Transient peripheral immune response and central nervous system leaky compartmentalization in a viral model for multiple sclerosis

Abstract

Theiler's virus-induced demyelination represents an important animal model to study the chronic-progressive form of multiple sclerosis (MS). The aim of the present study was to identify specific genes and pathways in the deep cervical lymph node (cLN) and spleen of experimentally infected SJL-mice, using DNA microarrays. Analyses identified 387 genes in the deep cLN and only 6 genes in the spleen of infected animals. The lymph node presented 27.4% of genes with fold changes +/-1.5 at 14 days post infection (dpi) and a reduced transcription at later time points. K-means clustering analyses resulted in five clusters. Accordingly, functional annotation revealed that the B-cell immune response pathway was the most up-regulated cluster at the early phase. Additionally, an increase of CD68- and lysozyme-positive cells in the deep cLN was observed by immunohistochemistry. Polioencephalitis was most intense at 14 dpi, and the spinal cord demyelinating leukomyelitis started at 42 dpi. In summary, early gene expression is indicative of virus-trigged immune responses in the central nervous system (CNS)-draining lymph node. The decreased gene transcription in the deep cLN during the chronic phase and the low number of spleen genes supports the hypothesis of a compartmentalized inflammation within the CNS, as described in progressive MS.

Figure 1

Pathohistological changes in brain and spinal cord in Theiler's murine encephalomyelitis virus‐infected mice. (A) Transversal section of hippocampus displaying disseminated perivascular infiltration (arrows) at 14 days post infection (dpi). (B) Higher magnification from (A) displaying infiltration of immune cells in vessels (arrows). Hematoxylin and eosin. (C) Transversal section of the spinal cord displaying perivascular infiltration (arrows) and demyelination in the white matter (arrow head) at 98 dpi. (D) Higher magnification from (C) displaying infiltration of immune cells in vessels in the white matter. Hematoxylin and eosin. (E) Demyelinated area (arrow) and normal white matter (arrow head) in spinal cord at 196 dpi. (F) Higher magnification from (E) displaying macrophages/microglia with Gitter cell morphology (arrows) within the demyelinated white matter lesions. Luxol fast blue‐cresyl violet. Scale bars = (A) 500 µm; (B) 50 µm; (C) 100 µm; (D) 25 µm; (E) 250 µm; (F) 50 µm.

Figure 2

Semiquantitative assessment of pathohistological changes in brain during Theiler's murine encephalomyelitis. Increased perivascular infiltration is observed during the acute phase of the disease, progressively declining until 98 dpi, and moderately increasing during the late chronic phase in Theiler's murine encephalomyelitis virus‐infected mice. Columns show the semi‐quantitative score average and standard deviation. A significant difference between the groups, infected (n = 47) and sham‐infected animals (n = 48), as detected by the Mann–Whitney U‐tests is marked as follows: *P < 0.05. Significances between time points were analyzed with one‐way ANOVA and Tukey post‐hoc test; different bars denote statistically significant groups at a 0.05%.

Figure 3

Semiquantitative assessment of pathohistological changes in the spinal cord during Theiler's murine encephalomyelitis. (A) Perivascular infiltrates in the white matter revealed significant differences between infected (n = 47) and sham‐infected animals (n = 48) all through the study period, showing a progressive demyelination (B) in Theiler's murine encephalomyelitis virus‐infected mice, starting at 42 dpi until the last time period. Columns show the semiquantitative score average and standard deviation. A significant difference between the groups as detected by the Mann–Whitney U‐tests is marked as follows: *P < 0.05. Significances between time points were analyzed with one‐way ANOVA and Tukey post‐hoc test; different bars denote statistically significant groups at a 0.05%.

Figure 4

Lysozyme‐positive cells in the central nervous system‐draining lymph node of Theiler's murine encephalomyelitis virus (TMEV)‐ and sham‐infected mice. (A) Increased numbers of lysozyme‐positive cells (arrows) in the follicular area of the deep cervical lymph node of a TMEV‐infected mouse at 14 days post infection. (B) Note the comparatively low number of lysozyme‐positive cells (arrow) in the lymph node follicle of a sham‐infected control animal at the same time point. Immunohistochemistry counterstained with Mayer's hemalaun. Scale bars = 50 µm.

Figure 5

Quantification of CD68‐ and lysozyme‐positive cells in the follicle of the deep cervical lymph node of Theiler's murine encephalomyelitis virus (TMEV)‐ and sham‐infected mice. Immunohistochemistry revealed a significant increase of the percentage of CD68‐ (A) and lysozyme‐positive cells (B) in TMEV‐infected animals in comparison to sham‐infected mice at 14 days post infection. Columns display average values and standard deviations. Significant difference (P < 0.05) between the groups as detected by two‐way ANOVA with post‐hoc test for multiple pairwise comparisons with Bonferroni adjustment is marked as follows: *P < 0.05.

Figure 6

Expression profile of differentially expressed genes in the deep cervical lymph node. The fold changes of the 387 differentially genes were grouped by k‐means cluster analysis to reveal similar expression patterns. Each row represents one of the 387 genes and each column one of four experimental days (14, 42, 98 and 196 days post infection). The log₂‐transformed fold changes are indicated by a color scale ranging from –2 [relative low expression in Theiler's murine encephalomyelitis virus (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 cluster (I‐V). (A) Represents k‐means cluster I, showing gene up‐regulation in the acute phase of TME. (B) K‐means cluster II, consisted of down‐regulated genes during the acute phase. (C) K‐means cluster III, presented gene up‐regulation at the acute phase. (D) and (E) are k‐means cluster IV and V, respectively, presenting down‐regulation of genes during the acute phase.