Cell Viability, Cytoskeleton Organization and Cytokines Secretion of RAW 264.7 Macrophages Exposed to Gram-Negative Bacterial Components
Nancy Bigley (Advisor), Barbara Hull (Committee Member), Marjorie Markopoulos (Committee Member)
Master of Science (MS)
Macrophages play an important role in innate immunity by controlling cellular responses. In this study, the effects of gram-negative bacterial components (Flagellin, lipoprotein, lipopolysaccharides (LPS), outer membrane proteins-A (OMP-A) and peptidoglycan) were determined on cell viability, morphology, cytoskeletal filament and cytokines secretion of murine RAW 264.7 macrophages at 24 hours. The effect of LPS, flagellin and peptidoglycan from gram negative bacteria on viability murine RAW 264.7 macrophages were evaluated using different concentrations (1, 5 and 10 µg/ml). Cells stimulated with LPS displayed ~ 2-fold decrease (P=0.001) in cell viability compared to control cells at 24 hours whereas cells stimulated with flagellin displayed gradual and significant decrease (P = 0.01; P = 0.05) in cell viability with concentrations 5 and 10 µg/ml of this product. Effects of gram-negative bacteria components on the organization of F-actin and microtubule (tubulin) in murine RAW 264.7 macrophages were monitored via immunofluorescent staining to study distribution of cytoskeleton such as elongation cell with cells stimulated with LPS. By using ImageJ analysis, the fluorescent intensity of immunofluorescent images were quantified to evaluate F-actin and tubulin rearrangement. Cells stimulated with Flagellin, lipoprotein, LPS, OMP-A or peptidoglycan show morphological changes as elongation and flatten with some components from gram negative bacteria with different concentrations (1, 5 and 10 µg/ml). LPS at all concentrations displayed a significant increase (p=0.001) in F-actin staining compared to control cells at 24 hours. The cells appeared flattened with irregular shapes compared to control cells appeared rounded. High level of TNF- a were secreted by murine RAW264.7 macrophages exposed to LPS (2300 pg/ml) and flagellin (300 pg/ml) with 10 µg/ml concentration. These observations suggest that activation of TLRs leading to production of inflammatory cytokines such as TNF-a may account for our observed decreases in cell viability since LPS activates TLR4 and flagellin activates TLR5.
Department or Program
Microbiology and Immunology
Year Degree Awarded
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