GB2493142A - Ceramide and ceramide synthase in the diagnosis and treatment of multiple sclerosis - Google Patents

Abstract

A method of diagnosis and/or monitoring the progression of autoimmune disease, specifically 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. Also claimed are inhibitors of ceramide synthase and antagonists of ceramide for the treatment of multiple sclerosis, a method for screening compounds capable of preventing and/or alleviating the clinical symptoms of an autoimmune disease as well as compounds identifed by the screening method and their use in treating multiple sclerosis.

Description

Ceramide C16-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 eeramide and/or ceramide synthase arc described which are useful for the treatment of multiple sclcrosis. Also described is a method for screening compounds or combination of compounds capable of preventing and/or allcviating thc clinical symptoms of an autoimmune disease based on thc 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 in-flammatory 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 sys-tem, and alterations in the gcnome 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 evi-dent. 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 mani-

I

fests itself as sensory and/or motor impairment which constitute the clinical manifestations of the disease.

Multiple sclerosis (MS) and its prototypc animal model, the experimental autoimmune reac- tive encephalomyelitis (EAE), are induced by autoimmune responses against myelin compo-nents in the central ncrvous system (CNS). Activated autorcactive T-cells p'ay an essential role in thc development of the disease. These activated T-cells proliferate and secrete pro-inflammatory cytokines, which in turn stimulate microglia, macrophages and astrocytes, and recruit B cells, ultimately resulting in damage to myclin, thc myelin forming oligodendrocytes and axons (McFarland and Martin, 2007). One hypothesis concerning the development of multiple sclerosis is that interferon-gamma (INF-y) secreted from activated T-cell induces NO/TNF-u rclcase in activated macrophagcs which in turn induces apoptosis of oligodendro- cytes leading to the devastating demyelination process (Neuhaus et al., 2006). Oligodendro-cyte apoptosis is onc of the critical events in the dcvclopmcnt of multiple sclerosis (Kassmann et al., 2007) followed by glial activation and infiltration of lcucocytcs (lymphocytes and macrophages). TNF-a (Renno et al., 1995) and NO (Rejdak et al., 2004) arc secreted by acti-vated glia cells. Furthermore in cerebrospinal fluid NO metabolites are associated with the progrcssion 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).

Unfortunatcly the limited diagnostic options for a clinical dctcction of MS and the lack of diagnostic laboratory tests are thc main causcs that impair an early diagnosis and hence of thc disease. Furthermore, currently no possibilities exist to predict the prognosis and progression of the disease. Ncw thcrapcutic and diagnostic options investigated include thc usc of anti-myclin antibodies, microarray gcnc cxprcssion and studies with scrum and cerebrospinal fluid but none of them has yielded reliable positive results. Disease associated biomarkers investi-gated are interleukin-6, nitric oxide (NO) and nitric oxidc synthase (iNOS), ostcopontin, and fetuin-A. Sincc the disease progression is thc result of a neurodegencration 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 sphingolipid transduction pathway induces apoptosis, differentiation, proliferation, growth arrest and inflammation depending upon cell and recep-tor types and downstream targets (Hannun and Obeid, 2008). As the backbone of several complex sphingolipids (sphingomyelins, glycosyleeramides), ceramides are members of the

rapidly expanding field of bloactive lipids.

Ceramides can be generated by de novo synthesis or by degradation of complex sphingolip- ids. The two rate limiting steps in the biosynthesis of eeramides are the synthesis of sphin- ganine from L-serine and palmitoyl-CoA by L-serine palmitoyl transferase (SPT) and the at-tachment of various aeyl-CoA side chains by the ceramide synthases (CerS) to a sphingoide basc. The ceramide synthases (CcrSl-6) act chain length specific and introduce side chains form C14-C26. Thus, CerS] 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 supply 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. Ce-ramides 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 de-creased significantly (Riethmuller et al., 2009). SMases and their product ceramide induce an increase of TN F-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 cul- tire experiments reveal that pro-inflammatory stimuli such as lipopolysaccharide and cyto- kines activate macrophages and induce an increase of ecramides via the activation of sphin-gomyclinascs (MacKichan and DeFranco, 1999).

Antagonizing the pro-apoptotic effects of ceramides in the field of medicine is described in IJS2O 100278907, wherein immunogenic compositions containing ceramide or ceramide ana- logs 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 DEl 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 DE102008029734 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 muhiplc sclerosis. Furthermore, the lim-ited 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 thera- peutically active substances which may find their way into clinic application to tackle multi-ple 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 determin-ing the level of ceramide and/or ceramide synthase in a test sample.

The inventors surprisingly found that Cioo-Cer is involved in the induction of an animal model of MS (EAE) by activation of NO and TINE-a synthesis. Cioo-Cer and CerS6 are upregulated in the lesion site in activated microglia, astroglia and migrated leucocytcs in EAE mice. The inhibition of Cioo-Cer synthesis caused a reduced iNOS and TNF-a expres-sion and a remission of the disabilities of EAE mice. Moreover, it was demonstrated that Cio:oCer plays a critical role in the IINE-y induced iNOS/ TNF-a expression and NO/ TNF-a release. Accordingly, the anti-inflammatory substance methylprednisolonc prevents the in-crease of C 16:irCei in vivo and in vitro. Additionally, in the cerebrospinal fluid (CSF) of MS patients the C15o-Cer levels were increased as compared to control patients.

Since a significant upregulation of ceramide and ceramide synihase 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 eventu- ally leads to an immune reaction directed to the hosts own proteins and structures. In the con-text of the present invention an autoimmune disease is preferably an autoimmune discase of the central nervous system, more preferably a demyelinating disease, such as and most pre-ferred, multiple sclerosis.

