RESUMO
BACKGROUND: Fingolimod is a functional sphingosine-1-phosphate antagonist approved for the treatment of multiple sclerosis (MS). Fingolimod affects lymphocyte subpopulations and regulates gene expression in the lymphocyte transcriptome. Translational studies are necessary to identify cellular and molecular biomarkers that might be used to predict the clinical response to the drug. In MS patients, we aimed to clarify the differential effects of fingolimod on T, B, and natural killer (NK) cell subsets and to identify differentially expressed genes in responders and non-responders (NRs) to treatment. MATERIALS AND METHODS: Samples were obtained from relapsing-remitting multiple sclerosis patients before and 6 months after starting fingolimod. Forty-eight lymphocyte subpopulations were measured by flow cytometry based on surface and intracellular marker analysis. Transcriptome sequencing by next-generation technologies was used to define the gene expression profiling in lymphocytes at the same time points. NEDA-3 (no evidence of disease activity) and NEDA-4 scores were measured for all patients at 1 and 2 years after beginning fingolimod treatment to investigate an association with cellular and molecular characteristics. RESULTS: Fingolimod affects practically all lymphocyte subpopulations and exerts a strong effect on genetic transcription switching toward an anti-inflammatory and antioxidant response. Fingolimod induces a differential effect in lymphocyte subpopulations after 6 months of treatment in responder and NR patients. Patients who achieved a good response to the drug compared to NR patients exhibited higher percentages of NK bright cells and plasmablasts, higher levels of FOXP3, glucose phosphate isomerase, lower levels of FCRL1, and lower Expanded Disability Status Scale at baseline. The combination of these possible markers enabled us to build a probabilistic linear model to predict the clinical response to fingolimod. CONCLUSION: MS patients responsive to fingolimod exhibit a recognizable distribution of lymphocyte subpopulations and a different pretreatment gene expression signature that might be useful as a biomarker.
RESUMO
Cannabidiol (CBD) is one of the most important compounds in Cannabis sativa, lacks psychotropic effects, and possesses a high number of therapeutic properties including the amelioration of experimental autoimmune encephalomyelitis (EAE). The aim of this study was to analyse the relative efficacy of CBD in adoptively transferred EAE (at-EAE), a model that allows better delineation of the effector phase of EAE. Splenocytes and lymph nodes from mice with actively induced EAE were cultured in the presence of MOG35-55 and IL-12 and inoculated intraperitoneally in recipient female C57BL/6J mice. The effects of CBD were evaluated using clinical scores and magnetic resonance imaging (MRI). In the central nervous system, the extent of cell infiltration, axonal damage, demyelination, microglial activation and cannabinoid receptors expression was assessed by immunohistochemistry. Lymph cell viability, apoptosis, oxidative stress and IL-6 production were measured in vitro. Preventive intraperitoneal treatment with CBD ameliorated the clinical signs of at-EAE, and this improvement was accompanied by a reduction of the apparent diffusion coefficient in the subiculum area of the brain. Inflammatory infiltration, axonal damage, and demyelination were reduced, and cannabinoid receptor expression was modulated. Incubation with CBD decreased encephalitogenic cell viability, increasing early apoptosis and reactive oxygen species (ROS) and decreasing IL-6 production. The reduction in viability was not mediated by CB1, CB2 or GPR55 receptors. CBD markedly improved the clinical signs of at-EAE and reduced infiltration, demyelination and axonal damage. The CBD-mediated decrease in the viability of encephalitogenic cells involves ROS generation, apoptosis and a decrease in IL-6 production and may contribute to the therapeutic effect of this compound.
