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

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.

Theiler's murine encephalomyelitis virus as an experimental model system to study the mechanism of blood-brain barrier disruption.

Theiler's murine encephalomyelitis virus is a widely used model to study the initiation and progression of multiple sclerosis. Many researchers have used this model to investigate how the immune system and genetic factors contribute to the disease process. Current research has highlighted the importance of cytotoxic CD8 T cells and specific major histocompatibility complex (MHC) class I alleles. Our lab has adopted this concept to create a novel mouse model to study the mechanism of blood-brain barrier (BBB) disruption, an integral feature of numerous neurological disorders. We have demonstrated that epitope-specific CD8 T cells cause disruption of the tight junction architecture and ensuing CNS vascular permeability in the absence of neutrophil support. This CD8 T cell-initiated BBB disruption is dependent on perforin expression. We have also elucidated a potential role for hematopoietic factors in this process. Despite having identical MHC class I molecules, similar inflammation in the CNS, and equivalent ability to utilize perforin, C57BL/6 mice are highly susceptible to this condition, while 129 SvIm mice are resistant. This susceptibility is transferable with the bone marrow compartment. These findings led us to conduct a comprehensive genetic analysis which has revealed a list of candidate genes implicated in regulating traits associated with BBB disruption. Future studies will continue to define the underlying molecular mechanism of CD8 T cell-initiated BBB disruption and may assist in the development of potential therapeutic approaches to ameliorate pathology associated with BBB disruption in neurological disorders.

CD8 T cell-initiated blood-brain barrier disruption is independent of neutrophil support.

Blood-brain barrier (BBB) disruption is a common feature of numerous neurologic disorders. A fundamental question in these diseases is the extent inflammatory immune cells contribute to CNS vascular permeability. We have previously shown that CD8 T cells play a critical role in initiating BBB disruption in the peptide-induced fatal syndrome model developed by our laboratory. However, myelomonocytic cells such as neutrophils have also been implicated in promoting CNS vascular permeability and functional deficit in murine models of neuroinflammatory disease. For this reason, we evaluated neutrophil depletion in a murine model of CD8 T cell-initiated BBB disruption by employing traditionally used anti-granulocyte receptor-1 mAb RB6-8C5 and Ly-6G-specific mAb 1A8. We report that CNS-infiltrating antiviral CD8 T cells express high levels of granulocyte receptor-1 protein and are depleted by treatment with RB6-8C5. Mice treated with RB6-8C5, but not 1A8, display: 1) intact BBB tight junction proteins; 2) reduced CNS vascular permeability visible by gadolinium-enhanced T1-weighted magnetic resonance imaging; and 3) preservation of motor function. These studies demonstrate that traditional methods of neutrophil depletion with RB6-8C5 are broadly immune ablating. Our data also provide evidence that CD8 T cells initiate disruption of BBB tight junction proteins and CNS vascular permeability in the absence of neutrophil support.

Quantitative trait loci analysis reveals candidate genes implicated in regulating functional deficit and CNS vascular permeability in CD8 T cell-initiated blood-brain barrier disruption.

BACKGROUND: Blood-brain barrier (BBB) disruption is an integral feature of numerous neurological disorders. However, there is a relative lack of knowledge regarding the underlying molecular mechanisms of immune-mediated BBB disruption. We have previously shown that CD8 T cells and perforin play critical roles in initiating altered permeability of the BBB in the peptide-induced fatal syndrome (PIFS) model developed by our laboratory. Additionally, despite having indistinguishable CD8 T cell responses, C57BL/6J (B6) mice are highly susceptible to PIFS, exhibiting functional motor deficits, increased astrocyte activation, and severe CNS vascular permeability, while 129S1/SvImJ (129S1) mice remain resistant. Therefore, to investigate the potential role of genetic factors, we performed a comprehensive genetic analysis of (B6 x 129S1) F2 progeny to define quantitative trait loci (QTL) linked to the phenotypic characteristics stated above that mediate CD8 T cell-initiated BBB disruption. RESULTS: Using single nucleotide polymorphism (SNP) markers and a 95% confidence interval, we identified one QTL (PIFS1) on chromosome 12 linked to deficits in motor function (SNP markers rs6292954, rs13481303, rs3655057, and rs13481324, LOD score = 3.3). In addition we identified a second QTL (PIFS2) on chromosome 17 linked to changes in CNS vascular permeability (SNP markers rs6196216 and rs3672065, LOD score = 3.7). CONCLUSIONS: The QTL critical intervals discovered have allowed for compilation of a list of candidate genes implicated in regulating functional deficit and CNS vascular permeability. These genes encode for factors that may be potential targets for therapeutic approaches to treat disorders characterized by CD8 T cell-mediated BBB disruption.

