide level were determined by LC-MS/MS. The ceramide levels were related to the number of treated cells, (ns; not significant)

Figure 12: C16:0-Cer mediates INF-γ induced increase in TNF-a synthesis. A) The mRNA level of TNF-a is transiently increased in EAE mice. B/C) 70 μΜ fumonisin Bl (FBI), 500 μΜ L-cycloserine (Cyclo) or 1 μΜ methylprednisolone (MP) were preincubated for 30 min or 90 min, respectively, subsequently 10 ng/ml INF-γ were added for 16 h. The mRNA level of TNF-a (B) from the cell pellet and TNF-a release (C) from the supernatant was measured. The TNF-a was calculated as percentage value and the TNF-a level of interferon-gamma treated macrophages was set as 100% value. D/E) mRNA levels (D) and release of TNF-a (E) of scrambled (scr.) siRNA and siCerS6 treated RAW macrophages, which were co- incubated with 10 ng/ml INF-γ for 16 h, were determined. The relative mRNA expression of TNF-a was normalized to 18SRNA (EAE) and to GAPDH (RAW macrophages) and was calculated using the mRNA level of untreated mice at the same age or untreated cells as basal level. EAE data are mean ± s. e. m. of number of mice as indicated. Each measurement was achieved in triplicate. RAW macrophage data are mean ± s. e. m. of three independent experiments each achieved in duplicate. * (p < 0.05), * * (p < 0.01), *** (p < 0.001) indicate significant difference between INF-γ treated and inhibitor treated RAW macrophages; scrambled siRNA and siCerS6 treated macrophages; between EAE mice and untreated mice.

Figure 13: The serine palmitoyl transferase inhibitor L-cycloserine (Cyclo) prevents at least partly the development of disabilities in EAE mice, the increase in C16:0-Cer, the iNOS and TNF-a expression. A) Clinical score of EAE mice treated daily with 75 mg/kg L- cycloserine or saline (control) by i.p. injection. Mice were immunized with MOG35-55- peptide dissolved in complete Freud^'s adjuvant at day 0 and received pertussis toxin at day 0 and day 1. The EAE and CFA-treated mice were medicated with L-cycloserine or saline when the EAE mice reached scO.5. B) Ceramide level of the mice were determined by LC-MS/MS. The ceramide levels of the L-cycloserine- or saline-medicated EAE mice and CFA-treated mice were related to the ceramide level of age matched control mice. C/D) The relative mRNA expression of iNOS (C) and TNF-a (D) was normalized to 18SRNA and was calcu- lated using untreated age matched control mice as basal level. Data are mean ± s. e. m. of number of mice as indicated. * (p < 0.05) indicate significant difference between L- cycloserine- and saline-medicated EAE mice.

Figure 14: Methylprednisolone prevents the increase in C16:0-Cer in EAE mice and in INF-γ treated RAW macrophages. A/B) NO release (A) and ceramide level (B) in RAW macrophages treated with 10 ng/ml INF-γ, with 1 μΜ methylprednisolone (MP) or with a combination of both substances (MP was preincubated for 2 h) for 16 h. From the supernatant the NO release was measured and from the cell pellet the ceramide levels were determined by LC-MS/MS. The ceramide levels and the NO amounts were related to the number of treated cells. The relative increase in the specific ceramides was calculated using the ceramide level of untreated cells as 100% value. C) Clinical score of EAE mice treated daily with 10 mg/kg methylprednisolone or left untreated. Mice were immunized with MOG35-55-peptide dissolved in complete Freud^'s adjuvant on day 0 and received pertussis toxin on day 0 and day 1. The EAE and CFA-treated mice were treated with methylprednisolone when they reached sc2 - sc3. D) Ceramide levels of EAE mice and CFA-treated mice medicated with methylprednisolone or left untreated. The ceramide levels of the methylprednisolone medicated EAE and CFA-treated mice were related to the ceramide levels of untreated mice. Data are mean ± s. e. m. of number of mice as indicated.

Figure 15: C16:0-Cer mediates IFN-γ induced NO and TNF-a release from peritoneal macrophages. A) Time-dependent effect of IFN-γ treated peritoneal macrophages on the expression of CerS6 mRNA. Peritoneal macrophages were treated or not with IFN-γ (20 ng/ml) for the time points indicated. The relative expression of CerS6 mRNA was normalized to GAPDH and was calculated using untreated cells at the same time point as basal level. Data are means ± s. e. m. of two independent experiments, each performed in duplicate. ** (p < 0.01) indicates a significant difference between IFN-γ stimulated and unstimulated peritoneal macrophages. B-D) FBI (70 μΜ) and L-cycloserine (500 μΜ) prevented the IFN-γ (20 ng/ml) induced increase in NO and TNF-a synthesis in peritoneal macrophages. The amount of C16:0-Cer (B), release of NO (C) and TNF-a (D) were measured in peritoneal macrophages (after 16h). The relative expression of CerS6 mRNA was normalized to GAPDH. Ceramide, NO and TNF-a levels were normalized to the number of treated cells. Ceramide levels of untreated cells were set at 100%. Data are means ± s. e. m. of three independent experiments each performed in duplicate. ** (p < 0.01), *** (p < 0.001) indicate significant dif- ferences between IFN-γ stimulated and IFN-y/specific inhibitor co-stimulated peritoneal macrophages. E-H) EAE experiment using IFN-γ KO mice. IFN-γ KO or wild-type (WT) mice were immunized on day 0 with MOG35-55-peptide dissolved in complete Freund^'s adjuvant and received pertussis toxin on day 0 and day 1. CFA-treated mice received an injection of CFA only. The results were obtained from a single study. E) The relative expression of mRNA for CerS6, IFN-γ, TNF-a and iNOS from EAE/IFN-γ KO and EAE/WT mice at the onset of the disease (scO.5) was calculated using the mRNA levels of untreated mice of the same age as those at baseline. Data are means ± s. e. m. of number of mice as indicated; each measurement was done in triplicate. ** (p < 0.01), *** (p < 0.001) indicate significant differences between EAE/IFN-γ KO and EAE/WT mice. F-H) C16:0-Cer (F), TNF-a (G), CerS6 (H) and iNOS (H) expression in the lumbar spinal cord of EAE/IFN-γ KO and EAE/WT at the onset of the disease (scO.5). The ceramide levels of untreated mice were set at 100%. H) Western blots from one EAE/WT (scO.5) and two EAE/IFN-γ KO (scO.5) mice is shown. The western blot analysis shows one representative blot of two analyses. Data are means ± s. e. m. of the number of mice indicated. Each measurement was done in duplicate.

