The online version of this article (https://doi.org/10.1186/s12974-017-1049-5) contains supplementary material, which is available to authorized users.
It is debated whether multiple sclerosis (MS) might result from an immunopathological response toward an active Epstein-Barr virus (EBV) infection brought into the central nervous system (CNS) by immigrating B cells. Based on this model, a relationship should exist between the local immune milieu and EBV infection status in the MS brain. To test this hypothesis, we analyzed expression of viral and cellular genes in brain-infiltrating immune cells.
Twenty-three postmortem snap-frozen brain tissue blocks from 11 patients with progressive MS were selected based on good RNA quality and prominent immune cell infiltration. White matter perivascular and intrameningeal immune infiltrates, including B cell follicle-like structures, were isolated from brain sections using laser capture microdissection. Enhanced PCR-based methods were used to investigate expression of 75 immune-related genes and 6 EBV genes associated with latent and lytic infection. Data were analyzed using univariate and multivariate statistical methods.
Genes related to T cell activation, cytotoxic cell-mediated (or type 1) immunity, B cell growth and differentiation, pathogen recognition, myeloid cell function, type I interferon pathway activation, and leukocyte recruitment were found expressed at different levels in most or all MS brain immune infiltrates. EBV genes were detected in brain samples from 9 of 11 MS patients with expression patterns suggestive of in situ activation of latent infection and, less frequently, entry into the lytic cycle. Comparison of data obtained in meningeal and white matter infiltrates revealed higher expression of genes related to interferonγ production, B cell differentiation, cell proliferation, lipid antigen presentation, and T cell and myeloid cell recruitment, as well as more widespread EBV infection in the meningeal samples. Multivariate analysis grouped genes expressed in meningeal and white matter immune infiltrates into artificial factors that were characterized primarily by genes involved in type 1 immunity effector mechanisms and type I interferon pathway activation.
These results confirm profound in situ EBV deregulation and suggest orchestration of local antiviral function in the MS brain, lending support to a model of MS pathogenesis that involves EBV as possible antigenic stimulus of the persistent immune response in the central nervous system.
Additional file 1: Sample cohort selected for RNA quality control and neuropathological assessment. The table shows the steps of the quality control analysis performed on fresh frozen postmortem MS brain tissue blocks that led to identify a smaller sample cohort suitable for laser capture microdissection and gene expression analysis. (PDF 353 kb)12974_2017_1049_MOESM1_ESM.pdf
Additional file 2: Neuropathological characterization of white matter lesions and meningeal immune infiltrates in postmortem MS brain samples. The figure shows representative images of white matter lesions and areas with different degrees of demyelination and inflammation from which perivascular immune infiltrates were microdissected and of ectopic B cell follicles and diffuse immune infiltrates characterized in the meninges. (PDF 843 kb)12974_2017_1049_MOESM2_ESM.pdf
Additional file 3: List of Taqman inventoried assays used to study cellular gene expression. The table lists the immune-related cellular genes and the corresponding Taqman inventoried gene expression assays used in this study. (PDF 358 kb)12974_2017_1049_MOESM3_ESM.pdf
Additional file 4: List of Taqman self-designed primers and probes used to study EBV gene expression. The table lists the EBV genes, the GenBank nucleotide sequence accession numbers, and the self-designed primers and probes used in this study to analyze EBV gene expression. (PDF 322 kb)12974_2017_1049_MOESM4_ESM.pdf
Additional file 5: Assessment of the specificity of the EBV gene expression assays using droplet digital (dd) PCR. The figure shows the results of a representative experiment performed in EBV+ and EBV− cell lines to verify the specificity of the self-designed EBV gene expression assays in a ddPCR setting. (PDF 669 kb)12974_2017_1049_MOESM5_ESM.pdf
Additional file 6: Quantification of EBV transcripts in an EBV+ lymphoblastoid cell line by PreAmp droplet digital (dd) PCR compared to real-time PCR. The figure shows the results of an experiment to verify whether EBV gene expression data obtained using ddRT-PCR were comparable with those obtained using real-time RT-PCR. (PDF 345 kb)12974_2017_1049_MOESM6_ESM.pdf
Additional file 7: EBV gene expression in laser-cut immune infiltrates from the MS brain. The table shows the EBV latent and lytic transcripts detected in individual immune infiltrates isolated from brain sections of 9 of the 11 MS cases analyzed. (PDF 339 kb)12974_2017_1049_MOESM7_ESM.pdf
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- Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis
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