Max Eberle

Ceramide Synthase 6 Plays a Critical Role in the Development of Experimental Autoimmune Encephalomyelitis

Ceramides are mediators of apoptosis and inflammatory processes. In an animal model of multiple sclerosis (MS), the experimental autoimmune encephalomyelitis (EAE) model, we observed a significant elevation of C16:0-Cer in the lumbar spinal cord of EAE mice. This was caused by a transiently increased expression of ceramide synthase (CerS) 6 in monocytes/macrophages and astroglia. Notably, this corresponds to the clinical finding that C16:0-Cer levels were increased 1.9-fold in cerebrospinal fluid of MS patients. NO and TNF-α secreted by IFN-γ–activated macrophages play an essential role in the development of MS. In murine peritoneal and mouse-derived RAW 264.7 macrophages, IFN-γ–mediated expression of inducible NO synthase (iNOS)/TNF-α and NO/TNF-α release depends on upregulation of CerS6/C16:0-Cer. Downregulation of CerS6 by RNA interference or endogenous upregulation of C16:0-Cer mediated by palmitic acid in RAW 264.7 macrophages led to a significant reduction or increase in NO/TNF-α release, respectively. EAE/IFN-γ knockout mice showed a significant delay in disease onset accompanied by a significantly less pronounced increase in CerS6/C16:0-Cer, iNOS, and TNF-α compared with EAE/wild-type mice. Treatment of EAE mice with l-cycloserine prevented the increase in C16:0-Cer and iNOS/TNF-α expression and caused a remission of the disease. In conclusion, CerS6 plays a critical role in the onset of MS, most likely by regulating NO and TNF-α synthesis. CerS6 may represent a new target for the inhibition of inflammatory processes promoting MS development.

Lack of ceramide synthase 2 suppresses the development of experimental autoimmune encephalomyelitis by impairing the migratory capacity of neutrophils

Ceramide synthases (CerS) synthesise ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed a significant elevation of CerS2 and its products, C24-ceramides, in CD11b(+) cells (monocytes and neutrophils) isolated from blood. This result correlates with the clinical finding that CerS2 mRNA expression and C24-ceramide levels were significantly increased by 2.2- and 1.5-fold, respectively, in white blood cells of MS patients. The increased CerS2 mRNA/C24-ceramide expression in neutrophils/monocytes seems to mediate pro-inflammatory effects, since a specific genetic deletion of CerS2 in blood cells or a total genetic deletion of CerS2 significantly delayed the onset of clinical symptoms, due to a reduced infiltration of immune cells, in particular neutrophils, into the central nervous system. CXCR2 chemokine receptors, expressed on neutrophils, promote the migration of neutrophils into the central nervous system, which is a prerequisite for the recruitment of further immune cells and the inflammatory process that leads to the development of MS. Interestingly, neutrophils isolated from CerS2 null EAE mice, as opposed to WT EAE mice, were characterised by significantly lower CXCR2 receptor mRNA expression resulting in their reduced migratory capacity towards CXCL2. Most importantly, G-CSF-induced CXCR2 expression was significantly reduced in CerS2 null neutrophils and their migratory capacity was significantly impaired. In conclusion, our data strongly indicate that G-CSF-induced CXCR2 expression is regulated in a CerS2-dependent manner and that CerS2 thereby promotes the migration of neutrophils, thus, contributing to inflammation and the development of EAE and MS.

Ceramide metabolism in mouse tissue.

Ceramides with different N-acyl chains can act as second messengers in various signaling pathways. They are involved in cell processes such as apoptosis, differentiation and inflammation. Ceramide synthases (CerS) are key enzymes in the biosynthesis of ceramides and dihydroceramides. Six isoenzymes (CerS1-6) catalyze the N-acylation of the sphingoid bases, albeit with strictly acyl-Coenzyme A (CoA) chain length specificity. We analyzed the mRNA expression, the protein expression, the specific activity of the CerS, and acyl-CoA, dihydroceramide and ceramide levels in different tissues by LC-MS/MS. Our data indicate that each tissue express a distinct composition of CerS, whereby the CerS mRNA expression levels do not correlate with the respective protein expression levels in the tissues. Furthermore, we found a highly significant negative correlation between the protein expression level of CerS6 and the C16:0-acyl-CoA amounts as well as between the protein expression of CerS2 and C24:0-acyl-CoA amounts. These data indicate that in mouse tissues low substrate availability is compensated by higher CerS protein expression level and vice versa. Apart from the expression level and the specific activity of the CerS, other enzymes of the sphingolipid pathway also influence the composition of ceramides with distinct chain lengths in each cell. Acyl-CoA availability seems to be less important for ceramide composition and might be compensated for by CerS expression/activity.