A further embodiment of the invention relates to the above method, wherein the increased level of ceramidc 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 ccramide 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 cerainide is Cioo-Cer and/or the ceramide synthase is CerS6. The present invention surprisingly found that Cioo-Cer and/or CerS6 can be used as biomarkers in the diagnosis or iii 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 con-tains preferably tissue, cells or ccrcbrospinal 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 cenfral 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, lym-phocytes), microglia and astroglia.

In thc context of this invention a control sample corresponds to the test sample with the dif-ference 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 the control sample is preferably a sample from a subject not having said autoimmune disease. Alternatively the level of ceramide andior ceramide synthase observed in the test sample may be compared with a reference value. The reference value represcnts the level of ceramide and/or ceramide synthase in a subject not having said autoimmune disease.

For determining the level of ccramidc and/or ceramide synthase in thc 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 mRNA 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 C16: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 sam-ple 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 an-tibody which is specifically directed to ceramide, preferably Cioo-Cer and/or to a eeramide 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-rnent 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 arc 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 ex- pression of a ceramide synthase for use in the treatment of an inflammatory disease, prefera-bly multiple sclerosis. In a one embodiment of the aspect thc inhibitor is an inhibitor of CerS6.

Yet further preferred is that the described inhibitor of the invention is selected from an inhibi- tory nucleic acid, such as an siRNA, an inhibitory CerS6 antibody, or a small molecule capa-ble of binding and inhibiting the enzymatic activity of CerS6. A nucleic acid based inhibitor is preferaNy an RNA molecule comprising a sequence which is comp'ementary and specific to the gene sequence of CerS6. In a preferred embodiment of this aspect, the RNA sequence of the RI4Ai molecule should be selected to allow a specific targeting of CerSô expression and not of any other ccramidc synthase.

In a prcfcrrcd embodiment the inhibitory nucleic acid comprises the sequence of SEQ ID No.1, or a variant thereof having preferably 50%, 60%, 70%, 80%, 90%, 95% or 99% se- quence identity to the sequence shown in SEQ ID No. 1. Another preferred embodiment re-lates to a RNAi construct, such as an siRNA, comprising the sequence of SEQ ID No.1, 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. I are exchanged with Uracil in the RNA sequence. Preferably the siRNA molecule for usc 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 #s109529, having the sequence 5'-GAC CUD 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 C16-Cer, and impairs it's flinetion as a second messenger. Such antago-nists arc preferably inhibitory antibodies which bind specifically to ceramide resulting in the formation of an inactive ceramide-antibody complex. Preferably the ceramide is C16-Cer.

Another aspect of the invention relates to a method for screening compounds or combination of compounds capable of preventing andlor alleviating the clinical symptoms of an autoim-rnune 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 ccramidc or ccramide synthase in said cell, wherein an reduced level of ceramide or ceramide synthase compared to a control is indica-tive 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 thc cvcl ccramide or ceramidc synthase in said cell.

Yet a further embodiment of the above method of the invention comprises the optional, alter-native 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-ct re- lease compared to a control is indicative for the capability of the candidate compound or can-didate 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-ct release compared to a control and a reduced level of ccramidc 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.

a preferred embodiment of the invention the cell for use in the method is a cell capable of expressing eeramide 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 inven-tion is that said cell is a macrophage.

In another embodiment said autoimmunc disease is preferably a demyelinating disease, such as multipk sclerosis.

In macrophages, one of the key indueers of the formation of NO and/or TNF-a is Interferon-y.

Hence, it is further preferred that in one embodiment step b) of the inventive method com-prises the use of lnterferon-y (lNF-y. INF-y acts according to the invention as an upstream factor of ecramide 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 Cl 6-Cer and/or the eeramidc syn-thase 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 ncxt 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 Bi, a flimonisin Bi derivative, L-cycloserine, L-cycloserine derivatives, myrioein, -10 - rnyriocin derivatives, FTY72O (fingolimod) and its derivatives or a pharmaceutically accept-able salt of these compounds.

The present invention will now be explained in the following examples with reference to the accompanying figures, without being limited thereto. For the purposes of the rresent inven- tion, aLl references as cited herein are incorporated by refirence in their entireties. In the Fig-tires, Figure 1: C16:0-Cer level is altered in EAE mice. A) Clinical score of EAE mice. Mice were immunized with MOG3S-55-peptide dissolved in complete Freud's adiuvant on day 0 and received pertussis toxin on day 0 and day 1. Onset of disease comprises mice with clinical sc0.5 -sci.5. The acute phase included mice with a clinical sc2 -sc3. B) Cerarnide levels of untreated mice, of CFA-fteated mice alone or FAF mice were determined by [C-MS/MS.

The ceramide level of the EAE mice and the CFA-trcated mice were related to the ceramide levels of untreated mice. The eerarnide level of untreated mice were for C16:0-Cer 0.47 ± 0.13 pnio]/mg tissue, for Ci8:0-Cer 10.05 ± 3.76 rnol/mg tissue, for C20:0-Cer 14,41 ± 6.44 mol/mg tissue and for C24:0-Cer 19.06 8.91 imol/mg tissue. The ceramide levels of un- treated mice were set as 100%. S (p < 0.05) " (p < 0.01) indicate significant difference be-tween EAL and CFA-*treated mice.