Preserved vascular integrity and enhanced survival following neuropilin-1 inhibition in a mouse model of CD8 T cell-initiated CNS vascular permeability.

BACKGROUND: Altered permeability of the blood-brain barrier (BBB) is a feature of numerous neurological conditions including multiple sclerosis, cerebral malaria, viral hemorrhagic fevers and acute hemorrhagic leukoencephalitis. Our laboratory has developed a murine model of CD8 T cell-initiated central nervous system (CNS) vascular permeability in which vascular endothelial growth factor (VEGF) signaling plays a prominent role in BBB disruption. FINDINGS: In this study, we addressed the hypothesis that in vivo blockade of VEGF signal transduction through administration of peptide (ATWLPPR) to inhibit neuropilin-1 (NRP-1) would have a therapeutic effect following induction of CD8 T cell-initiated BBB disruption. We report that inhibition of NRP-1, a co-receptor that enhances VEGFR2 (flk-1) receptor activation, decreases vascular permeability, brain hemorrhage, and mortality in this model of CD8 T cell-initiated BBB disruption. We also examine the expression pattern of VEGFR2 (flk-1) and VEGFR1 (flt-1) mRNA expression during a time course of this condition. We find that viral infection of the brain leads to increased expression of flk-1 mRNA. In addition, flk-1 and flt-1 expression levels decrease in the striatum and hippocampus in later time points following induction of CD8 T cell-mediated BBB disruption. CONCLUSION: This study demonstrates that NRP-1 is a potential therapeutic target in neuro-inflammatory diseases involving BBB disruption and brain hemorrhage. Additionally, the reduction in VEGF receptors subsequent to BBB disruption could be involved in compensatory negative feedback as an attempt to reduce vascular permeability.

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

BACKGROUND: 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. METHODS: PIFS was induced by intravenous injection of VP2121-130 peptide 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. RESULTS: C57BL/6 and 129 SvIm mice had similar inflammation in the CNS during acute infection. After administration of VP2121-130 peptide, 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. CONCLUSION: 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.

Review and Meta-analysis on the Impact of the ADRA2A Variant rs1800544 on Methylphenidate Outcomes in Attention-Deficit/Hyperactivity Disorder.

Background: Methylphenidate is among the most prescribed medications for treating attention-deficit/hyperactivity disorder (ADHD). However, nearly half of pediatric patients with ADHD do not respond to methylphenidate treatment. Pharmacogenetic testing can aid in identifying patients for whom methylphenidate is unlikely to be safe or effective, leading to improved methylphenidate outcomes and increased use of alternative treatment options for ADHD. This article aimed to summarize findings from studies of the ADRA2A gene variant, rs1800544, and its association with methylphenidate outcomes in ADHD. Methods: We systematically reviewed and meta-analyzed available literature on the impact of rs1800544 on methylphenidate outcomes in ADHD. Results: Fourteen studies met inclusion criteria for review, 9 of which were eligible for meta-analysis. The included studies compared methylphenidate outcomes in patients with ADHD categorized by rs1800544 genotype. G-allele carriers experienced significantly greater improvements in ADHD symptom scores (Swanson, Nolan, and Pelham Version-IV Scale or ADHD Rating Scale-IV) relative to noncarriers (odds ratio 3.08, 95% confidence interval 1.71-5.56, p = .0002) and greater response rates as measured by a ≥50% improvement in symptom scores (odds ratio 2.68, 95% confidence interval 1.23-5.82, p = .01); no significant difference in response rate as measured by Clinical Global Impressions score ≤2 was found. Stouffer's z-score method showed significant improvement across all methylphenidate outcomes in G-allele carriers relative to noncarriers (z = 3.03, p = .002). Conclusions: These findings suggest that carriers of rs1800544 may have improved ADHD outcomes following methylphenidate treatment. However, the extent to which these improvements are clinically impactful remain unclear. Additional studies are required to determine if rs1800544 carrier status should influence clinical recommendations for treatment of ADHD symptoms.

Rapid formation of extended processes and engagement of Theiler's virus-infected neurons by CNS-infiltrating CD8 T cells.

