CNS expression of B7-H1 regulates pro-inflammatory cytokine production and alters severity of Theiler's virus-induced demyelinating disease

Abstract

The CNS is a unique organ due to its limited capacity for immune surveillance. As macrophages of the CNS, microglia represent a population originally known for the ability to assist neuronal stability, are now appreciated for their role in initiating and regulating immune responses in the brain. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a mouse model of multiple sclerosis (MS). In response to TMEV infection in vitro, microglia produce high levels of inflammatory cytokines and chemokines, and are efficient antigen-presenting cells (APCs) for activating CD4(+) T cells. However, the regulatory function of microglia and other CNS-infiltrating APCs in response to TMEV in vivo remains unclear. Here we demonstrate that microglia increase expression of proliferating cell nuclear antigen (PCNA), and phenotypically express high levels of major histocompatibility complex (MHC)-Class I and II in response to acute infection with TMEV in SJL/J mice. Microglia increase expression of the inhibitory co-stimulatory molecule, B7-H1 as early as day 5 post-infection, while CNS-infiltrating CD11b(+)CD11c(-)CD45(HIGH) monocytes/macrophages and CD11b(+)CD11c(+)CD45(HIGH) dendritic cells upregulate expression of B7-H1 by day 3 post-infection. Utilizing a neutralizing antibody, we demonstrate that B7-H1 negatively regulates TMEV-specific ex vivo production of interferon (IFN)-γ, interleukin (IL)-17, IL-10, and IL-2 from CD4(+) and CD8(+) T cells. In vivo blockade of B7-H1 in SJL/J mice significantly exacerbates clinical disease symptoms during the chronic autoimmune stage of TMEV-IDD, but only has minimal effects on viral clearance. Collectively, these results suggest that CNS expression of B7-H1 regulates activation of TMEV-specific T cells, which affects protection against TMEV-IDD.

Figure 1. TMEV infection induces increased microglia cell numbers associated with elevated levels of PCNA expression.

A. Microglia were isolated from sham-infected and TMEV-infected SJL/J mice at day 3, 5, and 7 post-infection and identified by CD11b and CD45 expression via flow cytometric analysis using the illustrated gating strategy. The data is representative of 2–3 independent experiments with an average of 3–5 mice per group per experiment. B. The percentage of cells expression Proliferation Cell Nuclear Antigen (PCNA) was measured in microglia (CD11b⁺CD45^LOW) isolated during the acute phase of TMEV infection using flow cytometry. C. Mean Fluorescence Intensity (MFI) of PCNA expression in microglia isolated from TMEV-infected mice compared to sham-infected mice. Microglia isolated from LPS-treated mice (200 µg/for 15 hours) served as a positive control. These data are representative of four independent experiments with n = 4–5/group expressed as the average ± SEM.

Figure 2. TMEV induces differential expression of antigen presentation molecules on microglia in vivo.

A. Microglia (CD11b⁺CD45^LOW) from sham- and TMEV-infected mice at days 3, 5, and 7 post-infection and analyzed for H-2K^s and I-A^s expression using flow cytometry. A&B. Histograms are representative of H-2K^s expression. B. Quantification of percent positive for H-2K^s are reflected based of negative staining from isotype controls and sham-infected microglia. C&D. Histograms are representative of I-A^s expression. Quantification of percent positive for I-A^s are reflected based of negative staining from sham-infected microglia. The data is representative of three independent experiments with n = 4–5 mice/group expressed as the average ± SEM. *p<0.05 compared to sham-infected mice.

Figure 3. TMEV induces differential expression of positive and negative co-stimulatory molecules on microglia in vivo.

A. Microglia (CD11b⁺CD45^LOW) from sham- and TMEV-infected mice at days 3, 5, and 7 post-infection were analyzed for CD80, CD86, and B7-H1 expression using flow cytometry. A&B. Histograms are representative of CD80 expression. C&D. Histograms are representative of CD86 expression. E&F. Histograms are representative of B7-H1 expression. The data is representative of three independent experiments with n = 4–5 mice/group expressed as the average ± SEM. *p<0.05 compared to sham-infected mice.

Figure 4. Expression of B7-H1 by CNS-Infiltrating APCs following TMEV infection in vivo.

