Charles L. Howe, Ph.D. - US grants

DESCRIPTION (provided by applicant): Foodborne and waterborne picornaviruses such as enterovirus 71 are a global health issue. Neurologic complications associated with neurovirulent non-polio picornavirus infection are a serious ongoing health problem, especially in children. Unfortunately, the mechanisms of picornavirus-induced injury to the central nervous system (CNS) are unclear. We propose that the innate immune response is an important cause of neuron death during acute infection. This is in contrast to the prevailing hypothesis that neuron loss is mediated solely by virus. While we do not doubt that some neurons die directly as the result of viral infection, our preliminary findings suggest that certain populations, such as CA1 pyramidal neurons in the hippocampus, are killed by the innate immune response rather than by the virus. We have established a mouse model of picornavirus infection of the CNS using the Theiler's murine encephalomyelitis virus to directly test the role of neutrophils in the initiation of neuronal apoptosis. Our preliminary evidence indicates that during acute picornaviral infection of the CNS, many uninfected CA1 pyramidal neurons undergo apoptotic death associated with oxidative injury, calpain activity, and caspase activity;this injury severely reduces cognitive performance in a spatial memory test. We have further observed that neutrophils infiltrate the hippocampus within hours of infection. Reduced neutrophil infiltration is neuroprotective, while adoptive transfer of activated neutrophils into mice with a defective neutrophil response induces hippocampal injury. Finally, treatment with calpain inhibitors protects hippocampal neurons from death and preserves cognitive function without constraining the inflammatory response that is necessary to mediating host defense and viral clearance. On the basis of these observations we hypothesize that neutrophils kill hippocampal neurons via a calpain-dependent mechanism during acute picornaviral infections of the CNS. We intend to address the following experimental questions: 1) are neutrophils necessary and sufficient to kill hippocampal neurons?;2) is calpain the key executioner of hippocampal neurons during death induced by the neutrophil response to acute CNS infection? We propose several innovations, including the use of live animal imaging and adoptive transfer of neutrophils, to address these questions. The key concept of our proposal is that while inflammation critically mediates host defense to virus infection, the inflammatory response may indirectly kill neurons, and therefore therapeutic interventions aimed at preventing neuronal death without thwarting inflammatory control of virus may preserve host function. PUBLIC HEALTH RELEVANCE: Certain foodborne and waterborne viruses have the ability to infect the brain. Although adults are susceptible, children are at particular risk for such neurovirulent infections. We have evidence from a mouse model that cognitive function is lost concomitantly with the death of hippocampal neurons. We also have evidence that this neuronal death is caused by a specific population of immune cells called neutrophils that are trying to clear the virus from the brain. Importantly, we have found that treatment with an FDA-approved drug protects neurons and cognitive function without altering the ability of the immune system to clear the virus from the brain.