Figure 16: The mRNA expression of ceramide synthase 6 and C16:0-Ceramide levels are increased in the lumbar spinal cord of RR-EAE mice (female and male). A) The relative mRNA expression levels of CerS6 from RR-EAE mice with no disease (scO) (control), at the onset (sc0.5-scl), progression (sc2), peak (sc3) and in the chronic phase (sc3) of the disease. The mRNA levels were normalized to the geometric mean of eEF2 and PPIA and related to a basal level. Baseline data were taken from age-matched control animals. Data are means ± s. e. m. of number of mice as indicated; each measurement was made in triplicate. *(p < 0.05) **(p < 0.01) indicate significant differences between RR-EAE and control mice. B) Western blot analysis of the CerS6 protein expression in the lumbar spinal cord of control and RR- EAE mice (female) (scl, sc2, sc3). The CerS6 expression was related to the expression level of the loading control GAPDH. C) Ceramide levels are shown from RR-EAE mice (female) with no disease (scO) (control), at the onset (sc0.5-scl), progression (sc2) and peak (sc3) of the disease. The ceramide levels of RR-EAE mice were related to mice with no disease. Data are means ± s. e. m. of the number of mice indicated.

Figure 17: C16:0-Ceramide levels are upregulated in the cerebrospinal fluid of PP-EAE mice as compared to mice treated only with CFA. Data are means ± s. e. m. of the number of mice indicated. Each measurement was done in duplicate. * (p < 0.05) indicates significant differences between EAE and CFA mice.

Figure 18: The putative role of C16:0-Cer in the INF-γ induced NO and TNF-a release. (CerS, ceramide synthase; INF-γ, interferon-gamma; iNOS, inducible nitric oxide synthase; NO, nitric oxide; TNF-a, tumor necrosis factor alpha); see references Steinman et al. 2001 and Hendriks et al. 2005.

Examples

The following materials and methods were used in context of the presented examples of the invention:

Cells and reagents

RAW 264.7 mouse macrophages were cultured and incubated in RPMI 1640 medium containing 10% FCS and 1% penicillin/streptomycin. Cells were cultured at 37 °C in an atmosphere containing 5% C0₂. L-cycloserine (Cyclo), fumonisin Bl (FBI), palmitic acid (PA) and 6a-methylprednisolone (MP) were purchased from Sigma-Aldrich (Schnelldorf, Germany). INF-γ, TNF-alpha, and IL-1B were purchased from PeproTech (Hamburg, Germany). siR As CerS6 (sl09529) were purchased from Ambion (Darmstadt, Germany). The sphingolipids were purchased either from Avanti Polar Lipids (Alabaster, USA) or Matreya LLC (Pleasant Gap, USA). The EAE (EK-01 15) and the control (CK-01 15) kit were purchased from Hooke Laboratories (Lawrence, USA). The INF-γ ELISA kit was purchased from Biolegend (Uithoorn, Netherlands).

EAE induction

In all experiments, the ethics guidelines for investigations in conscious animals were obeyed and the experiments were approved by the local Ethics Committee for Animal Research. Eight- to ten- week-old female C57 BL/6 or IFN-γ KO (C57BL6 background) weighing 18 g - 20 g were obtained from Harlan Laboratories (Horst, Netherlands). The procedure used for the induction of EAE was conducted as recommended by Hooke Laboratories (Lawrence, USA). Briefly, EAE mice received a subcutaneous injection in the upper and lower back with MOG₃₅-55 peptide emulsified in complete Freund^'s adjuvant (CFA) containing Mycobacterium tuberculosis. 2 hours thereafter, and again 24 hours later, the mice received an intraperitoneal injection of pertussis toxin (PTX). The CFA-treated mice were treated with CFA containing M. tuberculosis and twice with PTX. One week after the injection, the mice were examined daily for developing disabilities. 90 % of the EAE mice develop after 13 ± 2 days first signs of disabilities. Mice which develop no clinical score were excluded. The control mice were led untreated. Clinical scores were defined as follows: 0) no signs, 0.5) limp tail, 1) limp tail and weakness of hind legs, 2) limp tail and paresis of hind legs, 3) limp tail and paralysis of hind legs. RR-EAE model

For the RR-EAE model TCR(T-cell receptor) 1640 SJL mice, which overexpress a TCR against a component of the myelin sheath, were used. The mice were kindly allocated from Prof Wekerle (MPI Martinsried).

EAE mice treated with L-cycloserine or methylprednisolone

EAE and CFA-treated mice were treated with 75 mg/kg L-cycloserine dissolved in saline by a daily i.p. injection when the mice reached scO.5. As control EAE and CFA-treated mice were medicated daily with saline by an i.p. injection. EAE and CFA-treated mice were medicated with 10 mg/kg methylprednisolone. Methylprednisolone was dissolved in ethanol and then added to the drinking water when the mice reached sc2. Every second day the drinking water supplemented with methylprednisolone was replaced. As control, EAE and CFA-treated mice were left untreated.

Cerebrospinal fluid from patients

Cerebrospinal fluid (CSF) from patients were collected over one year with informed consent from patients from the Department of Neurology (Goethe-University, Frankfurt/Main). The protocol was approved from the local Ethics committee. All samples were stored at -80°C. Control CSF were collected from patients who suffer from non-autoimmune diseases, like dementia, headache, somatoform disorders.

