Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis

Abstract

Theiler's murine encephalomyelitis is an experimentally virus-induced inflammatory demyelinating disease of the spinal cord, displaying clinical and pathological similarities to chronic progressive multiple sclerosis. The aim of this study was to identify pathways associated with chronic demyelination using an assumption-free combined microarray and immunohistology approach. Movement control as determined by rotarod assay significantly worsened in Theiler's murine encephalomyelitis -virus-infected SJL/J mice from 42 to 196 days after infection (dpi). In the spinal cords, inflammatory changes were detected 14 to 196 dpi, and demyelination progressively increased from 42 to 196 dpi. Microarray analysis revealed 1001 differentially expressed genes over the study period. The dominating changes as revealed by k-means and functional annotation clustering included up-regulations related to intrathecal antibody production and antigen processing and presentation via major histocompatibility class II molecules. A random forest machine learning algorithm revealed that down-regulated lipid and cholesterol biosynthesis, differentially expressed neurite morphogenesis and up-regulated toll-like receptor-4-induced pathways were intimately associated with demyelination as measured by immunohistology. Conclusively, although transcriptional changes were dominated by the adaptive immune response, the main pathways associated with demyelination included up-regulation of toll-like receptor 4 and down-regulation of cholesterol biosynthesis. Cholesterol biosynthesis is a rate limiting step of myelination and its down-regulation is suggested to be involved in chronic demyelination by an inhibition of remyelination.

Fig 1

Rotarod analysis. Rotarod analysis revealed a chronic progredient reduction of motor strength and control in TMEV-infected mice compared with mock-infected mice. Data are presented as the mean ± standard error of mean of the percent change compared with the pre-injection baseline measurement on day 0. The following number of animals was evaluated per group: n= 39 at 14 dpi; n= 33 at 28 and 42 dpi; n= 27 at 56, 70 and 98 dpi; n= 21 at 147 dpi; n= 15 at 196 dpi. A significant difference between the groups as detected by two-way repeated-measure anova with post hoc independent t-tests for the different time-points is marked as follows: *P≤ 0.05.

Fig 2

Inflammatory demyelination in the spinal cord 98 days after TMEV infection. (A) Transversal section of the thoracic spinal cord displaying multifocal mononuclear inflammatory infiltrates (arrowheads) within the meninges and perivascular spaces. Multiple demyelinated areas (arrows) are mainly present in the ventral and lateral funiculi of the thoracic spinal cord. (B) Higher magnification from (A) displaying perivascular mononuclear infiltrates (arrowheads) and macrophages/microglia displaying Gitter cell morphology (arrow) within the demyelinated white matter lesions. Luxol fast blue-cresyl violet. Scale bars = (A) 500 μm; (B) 20 μm.

Fig 3

Pathohistological changes in the spinal cords. Semi-quantitative assessment of pathohistological changes in Theiler’s murine encephalomyelitis revealed early onset inflammatory changes followed by a chronic progressive demyelination in TMEV-infected mice. Box and whisker plots show the median and quartiles of the semi-quantitative scores. Extreme values are shown as circles. Six mice were evaluated per group and time-point, except for five TMEV-infected mice at 98 dpi. A significant difference between the groups as detected by the Mann-Whitney U-tests is marked as follows: *P≤ 0.05.

Fig 4

Expression profile of differentially expressed genes. The fold changes of the 1001 differentially expressed genes were grouped by k-means cluster analysis to reveal similar expression patterns. (A) Each row represents one of 1001 genes and each column one of the four experimental days. The log₂-transformed fold changes are indicated by a colour scale ranging from –2 (relative low expression in TMEV-infected mice) in green to 2 (relative high expression in TMEV-infected mice) in red. The majority of the differentially expressed genes were organized into five k-means clusters (I-V) with increasing transcription and up-regulation in the chronic phase of Theiler’s murine encephalomyelitis. Only one k-means cluster (VI) consisted of genes with a decreasing expression during the study period and a down-regulation mainly in the chronic phase of Theiler’s murine encephalomyelitis. (B) Higher magnification from (A) displaying the log₂-transformed fold change of the 67 genes of k-means clusters I and II on a broader scale ranging from –5 (relative low expression in TMEV-infected mice) in green to 5 (relative high expression in TMEV-infected mice) in red. The k-means clusters I and II include the genes with the highest up-regulations. The k-means cluster I showed a more constantly up-regulated expression from 42 to 196 dpi, whereas k-means cluster II displayed a peaking up-regulation at 98 dpi.

Fig 5

Temporal changes of leucocytic infiltrates and demyelination in the spinal cord. Box and whiskerplots show the median and quartiles of the density of (A) CD3, (B) CD45R/B220, (C) IgG, (D) CD107b⁺ cells and (E) the MBP⁺ area. Extreme values are shown as circles. Six mice were evaluated per group and time-point, except for five TMEV-infected mice at 98 dpi. A significant difference between the groups as detected by the Mann-Whitney U-test is marked as follows: *P≤ 0.05.

Fig 6

Immunohistological characterization of a demyelinating lesion at 98 dpi. Serial sections from the spinal cord of a TMEV-infected mouse from 98 dpi were immunolabelled for CD3 (A), CD45R/B220 (B), IgG (C), CD107b (D) and MBP (E). (A) A high amount of CD3⁺ T lymphocytes in the perivascular spaces and fewer cells in the white matter (arrows). (B) Few perivascular CD45R/B220⁺ B lymphocytes (arrow). (C) A high amount of IgG⁺ cells in the perivascular spaces (arrown). (D) CD107b⁺ macrophages with Gitter cell morphology (arrows) comprise the majority of parenchymal cellular infiltrates (E) Multifocally decreased MBP immunoreactivity (arrow) in the white matter. m = meninges, v = vessel, wm = white matter. Avidin-biotin-peroxidase complex method, haematoxylin counter-stain, Nomarski differential interference contrast. Scale bars = 20 μm.