Exacerbation of experimental autoimmune encephalomyelitis in ceramide synthase 6 knockout mice is associated with enhanced activation/migration of neutrophils

Ceramides are mediators of inflammatory processes. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that CerS6 mRNA expression was upregulated 15‐fold in peripheral blood leukocytes before the onset of EAE symptoms. In peripheral blood leukocytes from MS patients, a 3.9‐fold upregulation was found. Total genetic deletion of CerS6 and the selective deletion of CerS6 in peripheral blood leucocytes exacerbated the progression of clinical symptoms in EAE mice. This was associated with enhanced leukocyte, predominantly neutrophil infiltration and enhanced demyelination in the lumbar spinal cord of EAE mice. Interferon‐gamma/tumor necrosis factor alpha (IFN‐γ/TNF‐α) and granulocyte colony‐stimulating factor (G‐CSF) both drive EAE development and induce expression of the integrin CD11b and the chemokine receptor C‐X‐C motif chemokine receptor 2 (CXCR2), and we found they also induce CerS6 expression. In vivo, the genetic deletion of CerS6 enhanced the activation/migration of neutrophils, as reflected by an enhanced upregulation of CD11b and CXCR2. In vitro, the genetic deletion of CerS6 enhanced the activation status of IFN‐γ/TNF‐α‐stimulated neutrophils, as shown by increased expression of nitric oxide and CD11b and an increased adhesion capacity. In G‐CSF‐stimulated neutrophils, the migration status was enhanced, as reflected by an elevated level of CXCR2 and an increased migration capacity. These data suggest that CerS6/C16‐Cer mediates feedback regulation by inhibiting the formation of CD11b and CXCR2, which are induced either by IFN‐γ/TNF‐α or by G‐CSF, respectively. We conclude that CerS6/C16‐Cer mediates anti‐inflammatory effects during the development of EAE and MS possibly by suppressing the migration and deactivation of neutrophils.

PGE2/EP4 signaling in peripheral immune cells promotes development of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory autoimmune disease model of multiple sclerosis (MS). The inflammatory process is initiated by activation and proliferation of T cells and monocytes and by their subsequent migration into the central nervous system (CNS), where they induce demyelination and neurodegeneration. Prostaglandin E2 (PGE2) - synthesized by cyclooxygenase 2 (COX-2) - has both pro- and anti-inflammatory potential, which is translated via four different EP receptors. We hypothesized that PGE2 synthesized in the preclinical phase by peripheral immune cells exerts pro-inflammatory properties in the EAE model. To investigate this, we used a bone marrow transplantation model, which enables PGE2 synthesis or EP receptor expression to be blocked specifically in peripheral murine immune cells. Our results reveal that deletion of COX-2 or its EP4 receptor in bone marrow-derived cells leads to a significant delay in the onset of EAE. This effect is due to an impaired preclinical inflammatory process indicated by a reduced level of the T cell activating interleukin-6 (IL-6), reduced numbers of T cells and of the T cell secreted interleukin-17 (IL-17) in the blood of mice lacking COX-2 or EP4 in peripheral immune cells. Moreover, mice lacking COX-2 or EP4 in bone marrow-derived cells show a reduced expression of matrix metalloproteinase 9 (MMP9), which results in decreased infiltration of monocytes and T cells into the CNS. In conclusion, our data demonstrate that PGE2 synthesized by monocytes in the early preclinical phase promotes the development of EAE in an EP4 receptor dependent manner.