Preparation of tissue for histology studies

Animals were sacrificed and perfused transcardially with phosphate buffered saline (PBS) (for mRNA, protein and sphingolipid analysis) and followed by 4 % paraformaldehyde (for immunohistochemistry). Brain (cerebellum) and spinal cord (lumbar, thoracal and cervical segments) were extracted and stored at -80 °C for mRNA, protein and sphingolipid analysis. The tissues for immunohistochemistry (spinal cord) was kept in 4 % paraformaldehyd for 1 h, placed overnight in 30 % sucrose, embedded in tissue freezing medium (Jung, Leica Microsystems GmbH, Nussloch, Germany), then quick frozen on dry ice and stored at -80 °C.

Immunohistochemistry

14 micrometer sections were permeabilized in PBS containing 0.1 % Triton X-100 for 10 min, blocked in PBS containing 3 % bovine serum albumin and 0.1 % Triton X-100 for 30 minutes at RT. The sections were incubated with the primary antibody at 4 °C overnight, followed by fluorescence labeled antibodies diluted 1 :800 for 2 h in PBS containing 0.1 % Triton X-100. The following antibodies in the indicated dilution were used: CerS6 (1 : 100), Ionized calcium-binding adaptor molecule 1 (Ibal) (1 :200), Anti-Glial Fibrillary Acidic Protein (GFAP) (1 : 1000), Cdl lb (1 : 100), CD45 (1 : 100), iNOS (1 : 100), death receptor 5 (DR5) (1 :50). The CerS6 (goat polyclonal) and DR5 (rat polyclonal) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), while GFAP (rabbit polyclonal) and CD45 (rat polyclonal) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Ibal (rabbit polyclonal) were purchased from Wako Chemicals GmbH (Neuss, Germany). The antibody against Cdl lb (rat polyclonal) was used from Serotec (Dusseldorf, Germany). The antibody against iNOS (rabbit polyclonal) was purchased from Becton Dickinson (Heidelberg, Germany).

Preparation of crude protein extracts

RAW macrophages were seeded in 5 cm dishes at a density of 5·10⁵ cells/dish. Cells were treated with 10 ng/ml or 0.5 ng/ml INF-γ, 5 ng/ml TNF-alpha, 1 ng/ml IL1B, 25 μΜ palmitic acid for the indicated time points. INF-γ (10 ng/ml) treated RAW macrophages were co- treated with 70 μΜ fumonisin Bl , 500 μΜ L-cycloserine or 1 μΜ methylprednisolone for 16 h. Methylprednisolone and L-cycloserine were preincubated for 90 min, while fumonisin Bl was preincubated for 30 min. INF-γ (0.5 ng/ml) treated RAW macrophages were co-treated with 25 μΜ palmitic acid for 20 h. Vehicle treated cells were used as control. At the end of the incubation period the crude extracts were prepared as already published elsewhere (Schiffmann et al., 2008). Tissue samples from spinal cord were homogenized in Tris- CHAPS-buffer (10 mM Tris-HCl/20 mM CHAPS, pH 7,4) supplemented with protease inhibitors. The homogenate was centrifuged and the pellet (CerS6) (resuspended in Tris- CHAPS-buffer) and the supernatant (iNOS) were collected and stored at -80 °C. Protein concentrations were assessed using the Bradford method.

Preparation of peritoneal macrophages

Eight- to ten-week-old female C57 BL/6 mice (Harlan Laboratories, Horst, Netherlands) received 0.5 ml of 10% thioglycollate (Sigma, St. Louis, MO) i.p. After 4 days, the mice were killed by inhalation with carbon dioxide. Peritoneal macrophages were collected by removing the skin from the abdomen and injecting 2 ml of lavage fluid (Hank's Buffered Salt Solution (HBSS)) and 2 ml air into the peritoneal cavity. The lavage fluid (enriched with macrophages) was collected from the peritoneal cavity using a 2 ml syringe. The step was repeated with 2 ml HBSS. Macrophages depleted of erythrocytes were cultured in RPMI 1640 medium containing 10% FCS and 1% penicillin/streptomycin. After 3h, the cell medium was replaced and the cells were used for further experiments. Peritoneal macrophages were incubated with 20 ng/ml IFN-γ for various time points. Peritoneal macrophages were co-stimulated with IFN-γ (20 ng/ml) and Fumonisin Bl (70 μΜ) and L-cycloserine (500 μΜ) for 16h. Cells were prein- cubated with Fumonisin Bl and L-cycloserine for 30 min and 90 min, respectively.

Western blot analysis

Immunoblotting was performed as described previously (Schiffmann et al, 2008). 30 μg proteins of cell lysates and 30 μg tissue homogenates were used. Membranes were analyzed on the Odyssey infrared scanner from LI-COR (Bad Homburg, Germany). The antibodies used were diluted as follows: primary antibody raised against iNOS (1 :200), CerS6 (1 : 100), Glyc- erinaldehyd-3-phosphat-Dehydrogenase (GAPDH (1 : 1000). The GAPDH antibody was purchased from Ambion (Darmstadt, Germany).

Real time qPCR

RAW macrophages and lumbar spinal cord were analyzed for mRNA levels by qPCR as previously described (Schiffmann et al, 2010). The expression levels of CerSl-6, GAPDH, β- actin and iNOS were determined by Taqman™ analysis using the SYBR Green Kit (ABgene Limited, Epsom, United Kingdom) with an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Austin, USA). Relative expression of CerS family genes, GAPDH and iNOS was determined using the comparative CT (cycle threshold) method, normalizing relative values to the expression level of 18SRNA (spinal cord samples) or GAPDH (RAW macrophages) as housekeeping gene. The designed primer sets for CerSs, iNOS and INF-γ were adopted from Laviad et al. (Laviad et al., 2008), from Chiang et al. (Chiang et al., 2009) and from Nath et al. (Nath et al., 2009), respectively. Linearity of the assays was determined by serial dilutions of the templates for each primer set separately.

