A hematopoietic contribution to microhemorrhage formation during antiviral CD8 T cell-initiated blood-brain barrier disruption

<p>Abstract</p> <p>Background</p> <p>The extent to which susceptibility to brain hemorrhage is derived from blood-derived factors or stromal tissue remains largely unknown. We have developed an inducible model of CD8 T cell-initiated blood-brain barrier (BBB) disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide-induced fatal syndrome (PIFS) model results in severe central nervous system (CNS) vascular permeability and death in the C57BL/6 mouse strain, but not in the 129 SvIm mouse strain, despite the two strains' having indistinguishable CD8 T-cell responses. Therefore, we hypothesize that hematopoietic factors contribute to susceptibility to brain hemorrhage, CNS vascular permeability and death following induction of PIFS.</p> <p>Methods</p> <p>PIFS was induced by intravenous injection of VP2_121-130peptide at 7 days post-TMEV infection. We then investigated brain inflammation, astrocyte activation, vascular permeability, functional deficit and microhemorrhage formation using T2*-weighted magnetic resonance imaging (MRI) in C57BL/6 and 129 SvIm mice. To investigate the contribution of hematopoietic cells in this model, hemorrhage-resistant 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Gadolinium-enhanced, T1-weighted MRI was used to visualize the extent of CNS vascular permeability after bone marrow transfer.</p> <p>Results</p> <p>C57BL/6 and 129 SvIm mice had similar inflammation in the CNS during acute infection. After administration of VP2_121-130peptide, however, C57BL/6 mice had increased astrocyte activation, CNS vascular permeability, microhemorrhage formation and functional deficits compared to 129 SvIm mice. The 129 SvIm mice reconstituted with C57BL/6 but not autologous bone marrow had increased microhemorrhage formation as measured by T2*-weighted MRI, exhibited a profound increase in CNS vascular permeability as measured by three-dimensional volumetric analysis of gadolinium-enhanced, T1-weighted MRI, and became moribund in this model system.</p> <p>Conclusion</p> <p>C57BL/6 mice are highly susceptible to microhemorrhage formation, severe CNS vascular permeability and morbidity compared to the 129 SvIm mouse. This susceptibility is transferable with the bone marrow compartment, demonstrating that hematopoietic factors are responsible for the onset of brain microhemorrhage and vascular permeability in immune-mediated fatal BBB disruption.</p

CMV Infection Attenuates the Disease Course in a Murine Model of Multiple Sclerosis

Recent evidence in multiple sclerosis (MS) suggests that active CMV infection may result in more benign clinical disease. The goal of this pilot study was to determine whether underlying murine CMV (MCMV) infection affects the course of the Theiler's murine encephalitis virus (TMEV) induced murine model of MS. A group of eight TMEV-infected mice were co-infected with MCMV at 2 weeks prior to TMEV infection while a second group of TMEV-infected mice received MCMV two weeks post TMEV. We also used 2 control groups, where at the above time points MCMV was replaced with PBS. Outcome measures included (1) monthly monitoring of disability via rotarod for 8 months; (2) in vivo MRI for brain atrophy studies and (3) FACS analysis of brain infiltrating lymphocytes at 8 months post TMEV infection. Co-infection with MCMV influenced the disease course in mice infected prior to TMEV infection. In this group, rotarod detectable motor performance was significantly improved starting 3 months post-infection and beyond (p≤0.024). In addition, their brain atrophy was close to 30% reduced at 8 months, but this was only present as a trend due to low power (p = 0.19). A significant reduction in the proportion of brain infiltrating CD3+ cells was detected in this group (p = 0.026), while the proportion of CD45+ Mac1+ cells significantly increased (p = 0.003). There was also a strong trend for a reduced proportion of CD4+ cells (p = 0.17) while CD8 and B220+ cell proportion did not change. These findings support an immunomodulatory effect of MCMV infection in this MS model. Future studies in this co-infection model will provide insight into mechanisms which modulate the development of demyelination and may be utilized for the development of novel therapeutic strategies

Contrasting roles for CD4 vs. CD8 T-cells in a murine model of virally induced "T1 black hole" formation.