Figure 2: Cl 6:0-Cer level is upregulated in eerehrospinal fluid of MS patients (n= 18) as compared to control patients (n=13). Cl6:0-Cer levels were determined by IC-MS/MS. * (p <0.05) indicate ignificant difference between MS patients and control patients.

Figure 3: CerS6 is elevated in the onset of the disease. A) The relative mRNA expression of CerS 1, CerS2, CerS4, CerSS, CerS6 and GAPDH (control) was normalized to 1 8SRNA 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 CerSó 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 load-ing control GAPDH was used (one of three independent experiments is shown). (p < 0.05), -11 - ** (p < 0.01), (p c 0.001) indicate significant difference between EAE and CEA-treated mice Figure 4: Expression of CcrS6 in the lumbar spinal cord. A) Exprcssion of oligodendro-cyte 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 CerSô (red channel) and specific cell type markers (green channel) (activated microglia (Cdl Ib/Ibal), migrated lymphocytes and macrophages (CD4S), activated astroglia (GFAP)) in sd.5 mice. D) 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 im-ages of three independent experiments. Scale bar, 10 tm (A1C, 60x), Scale bar, 100 RM (B, 5x) Scale bar, 25 tM (B, 20x).

Figure 5: 1INF-' induced in RAW macrophages an alteration of the eeramide levels and an increase of CerSô A) RAW macrophages were treated for 16 h with 10 ng/ml INF-y, 5 ng!ml TNF-alpha and I ng/ml IL-lB. The ceramide levels were determined by LC-MS/MS and normalized to the number of treated cells. The relative increase of the specific ceramides was calculated using the eeramide 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 differ-ence between eytokine-treated and untreated RAW maerophages. B/C/D) The time dependent effect of INF-y treated RAW maerophages 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-y 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 of 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 C 0.01) indicates significant difference between INF-y 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 C 0.01), (p C 0.001) indicate significant difference between INF-y treated and untreated cells. D) Densitometric analysis of the CerS6 expression. The CerSb expression was related to the expression level of GAPDH. * (p C 0.05) indicate significant -12 - difference between INF-y treated and untreated cells. Western blot analysis of CerS6 at sev-eral time points as indicated. As Loading control GAPDH was used (one of two independent experiments is shown).

Figure 6: The IN F-y expression correlates with disease progress. A) The relative mRNA levels were normalized to I8SRNA 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-y expression was determined by mouse INF-y ELISA Kit (Bioleg-end, Uithoom, Netherlands). (p < 0.05) indicate significant difference between EAF and untreated mice (control).

Figure 7: FBI and L-cycloscrine prevent the INF-y induced increase of NO synthesis. A) The mRNA level of iNOS is transiently increased in EAE mice and in INF-y (10 ng/ml) time dependently stimulated RAW macrophages. The relative mRNA expression of iNOS was normalized to 1SSRNA (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 IN F-y treated RAW macrophages. Western blot analysis of iNOS in EAE mice (in vivo) and in RAW macrophages (in vitro). As loading control GAPDI-1 was used (one of two independent experiments is shown). C) The NO level is time dependent increased in INF-y (10 ng/ml) in-duced RAW maerophages. The NO amount was determined from the supernatant and related to the number of cells. D/E)70 RM fumonisin Bl (FBI), 500 tM L-cycloserine (Cyclo) or 1 RM methylprednisolone (MP) were preincubated for 30 olin or 90 mm, respectively, subse-quently 10 ng/ml INF-y 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 GAPDFI 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 c 0.01), (p < 0.001) indicate significant difference between INF-y treated and untreated RAW macrophages as well as between EAE mice and untreated mice.

-13 - Figure 8: L-Cycloserine arid Fumonisin B I prevent the INF--y induced increase of cera-mides. 500 aM L-cycloserine (Cyclo) or 70 tN1 fumonisin Bi (FBI) or DMSO (control) were preincubated for 2 h, subsequently lOng/mI INF-y were added for 16 h. From thc cell pellet the ceramide level were determined by LC-MS/MS. The ceramide level wcre related to the number of treated cells. The ceramide amount of the control cells was set as 100% value. * p<O.O5, ** p<O.OI, p<O.OOl indicate significant difference between INF-y treated cells and cells co-treated with INF-y and an inhibitor of the sphingolipid synthesis.

Figure 9: Co-stimulation of INF-y and L-cycloserinc (Cyclo) or fumonisin BI (FBI) or methylprednisolone (MP) didn't reduce macrophage viability. RAW macrophages were treated for 16 h with INF-y (10 nglml) alone or co-treated with L-cycloserine (500 tM), fu-monisin B 1 (70 RM) or methylprcdnisolonc (1 tM). The cell viability was determined with the WST proliferation assay (Roche Diagnostics). The cell viability was determined by using TNF-y treated cells as 100 % value.

Figure 10: Down-regulation of CerS6 prevents at least partly the INF-y induced NO re-lease and exogenously added palmitic acid amplified the INF-y induced NO synthesis. A-C) mRNA levels of CerS5, CerS6 and iNOS (A), ceramide levels (B) and NO release (C) in scrambled (scr.) s1RNA and siCerSô treated RAW macrophages, which were co-incubated with 10 ng!ml INF-y 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 ccramide lev-els 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 < 0.00 1) indicate sig-nificant difference between siCerS6 treated and scrambled siRNA treated RAW macrophages.

D/E) NO release (D) and CI 6:0-Cer level (E) of RAW macrophages stimulated simultane-ously with 25 jiM palniitic acid and 0.5 ng/ml INF-y for 16 h. Ceramide levels were detected by LC-MS/MS. The relative increase of the Cl6:0-Cer was calculated using the ceramide level of untreated ccl Is 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 c 0.0), (p < 0.001) indicate significant difference between INF-y stimulated and IINF-y!palmitic acid co-stimulated RAW macrophages.