Silencing of CerS with siRNA

RAW macrophages were transfected with 150 pmol CerS6 siRNA or 100 pmol scrambled siRNA as control. siPort Amine (Ambion, Darmstadt, Germany) was used for transfection according to the manufacturer's protocol and as described previously (Schiffmann et al., 2010). Briefly, Opti-MEM medium with transfection reagent were incubated for 10 min at RT, then added to the siRNA solution consisting of Opti-MEM medium and siRNA, followed by an incubation for 10 min at RT. 3.5xl0⁵ RAW macrophages were incubated with siRNA transfection solution, and the process was repeated after 24 h. After 41 h the transfected RAW macrophages were either harvested for mRNA isolation (RNA isolation kit (Qiagen, Hilden, Germany)) or treated with INF-γ for 16 h and subjected to sphingolipid analysis and NO determination. The effectiveness of the siRNA knock-down was verified using quantitative PCR.

Determination of sphingolipid concentrations (cells, tissue, human cerebrospinal fluid)

The quantification of sphingolipid amounts in RAW macrophages was achieved as previously published (Schiffmann et al, 2009a). Briefly, cells were seeded at a density of 0.5xl0⁶ 15 cm dish and incubated for 24 h. Subsequently, cells were treated with various substances. Cells were counted in a Neubauer chamber and stored at -80 °C. After thawing, the internal standards (Ci7:o-Cer) were added and the lipids were extracted twice with methanol. The organic phases were collected, dried under a stream of nitrogen at 45 °C and redissolved in methanol. For the quantification of sphingolipid concentrations in tissue samples, about 20-100 mg of tissue was dounced in PBS on ice. 20 μΐ of tissue suspension (0.02 mg/μΐ) was extracted in 600 μΐ chloroform/methanol (7: 1) after the addition of the internal standards (C_17:0-Cer)) and 80 μΐ water. The suspension was vigorously vortexed at 25 °C for 1 min and centrifuged for 5 min at 25 °C and 14,000 rpm. The supernatants were collected and the extraction step was repeated. The combined organic phases were dried under a stream of nitrogen at 45 °C and redissolved in 50 μΐ of methanol for quantification. Ci4:o-Cer, Ci6:o-Cer, Ci8:o-Cer, C2o_:o-Cer, C₂4:i-Cer and C₂4:o-Cer and the internal standards were determined by LC-MS/MS as described previously (Schiffmann et al., 2009b; Schiffmann et al., 2009a). Concentrations of the calibration standards, quality controls and unknowns were evaluated using the Analyst software version 1.5 (Applied Biosystems). Linearity of the calibration curve was proven for Ci4:o-Cer from 0.3 - 500 ng/ml, Ci6:o-Cer/C₂4:o-Cer from 3 - 5000 ng/ml, for Ci8:o-Cer from 0.9 - 1500 ng/ml and for C₂o:o-Cer/C₂4:i-Cer from 1.2 - 2000 ng/ml. The coefficient of correlation for all measured sequences was at least 0.99. Variations in accuracy and intraday and interday precision (n=2 for each concentration in mouse tissue and cells) were less than 15 % over the range of calibration.

Determination of sphingolipid concentrations (murine cerebrospinal fluid) A standard mixture of 2.4 ng/ml in methanol was prepared by mixing equal volumes of cera- mide stock solutions (50 ng/ml). This mixture was diluted to obtain various calibration standard solutions in the range from 0.006 to 2.4 ng/ml. 50 μΐ of every calibration standard solution were mixed with 2 μΐ PBS and 8 μΐ C 17 ceramide (30 ng/ml in methanol) as an internal standard in polypropylene tubes. The mixture was vortexed for 1 minute and centrifuged at 20,238 x g for 3.5 min (Eppendorf Microcentrifuge 5424, Wessling-Berzdorf, Germany). 40 μΐ of the supernatant of each sample were transferred to a new tube and the solvent was evaporated under nitrogen at 45 °C. Samples were reconstituted in 40 μΐ solvent A (methanol- water 80:20 (v/v) containing 5 mmol/1 ammonium formate and 0.1% formic acid). CSF samples were prepared by mixing 2 μΐ of CSF with 50 μΐ of methanol and 8 μΐ of the internal standard CI 7 ceramide (30 ng/ml in methanol). Extraction procedure was the same as mentioned above. Standard and CSF samples were analyzed in an eksigent nano LC 2D Ultra system (AB Sciex, Darmstadt, Germany) equipped with an AS-2 autosampler with a 10 μΐ sample loop. The LC-System was connected to a hybrid triple quadrupole - ion trap mass spectrometer model 5500 QTRAP (AB Sciex, Darmstadt, Germany). Analyte ions were generated by nanospray ionization using a Silica Pico Tip emitter (10 μιη tip, New Objective, distributed by DNU-MS GbR, Berlin, Germany). Chromatographic separation was performed under gradient conditions using a 75 μιη inner diameter reversed-phase C8 column packed in-house (particle size 5 μιη). 3 μΐ of samples were injected and via a 10 μΐ sample loop directly loaded into the analytical column. They were eluted at 350 nl/min with solvent A (methanol- water 80:20 (v/v) containing 5 mmol/1 ammonium formate and 0.1% formic acid) and solvent B (methanol-aceton 80:20 (v/v) containing 10 mmol/1 ammonium formate and 0.1% formic acid). Gradient started with 90% B for 1 min, was linearly increased within 2 min to 100% B and maintained for 9 min. Then, it was reverted to the initial conditions within 0.5 min and the column was re-equilibrated for 3.5 min. The overall runtime was 16 min. All solvent mixtures were sonicated for degassing before using them (USR 90 H Merck Eurolab/VWR, Darmstadt, Germany). The mass spectrometer was operated in positive multiple reaction monitoring mode (MRM) at unit resolution.

