Reghann G. LaFrance-Corey

Apoptosis of hippocampal pyramidal neurons is virus independent in a mouse model of acute neurovirulent picornavirus infection.

Many viruses, including picornaviruses, have the potential to infect the central nervous system (CNS) and stimulate a neuroinflammatory immune response, especially in infants and young children. Cognitive deficits associated with CNS picornavirus infection result from injury and death of neurons that may occur due to direct viral infection or during the immune responses to virus in the brain. Previous studies have concluded that apoptosis of hippocampal neurons during picornavirus infection is a cell-autonomous event triggered by direct neuronal infection. However, these studies assessed neuron death at time points late in infection and during infections that lead to either death of the host or persistent viral infection. In contrast, many neurovirulent picornavirus infections are acute and transient, with rapid clearance of virus from the host. We provide evidence of hippocampal pathology in mice acutely infected with the Theilers murine encephalomyelitis picornavirus. We found that CA1 pyramidal neurons exhibited several hallmarks of apoptotic death, including caspase-3 activation, DNA fragmentation, and chromatin condensation within 72 hours of infection. Critically, we also found that many of the CA1 pyramidal neurons undergoing apoptosis were not infected with virus, indicating that neuronal cell death during acute picornavirus infection of the CNS occurs in a non-cell-autonomous manner. These observations suggest that therapeutic strategies other than antiviral interventions may be useful for neuroprotection during acute CNS picornavirus infection.

Febrile infection‐related epilepsy syndrome treated with anakinra

Febrile infection‐related epilepsy syndrome (FIRES) is a devastating epileptic encephalopathy with limited treatment options and an unclear etiology. Anakinra is a recombinant version of the human interleukin‐1 receptor antagonist used to treat autoinflammatory disorders. This is the first report of anakinra for treatment of a child with super‐refractory status epilepticus secondary to FIRES. Anakinra was well tolerated and effective. Cerebral spinal fluid analysis revealed elevated levels of proinflammatory cytokines before treatment that normalized on anakinra, suggesting a potential pathogenic role for neuroinflammation in FIRES. Further studies are required to assess anakinra efficacy and dosing, and to further delineate disease etiology. Ann Neurol 2016;80:939–945

Inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain.

BackgroundNeuropathology caused by acute viral infection of the brain is associated with the development of persistent neurological deficits. Identification of the immune effectors responsible for injuring the brain during acute infection is necessary for the development of therapeutic strategies that reduce neuropathology but maintain immune control of the virus.MethodsThe identity of brain-infiltrating leukocytes was determined using microscopy and flow cytometry at several acute time points following intracranial infection of mice with the Theilers murine encephalomyelitis virus. Behavioral consequences of immune cell depletion were assessed by Morris water maze.ResultsInflammatory monocytes, defined as CD45hiCD11b++F4/80+Gr1+1A8-, and neutrophils, defined as CD45hiCD11b+++F4/80-Gr1+1A8+, were found in the brain at 12 h after infection. Flow cytometry of brain-infiltrating leukocytes collected from LysM: GFP reporter mice confirmed the identification of neutrophils and inflammatory monocytes in the brain. Microscopy of sections from infected LysM:GFP mice showed that infiltrating cells were concentrated in the hippocampal formation. Immunostaining confirmed that neutrophils and inflammatory monocytes were localized to the hippocampal formation at 12 h after infection. Immunodepletion of inflammatory monocytes and neutrophils but not of neutrophils only resulted in preservation of hippocampal neurons. Immunodepletion of inflammatory monocytes also preserved cognitive function as assessed by the Morris water maze.ConclusionsNeutrophils and inflammatory monocytes rapidly and robustly responded to Theilers virus infection by infiltrating the brain. Inflammatory monocytes preceded neutrophils, but both cell types were present in the hippocampal formation at a timepoint that is consistent with a role in triggering hippocampal pathology. Depletion of inflammatory monocytes and neutrophils with the Gr1 antibody resulted in hippocampal neuroprotection and preservation of cognitive function. Specific depletion of neutrophils with the 1A8 antibody failed to preserve neurons, suggesting that inflammatory monocytes are the key effectors of brain injury during acute picornavirus infection of the brain. These effector cells may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing central nervous system pathology associated with acute viral infection.