DESCRIPTION (provided by applicant): Brain-and spinal cord-infiltrating inflammatory monocytes and neutrophils contribute to pathogenesis, injury, and repair/regeneration in a wide array of neurologic diseases, including stroke, epilepsy, demyelinating disease, Alzheimer disease, ALS, cancer, pain, TBI, spinal cord injury, and infection. While a number of surface markers exist that provide variable and overlapping resolution of different myelomonocytic populations, these tools are suboptimal for identifying, tracking, and quantitating monocytes and neutrophils in target tissues such as the brain. The development of the LysM-eGFP mouse by Graf, in which only cells of myelomonocytic lineage express GFP, has provided a far more sophisticated tool for following monocytes and neutrophils. At the same time, a burgeoning but conflicted literature indicates that neutrophil and monocyte recruitment to the CNS is quite complex and dependent upon a variety of chemokines and chemokine receptor interactions. For example, in general, neutrophil trafficking may depend upon signaling through the CXCR2 and CXCR3 axis, while monocyte trafficking may depend upon CCR2 and CCR5 receptors. Based on this concept, and in an effort to dissect the role of specific chemokine receptors in the trafficking of myelomonocytic cells to the CNS, our first objective in this small grant proposal is to cross LysM-eGFP mice with mice that are homozygously deficient in CCR2, CCR5, CXCR2, or CXCR3. Our second objective is to characterize the kinetics and spatial distribution of myelomonocytic cells infiltrating the brain in mice infected with the Theiler's murine encephalomyelitis virus. To accomplish this objective, we intend to use a fiber optic-based fluorescence endoscope to acquire deep tissue images of GFP-positive neutrophil and inflammatory monocyte trafficking in live animals and determine whether chemokine receptor deficiency alters the trafficking of the cells. Our long- term goals are to use these four lines to identify the factors responsible for leukocyte infiltration into the CNS and to assess the temporal inter-relation between myelomonocytic cells by thwarting infiltration of one population (for example neutrophils via CXCR2 deficiency) and quantifying the infiltration of other populations (for example monocytes). We intend to use these mouse models to determine basic aspects of leukocyte trafficking into the brain in our specific virus model and to make these lines available to other investigators studying stroke, TBI, spinal cord injury, etc. This project is innovative because it will generate new mouse models for more carefully studying neutrophil and inflammatory monocyte trafficking into the CNS and because it employs a fiber optic microscope to observe the trafficking of these cells within deep brain structures in living animals. Our proposed project is significant because it is expected to provide tools that will resolve a number of conflicting concepts regarding the mechanisms of leukocyte trafficking to the CNS. By extending our knowledge of neutrophil and monocyte trafficking mechanisms, these tools have the potential to greatly impact the development of therapeutic strategies for ameliorating human disease.

DESCRIPTION (provided by applicant): The cell type responsible for transmission of immunologically relevant information from the central nervous system (CNS) to the peripheral immune system is currently unclear. The identification of this cell is central to understanding the genesis of innate and adaptive immune responses to CNS infection, the maladaptive immunological response to self that occurs in CNS autoimmune disease, and the escape from immunological assault that protects CNS tumors. The field has largely focused on dendritic cells and microglia as antigen presenting cells within the CNS that drive T cell restimulation, but the capacity of these cells to transmit information to the lymph nodes for original T cell priming is limited or unstudied. A long-term goal is to identify cells that carry neural antigens to the peripheral lymph nodes in order to therapeutically modify, modulate, or abrogate their function. The specific objective of this proposal is to test the ability of CD45hiCD11bhiLy6Chi Ly6Gneg inflammatory monocytes to transmit antigens from the acutely infected brain to the peripheral immune system. The central hypothesis is that inflammatory monocytes which infiltrate the brain in response to specific chemokine signals are a critical antigen-presenting cell with the capacity to exit the brain and carry antigen to the cervical lymph nodes (CLNs). The hypothesis is based upon preliminary data showing that brain-infiltrating inflammatory monocytes acquire protein antigens and virus within the brain in the first 18-24 hours after infection and carry this information to the CLNs. The main hypothesis will be tested by pursuing two specific aims: 1) To identify the mechanisms responsible for recruitment and infiltration of inflammatory monocytes into the brain during the first 48 hours of virus infection; and 2) To determine if brain-infiltratng inflammatory monocytes acquire a dendritic cell phenotype, exit the brain carrying viral antigens, and function as antigen- presenting cells in the CLNs. The experimental approach will utilize LysM:GFP reporter mice acutely infected with the Theiler's murine encephalomyelitis virus and will exploit genetic and immunological manipulation of chemokines, chemokine receptors and adhesion factors as well as adoptive transfers to test the main hypothesis. The approach is innovative because it uses a model system in which brain-restricted antigens are acquired by infiltrating inflammatory monocytes that then carry these antigens to the periphery to drive T cell responses. The proposed research is significant because it identifies an inflammatory responder that infiltrates the brain, acquires immunological information, and transmits that information to the peripheral immune system, providing an entirely new target for therapeutic and pharmacological modulation of CNS immune responses.

