Anti-inflammatory mechanisms of the alkyl-lysophospholipid edelfosine in the murine experimental autoimmune encephalomyelitis and in human cells.

Abstract

Edelfosine was tested in EAE-mouse models to define effective concentrations which ameliorate the clinical course in the absence of side effects. Consequently, 10 mg/kg edelfosine were used to treat EAE in SJL mice in a preventive as well as in a therapeutic setting. 1 mg/kg edelfosine was used as a reference regimen. Trials additionally included a PBS-treated control cohort. To link the detected amelioration of EAE to mechanistic changes upon edelfosine treatment, secondary lymphoid organs (lymph nodes, spleens) were investigated at day 9 after immunization by flow cytometry (start of treatment: day of immunization). A significant higher frequency of T cells which showed signs of activated caspase-3 was found in lymphoid organs of mice that were treated with 10 mg/kg edelfosine. Here, also significantly more naïve T cells were detected. At day 9 after immunization, the proliferative capacity of lymph node cells as well as splenocytes was tested in ex vivo thymidine-incorporation assays. Upon edelfosine treatment in EAE mice, lymphocytes retained their proliferative capacity irrespective of the recall stimulus (mitogenic, polyclonal or disease-relevant). Flow cytometry was used to investigate treatment-dependent changes in the composition of immune cells infiltrating into the CNS of immunized mice in the acute phase of EAE. The treatment with 10 mg/kg edelfosine led to a significantly reduced frequency of infiltrating CD4+ T cells. Within the CD4+ T-cell population the frequency of T cells with activated caspase-3 was significantly increased. The immunohistochemical evaluation of NeuN+ neurons in the acute phase of EAE using cervical spinal cord sections revealed a significant loss of neurons if mice were treated with PBS compared to non-immunized control mice. In comparison, no significant loss was found when comparing non-immunized mice to mice that were treated with 10 mg/kg edelfosine. The treatment of human CD4+ T cells with edelfosine led to an edelfosine dose-dependent decrease of the proliferative response in thymidine-incorporation assays. Subsequently, CD4+ T cells were used to perform gene-expression analysis. Here, an edelfosine-induced decrease of MHC class II molecules and molecules involved in MHC class II-associated processing and presentation was found in unstimulated CD4+ T cells. Furthermore, type I interferon-associated genes were found to be upregulated if cells were stimulated in the presence of edelfosine. Interestingly, those genes were also identified to be upregulated in a published longitudinal study of gene-expression profiles, in which PBMCs from MS patients under IFN-β treatment were investigated. Both observations are novel and might offer the opportunity to develop edelfosine further for clinical use. Since the drug is orally available and well tolerated, this remains an important and interesting goal.