Figure II: Palmitic acid don't increase C14:0-Cer, C24:l-Cer and C24:0-Cer neither in control nor in INF-y treated RAW macrophages. DMSO (control), 25 jiM palmitic acid, 0.5 -14 -ng/ml INF-y or 25 gM palmitie acid and 0.5 ng/ml INF-y were incubated for 20 It From the cell pellet the ceramide level were determined by LC-MS/MS. The ceramide level were re-lated to the number of treated cells. (ns; not significant) Figure 12: C16:0-Cer mediates INF-y induced increase of TNF-u synthesis. A) The mRNA level of TNF-a is transiently increased in EAE mice. B/C) 70 RM fumonisin El (FE 1), 500 RM L-cycloserine (Cyelo) or 1 jiM methylprednisolone (MP) were preincubated for 30 mm or mm, respectively, subsequently 10 ng/ml INF-y were added for 16 h. The mRNA level of TNF-ci (B) from the cell pellet and TNF-a release (C) from the supernatant was measured.

The TINE-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 siCcrS6 treated RAW macrophagcs, which were co-incubated with 10 ng/ml INF-y for 16 h, were determined. The relative mRNA expression of TINE-a was normalized to I8SRNA (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 ex-periments each achieved in duplicate. * (p < 0.05), ** (p < 0.01), (p < 0.001) indicate significant difference between fNF-y treated and inhibitor treated RAW macrophages; scram-bled 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 thc development of disabilities in EAB mice, the increase of 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 M0G35-55-peptide dissolved in complete Freud's adjuvant at day 0 and received pertussis toxin at day 0 and day 1. The EAE and CFA-trcatcd mice were medicated with L-cycloserine or saline when the EAE mice reached sc0.5. B) Ceramide level of the mice were determined by LC-MS/MS.

The eeramide levels of the L-cycloserine-or saline-medicated EAE mice and CFA-trcated mice were related to the ceramide level of age matched control mice. CD) The relative mRNA expression of iNOS (C) and TNF-ri (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 c 0.05) indicate significant difference between L-cycloserine-and saline-medicated EAE mice.

-15 -Figure 14: Methylprcdnisolone prevents the increase of 06:0-Cer in EAE mice and in TNF-y treated RAW macrophages. A/B) NO release (A) and ceramide level (B) in RAW macrophages treated with 10 ng/ml IINF-y, with 1 jiM methyiprednisolone (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 of the specific ceramides were calculated using the ceramide level of untreated cells as 100% value. C) Clinical score of EAF mice treated daily with 10 mg/kg methylprednisolone or left untreated. Mice were immunized with MOG3S-55-peptide dis-solved 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 methylprednisolonc when they reached sc2 -sc3. D) Ceramide levels of EAE mice and CFA-treated mice medicated with methylpredni-solone 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: The putative role of C16:0-Cer in the INF-y induced NO and TNF-ri release.

(CerS, ceramide synthase; INF-y, interferon-gamma; INFGR, interferon-gamma receptor; iNOS, inducible nitric oxide synthase; NO, nitric oxide; TNF-a, tumor necrosis factor alpha) -16 -

Examples

The following materials and methods were used in context of the presented examples of the invention: Cells and reaents RAW 264.7 mouse macrophages were cultured and incubated in RPM! 1640 medium con- taining 10% FCS and 1% penicillin/streptomycin. Cells were cultured at 37 °C in an atmos-phere containing 5% CO2. L-eycloserine (Cyclo), flimonisin Hi (FBI), palmitic acid (PA) and 6a-methylprednisolone (MP) were purchased from Sigma-Aldrich (Sehnel!dorf, Germany).

INF--y, TNF-alpha, and IL-lB were purchased from PeproTeeh (Hamburg, Germany). siRNAs CerS6 (s109529) 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-Ol 15) and the control (CK-01 15) kit were purchased from Hooke Laboratories (Lawrence, USA). The INF-y 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 weighing 18 g -20 g were obtained from Harlan Laboratories (I-lorst, Netherlands). The procedure used for the induction of EAE was con- ducted as recommended by Hookc Laboratories (Lawrence, USA). Briefly, EAE mice re- ceived a subcutaneous injection in the upper and lower back with MO03555 peptide emulsi-fied in complete Freund's adjuvant (CFA) containing Mycobucterium tuberculosis. 2 hours thereafter, and again 24 hours later, the mice rcccivcd an intraperitonea! injection of pcrtussis toxin (PTX). The CFA-treated mice were treated with CFA containing Al. 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!ed 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.

EAE mice treated with L-cycloserine or methylprednisolone -17 -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 sc0.5. As control EAE and CFA-trcated mice were medicated daily with saline by an i.p. injection. EAE and CFA-treated mice were mcdicated with 10 mg/kg methyiprednisolone. Methyiprednisolone was dissolved in ethanol and then added to the drinking water when the mice reached sc2. Every second day the drinking water supplemented with mcthylprcdnisolone 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 perthsed 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 Micro-systems GmbH, Nussloch, Germany), then quick frozen on dry icc and stored at -80 °C.

