Type I interferon signals control Theiler's virus infection site, cellular infiltration and T cell stimulation in the CNS

Abstract

Theiler's murine encephalomyelitis virus (TMEV) establishes a persistent infection in the central nervous system (CNS). To examine the role of type I interferon (IFN-I)-mediated signals in TMEV infection, mice lacking a subunit of the type I IFN receptor (IFN-IR KO mice) were utilized. In contrast to wild type mice, IFN-IR KO mice developed rapid fatal encephalitis accompanied with greater viral load and infiltration of immune cells to the CNS. The proportion of virus-specific CD4(+) and CD8(+) T cell responses in the CNS was significantly lower in IFN-IR KO mice during the early stage of infection. Levels of IFN-γ and IL-17 produced by isolated primed CD4(+) T cells in response to DCs from TMEV-infected IFN-IR KO mice were also lower than those stimulated by DCs from TMEV-infected wild type control mice. The less efficient stimulation of virus-specific T cells by virus-infected antigen-presenting cells is attributable in part to the low level expression of activation markers on TMEV-infected cells from IFN-IR KO mice. However, due to high levels of cellular infiltration and viral loads in the CNS, the overall numbers of virus-specific T cells are higher in IFN-IR KO mice during the later stage of viral infection. These results suggest that IFN-I-mediated signals play important roles in controlling cellular infiltration to the CNS and shaping local T cell immune responses.

Figure 1

IFN-IR KO mice infected with TMEV show a severely decreased survival rate and increased viral load during TMEV infection. (A) IFN-IR KO mice, but not control WT (129S2/SP) mice, develop fatal encephalitis following intracerebral infection with TMEV. (B) Virus levels in the CNS (brains and spinal cords) of infected mice were determined by plaque assays at 6 and 12 days post-infection. Brains and spinal cords from 3 mice per each group were pooled for plaque assay. The levels of viral load in the CNS of IFN-IR mice are significantly higher than that in wild type mice. ***, p<0.001 based on Student’s t test.

Figure 2

Histopathology of TMEV-infected WT and IFN-IR KO mice. (A) Mononuclear cell infiltration in meninges and cerebral cortex of wild type mouse at 6 dpi. Hematoxylin and eosin (H&E) stain. (B) Severe acute inflammatory cell infiltration composed of both polymorphonuclear and mononuclear cells in meninges and cerebral cortex of IFN-IR KO mouse. H&E stain. (C) Normal appearing choroid plexus of wild type mouse. H&E stain. (D) Severe polymorphonuclear and mononuclear cells infiltration in choroid plexus and around fourth ventricular area of IFN-IR KO mouse. H&E stain. (E and F): Infiltration of CD45⁺ cells (Green, FITC stain) in hippocampus (HP) corpus callosum (CC) and cerebral cortex (CTX) of wild type (E) and IFN-IR KO mice (F). Blue, DAPI stain. (G and H): Infiltration of CD11b⁺ cells to hippocampus (HP), corpus callosum (CC) and cerebral cortex (CTX) of wild type (G) and IFN-IR KO mice (H). (I and J): TMEV antigens (Red, Cy3 stain) in confined regions of hippocampus (HP) in wildtype mice (I) and wide distribution in hippocampus (HP), corpus callosum (CC) and cerebral cortex (CTX) in IFN-IR KO mice (J). Bar= 200 µm

Figure 3

Proportion and number of cell types in the CNS during the course of TMEV infection in WT and IFN-IR KO mice. (A) A representative flow cytometric analysis of CNS-infiltrating mononuclear cells at 6 and 12 days post-infection. (B) Numbers of mononuclear cell types in the CNS of WT and IFN-IR KO mice at 6 and 12 days post-infection. Microglia (MG, CD45^int CD11b⁺), Macrophage (MP, CD45^hi CD11b⁺), Natural killer cells (NK, DX5⁺). The data represent three separate experiments. (C) Chemokine mRNA expression in the brain (BR) and spinal cord (SC) of virus-infected WT and IFN-IR KO mice at 6 d post-infection. Brains and spinal cords from 3–5 mice per group were pooled for RNA preparations. mRNA levels were determined by real-time PCR in triplicate samples. (D) Chemokine mRNA expression in mock or 10 MOI in vitro TMEV infected microglia for 15hr. The data represent three separate experiments and were determined by realtime PCR in triplicate samples. Fold expression represents fold expressions relative to the lowest value among samples for each chemokine message. **, p<0.01 and ***, p<0.001 based on Student’s t test.

