Perforin Expression by CD8 T Cells Is Sufficient To Cause Fatal Brain Edema during Experimental Cerebral Malaria

Abstract

Human cerebral malaria (HCM) is a serious complication of Plasmodium falciparum infection. The most severe outcomes for patients include coma, permanent neurological deficits, and death. Recently, a large-scale magnetic resonance imaging (MRI) study in humans identified brain swelling as the most prominent predictor of fatal HCM. Therefore, in this study, we sought to define the mechanism controlling brain edema through the use of the murine experimental cerebral malaria (ECM) model. Specifically, we investigated the ability of CD8 T cells to initiate brain edema during ECM. We determined that areas of blood-brain barrier (BBB) permeability colocalized with a reduction of the cerebral endothelial cell tight-junction proteins claudin-5 and occludin. Furthermore, through small-animal MRI, we analyzed edema and vascular leakage. Using gadolinium-enhanced T1-weighted MRI, we determined that vascular permeability is not homogeneous but rather confined to specific regions of the brain. Our findings show that BBB permeability was localized within the brainstem, olfactory bulb, and lateral ventricle. Concurrently with the initiation of vascular permeability, T2-weighted MRI revealed edema and brain swelling. Importantly, ablation of the cytolytic effector molecule perforin fully protected against vascular permeability and edema. Furthermore, perforin production specifically by CD8 T cells was required to cause fatal edema during ECM. We propose that CD8 T cells initiate BBB breakdown through perforin-mediated disruption of tight junctions. In turn, leakage from the vasculature into the parenchyma causes brain swelling and edema. This results in a breakdown of homeostatic maintenance that likely contributes to ECM pathology.

Keywords: CD8 T cell; blood-brain barrier; experimental cerebral malaria; perforin.

FIG 1

CD8 T cell trafficking to the brain and parasitemia are independent of perforin during experimental cerebral malaria. (A) Survival curve showing fatal disease progression in C57BL/6 mice 6 days after infection with 10⁶ parasitized erythrocytes containing Plasmodium berghei ANKA compared with perforin-deficient mice (pfp^−/−) that are fully protected from ECM (P < 0.0001). Survival curve statistical analysis was done by log rank test, with 10 mice, with results pooled from two independent experiments. ****, P < 0.0001. (B) Representative plot illustrating gating strategy for quantifying brain-infiltrating immune cells. (C and D) CD8 T cells infiltrating the brain at 6 dpi are at the same frequencies (C) and total numbers (D) in ECM-protected pfp^−/− and WT C57BL/6 mice. Ten mice per group were used, and results were pooled from two independent experiments. (E) Cumulative percentages of parasitemia postinfection show no difference between pfp^−/− and WT C57BL/6 mice. Giemsa-stained peripheral blood smears were analyzed at ×100 magnification from P. berghei ANKA-infected mice. Error bars represent standard errors of the mean (SEM), and P values were determined by an unpaired two-tailed t test.

FIG 2

Vascular leakage associates with perforin-dependent tight-junction disruptions during ECM. (A to C) Representative immunofluorescent confocal microscopy images of occludin, claudin-5, and FITC-albumin in mouse brain sections. (A) PBS-injected WT control mice show FITC-albumin restricted within the vasculature. (B) P. berghei ANKA (PbA)-infected C57BL/6 animals show increased FITC-albumin leakage from vessels into the parenchyma and claudin-5 and occludin disruption. (C) P. berghei ANKA-infected pfp^−/− mice maintain claudin-5 and occludin tight-junction integrity, and FITC-albumin is again constrained within the microvasculature. Fresh frozen sections were taken from mice 6 dpi. (D) Leakage of intravenously injected FITC-albumin into the CNS was significantly elevated 6 dpi in WT C57BL/6 mice infected with P. berghei ANKA compared to that in PBS-injected controls (P = 0.003) and infected pfp^−/− mice (P = 0.002). (E and F) Quantification of claudin-5 (E) and occludin (F) show reductions in the area of tight-junction proteins within the brains of C57BL/6 mice but not pfp^−/− mice at 6 dpi compared to those in uninfected controls. (G) Densitometry quantification of Western blots of brain protein lysate shows no detectable elevation of active cleaved caspase-3 in the cortex or olfactory bulb 6 days after P. berghei ANKA infection or PBS injection (P = 0.20). GL261 brain tumor samples show increased cleavage of caspase-3, indicating an active apoptotic pathway. Four mice per experiment were used, and results are representative of three independent experiments. Error bars represent SEM, and P values were determined by analysis of variance (ANOVA) with Bonferroni's post hoc analysis. **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

FIG 3

Vascular permeability during ECM is localized to specific vital brain regions. (A to F) Representative T1-weighted gadolinium-enhanced MRI axial sections comparing levels of vascular permeability in the CNS of C57BL/6 (A and E) and pfp^−/− mice (C and F). 3D volumetric renderings show gadolinium leakage (red) in C57BL/6 mice (B) that is elevated compared to that in pfp^−/− mouse brain scans (D). Midsagittal sections show gadolinium enhancement in the olfactory bulb (arrow a), ventricles (arrow b), and brainstem (arrow c) of C57BL/6 mice (E) but not in pfp^−/− mice (F). MRIs were analyzed with Analyze 10.0 (Biomedical Imaging Resource, Mayo Clinic) to render 3D masks of gadolinium leakage volume. All images are representative of three independent experiments of 5 mice per group.

FIG 4

Elevated brain edema during ECM correlates with regions of BBB disruption. (A to C) Axial sections from representative T2-weighted MRIs of murine brains during ECM. T2 hyperintensity is seen in ventricles of all samples but also in the cortex of P. berghei ANKA (PbA)-infected C57BL/6 mice. (D) Quantification of hyperintensity volumes illustrates brain edema during ECM in C57BL/6 mice. Hyperintensity in P. berghei ANKA-infected C57BL/6 mice is significantly increased compared to that in both uninfected C57BL/6 (P = 0.0015) and infected pfp^−/− (P = 0.0016) animals. *, P < 0.05. (E and F) Sagittal sections of brains 6 dpi demonstrate regions of meningeal hyperintensity (asterisks) in C57BL/6 animals (E) that do not appear in pfp^−/− mice (F). Images were analyzed using Analyze 10.0 (Biomedical Imaging Resource, Mayo Clinic). Five mice per group were used, and data are representative of two individual experiments. Error bars represent SEM, and P values were determined using ANOVA with Bonferroni's post hoc analysis.

FIG 5

Perforin secretion is required for CD8 T cells to cause brain edema during ECM. (A) Survival curve demonstrating that 6 days after adoptive transfer of purified CD8 T cells from WT C57BL/6 mice into pfp^−/− recipients, animals develop ECM. In contrast, the control group of pfp^−/− animals receiving pfp^−/− CD8 T cells remains protected from disease (P = 0.0008). Survival was analyzed using a log rank test with 8 animals, and results were pooled from two independent experiments. ***, P < 0.001. (B) Representative T2-weighted MRIs of sagittal sections from mice 6 days after adoptive transfer and P. berghei ANKA infection. (C) Volumetric quantification of hyperintensity from T2-weighted MRI illustrates brain edema during ECM in pfp^−/− mice with adoptively transferred C57BL/6 CD8 T cells but not in pfp^−/− donors (P = 0.0213). Error bars represent SEM, and the P value was obtained using an unpaired t test. *, P < 0.05.