Publication Date
2014
Document Type
Thesis
Committee Members
Nancy Bigley (Advisor), Barbara Hull (Committee Member), Javier Leefmans (Committee Member)
Degree Name
Master of Science (MS)
Abstract
Macrophages are specialized phagocytic cells derived from the peripheral blood mononuclear cells. Macrophages can assume M1 or M2 cellular states upon activation with specific cytokines. Upon activation, macrophages produce either pro- or anti- inflammatory cytokines and thereby perform their functions of antigen engulfment and debris clearance. Macrophages have several cell surface cluster differentiation (CD) markers including CD80, CD163 and CD200R that differentiate M1 macrophages from M2. Downstream signaling and cytokine production varies based on the interaction between the specific CD marker and antigen.
The present study was aimed at analyzing and comparing the effects of HSV-1 infection on un-polarized and polarized macrophages at 18, 24, and 48 hours. RAW 264.7 murine macrophage cell lines were cultured under standard conditions and activated appropriately for obtaining M1 and M2 cells. The expression of CD markers and cell viability prior to and post infection was determined by immunofluorescence staining and flow cytometry, respectively. Data were analyzed using Student's t test and one-way ANOVA.
Polarization led to morphological changes in RAW264.7 macrophages. M1 cells were large, elongated, vacuolated and strongly adherent, and showed a significant decrease in cell viability over time after cytokine treatment. M2 macrophages retained more of their original M0 phenotype, appearing rounded, and they exhibited a slight decrease in cell viability. Before viral infection, CD80 expression was highest in M1 cells and CD200R expression was highest in M2 cells. CD163 expression was high in both M1 and M2 (IL-4 activated), but not in M2 (IL-10 and IL-13 activated) cells. Following HSV-1 challenge (MOI 0.1), morphologies of both uninfected and infected M1 and M2 cells were similar. Virus infected M1 macrophages showed further decreases in cell viability compared to uninfected M1 macrophages. The levels of CD markers altered depending on their pro- or anti-inflammatory nature. CD80 expression decreased, although not significantly, in M1 cells infected with HSV-1 compared to uninfected M1 phenotype, suggesting that this is a specific M1 macrophage cell-surface marker that promotes pro-inflammatory immune responses. CD163 expression was higher in IL-10 activated M2 macrophages after viral infection; however, this was also not statistically significant. The only statistical significance was found with CD200R expression. CD200R was found at significantly higher levels in M2 macrophages compared with the M1 phenotype. This suggested that higher expression of CD200R might serve to block the pro-inflammatory pathway.
Page Count
97
Department or Program
Microbiology and Immunology
Year Degree Awarded
2014
Copyright
Copyright 2014, some rights reserved. My ETD may be copied and distributed only for non-commercial purposes and may be modified only if the modified version is distributed with these same permissions. All use must give me credit as the original author.
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This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.
