Infiltrating macrophages are key to the development of seizures following virus infection

Abstract

Viral infections of the central nervous system (CNS) can trigger an antiviral immune response, which initiates an inflammatory cascade to control viral replication and dissemination. The extent of the proinflammatory response in the CNS and the timing of the release of proinflammatory cytokines can lead to neuronal excitability. Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), two proinflammatory cytokines, have been linked to the development of acute seizures in Theiler's murine encephalomyelitis virus-induced encephalitis. It is unclear the extent to which the infiltrating macrophages versus resident CNS cells, such as microglia, contribute to acute seizures, as both cell types produce TNF-α and IL-6. In this study, we show that following infection a significantly higher number of microglia produced TNF-α than did infiltrating macrophages. In contrast, infiltrating macrophages produced significantly more IL-6. Mice treated with minocycline or wogonin, both of which limit infiltration of immune cells into the CNS and their activation, had significantly fewer macrophages infiltrating the brain, and significantly fewer mice had seizures. Therefore, our studies implicate infiltrating macrophages as an important source of IL-6 that contributes to the development of acute seizures.

Fig 1

Flow cytometry analysis of CD45⁺ CD11b⁺ monocytes/macrophages in the periphery of TMEV-infected mice. (A) Peripheral blood was collected from a single representative mouse by cheek bleed prior to infection (upper panel) and 48 h after TMEV infection (lower panel). (B) Graph of the flow cytometric data of each of 4 mice before infection (baseline; circles) and 48 h p.i. (red triangles). *, P < 0.05, Student's paired t test.

Fig 2

Numbers of specific cell types in the brains of TMEV-infected mice. Representative flow cytometry histograms (left panels) of cells isolated from the brains of either mock-infected (gray) or TMEV-infected (blue) mice. The cell types shown are monocytes/macrophages (CD11b⁺) (A), B cells (CD45⁺ CD19⁺) (B), T cells (CD45⁺ CD3⁺) (C), and natural killer cells (CD45⁺ NK1.1⁺) (D). Control responses (black dotted lines) were determined by the FMO method as described in Materials and Methods. Quantification of flow cytometry data (right panels) from three different experiments are presented as the mean + standard error of the mean with a total of 4 mice per group. Mock-infected mice were injected in parallel with TMEV-infected mice for each experiment. *, P < 0.05, Student's paired t test.

Fig 3

Detection of markers of activation of CD11b⁺ monocyte-derived cells in the brains of TMEV-infected mice. (A) Representative flow cytometry plots of CD11b⁺ cells from the brains of either mock-infected (top panels) or TMEV-infected (lower panels) mice. Activation markers examined included MHC II and CD86. Gates (small boxes with percent of total cells found within gate) were set according to FMO, as described in Materials and Methods. (B) Quantification of flow cytometry data from three separate experiments, presented as the means + standard errors of the means of 4 mice per group. *, P < 0.05, Student's paired t test.

Fig 4

Differentiation between microglia and macrophages in the brains of TMEV-infected mice. (A) Representative flow cytometry plots of microglia (CD45^lo/int CD11b⁺; R1) and macrophages (CD45^hi CD11b⁺; R2) isolated from the brain of either a mock-infected mouse (left panel) or a TMEV-infected mouse (right panel). Gates were set according to FMO, as described in Materials and Methods. (B) Quantification of flow cytometry data from three separate experiments, presented as the means + standard errors of the means for 4 mice per group. *, P < 0.05, Student's paired t test. (C) Inflammatory monocytes (CD45^hi CD11b⁺ Gr-1⁺) were analyzed by fluorescence-activated cell sorting. (D) Photomicrograph of cells stained with hematoxylin and eosin after they were cytospun.

Fig 5

Bone marrow-derived GFP⁺ cells (macrophages) infiltrate the brains of TMEV-infected mice. Brains were harvested 72 h p.i. The generation of GFP chimeric mice is described in Materials and Methods. (A) Representative flow cytometry plots of cells obtained from the brain of a TMEV-infected chimeric mouse in which the microglial (R1) and macrophage (R2) cell populations were assayed for GFP expression. (B) Representative flow cytometry plots of cells, obtained from the brains of either a mock-infected chimeric mouse (left panel) or a TMEV-infected chimeric mouse (right panel), that were assayed for the presence of the following cell surface markers: CD11c⁻ CD11b⁺ GFP^+/−. (C) Quantification of flow cytometry data from three separate experiments, presented as the means + standard errors of the means of 5 mice per group. **, P < 0.005, Student's paired t test.