Determination of INF-y, TNF-q and nitrite/nitrate

RAW macrophages were treated with INF- γ and various substances (L-cycloserine, fu- monisin Bl, methylprednisolon, palmitic acid). After centrifugation the supernatant was harvested, centrifuged (1,200 rpm, 3 min, 4° C) and stored at -20 °C. The release of NO was assessed by measuring concentrations of nitrite and nitrate in the supernatant (1 ml) using the Griess method (Green et al, 1982). The release of TNF-a was determined using the mouse TNF-a ELISA detection kit from Biolegend (Uithoorn, The Netherlands). The amounts of INF-γ in the lumbar spinal cord of EAE, CFA-treated and control mice were determined using the mouse INF-y ELISA detection kit from Biolegend (Uithoorn, The Netherlands). The determination of the concentration of INF-y and TNF-a was achieved according to the manufacturer^'s protocol.

Statistics

Results are presented as a mean ± s.e.m (standard error of the mean). Cell culture data and animal data were analyzed using oneway ANOVA. Significant differences were analyzed by the Bonferroni post hoc-test (PASW Statistics 18 software). The clinical score data from L- cycloserine- and saline-treated EAE mice were analyzed by calculating the area under the curve. The AUC values were analyzed using the t-test. The level of significance was set at p<0.05.

Example I: Ci_6:o-Cer level is increased in the onset of EAE

EAE is induced in C57BL6 mice by injection of the myelin oligodendrocyte protein(MOG)35_ 55-peptide emulsified in complete Freund^'s adjuvant (CFA). The mice developed after 13 ± 2 days first signs of reduced motor skills which strengthen after 4 ± 1 days to a complete paralysis of the hind paws (Figure 1 A). The ceramide levels of the lumbar spinal cord were determined in untreated, CFA-treated (only treated with CFA-emulsion and PTX) and EAE mice. The EAE mice were divided in two groups: scO.5 - scl .5 (onset of the disease) and sc2 - sc3 (acute phase of the disease). The inventors determined the amounts of C_14:0-, C^o-, C_18:0-, C₂o:o-, Ci8:i-, C_24:1- and C₂4:o-Cer. The amount of C_14:0-, C_18:1- and C₂4:i-Cer was under the detection limit (data not shown). The lumbar spinal cord reveals a significant increase in Ci6:o-Cer at the onset (sc0.5-scl .5) of the disease, which was persistent in the acute phase (sc2-sc3) of the disease (Figure IB). All other ceramide levels were not altered in these mice. CFA-treated mice (control) show no alteration in the ceramide level (Figure IB).

Example II: Ci_6:o-Cer levels are increased in the cerebrospinal fluid of MS patients

Since specifically Ci6:o-Cer was elevated in EAE mice, the inventors investigated whether or not also in MS patients ceramides are regulated. The ceramide levels were measured in cerebrospinal fluid because this human sample is routinely collected in patients who were diag- nosed with MS. Interestingly, also in the cerebrospinal fluid of MS patients the Ci₆:o-Cer levels are significantly upregulated as compared to control patients (Figure 2) indicating that Ci6:o-Cer may play a role in the diagnosis of MS. Control patients suffered from non- autoimmune diseases like headache, dementia, somatoform disorders.

Example III: Ci_6:o-Cer increase in EAE mice is linked to a raised CerS6 expression

Next the inventors studied, whether or not the raised Ci₆:o-Cer level is due to an increased expression of a specific CerS. For this purpose the inventors determined the mRNA level of all CerSs -besides CerS3; which was not detectable- in the lumbar spinal cord of untreated mice, CFA-treated (control), scO.5, scl .5 and sc3 mice. Quantitative PCR results revealed that the mRNA of CerS6 was significantly increased in mice at the onset of EAE (Figure 3 A). The inventors confirmed the mRNA data on protein level by western blot analysis. The protein expression of CerS6 from the homogenate of the lumbar spinal cord of untreated, CFA- treated, scO.5, scl .5 and sc3 mice were determined. Scl .5 mice showed a significant increase in CerS6 as compared to CFA-treated mice, while in sc3 mice CerS6 expression was reduced to the expression level of CFA-treated mice (Figure 3B/3C). Thus, the mRNA and protein expression profile suggest a transient increase in CerS6 and Ci₆:o-Cer during development of EAE.

Example IV: CerS6 is expressed in inflammatory cells in EAE mice

Then the inventors studied in which cells CerS6 and Ci₆:o-Cer is increased. Double immu- nolabeling analyses showed that in the lumbar spinal cord of untreated mice and EAE scl .5 mice (Figure 4A, data shown for untreated mice) CerS6 is colocalized with the oligodendrocyte specific protein (OSP). In EAE mice (scl .5) CerS6 is additionally expressed in the lesion site in the white matter of the lumbar spinal cord (Figure 4B). The lesion site consists among others of infiltrating macrophages, activated microglia and astroglia. Therefore the inventors investigated whether CerS6 is localized in these cell types. CerS6 is expressed in microglia (Ibal and CD 1 lb staining); specifically in microglia with a round morphology indicating an activated form of these cell type (Figure 4C). CerS6 is also expressed in astroglia (GFAP staining) and in macrophages/lymphocytes (CD45 staining) (Figure 4C). Since Ci₆:o-Cer is discussed to be involved in apoptotic processes, the inventors investigated whether or not CerS6 is detectable in apoptotic cells. CerS6 was not expressed in death receptor 5 (DR5) positive cells (Figure 4D).

Example V: INF-γ induces increase in ceramides in macrophages

Next the inventors wanted to investigate whether or not Ci6_:o-Cer plays a role in the induction of EAE. Therefore, the inventors switched to the cell culture system. Since CerS6 was not expressed in apoptotic cells, but in inflammatory cells the inventors used the central cell type for inflammation macrophages. Recent studies revealed an important role of the Thl- mediated response in the development of EAE (Domingues et al, 2010). Thl-cells secrete predominantly INF-γ, IL-IB and TNF-alpha which in turn activate macrophages. Therefore the inventors studied the influence of INF-γ, IL-IB and TNF-alpha on the ceramide levels in macrophages. Figure 5A demonstrates that only INF-γ induced in macrophages a significant elevation of Ci₆:o-Cer after 16 h treatment. INF-γ led to a time dependent predominant increase in Ci4:o-Cer and Ci₆:o-Cer and to a slight increase in C₂4:o-Cer (Figure 5B).