Cognitive decline, whether in the context of neurodegeneration and dementia or in the more colloquial setting of normal aging, is the result of multifactorial processes that operate decades before overt manifestations. While some of the genetic risk factors that contribute to these processes are understood, it is not clear why individuals decline at different rates and why some people experience more cognitive impairment simply as a result of getting older. Likewise, while neuroinflammation is known to play a significant role in neurodegeneration, the factors that precipitate such neuroinflammation are unclear, and it is unknown whether neuroinflammation begets neurodegeneration or vice versa. Finally, the role of peripheral inflammatory cell infiltration into the brain relative to the role of local neuroinflammation in driving or exacerbating neurodegeneration is poorly defined. A provocative hypothesis is that mild brain inflammation associated with numerous CNS and peripheral infections - infections that in themselves are common, acute, self-resolving, and subclinical - is an environmental trigger for the inexorable accumulation of neuronal injury and loss that culminates in memory impairment later in life. The specific hypothesis tested in this proposal is that infiltration of inflammatory monocytes into the brain during subclinical viral encephalitis in young adult mice accelerates neurodegeneration mediated by mutant tau expression. A well-characterized picornavirus model of acute encephalitis will be used to study infections that exhibit mild neuroinvasive properties and intranasal inoculation with influenza virus will be used to model peripheral infections that drive brain inflammation despite the absence of tropism to the CNS. In order to rapidly exploit current viral models and immune targeted mouse strains without extensive backcrossing and to alleviate issues surrounding MHC haplotype-dependent responses to infection, mice will be transduced via intracerebral injection of adeno-associated viruses encoding neuron-specific P301S mutant tau to drive neurodegeneration. These experiments are significant because they will uncover interactions between viral infection-induced brain inflammation and later life cognitive decline within the context of a genetic predisposition to such impairment. This may reveal novel therapeutic strategies that will ameliorate aging-related degradation of cognitive performance. The hypothesis that mild viral encephalitis in young adulthood accelerates tauopathy-associated neurodegeneration is conceptually innovative and the toolbox employed to test this hypothesis is technically innovative. Findings in this study will provide a sustained, powerful influence on the field by revealing the interactions of virus infection, microglial activation, inflammatory monocyte infiltration into the brain, and the evolution of neurodegeneration. This work will also support future efforts to determine whether multiple mild infections over the lifetime of an individual result in accumulation of neuroinflammatory insults and neural injury that culminates in age-related cognitive decline in the absence of genetic drivers.

There is a critical unmet need to identify new strategies to control seizures in individuals with epilepsy who fail to respond to currently available drugs. Many of these individuals undergo invasive surgical resection of electroencephalographically aberrant tissue. However, seizures are likely to recur in up to half of these subjects within 5 years of surgery. Resistance to therapies that target electrophysiological mechanisms of aberrant neural activity coupled to post-surgical recurrence of seizures in previously non-ictal tissue may indicate a role for epileptogenic drivers that are non-neural and self-amplifying. Multiple studies have demonstrated changes in peripheral inflammatory factors in individuals with epilepsy, and steroids and other immunomodulatory therapies have proven effective in some patients. Likewise, evidence from animal models clearly supports a role for cytokines such as TNF? and IL1? in seizure activity. Therefore, neuroinflammation may be a critical driver of drug-resistant epilepsy. The central hypothesis of this proposal is that aberrant neural activity triggers local release of chemokines and cytokines that promote infiltration of innate inflammatory effector cells, production of additional inflammatory mediators, and further disruption of neural circuitry. Breaking this cycle may stop ictogenesis and/or epileptogenesis. The specific hypothesis of this proposal is that levels of the chemoattractant CCL2 and the effector cytokines TNF?, IL1?, and/or IL6 are elevated in spatial and temporal association