Fig 6

Mice that have seizures have a significantly higher number of GFP⁺ infiltrating macrophages in the brain. Mice were observed for seizures daily as described in Materials and Methods. (A) Representative immunofluorescence of brain sections from mice that either had seizures (right panels) or did not have seizures (left panels). GFP⁺ cells (infiltrating macrophages) are shown in green. (B) Quantification of GFP⁺ cells. Whole-tissue slides were quantified for the right hemisphere (site of injection). *, P < 0.05, t test.

Fig 7

TNF-α and IL-6 cytokine levels in GFP chimeric mice. TNF-α and IL-6 cytokine levels were assessed in GFP⁻ and GFP⁺ cells isolated from TMEV-infected mouse brains. (A) TNF-α levels; (B) IL-6 levels. The red histograms are the FMO controls. The black line is representative of CD11c⁻ CD11b⁺ GFP⁻ cells. The green line is representative of CD11c⁻ CD11b⁺ GFP⁺ cells. Each symbol in the far right graphs is representative of one mouse. The mean value is shown by the black line. *, P < 0.05, Student's paired t test.

Fig 8

IL-6⁺ cells in GFP chimeric mice, shown in representative hippocampal tissue sections immunohistochemically stained for IL-6. All representative brain tissue sections were obtained from TMEV-infected mice at day 7 p.i. (A to D) Sections from a mouse that did not experience seizures. (E to H) Sections from a mouse that experienced seizures. (I to L) Sections from a C57BL/6 IL-6^−/− mouse. Arrows point to IL-6⁺ cells. Magnification, ×20; for insets, magnification is ×40. Control (IL-6^−/−) TMEV-infected mice were stained in conjunction with the experimental tissue sections. CA, cornu ammonis; DG, dentate gyrus; PC, parietal cortex.

Fig 9

TNF-α⁺ cells in GFP chimeric mice, shown in representative hippocampal tissue sections immunohistochemically stained for TNF-α. All representative brain tissue sections were obtained from TMEV-infected mice at day 7 p.i. (A to D) Sections from a mouse that did not experience seizures. (E to H) Sections from a mouse that experienced seizures. (I to L) Sections from a mouse that experienced seizures with no primary antibody (Ab). Brown indicates TNF-α staining. Magnification, ×20; for insets, magnification is ×40. Control (no primary Ab) TMEV-infected mice were stained in conjunction with the experimental tissue sections. CA, cornu ammonis; DG, dentate gyrus; PC, parietal cortex.

Fig 10

TMEV-infected C57BL/6 mice treated with minocycline and wogonin have a significantly lower number of infiltrating macrophages in the brain than do vehicle-treated mice. (A) Representative flow cytometry plots of cells obtained on day 3 p.i. from the brains of TMEV-infected mice treated with vehicle (DMSO), wogonin, or minocycline. Microglial cells are CD45^lo/int CD11b⁺ (R1). Macrophages are CD45^hi CD11b⁺ (R2). (B) No significant differences in the numbers of microglia (R1) were detected. Minocycline- and wogonin-treated mice had significantly fewer macrophages (R2) that infiltrated into the brain than did vehicle-treated mice. Data are means + standard errors of the means for 5 mice per group. *, P < 0.05, Student's paired t test.

Fig 11

Seizure frequencies (Racine scale, stages 3 to 5) in wogonin- and vehicle-treated mice. C57BL/6 mice infected with TMEV were treated, as described in Materials and Methods, with either vehicle (DMSO) or wogonin and monitored for seizures. Wogonin-treated mice had significantly fewer seizures (35%) than vehicle-treated mice (63%). ‡, P < 0.05, chi-square test. The total number of mice infected is shown above each individual bar in the graph. The percentage of mice was determined based on the number of mice with seizures divided by the total number of mice infected for each group, × 100.