The fact that dihydroceramides are elevated (data not shown) indicate an activation of the sphingolipid de novo synthesis. The mRNA levels of the various CerSs in INF-γ treated macrophages support the LC-MS/MS data and revealed a significant increase in the mRNA level of CerS6 (Figure 5C). The upregulation of the mRNA expression of CerS6 in RAW macrophages is transient as in the lumbar spinal cord of EAE mice (Figure 3 A). The protein expression of CerS6 is also time dependent elevated in INF-γ treated macrophages with a significant increase already after 6 h (Figure 5D). The transient increase in CerS6 in the lumbar spinal cord and in INF-γ treated RAW macrophages and the subsequent predominant increase in Ci6:o-Cer in both experimental setups indicate that the in vitro experiment simulates very closely the in vivo situation.

Example VI: Ci_6:o-Cer mediate INF-γ induced NO release in macrophages

Macrophages and astrocytes contribute by NO release to oligodendrocyte degeneration in demyelinating diseases (Merrill et al, 1993; Mitrovic et al, 1994). One of the key events in macrophage responses to INF-γ stimuli is the expression of iNOS and the subsequent formation of NO. In EAE mice the mRNA and protein expression of INF-γ is increased in the onset of the disease (Figure 6). Moreover, in the EAE mice the inventors observed also an increase in the mRNA and protein expression of iNOS correlating with the progress of the disease (Figure 7A/B). The mRNA level of CerS6 is already at scO.5 10 fold increased, while the mRNA level of iNOS is only about 2.5 fold increased suggesting that iNOS acts downstream of CerS6. RAW macrophages treated with 10 ng/ml INF-γ led to an increase in the mRNA and protein expression of iNOS and subsequently to NO release (Figure 7A/B/C). The upregulation of CerS6 starts at 6 h (Figure 5D) which is much earlier than the upregulation of the iNOS expression after 16 h (Figure 7B) indicating that CerS6 acts upstream of iNOS. When the NO synthesis is regulated by Ci₆:o-Cer, the INF-γ induced NO level should be reduced by the treatment with specific inhibitors of the sphingolipid synthesis. L-cycloserine (inhibitor of the serine palmitoyl transferase) and fumonisin Bl (FBI) (inhibitor of the CerSs) prevent the increase in ceramides induced by INF-γ in RAW macrophages (Figure 8). The effectiveness of L-cycloserine in inhibiting of INF -γ induced ceramide synthesis points to that INF-γ induces rather de novo synthesis than the salvage pathway of ceramide synthesis. Importantly, L-cycloserine and FB 1 inhibit significantly the INF-γ induced NO release (Figure 7D) as effective as the anti-inflammatory glucocorticoide methylprednisolone (Figure 7D). Additionally, the inventors demonstrated that L-cycloserine and FB 1 prevent the INF-γ induced upregulation of the mRNA expression of iNOS (Figure 7F) excluding that the inhibitors unselectively inhibit the activity of iNOS. To exclude that the reduced NO release induced by FBI and L-cycloserine in INF-γ treated macrophages is due to reduced cell viability, the inventors achieved a cytoxicity test. Neither FB I nor L-cycloserine reduced the cell viability in INF-γ pretreated macrophages (Figure 9). These data suggest that ceramides mediate the INF-γ induced NO release.

To verify that predominantly Ci6:o-Cer and CerS6 are responsible for the mediation of the NO increase in INF-γ treated macrophages, CerS6 was down-regulated by RNAi. siCerS6 pretreated RAW macrophages were stimulated with 10 ng/ml INF-γ for 16 h. siCerS6 prevent in INF-γ treated cells CerS6 upregulation, which is observable in INF-γ treated with scrambled siRNA pre-incubated RAW macrophages (Figure 10A). CerS5, which has a similar substrate specificity than CerS6, is not regulated by the siCerS6 (Figure 10A). The down regulation of CerS6 prevent an increase in specifically Ci4:o-Cer (data not shown) and Ci₆:o-Cer (Figure 10B), but not of C₂4:o-Cer (data not shown), which is also slightly upregulated in INF-γ treated cells (Figure 5B). As expected, siCerS6 prevented INF-γ induced iNOS mRNA expression (Figure 10A) and NO release (Figure IOC) in RAW macrophages as compared to scrambled siRNA treated RAW macrophages. These findings indicate that CerS6 and Ci₆:o-Cer mediate the activation of iNOS expression in INF-γ stimulated RAW macrophages. Since the down regulation of Ci₆:o-Cer prevents INF-γ induced NO synthesis, a specific endogenous upregula- tion of Ci6:o-Cer should increase the NO release. Importantly, exogenously added palmitic acid (25 μΜ), which led to a specific increase in Ci6:o-Cer (Figure 10D, Figure 1 1) amplifies the INF-γ (0.5 ng/ml) induced NO synthesis significantly in RAW macrophages (Figure 10E).

Example VII: Ci_6:o-Cer mediate INF-γ induced TNF-a release in macrophages

Besides NO also TNF-a is a mediator of oligodendro glial death, therefore the inventors studied whether TNF-a is also regulated by Ci6:o-Cer. As expected, TNF-a is upregulated disease dependently in EAE mice (Figure 12 A) and INF- γ induces beside the NO release also the synthesis of TNF-a in macrophages (Figure 12B). Interestingly, INF- γ induced TNF-a mRNA expression and TNF-a release is inhibited by FB I and L-cycloserine (Figure 12 B, C). As expected, siCerS6 prevented INF-γ induced TNF-a mRNA expression (Figure 12D) and TNF-a release (Figure 12E) in RAW macrophages as compared to scrambled siRNA treated RAW macrophages. Moreover, exogenously added palmitic acid (25 μΜ) amplifies the INF-γ (0.5 ng/ml) induced TNF-a synthesis in RAW macrophages (Figure 12C).