MRI is sensitive to tissue pathology in multiple sclerosis (MS); however, most lesional MRI findings have limited correlation with disability. Chronic T1 hypointense lesions or "T1 black holes" (T1BH), observed in a subset of MS patients and thought to represent axonal damage, show moderate to strong correlation with disability. The pathogenesis of T1BH remains unclear. We previously reported the first and as of yet only model of T1BH formation in the Theiler's murine encephalitis virus induced model of acute CNS neuroinflammation induced injury, where CD8 T-cells are critical mediators of axonal damage and related T1BH formation. The purpose of this study was to further analyze the role of CD8 and CD4 T-cells through adoptive transfer experiments and to determine if the relevant CD8 T-cells are classic epitope specific lymphocytes or different subsets. C57BL/6 mice were used as donors and RAG-1 deficient mice as hosts in our adoptive transfer experiments. In vivo 3-dimensional MRI images were acquired using a 7 Tesla small animal MRI system. For image analysis, we used semi-automated methods in Analyze 9.1; transfer efficiency was monitored using FACS of brain infiltrating lymphocytes. Using a peptide depletion method, we demonstrated that the majority of CD8 T-cells are classic epitope specific cytotoxic cells. CD8 T-cell transfer successfully restored the immune system's capability to mediate T1BH formation in animals that lack adaptive immune system, whereas CD4 T-cell transfer results in an attenuated phenotype with significantly less T1BH formation. These findings demonstrate contrasting roles for these cell types, with additional evidence for a direct pathogenic role of CD8 T-cells in our model of T1 black hole formation

MRI results.

<p>Ventricular volumetry at 8 months post TMEV infection. Lower numbers represent less atrophy. The observed close to 30% lower atrophy in the MCMEV/TMEV group only showed a trend (p = 0.19) due to low statistical power, which was the consequence of the relatively high standard deviation observed in these groups. Of note, ventricular volumetry of SJL/J mice undergoing normal aging does not demonstrate the development of significant atrophy as demonstrated earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032767#pone.0032767-Pirko2" target="_blank">[15]</a>.</p

Flow cytometric analysis of brain infiltrating immune cells in 8 months TMEV infected mice that were either sham/PBS injected, pre- or post-infected with MCMV.

<p>Shown are CD45+ cells that are: A.) CD3+, B.) CD4+, and C.) Mac1+, respectively. The observed reduction in CD3+ cells compared to PBS controls was significant (p = 0.026), and so was the increase in MAC1+ cells (p = 0.003). The decrease in CD4+ T-cells demonstrated a trend (p = 0.17).</p

MCMV infection preserves motor function in SJL/J mice undergoing TMEV induced demyelinating syndrome.

<p>SJL/J mice infected with MCMV two weeks prior to TMEV infection have significantly higher rotarod scores compared to controls that received PBS injection instead of MCMV The intergroup difference first reaches significance at 3 months, and remains significant beyond that time point (p≤0.024). The figure also shows data acquired in mice infected with MCMV two weeks after TMEV infection; the data in those mice is virtually identical to our PBS/TMEV control group. Error bars represent SD.</p

FACS analysis of brain infiltrating immune cells on day 7 following TMEV infection.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031459#pone-0031459-g003" target="_blank">Figure 3A</a> (top half) shows transfer efficiency following adoptive transfer of CD8+ splenocytes from C57BL/6 donors into RAG-1 deficient hosts. Note that with sham transfer, only 1.6% of BIL-s is CD8+ and only 0.2% recognizes VP2 epitopes, whereas transfer of CD8+ cells results in 12.6% CD8+ positivity and 2.6% positivity for VP2 peptide. The 3.3% VP2+ CD8− cells are overall unidentified, and potentially represent dead cells that bind tetramer, auto fluorescing CNS cells, dying CD8+ tetramer+ cells that have become CD8− tetramer+, or NK cells with receptors specific for class I. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031459#pone-0031459-g003" target="_blank">Figure 3B</a> (lower half) describes transfer efficiency in our CD4 transfer experiments. With sham transfer, only 0.2% of BIL-s is CD4+, whereas with CD4 transfer, 14.4% positivity is demonstrated.</p

MRI T1BH volumetry results in adoptive transfer experiments using RAG 1 −/− recipient mice.

<p>The bar chart shows the mean T1BH volumes in CD8 and CD4 adoptive transfer experiments. Controls represent sham transfer (PBS injection) following irradiation and infection. CD8 T cell transfer resulted in 3-fold increase of T1BH formation, while CD4 transfer resulted in a 2-fold decrease. Error bars represent SD.</p

Example for T1 black hole formation in 7-day TMEV infected C57BL/6J mice.

<p>Top row: axial images, bottom row: coronal images extracted from a representative mouse. To provide guidance, some (but not all) areas containing T1 black holes are indicated with red frames. These are located in diverse areas of the brain, but mostly in periventricular areas, in the thalamus, hippocampus, white matter, corpus callosum and even in the cortex.</p