Immunohistochemistry 14 micrometer sections were permeabilized in PBS containing 0.1 % Triton X-100 for mm, blocked in PBS containing 3 % bovine serum albumin and 0.1 % Triton X-l00 for 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: CerSb (1:100), Ionized calcium-binding adaptor molecule 1 (Ibal) (1:200), Anti-Glial Fibrillary Acidic Pro-tein (GFAP) (1:1000), Cdllb (1:100), CD4S (1:100), iNOS (1:100), death receptor 5 (DRS) (1:50). The CerS6 (goat polyclonal) and DRS (rat polyclonal) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), while GFAP (rabbit polyclonal) and CD45 (rat -18 - polyclonal) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Ibal (rabbit poly-clonal) were purchased from Wako Chemicals GmbH (Neuss, Germany). The antibody against Cdl lb (rat polyclonal) was used from Serotcc (Düsseldorf, Germany). Thc antibody against iNOS (rabbit polyclonal) was purchased from Becton Dickinson (Heidelberg, Ger-many).

Preparation of crude protein extracts RAW macrophages were seeded in 5 cm dishes at a density of siO cells/dish. Cells were treated with 10 ag/mI or 0.5 ng/ml 1INF-y, 5 ng/ml TNF-alpha, 1 ng/ml 1L113, 25 jiM palmitic acid for the indicated time points. INF-y (10 ng'ml) treated RAW macrophages were co-treated with 70 jiM fumonisin Bi, 500 jiM L-cycloserine or 1 jiM methylprednisolone for 16 h. Mcthylprcdnisolone and L-eycloscrinc were preincubated for 90 mi while fumonisin BI was preincubated for 30 mm. INF-y (0.5 ng/ml) treated RAW macrophages were co-treated with 25 jiM 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-I-1C1120 mM CHAPS, pH 7,4) supplemented with protease in- hibitors. 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 con-centrations were assessed using the Bradford method.

Western blot analysis Immunoblotting was performed as described previously (Sehiffmann et al., 2008). 30 jig pro-teins of cell lysates and 30 jig 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 pur-chased from Ambion (Darmstadt, Germany).

Real time uPCR RAW maerophages and lumbar spinal cord were analyzed for mRNA levels by qPCR as pre- viously described (Schiffmann et al., 2010). The expression levels of CerSl-6, GAPDH, 13-actin and iNOS were determined by TaqmanTM analysis using the SYBR Green Kit (ABgene Limited, Epsom, United Kingdom) with an ABI Prism 7500 Sequence Detection System (Ap- -19 -plied Biosystems, Austin, USA). Relative expression of CerS family genes, GAPDI-1 and iNOS was determined using the comparative CT (cycle threshold) method, normalizing rela-tive values to the expression level of I8SRNA (spinal cord samples) or GAPDH (RAW macrophages) as housekeeping gene. The designed primer sets for CerSs, iNOS and INF-y were adopted from Laviad et al. (Laviad et al., 2008), from Chiang eta!. (Chiang et al., 2009) and from Nath et a]. (Math et al., 2009), respectively. Linearity of the assays was determined by serial dilutions of the temp!ates for each primer set separately.

Silencing of CerS with siRNA RAW macrophages were transfected with 150 pmo! CerS6 siRNA or 100 pmo! scrambled 5iRNA 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 mm at RT, then added to the siRNA so!ution consisting of Opti-MEM medium and siRNA, fol!owed by an incubation for 10 mm at RT. 3.5x105 RAW macrophages were incubated with siRNA transfection so!ution, and the process was repeated after 24 h. After 41 h the transfected RAW macrophages were either harvested for mRNA isolation (RI'4A isolation kit (Qiagen, Hi!den, Germany)) or treated with IN F-y for 16 h and subjected to sphingo!ipid analysis and NO de-termination. The effectiveness of the siRNA knock-down was verified using quantitative PCR.

Determination of sohingolipid concentrations The quantification of sphingolipid amounts in RAW macrophages was achieved as previously pub!ished (Schiffmann et al., 2009a). Briefly, cells were seeded at a density of 0.Sx 106/5 cm dish and incubated for 24 h. Subsequently, cells were treated with various substances. Ce!!s were counted in a Neubauer chamber and stored at -80 °C. After thawing, the internal stan-dards (C17: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 samp!es, about 20-100 mg of tissue was dounced in PBS on ice. 20 p1 of tissue suspension (0.02 mgtjil) was extracted in 600 ul chloroform/methanol (7:1) after the addition of the internal standards (Ci7g-Cer)) and R1 water. The suspension was vigorously vortexed at 25 °C for 1 mill and centrifuged for 5 mm at 25 °C and 14,000 rpm. The supernatants were co!lected and the extraction step was repeated. The combined organic phases were dried under a stream of nitrogen at 45 °C and -20 -redissolved in 50 R' of methanol for quantification. C140-Cer, C16:oCer, Cig:crCer, C20:o Cer, C24:iCer and C24o-Cer and the internal standards were determined by LC-MS/MS as de-scribed previously (Schiffmann et al., 2009b; Schiffmann et al., 2009a). Concentrations of the calibration standards, quality controls and unknowns were evaluated using the Analyst soft-ware version 1.5 (Applied Biosystems). Linearity of the calibration curve was proven for C14:oCer from 0.3 -500 ng'ml, Ci&o-Cer/C24o-Cer from 3 -5000 ng/ml, for Ciso-Cer from 0.9-1500 ng/ml and for C20o-Cer/C24i-Cer from 1.2-2000 ng/ml. The coefficient of corre-lation for all measured sequences was at least 0.99. Variations in accuracy and intraday and interday precision (n2 for each concentration in mouse tissue and cells) were less than 1 5 % over the range of calibration.