Example VIII: Inhibition of Ci_6:o-Cer prevents the worsening of the clinical symptoms in the EAE model

Since L-cycloserine as well as FBI prevented the induction of NO and TNF-a in vitro and NO and TNF-a were related to the development of disabilities in the EAE model (Farias et al., 2007), the inventors treated EAE mice with L-cycloserine. The mice were treated with 75 mg/kg L-cycloserine by an i.p. injection once daily starting when the mice showed the first signs of disabilities (scO.5). The control EAE mice were treated with an i.p. injection of saline. For statistical analysis the area under the curve (AUC) was determined. The AUC of L- cycloserine treated EAE mice was 10.51 ± 1.8 score/day and from saline treated EAE mice 17.69 ± 2.3 score/day. Treatment with L-cycloserine prevents significantly (p=0.041) the development of disabilities (Figure 13 A). Figure 13B revealed that in L-cycloserine treated EAE mice compared to saline-treated EAE mice the Ci₆:o-Cer level is significantly lower. Moreover L-cycloserine prevents significantly the increase in the mRNA level of iNOS and TNF-a in the lumbar spinal cord (Figure 13C, D). These data indicate that Ci6:o-Cer is involved in the mediation of iNOS and TNF-a during the development of EAE. Next the inventors investigated whether or not an anti-inflammatory drug regulates the C^o- Cer level in EAE mice and in INF-γ stimulated RAW macrophages. If an anti-inflammatory drug could prevent the increase in Ci6_:o-Cer level in vivo and in vitro than Ci6_:o-Cer should be involved in the inflammatory process. Glucocorticoids are used for the treatment of MS patients. Moreover, in the EAE mouse model methylprednisolone resulted in a remission of the disabilities (Chan et al, 2008). The treatment of RAW macrophages with the glucocorticoid methylprednisolone (1 μΜ) led to an inhibition of the INF-γ induced NO release (Figure 14A). Importantly, the combined treatment of 1 μΜ methylprednisolone and 10 ng/ml INF-γ inhibit specifically the increase in Ci6_:o-Cer whereas the slight increase in C_24:1- and C24_:o-Cer was not affected by methylprednisolone (Figure 14B). EAE and control mice (CFA-treated) were medicated in stage sc2 - sc3 with a daily dose of 10 mg/kg methylprednisolone. As expected, the treatment with methylprednisolone resulted in a remission of the symptoms (Figure 14C). Interestingly, methylprednisolone led also to a specific decrease in the Ci₆:o-Cer level (Figure 14D) in the lumbar spinal cord as compared to untreated EAE mice. These data confirm the crucial role of Ci₆:o-Cer in the inflammatory process leading to the development of multiple sclerosis.

Example IX: C16:0-Cer mediates IFN-γ induced TNF-a and NO release in peritoneal macrophages (published)

In primary peritoneal macrophages, IFN-γ induced a time-dependent, transient increase in ceramide synthase 6, which resulted in a significant increase in C 16:0-Cer after 16h (Figure 15 A, 15B). Interestingly, the IFN-γ- induced release of NO and TNF-a was inhibited by FB I as well as by L-cycloserine (Figure 15C, 15D). These data indicate that in primary macrophages, the synthesis of NO and TNF-a is also C 16:0-Cer dependent.

Example X: C16:0-Cer concentrations and iNOS, TNF-a and CerS6 expression are reduced during the initial phase of disease in IFN-γ KO EAE mice:

The inventors furthermore investigated whether IFN-γ is relevant for the elevated C I 6:0- Cer/CerS6 levels in the PP-EAE model. For this purpose, PP-EAE was induced in IFN-γ KO mice. The inventors observed a significant delay in disease onset, as described by Pal et al. 2001 {J. Immunol 166:662). EAE/IFN-γ KO mice developed initial signs of clinical symptoms at day 12 ± 1 and EAE/WT mice at day 10 ± 1. Ceramide levels and mRNA expression were determined in EAE/WT and EAE/IFN-γ KO mice, when scO.5 was reached. Interestingly, the expression of mR As for CerS6, i OS and TNF-a were significantly reduced in EAE/IFN-γ KO mice when compared to EAE/WT mice (Figure 15E). Furthermore, in EAE/IFN-γ KO mice the C16:0-Cer levels were significantly lower when compared to EAE/WT mice (Figure 15F). Most importantly, CerS6 and iNOS protein was also clearly lower in EAE/IFN-γ KO mice than in EAE/WT mice (Fig. 15G). The reduced CerS6 and iNOS expressions in EAE/IFN-γ KO mice confirm our in vitro results showing that IFN-γ induces the synthesis of CerS6/C16:0-Cer which subsequently is relevant for the induction of iNOS.

Example XI: C16:0-Cei7CerS6 is also regulated in the RR-EAE model

For further experiments the inventors switched to a relapse remitting EAE (RR-EAE) model. To induce the PP-EAE model a peptide of the myelin oligodendrocyte protein emulsified in completes Freund^'s adjuvant was injected into the mice. For the RR-EAE model TCR (T-cell receptor) mice, which overexpress a TCR against a component of the myelin sheath, were used. The RR-EAE model is more similar to the situation in MS patients, because no CFA is used and the RR form is the most common form in MS patients. The development of MS is sex dependent. Females develop more likely the RR-MS form, while male develop more likely the primary progressive form. This situation is reflected in the RR-EAE model. Using the RR-EAE model the inventors observed also a transient increase in ceramide synthase 6 (mRNA and protein) in the lumbar spinal cord (Figure 16A, B). Furthermore, the C16:0- Ceramide levels were transiently increased in the lumbar spinal cord of RR-EAE mice (Figure 16C). Interestingly, ceramide synthase 6 was increased in female and male mice. Thus ceramide synthase 6/C16:0-Cearmide is sex independent and independently from the EAE form (PP or RR) regulated.