with chemically induced epileptiform activity. This hypothesis will be tested using a strategy based on simultaneous collection of intracortical EEG activity and large molecule microdialysis to measure inflammatory mediators in the extracellular fluid of the peri-electrode space in mice and pigs and in humans undergoing resective surgery for drug-resistant epilepsy. Despite circumstantial evidence in humans indicating a role for inflammation in seizure disorders and epilepsy, no study has yet measured the in situ inflammatory characteristics of the epileptic brain or assessed the relationship between epileptiform activity and local release of inflammatory molecules. Though brain microdialysis has been established as a technique in the neurocritical care setting for assessment of small molecules, this study will be the first to employ an innovative strategy that combines intracranial EEG collection and the use of high molecular weight cut-off membranes (100 kDa) for the capture of chemokines and cytokines in the peri- electrode space. These experiments are significant as they will provide novel insights into the role of inflammatory mediators as both cause and effect of neural circuit dysfunction and they may identify individual inflammatory drivers that can be targeted for personalized treatment strategies. Regardless of outcomes, this study will generate new, fundamental knowledge about the interplay between seizure activity and inflammation. Understanding this relationship may provide support for the use of immunomodulatory therapies in the millions of individuals with epilepsy that are currently underserved by current standards of care.

There is a critical unmet need to identify the pathogenic mechanisms that drive disease progression in patients with multiple sclerosis (MS). Accumulation of axon injury and functional disability in MS are not adequately impacted by current therapies. The long-term goal of this work is to discover new strategies to prevent or reverse disease progression in MS. Despite evidence that CD8+ T cells are associated with axon injury and progression in MS, the functional role for these cells and the relevant mechanisms required for recruitment of these cells to the demyelinated brain are unknown. The antigenic targets of neuron-specific CD8+ T cells are also unknown. The overall objectives of this study are to test the mechanistic role of neuron antigen-specific CD8+ T cells in the injury of demyelinated axons and to determine whether patients with MS have such cells. The rationale is that axon injury is the primary substrate of progression in MS, axonal MHC class I expression is upregulated by inflammation and demyelination, and cytotoxic CD8+ T cells directed against neuron-specific antigens injure demyelinated axons. The central hypothesis of this proposal is that neuron antigen-specific CD8+ T cells injure demyelinated axons. Guided by strong preliminary evidence, the hypothesis will be tested using AAV-mediated transduction of neurons to drive expression of the neoantigen ovalbumin (OVA) within the context of CNS demyelination induced by cuprizone toxicity or immunization against a myelin oligodendrocyte glycoprotein-derived peptide (MOG-EAE) in hosts that have transgenic CD8+ T cells directed against the OVA- derived peptide SIINFEKL (OT-I). The study will also use autologous T cells and fibroblast-derived iPSC- derived neurons grown in microfluidic chambers to determine whether MS patient CD8+ T cells injure their own axons. Three specific aims will be pursued: 1) determine the mechanisms of CD8+ T cell-mediated axon injury in the demyelinated CNS; 2) identify the cellular locus of MHC class I expression required for axon injury and determine how demyelination drives CNS infiltration of neuron antigen-specific CD8+ T cells; 3) determine whether MS patients have neuron-antigen specific CD8+ T cells. This approach is conceptually innovative because of the proposal that demyelination and inflammation induce axonal presentation of self-peptides on MHC class I. The approach is technically innovative based on the use of novel AAV vectors to drive neoantigens in neurons within the demyelinated CNS, selective deletion of brain-resident antigen presenting cells (APCs) vs peripheral APCs, use of multiple host manipulations coupled to adoptive transfer of traceable effector cells to temporally and spatially profile anti-neuronal T cell trafficking, and the use of patient-derived neurons and autologous CD8+ T cells. This work will make a significant, powerful impact on the field by revealing the capacity of CD8+ T cells directed against neuron-specific antigens to injure demyelinated axons and by identifying such T cells in patients with MS.