Determination of NE-v. TNF-a and nitrite/nitrate RAW macrophages were treated with IINF-y and various substances (L-eyeloserine, fu- monisin B I, methylprednisolon, palmitic acid). After eentriffigation the supernatant was har- vested, centrifuged (1,200 rpm, 3 mm, 4° C) and stored at -20 °C. The release of NO was as-sessed 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-ct ELTSA detection kit from Biolegend (IJithoom, The Netherlands). The amounts of INFj' in the lumbar spinal cord of EAE, CFA-treated and control mice were determined using the mouse INF-y ELISA detection kit from Biolegend (IJithoom, The Netherlands).The de- termination of the concentration of INE-y and TNF-ct was achieved according to the manufac-turer's protocol.

Statistics Results are presented as a mean ± s.c.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: Cio:trCer 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 (CPA). The mice developed after 13 ± 2 days first signs of reduced motor skills which strengthen after 4 + 1 days to a complete pa- ralysis of the hind paws (Figure 1A). The ceramide levels of the lumbar spinal cord were de-termined in untreated, CFA-treated (only treated with CFA-emulsion and PTX) and EAE mice. The EAB mice were divided in two groups: scO.5 -sd.5 (onset of the disease) and sc2 -sc3 (acute phase of the disease). The inventors determined the amounts of C140, C160, Ciso, C2o:o, Cisi_, C24:1 and C24:vCer. The amount of C14:0, Cis:i-and C24i-Cer was under the detection limit (data not shown). The lumbar spinal cord reveal a significant increase ofCioo-Cer at the onset (scO.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 1B).

Example II: C16:o-Cer levels are increased in the cerebrospinal fluid of MS patients Since specifically Cioo-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 cere- brospinal 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 Cioo-Cer lev-els are significantly upregulated as compared to control patients (Figure 2) indicating that Cio: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: C16:rCer increase in EAL mice is linked to a raised CerS6 expression Next the inventors studied, whether or not the raised Cioo-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, CEA-treated (control), scO.5, sd.5 and sc3 mice. Quantitative PCR results revealed that the mRNA of CerS6 was significantly increased in mice at the onset of EAE (Figure 3A). 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, sd.5 and sc3 mice were determined. Sd.5 mice showed a significant increase of CerS6 as compared to CFA-treated mice, while in se3 mice CerS6 expression was reduced -22 -to the expression level of CFA-treated mice (Figure 3B!3C). Thus, the mRNA and protein expression profile suggest a transient increase of CerS6 and C16ij-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 sd.5 mice (Figure 4A, data shown for untreated mice) CerS6 is colocalized with the oligodendro-cyte specific protein (OSP). In FAE mice (sd.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. CerSb is expressed in mieroglia (Ibal and CDIIb 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 Cioo-Cer is discussed to be involved in apoptotic processes, the inventors investigated whether or not CerS6 is detectable in apoptotie cells. CerS6 was not expressed in death receptor 5 (DRS) positive cells (Figure 4D).

Example V: INF-y induces increase of ceramides in macrophages Next the inventors wanted to investigate whether or not C16o-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 Thi-mediated response in the development of EAE (Domingues et al., 2010). ThI-cells secrete predominantly INF-y, IL-I B and TNF-alpha which in tum activate macrophages. Therefore the inventors studied the influence of INF-y, IL-lB and TNF-alpha on the ceramide levels in macrophages. Figure SA demonstrates that only IINF-' induced in macrophages a significant elevation of C ioo-Cer after 16 h treatment. INF-y led to a time dependent predominant in-crease of Ct4o-Cer and Cioo-Cer and to a slight increase of C24;0-Cer (Figure SB).

-23 -The fact that dihydroceramides are elevated (data not shown) indicate an activation of the sphingolipid de novo synthesis. The mRNA levels of thc various CerSs in INF-y treated macrophages support the LC-M S/MS data and revealed a significant increase of the mRINA level of CerS6 (Figure SC). The upregulation of the mRNA expression of CerS6 in RAW macrophages is transient as in the lumbar spinal cord of EAE mice (Figure 3A). The protein expression of CerS6 is also time dependent elevated in TNF-y treated macrophages with a sig-nificant increase already after 6 h (Figure 5D). The transient increase of CerS6 in the lumbar spinal cord and in 1NF-y treated RAW macrophages and the subsequent predominant increase of Cio:o-Cer in both experimental setups indicate that the in vitro experiment simulates very closely the in vivo situation.

Example VI: C16:o-Cer mediate INF-y induced NO release in macrophages Macrophages and astroeytes contribute by NO relcase to oligodendrocyte degeneration in demyelinating diseases (Merrill et al., 1993; Mitrovic et al., 1994). One of the key events in macrophage responses to 1NF--y stimuli is the expression of iNOS and the subsequent forma-tion of NO. In EAE mice the mRNA and protein expression of INF-y is increased in the onset of the disease (Figure 6). Moreover, in the EAE mice the inventors observed also an increase of 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 sc0.5 10 fold increased, while the rnRNA level of iNOS is only about 2.5 fold increased suggesting that iNOS acts downstream of CerS6. RAW macrophages treated with 1 0 ng/ml INF-y led to an increase of 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 SD) which is much earlier than the upregulation of the iNOS expression after 16 Ii (Figure 7B) indicating that CerS6 acts upstream of iNOS.

