The long-term goals of this program are an understanding of central nervous system (CNS) demyelinating disease. To this end, this program contains a multidisciplinary approach to defining mechanisms of myelin loss using a well defined rnurine model of demyelination induced by the neurotropic JHM strain (MHV-4) of mouse hepatitis virus (JHMV). This model provides a means to understand the interactions between a pathogen and its natural host that result in demyelination during acute and persistent CNS infection. Although the host response is competent to control infectious virus, a persistent infection without detectable infectious virus is associated with chronic ongoing myelin loss, which has numerous similarities to multiple sclerosis, the most prevalent human demyelinating disease. This program is unique, offering a stable core of investigators addressing fundamental questions of viral persistence and immune responses, both as protective mechanisms and as inducers of demyelination. Funding is requested for continuation of Project 12 (Norman Marten), Project 2 (Stephen Stohlman) and Project 10 (Cornelia Bergmann) and for addition of a new project directed by Thomas Lane. Project 12 focuses on the initial events in CNS inflammation including the role of innate cytokines and neutrophils in shaping both the innate and adaptive CNS immune response, and an exciting new approach to understanding how T cells traffic through the CNS parenchyma. Project 2 continues an examination of the role of distinct CD8+ T cell effectors mechanisms in controlling virus replication in oligodendroglia and astrocytes, the mechanisms used by oligodendroglia to resist perforin mediated cytolysis and the negative co-receptors which limit T cell effector function leading to persistent infection. Project 10 analyzes the role of MHC and costimulatory molecule expression by individual CNS cell types in modulating both acute and chronic demyelination. Project 13 focuses on the role(s) of the chemokine IP-10. Specifically, molecular regulation of IP-10 expression in JHMV infected astrocytes will be examined. In addition, the cellular basis for IP-10 mediated attraction of both T cells and macrophages into the CNS is determined. Data obtained from these projects will add to the basic understanding of viral pathogenesis within the CNS and importantly, provide valuable information on the interactions of specific CNS cells involved in viral persistence and demyelination with the cellular and soluble mediators of the host immune response. PROJECT 1 P.I.: Norman Marten Title: Regulation of CNS Inflammatory Cell Entry and Trafficking Description (provided by applicant) JHM virus (JHMV) infection induces an acute inflammatory response restricted to the CNS, which controls viral replication. However, it fails to fully clear virus, thereby setting the stage for a persistent CNS infection. Chronic JHMV infection is associated with persisting viral antigen and viral RNA in conjunction with ongoing demyelination. T cells play a distinct role in both viral clearances during acute infection as well as contributing to demyelination during chronic infection. Thus, rapid T cell infiltration during acute disease is crucial to controlling virus replication in order to limit subsequent immune mediated pathogenesis. The mechanisms, which govern the trafficking of inflammatory cells into the CNS during acute viral induced encephalomyelitis, are relatively unknown. The goals of this project are to identify the mechanisms governing inflammatory cell entry into the CNS.
Aim 1 investigates the role of IFN-??? in mediating CNS inflammation. IFN-???R-/- mice will be compared to wt mice to determine the influence of IFN-???? in inducing pro and anti-inflammatory, genes, including cytokines, matrix metalloproteinases (MMPs) and tissue inhibiters of MMPs (TIMPs). The kinetics and quantity of inflammatory cell recruitment reveal the roles of IFN-???? in shaping both the innate and adaptive CNS responses to viral infection. Definition of the mechanisms of IFN-???? anti-viral activity are determined via analysis of mice deficient in the two major IFN induced anti-viral pathways.
Aim 2 examines the contribution of neutrophils to regulating both the loss of blood brain barrier integrity and in shaping the overall inflammatory response within the CNS. Transient depletion of neutrophils results in dramatic reduction in CNS recruitment of inflammatory cells. CXCL2-/- mice, defective in neutrophil trafficking, will be used to characterize the proinflammatory role of neutrophils. JHMV infection is associated with induction of MMP-9 protein, a protease involved in breakdown of basement membranes. The factors which mediates initial neutrophil infiltration will be examined via infection of MMP-9-/- mice.
Aim 3 investigates a novel model of impaired intra-parenchymal inflammatory cell trafficking. Infection of mice doubly deficient in performing and IFN-? (PKO/GKO) results in accumulation of inflammatory cells in the subarachnoid and perivascular areas. By contrast, infection of these mice following reconstitution with CD8+T cells from normal donors, allows trafficking of the inflammatory cells into the CNS parenchyma. Infection of PKO/GKO mice is associated with increased expression of the MMP inhibitor TIMP-1. We will test the hypothesis that unregulated virus growth increases expression of TIMP-1, preventing MMP mediated leukocyte migration through the CNS parenchyma.