Example XII: C16:0-Cer is regulated in the cerebrospinal fluid of PP-EAE mice

Some groups of MS patients show in the white matter an increase in C 16-Ceramide. In the white matter (lumbar spinal cord) of PP-EAE mice the C16:0-Ceramide levels are increased as compared to control (CFA) mice. Astonishingly, the C16:0-Ceramide levels were also significantly increased in the cerebrospinal fluid of PP-EAE mice (sc0.5-sc3) as compared to control (CFA) mice (Figure 17). These data indicate that the observed regulation of CerS6/C16:0-Ceramide in the EAE model can be transferred to the situation in MS patients. In summary, the present invention shows that Ci6_:o-Cer plays an important role in the initial phase of the inflammatory process in MS disease. The transient increase in Ci6_:o-Cer /CerS6 at the beginning of the inflammatory process points to a significant role of Ci₆:o-Cer in the initial phase of MS. Furthermore, the examples demonstrate for the first time a direct correlation of the de novo synthesized Ci₆:o-Cer and INF-γ induced NO/TNF-a synthesis (Figure 18). Up to now, it is only described, that ceramides synthesized by the salvage pathway or glycosylated ceramides induce NO and TNF-a synthesis (Yang et al, 2001 ; Pannu et al, 2004). In primary rat microglia LPS-induced activation of SMase led to NO release, which could not prevented by fumonisin Bl , in contrast to the findings of the present invention.

Claims

1. A method for screening compounds or combination of compounds capable of preventing and/or alleviating the clinical symptoms of an autoimmune disease, comprising

a) contacting a cell with a candidate compound or candidate combination of compounds,

b) inducing in said cell the expression of ceramide or ceramide synthase, c) monitoring the level of ceramide or ceramide synthase in said cell, wherein an reduced level ceramide or ceramide synthase compared to a control is indicative for the capability of the candidate compound or candidate combination of compounds to prevent and/or alleviate the clinical symptoms of said autoimmune disease.

2. The method according to claim 1, further comprising

d) monitoring the level of NO and/or TNF-a release in said cell, and wherein additionally an reduced level of NO and/or TNF-a release compared to a control is indicative for the capability of the candidate compound or candidate combination of compounds to prevent and/or alleviate the clinical symptoms of said autoimmune disease.

3. The method according to claims 1 or 2, wherein the cell is a cell capable of expressing ceramide or ceramide synthase, more preferably a cell selected from the group comprising oligodendrocytes, migrated leucocytes (macrophages, lymphocytes), microglia, preferably activated microglia and astroglia, preferably a macrophage.

4. The method according to claims 1 to 3, wherein said autoimmune disease is a de- myelinating disease, such as multiple sclerosis.

5. The method according to claims 1 to 4, wherein in step b) comprises the use of In- terferon-γ (INF-γ).

6. The method according to claims 1 to 5, wherein the ceramide is Ci₆-Cer and/or the ceramide synthase is CerS6.

7. A compound or combination of compounds capable of preventing and/or alleviating the clinical symptoms of a demyelinating disease identified with the method according to any of claims 1 to 6, for use in the treatment and/or prevention of said demyelinating disease, preferably wherein said demyelinating disease is multiple sclerosis.

8. The compound according to claim 7, wherein the compound is selected from fu- monisin B_{l s} a fumonisin Bi derivative, L-cyclo serine, L-cyclo serine derivatives, myriocin, myriocin derivatives or a pharmaceutically acceptable salt of these compounds.

9. A method of diagnosis and/or monitoring the progression of an autoimmune disease, comprising a step of determining the level of ceramide and/or ceramide synthase in a test sample.

10. The method according to claim 9, wherein an increased level of ceramide and/or ceramide synthase in said test sample compared to a control sample and/or reference value is indicative for an autoimmune disease.

1 1. The method according to any of claims 9 or 10, wherein said autoimmune disease is an autoimmune disease of the central nervous system, more preferably a demyelinating disease, such as multiple sclerosis.

12. The method according to claim 1 1 , wherein the increased level of ceramide and/or ceramide synthase is indicative for the onset of multiple sclerosis.

13. The method according to any of claims 9 to 12, wherein the ceramide is Ci₆-Cer and/or wherein the ceramide synthase is CerS6.

14. The method according to any of claims 9 to 13, wherein the test sample is a biological sample from a subject to be diagnosed.

15. The method according to claims 1 to 14, wherein the test sample is a fluid or tissue from a subject to be diagnosed, preferably blood, a serum sample or cerebrospinal fluid (CSF); or is a tissue biopsy, such as a biopsy of the central nervous system, preferably a biopsy of the white matter, preferably derived from the spinal cord.

16. The method according to claims 1 to 15, wherein the control sample is a sample from a subject not having said autoimmune disease.

17. The method according to claims 1 to 15, wherein the reference value represents the level of ceramide and/or ceramide synthase in a subject not having said autoimmune disease.

18. The method according to any one of the preceding claims, wherein the method is performed in-vitro.

19. A diagnostic kit, comprising means to perform a method according to any of claims 9 to 17, and instructions for their use.

20. An inhibitor of the activity and/or the expression of a ceramide synthase for use in the treatment of an inflammatory disease, preferably multiple sclerosis.

21. The inhibitor according to claim 20, wherein the ceramide synthase is CerS6.

22. The inhibitor according to claims 20 or 21 , wherein the inhibitor is selected from an R Ai molecule, an inhibitory CerS6 antibody, or a small molecule binding and inhibiting the enzymatic activity of CerS6.

23. The inhibitor according to claim 20 or 21 , wherein the inhibitor is an R A molecule comprising a sequence complementary to the gene sequence of CerS6.

24. An antagonist of a ceramide for use in the treatment of multiple sclerosis.

25. The antagonist of claim 24, wherein the ceramide is Ci₆-Cer.