When the NO synthesis is regulated by Cio:o-Cer, the INF-y induced NO level should be re-duced by the treatment with specific inhibitors of the sphingolipid synthesis. L-cyeloserine (inhibitor of the serine palmitoyl transferase) and fumonisin B! (FBi) (inhibitor of the CerSs) prevent the increase of ceramides induced by INF-y in RAW macrophages (Figure 8). The effectiveness of L-cycloserine in inhibiting of INF-y induced ceramide synthesis points to that INF--y induces rather de novo synthesis than the salvage pathway of ceramide synthesis. Im-portantly, L-eyeloserine and FB 1 inhibit significantly the INF-y induced NO release (Figure 7D) as effective as the anti-inflammatory glucocorticoide niethyiprednisolonc (Figure 7D).

Additionally, the inventors demonstrated that L-cycloserine and FBI prevent the INF-y in- -24 - duced upregulation of the mRNA expression of INOS (Figure 7F) excluding that the inhibi- tors unselectively inhibit thc activity of iNOS. To cxclude that the reduced NO rcleasc in-duced by FBI and L-cycloserine in INF-y treated macrophages is due to reduced cell viability, the inventors achieved a cytoxicity test. Neither FBi nor L-cycloserine reduced the cell vi-ability in INF--y pretreated maerophages (Figure 9). These data suggest that ceramides mediate the INF-y induced NO release.

To verify that predominantly C16o-Cer and CerS6 are responsible for the mediation of the NO increase in INF-y treated macrophages, CerS6 was down-regulated by RNAI. siCerS6 pre-treated RAW macrophages were stimulated with 10 ng'ml INF-1 for 16 h. siCerS6 prevent in INF--y treated cells CerS6 upregulation, which is observable in INF--y treated with scrambled siRNA prc-incubated RAW macrophagcs (Figure bA). CerS5, which has a similar substrate specificity than CerS6, is not regulated by the siCerS6 (Figure bA). The down regulation of CerS6 prevent an increase of specifically C14;o-Cer (data not shown) and C16o-Cer (Figure lOB), but not of C24o-Cer (data not shown). which is also slightly upregulated in INF-y treated cells (Figure SB). As expected, siCerS6 prevented INF-y induced iNOS mRNA ex-pression (Figure 1 OA) and NO release (Figure 1 OC) in RAW macrophages as compared to scrambled siRNA treated RAW macrophages. These findings indicate that CerS6 and Cio:o-Cer mediate the activation of iNOS expression in INF-y stimulated RAW macrophages. Since the down regulation of Cioo-Cer prevents INF-y induced NO synthesis, a specific endogenous upregulation of C i6xrCer should increase the NO release. Importantly, exogenously added palmitic acid (25 jtM), which led to a specific increase of Cio:o-Cer (Figure 1OD, Figure 11) amplifies the INF-y (0.5 ng/ml) induced NO synthesis significantly in RAW macrophages (Figure bE).

Example VH:C16:i,-Cer mediate INF-y induced TNF-a release in macrophages Besides NO also TNF-cz is a mediator of oligodendroglial death, therefore the inventors stud-ied whether TNF-a is also regulated by Cioo-Cer. As expected, TNF-a is upregulated disease dependently in EAE mice (Figure 12A) and INF-y induces beside the NO release also the synthesis of TNF-a in macrophages (Figure 12B). Interestingly, TNF-y induced TNF-a mRNA expression and TNF-a release is inhibited by FBI and L-cycloserine (Figure 12 B, C).

As expected, siCerS6 prevented INF-y induced TNF-a mRNA expression (Figure 12D) and TNF-a release (Figure 1 2E) in RAW macrophages as compared to scrambled s1RNA treated -25 -RAW macrophages. Moreover, exogenously added palmitie acid (25.tM) amplifies the INF-y (0.5 ng/mI) induced TNF-a synthesis in RAW macrophages (Figure 12C).

Example VIII: Inhibition of Cioo-Cer prevent the worsening of the clinical symptoms in the EAE model Since L-cycloserine as well as FBI prevented the induction of NO and TN F-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-eyeloserine. The mice were treated with 75 mg/kg L-cyeloserine 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 sa- line. For statistical analysis the area under the curve (AUC) was determined. The AUC of L-eycloserine treated EAE mice was 10.51 ± 1.8 score/day and from saline treated EAF mice 17.69 + 2.3 score/day. Treatment with L-eyeloserine prevents significantly (p0.04l) the de-velopment of disabilities (Figure 13A). Figure 13B revealed that in L-eyeloserine treated EAE mice compared to saline-treated EAE mice the C16:o-Cer level is significantly lower. More-over L-eyeloserine prevents significantly the increase of the mRNA level of iNOS and TNF-a in the lumbar spinal cord (Figure 13C, D). These data indicate that Ci6ij-Ccr is involved in the mediation of iNOS and TNF-ct during the development of EAE.

Next the inventors investigated whether or not an anti-inflammatory drug regulates the Cio:o-Cer level in EAE mice and in INF-y stimulated RAW macrophagcs. If an anti-inflammatory drug could prevent the increase of Cio:o-Cer level in vivo and in vitro than C16:o-Cer should be involved in the inflammatory process. Glucocorticoids are used for the treatment of MS pa-tients. Moreover, in the EAE mouse model methylprednisolone resulted in an remission of the disabilities (Chan et al., 2008). The treatment of RAW macrophages with the glucocorticoid mcthylprcdnisolone (1 jiM) led to an inhibition of the INF-y induced NO release (Figure 14A). Importantly, the combined treatment of I jiM methylprednisolone and 10 ng/ml INF-y inhibit specifically the increase of Cio:o-Cer whereas the slight increase of C24: and C24:crccr was not affected by methylprednisolonc (Figure 14B). EAE and control mice (CFA-treated) were medicated in stage se2 -sc3 with a daily dose of 10 mg/kg methylprednisolone. As ex- pected, the treatment with methylprednisolone resulted in an remission of the symptoms (Fig-ure 14C). Interestingly, methylprednisolone led also to a specific decrease of the C16o-Cer level (Figure 14D) in the lumbar spinal cord as compared to untreated EAE mice. These data -26 -confirm the crucial role of Cio:o-Cer in the inflammatory process leading to the development of multiple sclerosis.

