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Tracking viral entry into target cells by virological and immunological methods. Do differentiated macrophages display profoundly different metabolic profiles, reflecting their different functions?

Fitzpatrick, Martin (2011)
M.Res. thesis, University of Birmingham.

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Abstract

Epstein-Barr virus is a γ-herpesvirus endemic (>90%) in the human population and implicated in a variety of lymphomas and solid tumours including Hodgkin’s lymphoma, Burkitt’s lymphoma and post-transplant lymphoproliferative disorder. Despite the prevalence of EBV, little is known about the mechanisms of early infection, including the role of the various membrane glycoproteins to entry, fusion and escape. Additionally, although the long term immune response is well characterised, the contribution of CD4+ and CD8+ T cell recognition to surveillance of primary cellular infection is not known. In order to further understand the processes of virus entry and processing we have used a
panel of glycoprotein knockout viruses and viral epitope-specific CD4+ CD8+ T cell clones to study EBV infection of primary B cells. We have shown that following binding
uptake is rapid yet selective, with suggested roles for gp42 and gp85 in attachment and initiation of fusion. Endocytosis results in rapid up-regulation of primary resting B cell endosomal activity, although no further surface-bound EBV is internalised. Endosomal trafficking of endocytosed virus is rapid, reaching the late endosomal compartment within the first hour, and subsequent HLA class II loading and presentation within 8 hours of binding. Further, both membrane and capsid proteins were seen in the late endosome by confocal, indicating relatively low fusion efficiency. The endocytic/HLA class II pathway may therefore offer a promising target for CD4+ T cell immunotherapy offering a first line of defence during de novo infection.

Macrophages have a wide range of immunological and non-immunological functions in the host, ranging from clearance of apoptotic cells, tissue remodelling, and release of pro and anti-inflammatory mediators at site of tissue damage or infection. Within these wide-ranging functions however, subsets of macrophages show unique phenotypic adaptations to their role. Studies on monocyte-derived cell lines have shown that these phenotypic differences are matched by underlying metabolic signatures, with the potential to have profound effects on cell capabilities. In order to determine whether differentiated macrophages displayed similar profound metabolic profiles, and whether these differences affect function, we differentiated primary blood monocytes under a range of culture oxygenation conditions. Initial results show significant differences in the metabolic profiles of M1 vs. M2 macrophages undergoing differentiation, with M1s displaying much reduced lactate levels, and corresponding increases in glucose suggestive of gluconeogenesis via putative PFKFB3 (fructose-1,6-bisphosphatase) activity. M1s were demonstrated to be constitutively active under reperfusion conditions, with no corresponding metabolic changes following LPS stimulation. M2s, in contrast, showed an expected hypoxia profile of increased lactate levels under differentiation, and remained inactive in reperfusion conditions, however production of IL-10 following LPS stimulation was shown to be significantly reduced in hypoxic conditions. A model of permissive inflammation during M1 infiltration and under hypoxia is suggested, with reperfusion and reduced recruitment driving resolution in normal tissues. However, in persistently hypoxic tissues or regions of aberrant recruitment and proliferation, the potential exists for differentiating macrophages to drive and maintain a chronic inflammatory state.

Type of Work:M.Res. thesis.
Supervisor(s):Shannon-Lowe, Claire and Long, Heather
School/Faculty:Colleges (2008 onwards) > College of Medical & Dental Sciences
Department:School of Immunity and Infection
Subjects:RC Internal medicine
Institution:University of Birmingham
ID Code:2940
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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