Date of Award

Spring 2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Richard P. Ciavarra

Committee Member

Julie A. Kerry

Committee Member

Christopher J. Osgood

Committee Member

Edward M. Johnson

Abstract

Evidence is presented herein that intranasal application of vesicular stomatitis virus (VSV) caused acute infection of the murine central nervous system (CNS) with associated morbidity and significant mortality in mice. However, VSV encephalitis was not invariably fatal, suggesting that the CNS contained a professional antigen-presenting cell (APC) capable of inducing or propagating a protective antiviral immune response. To examine this possibility, we administered VSV via the intranasal route and then characterized the cellular elements that infiltrate the brain as well as the activation status of resident microglia, cells widely believed to represent the major APC population in the CNS. To exclude a contribution of peripheral dendritic cells (DCs), a population with documented APC activity in vivo and capable of infiltrating the inflamed CNS, studies were conducted in a conditional ablation transgenic mouse model that allowed for the selective depletion of these cells with diphtheria toxin (DT). Microglia isolated from infected brains rapidly upregulated expression of major histocompatibility complex (MHC) class I antigens, program death receptor 1 (PD-1) and underwent a microgliosis indicating virus-induced activation and expansion. Peripheral blood cells started to infiltrate the virus infected brain around days 4–5, peaked on day 8 and achieved basal trace levels at 21 days post infection. The infiltrate was composed primarily of myeloid cells (CD45highCD11b +), CD4+ and CD8+ T cells; the latter subset containing cells specific for the immunodominant VSV nuclear protein epitope. Although the CD45highCD11b+ phenotype suggests that these cells are macrophages, subsequent 4–5-color flow cytometry demonstrated that this was primarily a granulocytic response (CD45 highCD11b+Gr-1+F4/80−). The T cell infiltrate correlated temporally with a rapid and sustained upregulation of class I expression on microglia, whereas class II expression was markedly delayed. Ablation of peripheral DCs profoundly inhibited the inflammatory response as well as infiltration of virus-specific CD8+ T cells and this correlated with inefficient viral clearance in the brain and increased morbidity/mortality. Unexpectedly, the VSV-induced interferon-gamma (IFN-γ) response in the CNS remained intact in mice rendered deficient of DCs suggesting that a resident brain cell produced this cytokine. In summary, these studies expanded prior work on the changes in resident microglia and the composition of brain leukocytes in the encephalitic brain. In addition, data demonstrated that peripheral DCs play an essential role in vivo in the inflammatory and certain components of the adaptive primary antiviral immune response in the CNS and as a result, modulate viral clearance and survivability for this pathogen.

Because macrophages may also function as APCs in vivo, we used two different approaches to ablate peripheral macrophages in vivo to examine their role in VSV encephalitis and viral clearance in the CNS. Chemical ablation of macrophages using AP20187 in the MAFIA (macrophage fas-induced apoptosis) conditional ablation model did not diminish microglia numbers or activation and failed to alter the cellular composition of the infiltrate. In contrast, ablation of phagocytic cells by intravenous liposome-encapsulated clodronate administration markedly suppressed the influx of leukocytes into the brain including CD8+VSV-specific T cells and enhanced morbidity/mortality despite normal viral clearance from the brain. Intracerebroventricular (ICV) infusion of clodronate did not impair VSV encephalitis and eliminated a role for brain perivascular macrophages (PVM) and/or meningeal macrophages as contributors to viral clearance or antigen presentation.

In summary, VSV encephalitis was characterized by i) microglial activation and expansion, ii) a prominent and early myeloid infiltrate dominated by granulocytes with smaller contributions of CD11c+ DCs and macrophages, iii) a lymphoid infiltrate of CD4+ and CD8+ T cells including a subset specific for the immunodominant nuclear protein epitope and iv) the absence of significant numbers of pDCs, B cells, NK and NK T cells. A role for peripheral DCs in the development of innate and adaptive antiviral immunity in the CNS, viral clearance and survivability was demonstrated in vivo using a conditional ablation mouse model. Enhanced VSV encephalitis was observed following depletion of circulating or brain-resident macrophages in the MAFIA transgenic model and following ICV infusion of liposome-encapsulated clodronate. These data suggest that macrophages enhance pathogenesis during viral infections of the CNS.

Comments

A Dissertation Submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/jfp7-zk61

ISBN

9781124148106

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