Infection and Activation of B Cells by Theiler’s Murine Encephalomyelitis Virus (TMEV) Leads to Autoantibody Production in an Infectious Model of Multiple Sclerosis

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  Figure 1. Levels of viral replication in different primary splenic cell types. (A) Isolated primary splenic dendritic cells (DC), B cells, T cells, and macrophages (M) were infected with Theiler’s murine encephalomyelitis virus (TMEV) (MOI = 10) for 24 h, and then virus levels were assessed by infectious center assay on baby hamster kidney (BHK) monolayers. (B) The results of the infectious center assays of these cell types were compared with those of the levels of plaques in BHK cells infected with TMEV for 24 h. * p < 0.05; *** p < 0.001. (C) The levels of viral protein production in CD4+ and CD8+ T cells and B cells were determined by using green fluorescent protein (GFP) expression after infection with GFP-TMEV for 6 and 12 h. The GFP expression was assessed by flow cytometry in conjunction with CD4, CD8, and CD19 expression. The presented data are representative of three independent experiments.

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  Figure 2. Characterization of B cells productively infected with TMEV. (A) Spleen cells were infected with GFP-TMEV for 6, 12, and 24 h. B cells were then analyzed for their activation by the expression of CD69 and CD19 as well as CD69 and GFP of CD19+ gated cells using flow cytometry. (B) Spleen cells were infected with GFP-TMEV for 24 h and then analyzed for their expression of GFP molecules in conjunction with CD20+ and CD19+ B cells. (C) B cell subpopulations permissive to TMEV infection were examined using antibodies to CD23, CD27, and CD19. CD23+CD19+ cells were considered as follicular B cells, CD23−CD19+ cells as marginal zone B cells, CD27+CD19+ cells as memory B cells, and CD27−CD19+ cells as naïve B cells. Immunofluorescent staining of the brain of SJL mice infected with TMEV (D and E). (D) Brain sections of TMEV-infected mice at 14 dpi were stained with rabbit anti-TMEV antibody (green) and rat anti-mouse CD20 antibody (red) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). There were clustered CD20+ B cells in the brain. Scale bar = 50 µm (E) Immunofluorescent staining of CD20+ cells (green) and CD3+ T cells (red) and counterstaining with DAPI (blue) in the…

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  Figure 3. Select cytokine gene activation in B cells upon TMEV infection. (A) Isolated splenic B cells were infected with two different stocks of TMEV (MOI = 2) for 48 h, and then the levels of IFN-α4, IFN-β, and IL-6 were determined using RT-PCR. (B) The levels of an activation marker (CD69) of CD4 and CD19 in TMEV-infected or uninfected IFNα/βR-deficient and control 129S2 mice were determined using flow cytometry. The presented data are representative of three independent experiments (n = 5).

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  Figure 4. Upregulation of cytokine production and costimulatory molecule expression following TMEV infection. (A) The levels of major histocompatibility complex (MHC) and costimulatory molecules on CD19+ B cells, and CD11b+ macrophages were determined by flow cytometry following infection with TMEV for 24 h. Gray line-colored histogram, isotype control; black line open histogram, TMEV-infected cells; and black line-colored histogram, mock-infected cells. (B) Costimulatory molecules on B cells of spleen from normal and IFN-α/βR knockout mice were determined by flow cytometry following infection with TMEV for 24 h. (C) SJL splenic CD4+ T cells from PLP139–151-primed SJL mice at 10 d post-immunization were stimulated with B cells from naïve SJL mice after infection with TMEV or mock-infection (PBS) for 24 h in the presence of the immunizing PLP139–151 peptide (2 μM) (left panel). The proliferation rate was determined by [3H]-thymidine uptake assay, and the values in the absence of PLP139–151 were subtracted (right panel). Cytokine levels (interferon (IFN)-γ and IL-17) in the culture supernatants of PLP139–151-primed T cells in the presence of 1 × 105 mock-infected or TMEV-infected B cells…

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  Figure 5. Reactivity of antibodies from TMEV-infected mice to normal spinal cord components. (A) Cross sections of normal SJL spinal cords were stained with sera from mice infected with TMEV and control medium. The tissue sections were stained with the mouse sera followed with goat anti-mouse IgG secondary antibody (red), and cell nuclei were counterstained with DAPI (blue). There were some positive cells in the sections stained with 80 dpi serum compared with the cells stained with normal mice serum (NMS). Gray matter (GM), white matter (WM). Scale bar = 50 µm. (B) Spinal cord extracts from normal SJL mice were subjected to western blotting to further determine the reactivity of sera from normal uninfected and TMEV-infected SJL mice at various time points. (C) The immunoreactivity pattern of the spinal cord extract with sera from TMEV-infected mice was compared with the reactivity to commercial anti-MBP antibodies. The presented data are representative of at least three separate experiments.

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  Figure 6. Accelerated production of autoantibodies in systemic lupus erythematosus (SLE)-prone mice infected with TMEV. (A and B) Two-month-old female and male BXSB/MpJ mice were infected intracerebrally with TMEV (5 × 106 pfu). Splenic CD4+ T cells (A) and CD19+ B cells (B) were analyzed for the expression of an activation marker (CD69) using flow cytometry at 4 weeks post-infection. (C) The levels of indicated autoantibodies (ssDNA, dsDNA, histone, and nucleosome) in pooled sera (n = 5) from three mice per group were assessed using ELISA at 2 and 4 weeks post-infection. The statistical significance between M-TMEV and M-NAIVE was analyzed by one-way analysis of variance (ANOVA). p < 0.001 (D) Ten-week-old NZB/WF1 mice were infected intracerebrally with TMEV (5 × 106 pfu). At 2 weeks post-infection of 3-month-old and 10 weeks post-infection of 5-month-old mice, the levels of autoantibodies to ssDNA, dsDNA, and histone in pooled sera (n = 5) from the above groups (three mice per group) were assessed using ELISA. Similarly accelerated production of autoantibodies in the SLE-prone mice infected with closely related CoxB3 virus. The statistical significance between 5MON+TMEV and 5MON+NAIVE was…

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  Figure 7. Potential mechanisms underlying polyclonal B cell activation following viral infection. Infection of susceptible mice with neurotropic TMEV induces excessive levels of innate immune cytokines, including type I IFNs, IL-6, and IL-1, which promote inflammatory Th17 responses over protective Th1 responses, leading to high viral loads in the CNS. Various glias and antigen-presenting cells (APCs), including B cells, are permissive to the viral infection and participate in the innate immune responses and viral persistence. Interestingly, B cells are activated and stimulated to produce elevated levels of antibodies. Such high viral loads and innate cytokines as well as adaptive immune responses in the CNS led to CNS tissue damage releasing sequestered autoantigens. Although the role of autoimmune components in the pathogenesis of virus-induced demyelinating disease is unclear, many considerable circumstantial evidences suggest a potentially important role of microbial infection in the induction and/or progression of various autoimmune diseases.