CNS-infiltrating APCs from TMEV-infected mice at days 3, 5 and 7 post-infection were analyzed for B7-H1 expression. A. Gating strategy identifying CD11b⁺CD11c⁻ monocytes/macrophages and CD11b⁺CD11c⁺ dendritic cells based on CD45^HIGH expression to exclude microglia. Numbers reflect total percentages of each cell population. B. Histograms of B7-H1 expression on monocyte/macrophage and dendritic cell populations following infection. C. Quantification of percent positive for B7-H1 are reflected based of negative staining from isotype controls and sham-infected cell populations. Black bars represent CD11b⁺CD11c⁻ monocytes/macrophages, while white bars reflect CD11b⁺CD11c⁺ dendritic cells. The data is representative of three independent experiments with n = 4–5 mice/group expressed as the average ± SEM. **p<0.01, ***p<0.001 compared to CD11b⁺CD11c⁻ infiltrating populations.

Figure 5. B7-H1 and PD-1 expression are highly upregulated on CD4⁺ and CD8⁺ CNS T cells from TMEV-infected SJL/J mice.

CNS T cells from wild-type SJL/J mice at day 7 post-TMEV infection were analyzed for expression of B7-H1 and PD-1 molecules by flow cytometry. A. Histograms of B7-H1 and PD-1 expression on CD4⁺ and CD8⁺ T cells. Black shaded area represents isotype control and the grey line represents positive staining. A representative histogram from one individual mouse from three separate experiments is shown. B. Data is also represented as Δ mean fluorescence intensity (ΔMFI) of B7-H1 and PD-1 expression on CD4⁺ and CD8⁺ T cells. Expression of B7-H1 and PD-1 is significant greater (*p<0.05) on with CD8⁺ as compared to CD4⁺ T cells. Data are expressed as the average ± SEM.

Figure 6. Blockade of B7-H1 enhances cytokine production from TMEV-specific CNS-infiltrating CD4⁺ T cells ex vivo.

CNS (brain and spinal cord) mononuclear cells were isolated from TMEV-infected mice (n = 10/group) at day 7 post-infection and co-cultured in the presence of the CD4⁺ T cell restricted peptide-VP2_70–86. Control Ig (open bars) and anti-mouse B7-H1 (black bars) were added to the cultures. Unstimulated and anti-mouse CD3-stimulated CNS mononuclear cells served as negative and positive controls, respectively. A. Proliferation was analyzed by ³[H]-TdR uptake of triplicate wells per group. B–D. After 72 h, supernatants from triplicate wells of each group in (A), the levels of IFN-γ (B), IL-17 (C), and IL-2 (D) were measured using CBA technology. Data are representative of three separate experiments. *p<0.05 compared to cultures containing rat IgG2a control.

Figure 7. Blockade of B7-H1 enhances cytokine production from TMEV-specific CNS-infiltrating CD8⁺ T cells ex vivo.

CNS (brain and spinal cord) mononuclear cells were isolated from TMEV-infected mice (n = 10/group) at day 7 post-infection and co-cultured in the presence of increasing concentrations of the CD8⁺ T cell restricted peptide-VP3_159–66. Control Ig (open bars) and anti-mouse B7-H1 (black bars) were added to the cultures. Unstimulated and anti-mouse CD3-stimulated CNS mononuclear cells served as negative and positive controls, respectively. After 72 h, levels of IFN-γ (A), and IL-2 (B) were measured using CBA technology from the supernatants of triplicate wells. Data are representative of three separate experiments. *p<0.05 compared to cultures containing Rat IgG2a control.

Figure 8. In vivo neutralization of B7-H1 enhances clinical disease in chronic TMEV-IDD.

Wild-type SJL/J mice were infected with TMEV and monitored for clinical signs of disease until day 90 post infection following 100 µg/mouse injections of αRat IgG2b isotype control (○) and anti-B7-H1 mAb (•) at days 0 and +3 relative to infection. The x-axis represents days post-infection, while the y-axis represents the mean clinical score as defined in the materials and methods. The data is representative of 3 independent experiments with n = 5 mice/group expressed as the average ± SEM. *p<0.05 compared to mice receiving Rat IgG2b isotype control at each time point, 2-way ANOVA.

Figure 9. B7-H1 limits viral replication in TMEV infection.

A. TMEV viral loads in the brain following treatment with anti-B7-H1 mAb or isotype control at days 7 and 14 post-infection. Data are expressed as plaque forming units/gram of tissue (PFU/g). The x-axis represents days post-infection, while the y-axis represents PFU/g tissue. The data is representative of 2 independent experiments with n = 2–4/group expressed as the average ± SEM.