CNS recruitment of CD8+ T lymphocytes specific for a peripheral virus infection triggers neuropathogenesis during polymicrobial challenge

Abstract

Although viruses have been implicated in central nervous system (CNS) diseases of unknown etiology, including multiple sclerosis and amyotrophic lateral sclerosis, the reproducible identification of viral triggers in such diseases has been largely unsuccessful. Here, we explore the hypothesis that viruses need not replicate in the tissue in which they cause disease; specifically, that a peripheral infection might trigger CNS pathology. To test this idea, we utilized a transgenic mouse model in which we found that immune cells responding to a peripheral infection are recruited to the CNS, where they trigger neurological damage. In this model, mice are infected with both CNS-restricted measles virus (MV) and peripherally restricted lymphocytic choriomeningitis virus (LCMV). While infection with either virus alone resulted in no illness, infection with both viruses caused disease in all mice, with ∼50% dying following seizures. Co-infection resulted in a 12-fold increase in the number of CD8+ T cells in the brain as compared to MV infection alone. Tetramer analysis revealed that a substantial proportion (>35%) of these infiltrating CD8+ lymphocytes were LCMV-specific, despite no detectable LCMV in CNS tissues. Mechanistically, CNS disease was due to edema, induced in a CD8-dependent but perforin-independent manner, and brain herniation, similar to that observed in mice challenged intracerebrally with LCMV. These results indicate that T cell trafficking can be influenced by other ongoing immune challenges, and that CD8+ T cell recruitment to the brain can trigger CNS disease in the apparent absence of cognate antigen. By extrapolation, human CNS diseases of unknown etiology need not be associated with infection with any particular agent; rather, a condition that compromises and activates the blood-brain barrier and adjacent brain parenchyma can render the CNS susceptible to pathogen-independent immune attack.

Figure 1. Co-infection model and pathogenic outcome.

(A) NSE-CD46⁺ transgenic mice survive MV IC challenge and mount an immune response that peaks between 7–9 dpi (left panel) . LCMV, a natural mouse pathogen, is restricted to peripheral organs (spleen, liver, kidney, pancreas) following IP inoculation (middle panel); all mice survive, and peak immunity also occurs between 7–9 dpi . In contrast, ∼50% of mice infected with both MV IC and LCMV IP (doubly infected; DI) succumbed to virus-induced seizures (right panel). (B) Baseline weights of all mice were obtained, daily weights were calculated, and weight gain or loss was determined. Mice challenged with PBS (black triangles), MV alone (black circles), or LCMV alone (asterisks) showed modest increases in weight over the 34d observation period (>10 mice/group). The cohort of mice infected with both LCMV and MV were divided into two groups based on the severity of the illness that was observed: animals with mild illness (white diamonds) and with severe illness and mortality (white squares). Statistics were performed using the Wilcoxon Signed Rank Test. #: p = 0.0002; ##: p<0.0001.

Figure 2. Tissue distribution of LCMV and MV in co-infected mice.

(A) RNA was purified from the indicated tissues of co-infected mice at 4 dpi and amplified by quantitative RT-PCR for MV (black bars, scale on left) or LCMV (gray bars, scale on right). BD  =  below detection. (B) The extent of MV load was assessed in brains of mice infected with MV alone or with both MV and LCMV at 7 dpi. Data points represent individual mice, with averages shown as horizontal lines.

Figure 3. Immune response within the brains of co-infected mice.

(A) Brains of mice infected with LCMV IP alone (left panels), MV IC alone (center panels), or both viruses (right panels) were harvested 7 dpi and serial sections were immunostained for either CD4⁺ T cells (top panels) or CD8⁺ T cells (bottom panels) using DAB (red/brown staining) and counterstained with hematoxylin (gray/light blue staining). Original magnification = 100X. (B) Brains were removed from the indicated mice at 7 dpi and homogenized. Mononuclear cells were extracted and subjected to staining using fluorophore-conjugated antibodies, followed by flow cytometry. Using this flow cytometry data, together with cell counts obtained using a hemocytometer, the total number of CD4⁺ and CD8⁺ T cells, as well as the percent of total CNS infiltrate, was calculated. Values for individual mice are shown, with averages shown as horizontal bars. Statistics were performed using the Wilcoxon Signed Rank test. ##: p<0.0001; NS: not significant.

Figure 4. LCMV-specific CD8⁺ T cells are found within the brains of co-infected mice.

(A) T cells were isolated from brains of mice that were either uninfected (left panels), infected with MV IC alone (center panels), or infected with both LCMV IP and MV IC (right panels), as described in Figure 3. Cells were dual stained for CD8 (x-axis) and for one of two immunodominant LCMV epitopes (y-axis), using specific tetramers (GP33: top panels; NP396: bottom panels). (B) A standard ⁵¹Cr release assay was performed by incubating effector splenocytes from LCMV-infected mice (6 dpi) with uninfected (diamonds), MV-infected (squares), or LCMV-infected (triangles) target cells at the indicated ratios. Released ⁵¹Cr was measured in a gamma counter and specific release was calculated.

Figure 5. Postmortem examination of neuropathogenesis in co-infected mice shows signs of brain herniation.

(A) Representative mice infected with LCMV IC (left panel), MV IC (in NSE-CD46⁺ RAG-2 KO mice) (center panel), and both LCMV and MV (right panel) are shown exhibiting characteristic, post-mortem postures. (B) The scalp was removed from moribund co-infected mice or PBS treated controls to expose the skull; photographs were taken of the rear of the brain and cervical spinal cord. Note compression of white matter (cerebellum) against the posterior skull in co-infected animals. Mice were also examined for unilateral pupillary dilation (mydriasis) (seen here in mouse's left eye; white arrow). Photographs are representative of 5 to 10 mice per group.

Figure 6. Pathogenesis in co-infected mice is CD8⁺ T cell dependent and perforin independent.

(A) NSE-CD46⁺ mice were injected IP with 150 µg of CD8-depleting antibody (clone 2.43), or an isotype control antibody, and challenged with MV IC alone (closed/open squares), LCMV IP alone (closed/open circles), or both MV IC and LCMV IP (closed/open triangles). Mice were monitored daily for survival. (B) Mice of the indicated genotypes were challenged with MV IC alone (closed/open squares), LCMV IP alone (closed/open circles), or both MV IC and LCMV IP (closed/open triangles) and monitored daily for survival.

Figure 7. Edema, but not blood brain barrier, changes correlate with pathogenesis in co-infected mice and is dependent on CD8⁺ T cells.

(A) Brains were removed 7 dpi from mice infected as indicated and weighed before and after dehydration in a vacuum oven. Total water weight was determined for each mouse and normalized to values from PBS IC mice; averages are shown as grey bars, with standard deviation indicated. Statistics were performed using the Wilcoxon Signed Rank test. #: p = 0.03; NS: not significant. (B) Mice, infected and treated as indicated, were anesthetized at 7 dpi and injected with Evan's Blue, followed by saline perfusion. Total Evan's Blue that diffused into the brain was determined by spectrophotometry following brain homogenization and clarification. Data were normalized to values from mice inoculated with PBS IC, and averages are shown as gray bars, with standard deviation indicated. Statistics were performed using the Wilcoxon Signed Rank test. NS: not significant. (C) Representative photomicrographs were taken from brain sections of mice with severe disease following co-infection with LCMV IP and MV IC. Evidence of choroid plexus inflammation (i), meningitis (ii), and hemosiderin deposition, indicative of capillary bleeding (iii), are shown. Original magnifications: A, B  =  640X; C  =  800X.