Publication Date
2016
Document Type
Thesis
Committee Members
Nancy Bigley (Advisor), Barbara Hull (Committee Member), Dawn Wooley (Committee Member)
Degree Name
Master of Science (MS)
Abstract
The cell cytoskeleton, a unique intracellular matrix found in all eukaryotes, is composed of three main protein structures, microtubules, microlaments and intermediate laments. The cytoskeleton maintains cell shape and internal structures providing mechanical support that facilitates intracellular transport (Parker et al., 2014). Many viruses such as the herpesviruses use the cytoskeleton system of the cell for infectivity (Henry Sum, M. S. 2015). HSV-1 utilizes the cell cytoskeleton in many steps of its life cycle from entry through assembly to egress (Lyman et al., 2008). During infection, HSV-1 viral proteins cause drastic changes and rearrangement of the cellular actin and cell signaling during viral replication. The goal of this study was to determine the effects of HSV-1 challenge on cell viability, morphology, and levels of tubulin and F-actin in unpolarized and cytokine-polarized murine J774A.1 macrophages at 24, 48 and 72 hours. M1 macrophage phenotype was generated by treatment with interferon-gamma (IFN-) and bacterial lipopolysaccharide (LPS); three M2 phenotypes were produced by polarization with interleukin-4 (IL-4) or IL-10 or IL-13. The most prominent change in cell morphology caused by HSV-1 is the formation of small rounded cell shape from the transformed cells (Taylor et al., 2011) which was confirmed in this study. Immunofluorescence images of all macrophage subgroups (unpolarized M0, M1, the three M2 phenotypes) after HSV-1 infection were indistinguishable all showing smaller rounded cell shapes. Uninfected M1 cells were flattened, irregularly shaped cells containing visible intracellular vacuoles compared to the rounded cell shape in uninfected M0 or M2 polarized macrophages. Furthermore, uninfected M2 macrophages stimulated by IL-4 showed a combination of morphologies ranging from elongated to rounded cells. Uninfected M2 macrophages polarized by IL-10 or IL-13 displayed a rounded cell type which closely resembled unpolarized (M0) cells. In viability experiments, HSV-1-infected unpolarized cells and polarized M1 inflammatory macrophages exhibited rapid decline in cell numbers compared to unpolarized M0 and M2 anti-inflammatory macrophages polarized by IL-4, IL-10 and IL-13. This reduction in cell viability was expected due to the crucial role macrophages play in viral clearance by producing molecules toxic to both the infecting agent and the cell itself. The relative amounts of tubulin and F-actin microfilaments were evaluated by quantifying the fluorescent intensity of immunofluorescent microscopy images through ImageJ analysis. The majority of uninfected M1 and M2 macrophages subgroups displayed increased levels of tubulin and F-actin compared to the levels seen in uninfected M0 control cells. In contrast, infection by HSV-1 markedly reduced amounts of the immunofluorescent intensity of either tubulin or F-actin 48 hours after polarization and infection of M1 and M2. By 72 hours, the greater amounts of tubulin and F-actin were present in all polarized and infected cells (M0, M1, and the three M2 phenotypes) which may reflect an anti-apoptotic effect of HSV-1 on these cells as described by others (Roizman and Taddeo, 2007) and (Aubert et al.,2008).
Page Count
71
Department or Program
Microbiology and Immunology
Year Degree Awarded
2016
Copyright
Copyright 2016, some rights reserved. My ETD may be copied and distributed only for non-commercial purposes and may not be modified. All use must give me credit as the original author.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
ORCID ID
http://orcid.org/0000-0002-3197-9018
