Visualizing production of beta interferon by astrocytes and microglia in brain of La Crosse virus-infected mice

Abstract

Beta interferon (IFN-β) is a major component of innate immunity in mammals, but information on the in vivo source of this cytokine after pathogen infection is still scarce. To identify the cell types responsible for IFN-β production during viral encephalitis, we used reporter mice that express firefly luciferase under the control of the IFN-β promoter and stained organ sections with luciferase-specific antibodies. Numerous luciferase-positive cells were detected in regions of La Crosse virus (LACV)-infected mouse brains that contained many infected cells. Double-staining experiments with cell-type-specific markers revealed that similar numbers of astrocytes and microglia of infected brains were luciferase positive, whereas virus-infected neurons rarely contained detectable levels of luciferase. Interestingly, if a mutant LACV unable of synthesizing the IFN-antagonistic factor NSs was used for challenge, the vast majority of the IFN-β-producing cells in infected brains were astrocytes rather than microglia. Similar conclusions were reached in a second series of experiments in which conditional reporter mice expressing the luciferase reporter gene solely in defined cell types were infected with wild-type or mutant LACV. Collectively, our data suggest that glial cells rather than infected neurons represent the major source of IFN-β in LACV-infected mouse brains. They further indicate that IFN-β synthesis in astrocytes and microglia is differentially affected by the viral IFN antagonist, presumably due to differences in LACV susceptibility of these two cell types.

Fig 1

IFN-β-producing cells are found near LACV-infected cells in various brain regions. Brain sections from cortex (A, B, E, and F) and cerebellum (C, D, G, and H) of diseased IFN-β^+/Δβ-luc mice infected with LACV-ΔNSs or LACV-wt were simultaneously stained for luciferase, viral antigen (LACV-G2), and cell nuclei (DAPI). Note that although in close proximity to infected cells, luciferase-positive cells did not usually stain for viral antigen. Scale bar, 50 μm.

Fig 2

LACV-ΔNSs activates the IFN-β reporter gene more efficiently than LACV-wt although it replicates less well in the brain. Brains of infected IFN-β^+/Δβ-luc mice showing signs of neurological symptoms were collected and homogenized. Viral titers (A) and luciferase activity (B) in brain homogenates were determined. ***, P < 0.0005.

Fig 3

Astrocytes, microglia, and neurons of LACV-infected mouse brains express IFN-β reporter gene. Results of double staining of brain slices for luciferase (luc) and the astrocyte marker GFAP, microglia/macrophage marker F4/80, and neuron marker NeuN are shown. Luciferase-positive neurons were detectable only in LACV-ΔNSs-infected (E) but not in LACV-infected brains (F). Cells were counterstained with DAPI. Single-channel and merged pictures of the same frames are shown. Scale bar, 10 μm.

Fig 4

LACV replicates predominantly in neurons. Brains of mice infected with LACV-ΔNSs or LACV-wt were stained simultaneously for viral antigen (LACV-G2) and either the neuron marker NeuN or the astrocyte marker GFAP. Scale bar, 20 μm.

Fig 5

Conditional reporter mice express the luciferase reporter gene in the predicted cell types. (A) LysM-Cre^+/− IFN-β^+/floxβ-luc mice were infected with LACV-ΔNSs, and brains of diseased mice were simultaneously stained for luciferase (green) and the microglia/macrophage marker F4/80 (red). (B) Thy1-Cre^+/− IFN-β^+/floxβ-luc mice were infected with LACV-ΔNSs, and brains of diseased mice were simultaneously stained for luciferase (green) and the astrocyte marker GFAP (red). (C) Synapsin1-Cre^+/− IFN-β^+/floxβ-luc mice were infected with LACV-ΔNSs, and brain sections of diseased mice were simultaneously stained for luciferase (green) and the neuron marker NeuN (red). Scale bar, 50 μm.

Fig 6

Conditional reporter mice show no significant differences in susceptibility to LACV. Global reporter mice (Δβ-luc) and conditional Thy1-Cre^+/− IFN-β^+/floxβ-luc (thy), LysM-Cre^+/− IFN-β^+/floxβ-luc(lys), and Synapsin1-Cre^+/− IFN-β^+/floxβ-luc (syn) reporter mice infected with LACV-ΔNSs (A) or LACV-wt (B) were monitored for neurological symptoms. Diseased animals were killed, and brain titers were determined. No significant differences were observed between the different mouse strains. p.i., postinfection.

Fig 7

IFN-β synthesis by astrocytes and neurons but not microglia is repressed by LACV-encoded IFN-antagonistic factor NSs. Reporter mice in which the luciferase gene is expressed exclusively in astrocytes and neurons (thy), neurons only (syn), or microglia/macrophages (lysM) were infected with LACV-ΔNSs (A and B) or LACV-wt (C and D). Global IFN-β^+/Δβ-luc reporter mice (Δβ-luc) served as the reference. Brains of diseased animals with very similar virus loads were selected for further analysis. Mean luciferase activities (with standard deviations) in brain samples from the various mouse strains infected with either LACV-ΔNSs or LACV-wt are shown. The average contributions of different cell types to luciferase activity in brains of mice infected with LACV-ΔNSs or LACV-wt are shown as pie charts; the activity of global IFN-β^+/Δβ-luc reporter mice was set to 100%.