In summary, the present invention shows that Ci6o-Cer plays an important role in the initial phase of the inflammatory process in MS disease. The transient increase of Ciô:o-Cer /CerSG at the beginning of the inflammatory process points to a significant role of Ci6o-Cer in the initial phase of MS. Furthermore, thc examples demonstrate for the first time a direct correla-tion of the de novo synthesized C16o-Cer and INF-y induced NO/TNF-a synthesis (Figure 15).

Up to now, it is only described, that ceramides synthesized by the salvage pathway or glyco-sylated 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 prcvcnted by fumonisin BI, in contrast to thc findings of the prcscnt invention.

Claims (1)

1. <claim-text>-27 -Claims 1. A method of diagnosis and/or monitoring the progression of an autoimnmne dis- ease, comprising a step of determining the level of ceramide and/or ceramide syn-thase in a test sample.</claim-text> <claim-text>2. The method according to claim 1, wherein an increased level of ceramide and/or ceramide synthasc in said test sample compared to a control sample and/or refer-ence value is indicative for an autoimmune disease.</claim-text> <claim-text>3. The method according to any of claims I or 2, wherein said autoimmune disease is an autoimmune disease of the central nervous system, more preferably a demyel I-nating disease, such as multiple sclerosis.</claim-text> <claim-text>4. The method according to claim 2, wherein the increased level of ceramide and/or ceramide synthase is indicative for the onset of multiple sclerosis.</claim-text> <claim-text>5. The method according to any of claims 1 to 4, wherein the ceramide is C16-Cer and/or wherein the ceramide synthase is CerS6.</claim-text> <claim-text>6. The method according to any of claims ito 5, wherein the test sample is a biologi-cal sample from a subject to be diagnosed.</claim-text> <claim-text>7. The method according to claims ito 6, wherein the test sample is a fluid or tissue from a subject to be diagnosed, preferably blood, a serum sample or ccrcbrospinal 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.</claim-text> <claim-text>8. The method according to claims I to 7, wherein the control sample is a sample from a subject not having said autoimmune disease. -28 -</claim-text> <claim-text>9. The method according to claims Ito 6, wherein the reference value represents the level of ccramidc and/or ceramide synthasc in a subject not having said autoim-mune disease.</claim-text> <claim-text>10. The method according to any one of the preceding claims, wherein the method is performed in-vitro.</claim-text> <claim-text>11. A diagnostic kit, comprising means to perform a method according to any of claims I to 10, and instructions for their use.</claim-text> <claim-text>12. An inhibitor of the activity and/or the expression of a ceramide synthase for use in the treatment of an inflammatory disease, preferably multiplc sclerosis.</claim-text> <claim-text>13. The inhibitor according to claim 12, whercin the ccramide synthase is CerS6.</claim-text> <claim-text>14. The inhibitor according to claims 12 or 13, wherein the inhibitor is selected from an RJ'Ai molecule, an inhibitory CerS6 antibody, or a small molecule binding and inhibiting the enzymatic activity of CerS6.</claim-text> <claim-text>15. The inhibitor according to claim 12 or 13, wherein the inhibitor is an RNA mole-cule comprising a sequence complementary to the gene sequence of CcrS6.</claim-text> <claim-text>16. An antagonist of a ceramidc for usc in the treatment of multiplc sclerosis.</claim-text> <claim-text>17. The antagonist of claim 16, wherein the ceramide is C16-Cer.</claim-text> <claim-text>18. A method for screening compounds or combination of compounds capable of pre- venting and/or alleviating the clinical symptoms of an autoimmune disease, com-prising 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 ccramide synthase in said cell, -29 -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 autoim-mune disease.</claim-text> <claim-text>19. The method according to claim 18, further comprising d) monitoring the level of NO andlorTNF-ri release in said cell, and wherein additionally an reduced level of NO and/or TNF-ri 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.</claim-text> <claim-text>20. The method according to claims 18 or 19, wherein the cell is capable of expressing ceramide or ccramide synthase, more preferably wherein the cell is selected from the group comprising oligodendrocytes, migrated leucocytes (macrophages, lym-phocytes), microglia, preferably activated microglia and astroglia, preferably a macrophage.</claim-text> <claim-text>21. The method according to claims 18 to 20, wherein said autoimmune disease is a demyelinating disease, such as multiple sclerosis.</claim-text> <claim-text>22. The method according to claims 18 to 21, wherein in step b) comprises the use of Interferon-y (INF-y).</claim-text> <claim-text>23. The method according to claims 18 to 22, wherein the ceramide is C16-Cer and/or the ccramide synthase is CerS6.</claim-text> <claim-text>24. A compound or combination of compounds capable of preventing and/or alleviat-ing the clinical symptoms of a dernyelinating disease identified with the method according to any of claims 18 to 22, for use in the treatment and/or prevention of said demyelinating disease, preferably wherein said demyelinating disease is mul-tiple sclerosis.-30 - 25. The compound according to claim 24, wherein the compound is selected from Lu-monisin B1, a fumonisin B1 derivative, L-cycloscrine, L-cycloserine derivatives, myriocin, myriocin derivatives or a pharmaceutically acceptable salt of these com-pounds.</claim-text>