Inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination

Abstract

Infection of the central nervous system (CNS) by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces an acute encephalomyelitis associated with demyelination. To examine the anti-viral and/or regulatory role of interferon-gamma (IFN-gamma) signaling in the cell that synthesizes and maintains the myelin sheath, we analyzed JHMV pathogenesis in transgenic mice expressing a dominant-negative IFN-gamma receptor on oligodendroglia. Defective IFN-gamma signaling was associated with enhanced oligodendroglial tropism and delayed virus clearance. However, the CNS inflammatory cell composition and CD8(+) T-cell effector functions were similar between transgenic and wild-type mice, supporting unimpaired peripheral and CNS immune responses in transgenic mice. Surprisingly, increased viral load in oligodendroglia did not affect the extent of myelin loss, the frequency of oligodendroglial apoptosis, or CNS recruitment of macrophages. These data demonstrate that IFN-gamma receptor signaling is critical for the control of JHMV replication in oligodendroglia. In addition, the absence of a correlation between increased oligodendroglial infection and the extent of demyelination suggests a complex pathobiology of myelin loss in which infection of oligodendroglia is required but not sufficient.

Figure 1

Virus replication within the CNS. Virus replication in the CNS at different time points p.i. determined in brain homogenates prepared from individual Wt and Tg mice by plaque assay. Bars indicate the average, and the noncontinuous line represents the limit of detection the assay.

Figure 2

Composition of CNS inflammatory cells in JHMV-infected mice. Flow cytometric analysis shows inflammatory cells derived from dissociated brains isolated via Percoll step gradients. Bone marrow-derived inflammatory and resident CNS cells were distinguished by CD45 expression. Total CNS cells per brain; bars represent Wt and Tg mice. Percentage of CNS-infiltrating cells; lines represent CD45^hi cells for Wt and Tg mice. Data are derived from one representative of five experiments each containing a minimum of three mice per group.

Figure 3

Phenotypes within the CNS-infiltrating cells. The phenotype and frequencies of the cells contained within the CD45^hi inflammatory cells within the CNS were identified with mAbs specific for T and natural killer cells. Values are calculated as percentages of the specific population by setting CD45^hi cells equal to 100%. Bars depict the individual populations within the CNS. Data represent one of five similar experiments.

Figure 4

CNS-infiltrating virus-specific CD8⁺ T cells. Percentage of virus-specific S510 tetramer⁺/CD8⁺ T cells within the CNS-infiltrating CD8⁺ T cell population in Wt and Tg mice ± SD. Values are calculated as a percentage of the specific population by setting CD45^hi/CD8⁺-infiltrating cells equal to 100%. Data represent the mean of five separate experiments containing at least three mice per time point.

Figure 5

Influence of IFN-γ signaling on viral antigen distribution. Virus-infected cells in brains from infected Wt (A) and Tg mice (B) at day 5 p.i. detected with a mAb specific for the viral nucleocapsid protein are shown. Arrowheads indicate antigen-positive ependymal cells, arrows indicate antigen-positive parenchymal cells, and cp indicates the choroids plexus. Note virus dissemination into the parenchyma. Scattered infected cells with a morphology consistent with oligodendroglia in brain from Wt mice at 10 days p.i. (C) and Tg mice (D) at day 10 p.i. Shown are virus-infected cells in spinal cord white matter tracks of Wt (E) and transgenic mice (F) at 18 days p.i. Scale bar = 100 μm.

Figure 6

JHMV infection induced CNS inflammation and demyelination. Spinal cords from Wt (A, C, and E) and Tg mice (B, D, and F) 18 days p.i stained with H&E (A and B) or luxol fast blue (C and D). Extensive white matter inflammation associated with demyelination in both Wt (A and C) and Tg mice (B and D). Axons within the demyelinated lesions in Wt (E) and Tg (F) mice were visualized with anti-phosphoneurofilament antibody. Scale bar = 100 μm. Co-localization of viral antigen in oligodendrocytes (G) is shown, using virus-specific mAb J.3.3 (red) and anti-adenomatus ployposis coli (gray) at 18 days p.i. Scale bar = 50 μm. The inset shows higher magnification of double-positive cells in spinal cord of Tg mice.

Figure 7

Contribution of apoptosis and infiltrating macrophages to CNS demyelination. Apoptotic cells in the spinal cords of Wt (A) and Tg (B) mice at 14 days p.i. detected by TUNEL. Scale bar = 200 μm. Percentage of CD45^hi F4/80⁺ CNS-infiltrating macrophages (C) in JHMV-infected Wt (full bar) and Tg mice (empty bar). Values calculated as percentage F4/80⁺ by setting CD45^hi bone marrow-derived inflammatory cells equal to 100%. Data represent one of five similar experiments.