Abstract
BackgroundCD8 T cell-mediated blood-brain barrier (BBB) disruption is dependent on the effector molecule perforin. Human perforin has extensive single nucleotide variants (SNVs), the significance of which is not fully understood. These SNVs can result in reduced, but not ablated, perforin activity or expression. However, complete loss of perforin expression or activity results in the lethal disease familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). In this study, we address the hypothesis that a single perforin allele can alter the severity of BBB disruption in vivo using a well-established model of CNS vascular permeability in C57Bl/6 mice. The results of this study provide insight into the significance of perforin SNVs in the human population.MethodsWe isolated the effect a single perforin allele has on CNS vascular permeability through the use of perforin-heterozygous (perforin+/−) C57BL/6 mice in the peptide-induced fatal syndrome (PIFS) model of immune-mediated BBB disruption. Seven days following Theiler’s murine encephalomyelitis virus (TMEV) CNS infection, neuroinflammation and TMEV viral control were assessed through flow cytometric analysis and quantitative real-time PCR of the viral genome, respectively. Following immune-mediated BBB disruption, gadolinium-enhanced T1-weighted MRI, with 3D volumetric analysis, and confocal microscopy were used to define CNS vascular permeability. Finally, the open field behavior test was used to assess locomotor activity of mice following immune-mediated BBB disruption.ResultsPerforin-null mice had negligible CNS vascular permeability. Perforin-WT mice have extensive CNS vascular permeability. Interestingly, perforin-heterozygous mice had an intermediate level of CNS vascular permeability as measured by both gadolinium-enhanced T1-weighted MRI and fibrinogen leakage in the brain parenchyma. Differences in BBB disruption were not a result of increased CNS immune infiltrate. Additionally, TMEV was controlled in a perforin dose-dependent manner. Furthermore, a single perforin allele is sufficient to induce locomotor deficit during immune-mediated BBB disruption.ConclusionsPerforin modulates BBB disruption in a dose-dependent manner. This study demonstrates a potentially advantageous role for decreased perforin expression in reducing BBB disruption. This study also provides insight into the effect SNVs in a single perforin allele could have on functional deficit in neurological disease.
Highlights
CD8 T cell-mediated blood-brain barrier (BBB) disruption is dependent on the effector molecule perforin
Neuroinflammatory profile of perforin-null, perforin-heterozygous, and perforin-WT mice during acute central nervous system (CNS) Theiler’s murine encephalomyelitis virus (TMEV) infection In order to determine if differential perforin expression alters immune cell migration into the CNS, we TMEVinfected perforin-null, perforin-heterozygous, and perforin-WT C57BL/6 mice
We observed no significant difference between perforin-null, perforin-heterozygous, and perforin-WT mice pertaining to numbers or frequency of CD4 T cells (Fig. 1b, c), CD8 T cells (Fig. 1d, e), or natural killer (NK) cells (Fig. 1h, i) in the brains of mice with a 7-day TMEV infection
Summary
CD8 T cell-mediated blood-brain barrier (BBB) disruption is dependent on the effector molecule perforin. Human perforin has extensive single nucleotide variants (SNVs), the significance of which is not fully understood. These SNVs can result in reduced, but not ablated, perforin activity or expression. We address the hypothesis that a single perforin allele can alter the severity of BBB disruption in vivo using a well-established model of CNS vascular permeability in C57Bl/6 mice. Our lab has defined a role for CD8 T cells and perforin in mediating BBB tight junction alterations during neuroinflammation [14,15,16]. CD8 T cells can both recognize antigen and induce BBB disruption through perforin expression
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