Hippocampal protection in mice with an attenuated inflammatory monocyte response to acute CNS picornavirus infection

Neuronal injury during acute viral infection of the brain is associated with the development of persistent cognitive deficits and seizures in humans. In C57BL/6 mice acutely infected with the Theilers murine encephalomyelitis virus, hippocampal CA1 neurons are injured by a rapid innate immune response, resulting in profound memory deficits. In contrast, infected SJL and B6xSJL F1 hybrid mice exhibit essentially complete hippocampal and memory preservation. Analysis of brain-infiltrating leukocytes revealed that SJL mice mount a sharply attenuated inflammatory monocyte response as compared to B6 mice. Bone marrow transplantation experiments isolated the attenuation to the SJL immune system. Adoptive transfer of B6 inflammatory monocytes into acutely infected B6xSJL hosts converted these mice to a hippocampal damage phenotype and induced a cognitive deficit marked by failure to recognize a novel object. These findings show that inflammatory monocytes are the critical cellular mediator of hippocampal injury during acute picornavirus infection of the brain.

Demyelinated Axons and Motor Function Are Protected by Genetic Deletion of Perforin in a Mouse Model of Multiple Sclerosis

Axon injury is a major determinant of the loss of neurological function in patients with multiple sclerosis. It is unclear, however, whether damage to axons is an obligatory consequence of demyelination or whether it is an independent process that occurs in the permissive environment of demyelinated lesions. Previous investigations into the role of CD8+ T cells and perforin in the Theiler murine encephalomyelitis virus model of multiple sclerosis have used mouse strains resistant to Theiler murine encephalomyelitis virus infection. To test the role of CD8+ T cells in axon injury, we established a perforin-deficient mouse model on the H-2q major histocompatibility complex background thereby removing confounding factors related to viral biology in this Theiler murine encephalomyelitis virus-susceptible strain. This permitted direct comparison of clinical and pathological parameters between perforin-competent and perforin-deficient mice. The extent of demyelination was indistinguishable between perforin-competent and perforin-deficient H-2q mice, but chronically infected perforin-deficient mice exhibited preservation of motor function and spinal axons despite the presence of spinal cord demyelination. Thus, demyelination is necessary but insufficient for axon injury in this model; the absence of perforin protects axons without impacting demyelination. These results suggest that perforin is a key mediatorof axon injury and lend additional support to the hypothesisthat CD8+ T cells are primarily responsible for axon damage in multiple sclerosis.

CD8 + T Cells Cause Disability and Axon Loss in a Mouse Model of Multiple Sclerosis

Background The objective of this study was to test the hypothesis that CD8+ T cells directly mediate motor disability and axon injury in the demyelinated central nervous system. We have previously observed that genetic deletion of the CD8+ T cell effector molecule perforin leads to preservation of motor function and preservation of spinal axons in chronically demyelinated mice. Methodology/Principal Findings To determine if CD8+ T cells are necessary and sufficient to directly injure demyelinated axons, we adoptively transferred purified perforin-competent CD8+ spinal cord-infiltrating T cells into profoundly demyelinated but functionally preserved perforin-deficient host mice. Transfer of CD8+ spinal cord-infiltrating T cells rapidly and irreversibly impaired motor function, disrupted spinal cord motor conduction, and reduced the number of medium- and large-caliber spinal axons. Likewise, immunodepletion of CD8+ T cells from chronically demyelinated wildtype mice preserved motor function and limited axon loss without altering other disease parameters. Conclusions/Significance In multiple sclerosis patients, CD8+ T cells outnumber CD4+ T cells in active lesions and the number of CD8+ T cells correlates with the extent of ongoing axon injury and functional disability. Our findings suggest that CD8+ T cells may directly injure demyelinated axons and are therefore a viable therapeutic target to protect axons and motor function in patients with multiple sclerosis.

