Functional genomics reveals an essential and specific role for Stat1 in protection of the central nervous system following herpes simplex virus corneal infection

Abstract

Innate immune deficiencies result in a spectrum of severe clinical outcomes following infection. In particular, there is a strong association between loss of the signal transducer and activator of transcription (Stat) pathway, breach of the blood-brain barrier (BBB), and virus-induced neuropathology. The gene signatures that characterize resistance, disease, and mortality in the virus-infected nervous system have not been defined. Herpes simplex virus type 1 (HSV-1) is commonly associated with encephalitis in humans, and humans and mice lacking Stat1 display increased susceptibility to HSV central nervous system (CNS) infections. In this study, two HSV-1 strains were used, KOS (wild type [WT]), and Δvhs, an avirulent recombinant lacking the virion host shutoff (vhs) function. In addition, two mouse strains were used: strain 129 (control) and a Stat1-deficient (Stat1(-/-)) strain. Using combinations of these virus and mouse strains, we established a model of infection resulting in three different outcomes: viral clearance without neurological disease (Δvhs infection of control mice), neurological disease followed by viral clearance (Δvhs infection of Stat1(-/-) mice and WT infection of control mice), or neurological disease followed by death (WT infection of Stat1(-/-) mice). Through the use of functional genomics on the infected brain stems, we determined gene signatures that were representative of the three infection outcomes. We demonstrated a pathological signature in the brain stem of Stat1-deficient mice characterized by upregulation of transcripts encoding chemokine receptors, inflammatory markers, neutrophil chemoattractants, leukocyte adhesion proteins, and matrix metalloproteases. Additionally, there was a greater than 100-fold increase in the inflammatory markers interleukin 1β (IL-1β) and IL-6. Consistent with this gene signature, we demonstrated profound CNS inflammation with a concomitant lethal breach of the BBB. Taken together, our results indicated an essential role for normal Stat1-dependent signaling in mediating a nonpathological immune response to viral CNS infection.

Fig. 1.

Virological analysis of brain stems and livers. Control or Stat1^−/− mice were inoculated on the cornea with 2 × 10⁶ PFU/eye of HSV-1 WT or HSV-1 Δvhs. At the indicated time postinfection, brain stems (A) and livers (B) were harvested and mechanically disrupted for a plaque assay (n ≥ 8). Titers are presented as PFU/tissue and are the average of results for at least 8 animals. The dotted line indicates threshold of detection (LOD). Where average values fall below the LOD and error bars are shown, virus was detected in some samples and not in others. Where no error bars are shown, virus in all samples was below the LOD. Asterisks indicate ranges of statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001) for differences between bracketed bars. All bars on days 3 and 5 showed statistically significant differences from each other, with the exception of results for WT-infected control mice and Δvhs-infected Stat1^−/− mice, which were not significantly different.

Fig. 2.

Protective signature genes. Control or Stat1^−/− mice were inoculated on the cornea with 2 × 10⁶ PFU/eye of HSV-1 WT or HSV-1 Δvhs or treated with a mock lysate. At the indicated time postinfection, RNA was harvested and processed for array analysis. (A) Heat map showing ANOVA results for differentially expressed genes within the cutoff values of ≥2-fold change and ANOVA P ≤ 0.01 in brain stems comparing nondiseased (WT virus-infected strain 129 control mice) and diseased (WT virus-infected Stat1^−/− mice and Δvhs virus-infected Stat1^−/− mice) models at 5 dpi. Red and black colors indicate upregulated and unchanged genes, respectively, compared to a mock-treated sample. The numbers indicate fold changes compared to results for a mock-treated sample. (B) Functional network analysis of 5 dpi ANOVA results determined by Ingenuity Pathways Analysis. Arrows indicate functional interactions between genes. Yellow and blue colors indicate up- and downregulated genes, respectively.

Fig. 3.

Comparison of protective responses in brain stem and liver. Heat map comparing gene expression changes in the brain stems and livers of WT virus-infected control mice and WT or Δvhs virus-infected Stat1^−/− mice at 3 and 5 dpi. Differentially expressed genes identified in the protective gene signature analysis are shown. Red and black colors indicate upregulated or unchanged genes, respectively, compared to a mock-treated sample. The numbers indicate fold changes compared to results for a mock-treated sample.

Fig. 4.

Pathogenicity genes and BBB permeability. (A) Heat map showing selected, differentially expressed genes within the cutoff values of ≥2-fold change and ANOVA P ≤ 0.01 in brain stems from WT virus-infected control mice, WT virus-infected Stat1^−/− mice, or Δvhs virus-infected Stat1^−/− mice at 5 dpi. The numbers indicate fold changes compared to results for mock-infected mice. (B) Quantification of Evans blue dye in brain tissue of infected mice. WT or Δvhs virus-infected control and Δvhs virus-infected Stat1^−/− mice are shown at 7 dpi. WT virus-infected Stat1^−/− mice were moribund at 7 dpi, and so data are shown for 6 dpi. The amount of dye is reported in ng/brain. The dotted line indicates the background level of Evans blue dye. *, P < 0.01.

Fig. 5.

Bead-based cytokine analysis of brain stems. Control or Stat1^−/− mice were inoculated with 2 × 10⁶ PFU/eye of HSV-1 WT or HSV-1 Δvhs. At the indicated time postinfection, protein was extracted from brain stem tissue and analyzed by bead-based cytokine analysis (n ≥ 4). Cytokine concentrations are presented as pg/ml.

Fig. 6.

Histological analysis of brain stem tissue. (A) Map taken from the Gene Expression Nervous System Atlas (GENSAT) Project (www.gensat.org) shows the region of the brain examined in the subsequent panels. (B) Control mice infected with WT virus, 5 dpi. (C) Stat1^−/− brain infected with WT virus, 5 dpi. (D) Stat1^−/− mice infected with WT virus, 7 dpi. (E) Stat1^−/− mice infected with Δvhs virus, 5 dpi. (F) Stat1^−/− mice infected with WT virus, 7 dpi. Mice were inoculated with 2 × 10⁶ PFU/eye of virus per eye, and brain stems were harvested and fixed for histological analysis at times indicated. Tissue sections were stained for HSV-1 antigen (brown) and counterstained with hematoxylin. Representative images are shown of the pons area in the brain stem where the trigeminal ganglia connect to the brain stem.