Figure 4

Peripheral immune responses to TMEV in IFN-IR KO and WT mice. (A) Proliferative responses of pooled splenic T cells from 3 mice in response to predominant CD4⁺ (VP2_203–220 and VP4_25–38) or CD8⁺ T cell epitopes (VP2_121–130 and VP3_110–120) of TMEV (each 2 µM) were assessed at 6 and 12 d post-infection using ³H-TdR uptake assays. (B) Levels of T cell cytokines (IFN-γ, IL-13 and IL-17) produced in the above splenic T cell cultures stimulated with predominant viral epitopes were assessed using ELISA. (C) Levels of anti-TMEV antibodies in pooled sera from 3 mice per group were assessed using ELISA. The data represent three separate experiments. *, p<0.05, **, p<0.01 and ***, p<0.001, respectively.

Figure 5

CD4⁺ and CD8⁺ T cell responses to viral epitopes in TMEV-infected WT and IFN-IR KO mice. (A) Intracellular cytokine staining was performed on mononuclear cells isolated from the CNS of TMEV-infected mice at 6 and 12 d post-infection after stimulation with PBS, CD4⁺ (VP2_203–220 and VP4_25–38) or CD8⁺ T cell epitopes (VP2_121–130 and VP3_110–120) of TMEV (2 µM for each peptide) for 6 hrs. Numbers in cytometric plots represent % of IFN-γ producing CD4⁺ or CD8⁺ T cells out of the total CD4⁺ or CD8⁺ T cells, respectively. Numbers in the lower panels represent % of H-2D^b-VP2_121–130 tetramer-positive CD8⁺ cells from total infiltrating CD8⁺ cells. (B) The proportions (left panel) and total numbers (right panel) of IFN-γ producing CNS CD4⁺ T cells in WT and IFN-IR KO mice were assessed at 6 and 12 d post-infection following intracellular cytokine staining. Due to large variations in cell proportions and numbers among the experiments, these experiments were repeated 2–3 times to determine the reproducibility of the results. A representation of 3 separate similar experimental results is shown. The number of cells represents total IFN-γ producing CD4⁺ cells per CNS. (C) The proportion (upper left panel) and total numbers (upper right panel) of IFN-γ producing CNS CD8⁺ T cells of WT and IFN-IR KO mice determined by intracellular staining at 6 and 12 days post-infection. Lower panels show the percentages (lower left panel) and total numbers (lower right panel) of VP2_121–130-specific CD8⁺ cells in the CNS at 6 and 12 d post-infection, determined by staining with D^b-VP2_121–130 tetramers without further stimulation.

Figure 6

The number of DCs in the CNS of TMEV-infected IFN-IR KO mice is increased. (A) Frequencies of CD45^hiCD11c⁺ DCs among total CD45^hi CNS-mononuclear cells and (B) DC numbers in the CNS of TMEV-infected WT and IFN-IR KO mice were compared at 3 and 5 dpi. Differences in the frequency and number between WT and IFN-IR KO mice are statistically significant (***, p<0.001). (C) Proliferation of purified CD4⁺ T cells (1×10⁵ cells/well) from TMEV-infected WT and IFN-IR KO mice after stimulation with purified splenic DCs (2×10⁴ cells/well) from TMEV-infected WT or IFN-IR KO mice at 6 d post-infection. Cultures containing combinations of CD4⁺ T cells and DCs were stimulated for 3 days in the presence of UV-inactivated TMEV (UV-TMEV) and proliferation levels were assessed by ³H-TdR uptake. Cytokine levels in the culture supernatants were determined using ELISA. (D) Expression of antigen presenting function-associated markers of splenic DCs (gated with CD11c⁺) from naïve (open histogram) and TMEV-infected (filled histogram) WT and IFN-IR KO mice at 6 d post-infection. *, p<0.05, **, p<0.01 and ***, p<0.001 based on Student’s t test.