Neuromyelitis optica IgG stimulates an immunological response in rat astrocyte cultures.

Neuromyelitis optica (NMO) is a primary astrocyte disease associated with central nervous system inflammation, demyelination, and tissue injury. Brain lesions are frequently observed in regions enriched in expression of the aquaporin‐4 (AQP4) water channel, an antigenic target of the NMO IgG serologic marker. Based on observations of disease reversibility and careful characterization of NMO lesion development, we propose that the NMO IgG may induce a dynamic immunological response in astrocytes. Using primary rat astrocyte‐enriched cultures and treatment with NMO patient‐derived serum or purified IgG, we observed a robust pattern of gene expression changes consistent with the induction of a reactive and inflammatory phenotype in astrocytes. The reactive astrocyte factor lipocalin‐2 and a broad spectrum of chemokines, cytokines, and stress response factors were induced by either NMO patient serum or purified IgG. Treatment with IgG from healthy controls had no effect. The effect is disease‐specific, as serum from patients with relapsing–remitting multiple sclerosis, Sjögrens, or systemic lupus erythematosus did not induce a response in the cultures. We hypothesize that binding of the NMO IgG to AQP4 induces a cellular response that results in transcriptional and translational events within the astrocyte that are consistent with a reactive and inflammatory phenotype. Strategies aimed at reducing the inflammatory response of astrocytes may short circuit an amplification loop associated with NMO lesion development. GLIA 2014;62:692–708

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theilers murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 μm, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1(+), CD11b(+) and F4/80(+)cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

Neuroprotection mediated by inhibition of calpain during acute viral encephalitis

Neurologic complications associated with viral encephalitis, including seizures and cognitive impairment, are a global health issue, especially in children. We previously showed that hippocampal injury during acute picornavirus infection in mice is associated with calpain activation and is the result of neuronal death triggered by brain-infiltrating inflammatory monocytes. We therefore hypothesized that treatment with a calpain inhibitor would protect neurons from immune-mediated bystander injury. C57BL/6J mice infected with the Daniel’s strain of Theiler’s murine encephalomyelitis virus were treated with the FDA-approved drug ritonavir using a dosing regimen that resulted in plasma concentrations within the therapeutic range for calpain inhibition. Ritonavir treatment significantly reduced calpain activity in the hippocampus, protected hippocampal neurons from death, preserved cognitive performance, and suppressed seizure escalation, even when therapy was initiated 36 hours after disease onset. Calpain inhibition by ritonavir may be a powerful tool for preserving neurons and cognitive function and preventing neural circuit dysregulation in humans with neuroinflammatory disorders.

Neuropilin-1 modulates interferon-γ-stimulated signaling in brain microvascular endothelial cells

ABSTRACT Inflammatory response of blood–brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis; however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ)-induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1-dependent manner. Moreover, endothelial-specific NRP1-knockout mice, VECadherin-Cre-ERT2/NRP1flox/flox mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neuron demyelination, altered lymphocyte infiltration, preserved BBB function and decreased activation of the STAT1–CXCL10 pathway. Furthermore, increased expression of NRP1 was observed in endothelial cells of acute multiple sclerosis lesions. Our data identify a new molecular mechanism of brain microvascular endothelial inflammatory response through NRP1–IFNγ crosstalk that could be a potential target for intervention of endothelial cell dysfunction in neuroinflammatory diseases. Summary: This is the first study to define the role of neuropilin-1, a co-receptor of several structurally distinct cytokines, in the IFNγ-induced inflammatory response of endothelial cells in neuroinflammatory diseases.