A fundamental question in neuroimmunology is the extent to which CD8 T cells actively engage virus-infected neurons. In the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis, an effective central nervous system (CNS)-infiltrating antiviral CD8 T cell response offers protection from this demyelinating disease. However, the specific CNS cell types engaged by these protective CD8 T cells in TMEV-resistant strains remains unknown. We used confocal microscopy to visualize the morphology, migration, and specific cellular interactions between adoptively transferred CD8 T cells and specific CNS cell types. Adoptively transferred GFP+ CD8+ splenocytes migrated to the brain and became 93% specific for the immunodominant virus epitope D(b):VP2(121-130). These CD8 T cells also polarized T cell receptor, CD8 protein, and granzyme B toward target neurons. Furthermore, we observed CD8 T cells forming cytoplasmic processes up to 45 µm in length. Using live tissue imaging, we determined that these T cell-extended processes (TCEPs) could be rapidly formed and were associated with migratory behavior through CNS tissues. These studies provide evidence that antiviral CD8 T cells have the capacity to engage virus-infected neurons in vivo and are the first to document and measure the rapid formation of TCEPs on these brain-infiltrating lymphocytes using live tissue imaging.

Modulation of peripheral cytotoxic cells and ictogenesis in a model of seizures.

PURPOSE: A link between seizure susceptibility, blood-brain barrier (BBB) failure, and the activation of peripheral white blood cells has been recently proposed. However, the molecular players involved in this cascade of events are unknown. We tested the hypothesis that immunosupression by splenectomy or lack of perforin, a downstream factor of natural killer (NK) and cytotoxic T cells, could reduce seizure onset. METHODS: Pilocarpine was used to induce seizures in adult rats wild-type and perforin-deficient mice. Splenectomy was performed prior to pilocarpine injection. Seizure onset was evaluated by electroencephalography (EEG) and joint time-frequency analysis. Spleens from control and pilocarpine-treated groups were analyzed for anatomical changes and CD3+ cell content. BBB damage was assessed by measuring albumin parenchymal extravasation. Fluorescence-activated cell sorting (FACS) analysis was performed on spleen and brain tissue of wild-type and perforin-deficient mice treated, or not, with pilocarpine. KEY FINDINGS: Splenectomy significantly reduced seizure-associated mortality. Histologic analysis of the spleens exposed to pilocarpine revealed altered white and red pulp anatomy and an increase in CD3+ T cells. Onset of status epilepticus (SE) and mortality were significantly decreased in perforin-deficient mice. Pilocarpine significantly increased spleen NK 1.1 and CD8+ cell percentage; in contrast, the brain inflammatory cell profile remained unchanged at the time of pilocarpine SE. BBB damage was reduced in the perforin-deficient pilocarpine-treated mice. SIGNIFICANCE: Immunosuppressant maneuvers such as splenectomy or lack of perforin decrease the onset or the severity of pilocarpine SE. Our results suggest that cytotoxic lymphocytes, and specifically the cytolytic factor perforin, may be key molecular players involved in the axis between peripheral intravascular inflammation and seizures.

Developmental treatment with the dopamine D2/3 agonist quinpirole selectively impairs spatial learning in the Morris water maze.

Developmental exposure to the dopamine D2/3 receptor agonist quinpirole is reported to induce D2 priming, impair Morris water maze performance, reduce acoustic startle prepulse inhibition (PPI), and alter locomotor activity. We treated rats from postnatal days 1-21 with the dose reported to induce these effects, 1.0 mg/kg/day, and two higher doses, 2.0 and 4.0 mg/kg/day, or saline. Offspring were tested in the Morris water maze, PPI, exploratory locomotor activity, activity after quinpirole and (+)-methamphetamine challenge, elevated zero maze, light-dark box, marble burying, straight channel swimming, and Cincinnati water maze. In the Morris water maze, all quinpirole groups had longer latencies on test days 3-5 of acquisition, but no effects on reversal or shifted-reduced platform trials. The quinpirole 4.0 mg/kg group had significantly reduced mean search distances on probe trials when combined across the 3 phases of testing but not separately. The male 4.0 mg/kg quinpirole group showed a greater increase in methamphetamine-stimulated activity during the first 10 min after drug challenge but not in the remainder of the 2 h test. No quinpirole effects were found for light-dark box, marble burying, exploratory locomotor activity, straight channel, Cincinnati water maze, or locomotor activity after quinpirole challenge. No effects were found on most measures in the elevated zero maze however the quinpirole 4.0 mg/kg females had longer latencies to enter an open quadrant. The results partially support prior Morris maze deficits induced by developmental quinpirole treatment but little evidence of dopamine D2/3 priming was found using locomotor activity with quinpirole or methamphetamine challenge or acoustic startle/PPI. The limited comparability to published data using developmental quinpirole exposure may be attributable to differences in experimental procedures or may be the result of quinpirole having limited effects. The data suggest that caution is warranted concerning the developmental efficacy of quinpirole.