Regulation of ceramide synthase 6 in a spontaneous experimental autoimmune encephalomyelitis model is sex dependent

Ceramides (Cer) are mediators of inflammatory processes. In a chronic experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), we observed a significant elevation of C16-Cer and its synthesizing enzyme, ceramide synthase(CerS)6, in the lumbar spinal cord. In the present study, we have confirmed that C16-Cer and CerS6 are also upregulated in the lumbar spinal cord in a spontaneous relapse-remitting EAE model, using SJL mice overexpressing a transgenic T cell receptor (TCR1640). CerS6 was found to be expressed in macrophages, T cells and B cells in EAE lesions. In macrophages, we demonstrated that interferon gamma (IFN-γ)-induced CerS6 upregulation was amplified by 17ß-estradiol, an action that was further accompanied by increased upregulation of tumor necrosis factor alpha (TNF-α). Accordingly, CerS6 and TNF-α expression was upregulated predominantly in the spinal cord in female TCR1640 mice, which usually develop the relapse-remitting form of EAE, while male TCR1640 mice showed an attenuated regulation of CerS6 and TNF-α and exhibit mostly chronic disease progression. Furthermore, expression of TNFR2, one of two receptors of TNF-α, which is linked to neuroprotection and remyelination, was also upregulated to a greater extent during EAE in female TCR1640 mice in comparison to male TCR1640 mice. Taken together, our results confirm the upregulation of CerS6 and C16-Cer in an adjuvant-independent, physiological EAE model and further suggest an anti-inflammatory role of CerS6 in the regulation of the disease course in female TCR1640 mice via TNF-α/TNFR2.

Nano-LC–MS/MS for the quantitation of ceramides in mice cerebrospinal fluid using minimal sample volume

A new nano-liquid chromatography-ESI-MS/MS method has been developed for the sensitive quantitation of C8:0, C16:0, C18:0, C18:1, C20:0, C24:1 and C24:0 ceramide in cerebrospinal fluid of mice using minimal sample volume. Volumes of 2 µL CSF were undertaken a simple, fast extraction procedure involving protein precipitation with methanol and dilution. Ceramides were separated by nano-liquid chromatography with a reversed phase C8 column and detected with a triple quadrupole mass spectrometer. C17:0 ceramide was used as internal standard. The method has been validated in terms of linearity, lower limit of quantitation, precision, accuracy and autosampler stability. Calibration curves covered a range of 2.25-120 pg/µL for most ceramides (7.5-120 pg/µL for C24:0 ceramide). The lower limits of quantitation determined for C8:0, C16:0, C18:1, C18:0, C20:0 and C24:1 ceramide were 0.225 pg on column (2.25 pg/µL) and that for C24:0 ceramide 0.750 pg on column (7.5 pg/µL). Intra- and interday precision and accuracy values, expressed as relative standard deviation and relative error, respectively, were lower than 15% in all cases. Autosampler stability for calibration standards and CSF samples was proven for at least 24h for all analytes. The suitability of the method has been demonstrated by quantifying the analytes, except the non-endogenous C8:0 ceramide, in cerebrospinal fluid samples of 12 mice. Calculated concentrations ranged from 3 to 120 pg/µL in cerebrospinal fluid for all analytes, except for C24:0 ceramide, which could not be detected in any of the analyzed samples.

Alpha-methylacyl-CoA racemase deletion has mutually counteracting effects on T-cell responses, associated with unchanged course of EAE.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Altering the metabolism of immune cells is an attractive strategy to modify their activity during autoimmunity in MS. We investigated the effect of modulating fatty acid metabolism in an animal model of MS, EAE. Alpha‐methylacyl‐CoA racemase (AMACR) converts R‐configuration branched fatty acids into the S‐configuration, thereby preparing them for β‐oxidation. We observed a significant, disease‐dependent elevation of AMACR expression in monocytes and T cells from blood, draining lymph nodes and spleen of EAE mice during the preclinical phase. In vitro analysis revealed that the proliferation of T cells was inhibited in AMACR KO mice, but T‐cell polarization was switched toward a pathogenic state involving the production of more IFN‐γ and IL‐17, but less IL‐4. These opposing effects appeared to cancel out each other in vivo, because AMACR KO EAE mice showed a marginal increase in the severity of early clinical symptoms. AMACR was not regulated in the white blood cells of MS patients. Our data show that AMACR is regulated in immune cells during EAE, but it is not a suitable target for the treatment of MS due to its opposing effects.