Differential regulation of innate and adaptive immune responses in viral encephalitis

Abstract

Viral encephalitis is a global health concern. The ability of a virus to modulate the immune response can have a pivotal effect on the course of disease and the fate of the infected host. In this study, we sought to understand the immunological basis for the fatal encephalitis following infection with the murine coronavirus, mouse hepatitis virus (MHV)-JHM, in contrast with the more attenuated MHV-A59. Distinct glial cell cytokine and chemokine response patterns were observed within 3 days after infection, became progressively more polarized during the course of infection and with the infiltration of leukocytes. In the brain, MHV-JHM infection induced strong accumulation of IFNbeta mRNA relative to IFNgamma mRNA. This trend was reversed in MHV-A59 infection and was accompanied by increased CD8 T cell infiltration into brain compared to MHV-JHM infection. Increased apoptosis appeared to contribute to the diminished presence of CD8 T cells in MHV-JHM-infected brain with the consequence of a lower potential for IFNgamma production and antiviral activity. MHV-JHM infection also induced sustained mRNA accumulation of the innate immune response products interleukin (IL)-6 and IL-1. Furthermore, high levels of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and MIP-2 mRNA were observed at the onset of MHV-JHM infection and correlated with a marked elevation in the number of macrophages in the brain on day 7 compared to MHV-A59 infection. These observations indicate that differences in the severity of viral encephalitis may reflect the differential ability of viruses to stimulate innate immune responses within the CNS and subsequently the character of infiltrating leukocyte populations.

Fig. 1

Viral titers and disease scores. Mice were infected with MHV-JHM and MHV-A59 at doses indicated. Brain viral titers were determined by plaque assay. *By day 9, mice infected with MHV-JHM generally had succumbed to infection, whereas replicating virus was not detected in MHV-A59-infected brains. Mean PFU/ml ± SE are shown (n = 4–8 mice per group). Disease scores were assessed on day 7, the peak of acute encephalitis (n = 15 mice/group). On day 7, cells were isolated from infected brains as described in Materials and Methods. Live cells were identified by trypan blue dye exclusion.

Fig. 2

MHV-JHM infection induces sustained accumulation of IFNβ mRNA. Mice were infected intracranially with MHV-JHM, control virus, and saline. On days 3, 5, and 7 p.i, animals were perfused and brains were harvested. Total RNA was extracted and analyzed by RPA. “S” indicates saline-injected mice. Two representative samples shown from four experiments. Mean band densities were calculated using NIH Image 1.161 software reflective of signal intensity. Band densities for IFNγ and IFNβ were normalized against corresponding L32 (housekeeping) band densities and averaged. To compare the relative difference in IFNβ and IFNγ mRNA accumulation, the average band densities were expressed as ratios.

Fig. 3

Diminished CD8 T cell infiltration following MHV-JHM infection. Mice were inoculated with MHV-JHM and MHV-A59. On day 7, mononuclear cells were isolated from infected brains and stained for either anti-CD8, anti-CD4, or anti-NK1.1, and analyzed by FACS analysis. (A) One representative experiment for each of anti-CD8, anti-CD4, or anti-NK1.1 staining is shown. (B) Means ± SE for 4–6 mice/N experiments is shown.

Fig. 4

MHV-JHM infection results in reduced IFNγ production within the CNS. Cells isolated from infected brains were stimulated with viral peptides or anti-CD3 in the presence of brefeldin A (mean percents ± SE shown reflect 4–6 mice per five experiments). Cells were stained for the detection of CD8 and IFNγ. (A) Stimulation with S510 peptide, following MHV-A59 infection, and an irrelevant peptide, following both infections, results in negligible amounts of IFNγ+ cells. Percentages depict the IFNγ+ CD8 cells in the upper right-hand quadrant from one representative experiment. These negative controls were not run every experiment due to the number of cells isolated from brains. (B) Mean percentages are shown and are represented by the IFNγ+ CD8 cells in the upper right-hand quadrants of the graphs. (C) The percentage of IFNγ+ CD8 cells following viral peptide stimulation was normalized against the percentage of IFNγ+ CD8 cells following anti-CD3 stimulation, for example, 24.2/21.5 = 1.1. (D) The percentage of IFNγ+ total cells within the mononuclear gate was determined following anti-CD3 stimulation. Mean percentages ± SE are shown.

Fig. 5

Enhanced CD8 T cell apoptosis is seen following MHV-JHM infection. Cells isolated from infected brains were stained with anti-CD8 and Annexin V. Histograms shown were gated on CD8-positive cells. Control panel represents cell sample (from MHV-JHM-infected brains) in the absence of Annexin V staining. One representative experiment of three is shown. The intensity of Annexin V staining of negative population from control to sample is consistent with the assay parameters of the manufacturer.

Fig. 6

Fatal encephalitis is associated with elevated IL-6 synthesis. (A) Mice were infected intracranially with MHV-JHM or MHV-A59. On days 3, 5, and 7 p.i., animals were perfused and brains were harvested. Total RNA was extracted and analyzed by RPA. Representative samples shown from four experiments. (B) Cells were isolated from infected brains on day 7. Cells were incubated in the presence of UV-irradiated virus and monensin for 5 h at 37 °C, then stained for intracellular IL-6 as described in Materials and Methods. Experiment was repeated with similar results.

Fig. 7

MIP-1α, MIP-1β, and MIP-2 mRNA levels are enhanced in MHV-JHM-infected brains. Mice were infected with MHV-A59 or MHV-JHM as stated for Fig. 2. Total RNA was isolated from brains and analyzed by RPA. Representative samples shown from four experiments.

Fig. 8

MHV-JHM infection results in a greater infiltration of macrophages into the brain. (A) Isotype controls reflect percentages from one representative experiment (MHV-JHM samples). (B) Cells isolated from MHV-JHM- or MHV-A59-infected brains on day 7 were stained for either CD45 and FcR or CD45 and CD11b. Percentages above quadrants reflect the percentage of cells within the upper right-hand quadrant, that is, either CD45 high/FcR high or CD45 high/CD11b high. One representative experiment. (C) Means ± SE for N experiments is shown.