Effects of neonatal (+)-methamphetamine on path integration and spatial learning in rats: effects of dose and rearing conditions.

Postnatal day (P)11-20 (+)-methamphetamine (MA) treatment impairs spatial learning and reference memory in the Morris water maze, but has marginal effects on learning in a labyrinthine maze. A subsequent experiment showed that MA treatment on P11-15, but not P16-20, is sufficient to induce Morris maze deficits. Here we tested the effects of P11-15 MA treatment under two different rearing conditions on Morris maze performance and path integration learning in the Cincinnati water maze in which distal cues were unavailable by using infrared illumination. Littermates were treated with 0, 10, 15, 20, or 25mg/kg MA x 4/day (2 h intervals). Half the litters were reared under standard housing conditions and half under partial enrichment by adding stainless steel enclosures. All MA groups showed impaired Cincinnati water maze performance with no significant effects of rearing condition. In the Morris maze, the MA-25 group showed impaired spatial acquisition, reversal, and small platform learning. Enrichment significantly improved Morris maze acquisition in all groups but did not interact with treatment. The male MA-25 group was also impaired on probe trial performance after acquisition and on small platform trials. A narrow window of MA treatment (P11-15) induces impaired path integration learning irrespective of dose within the range tested but impairments in spatial learning are dependent on dose. The results demonstrate that a narrower exposure window (5 days) changes the long-term effects of MA treatment compared to longer exposures (10 days).

Perforin competent CD8 T cells are sufficient to cause immune-mediated blood-brain barrier disruption.

Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin-/- mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

Depletion of GR-1-Positive Cells Is Associated with Reduced Neutrophil Inflammation and Astrocyte Reactivity after Experimental Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is the stroke subtype with the highest mortality and morbidity with 25% of patients dying within the first 48 h and a high incidence of poor outcomes. Because of high early mortality rates, an understanding of acute brain injury mechanisms is essential. In this study, we have investigated the putative role of acute inflammation in brain injury after experimental ICH. We depleted GR-1(+) cells in mice by intraperitoneal administration of anti-GR-1 antibody or normal rat serum (control). We then induced ICH by infusion of autologous whole blood into the striatum and compared functional outcome and brain injury markers between the two groups. We found that administration of anti-GR-1 antibody led to a profound decrease in circulating GR-1(+) cells (1.5 ± 0.34% vs. 50.3 ± 8.3% of CD45(+) cells, p ≤ 0.01) and that brain neutrophils decreased by approximately 50% (p ≤ 0.05). We observed a reduction in astrocyte immunoreactivity in the GR-1(+) cell-depleted group (p ≤ 0.05). Conversely, we did not find attenuation of brain edema or differences in behavioral deficits between the two groups. In summary, our results are promising and suggest that larger studies or different neutrophil manipulations may produce greater attenuation of injury after ICH.

Unconjugated bilirubin contributes to early inflammation and edema after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a stroke subtype with significant mortality and morbidity. The role of unconjugated bilirubin (UBR) in ICH brain injury is not well understood. Therefore, we studied the effects of UBR on brain injury markers and inflammation, as well as mechanisms involved therein. We induced ICH in mice by infusion of autologous whole blood with vehicle (dimethyl sulfoxide) or UBR. We found that UBR led to an increase in edema (P≤0.05), but a decrease in nitrate/nitrite formation (7.0±0.40 nmol/mg versus 5.2±0.70 nmol/mg protein, P≤0.05) and no change in protein carbonyls. Unconjugated bilirubin was also associated with an increase in neutrophil infiltration compared with ICH alone, as determined by both immunofluorescence and flow cytometry (36%±3.2% versus 53%±1.3% of CD45(+) cells, P≤0.05). In contrast, we observed reduced perihematomal microglia immunoreactivity in animals receiving UBR (P≤0.05). Using in vitro techniques, we show neutrophil activation by UBR and also show that protein kinase C participates in this signaling pathway. Finally, we found that UBR was associated with an increased expression of the leukocyte adhesion molecule intercellular adhesion molecule-1. Our results suggest that UBR possesses complex immune-modulatory and antioxidant effects.