Date of Award
Bachelor of Science
The main force behind innate immunity, our primary defense against infection, is the clearance of pathogens, or phagocytosis. Protein Kinase C-epsilon (PKC-ε) is necessary for efficient phagocytosis of antibody (IgG)-coated particles. Previous studies used IgG-coated glass beads, which are larger and more rigid than bacteria and lack their complex surface protein expression. To test the hypothesis that PKC-ε is necessary for phagocytosis of pathogens, we compared the rate of uptake of common pathogens by macrophages from wild type and PKC- ε knock out mice. We tested two targets, a bacteria (E. coli) and a yeast (zymosan), both physiologically relevant phagocytic targets of different sizes. To link these studies with our previous work, we included IgG-opsonized E. coli, to determine the involvement of PKC-ε in uptake of bacteria through the Fcγ receptor, a macrophage surface receptor that targets the immune complex IgG. Furthermore, to determine if differentiation environment impacted the results, we tested two populations of macrophages, in vivo-differentiated (ie. thioglycollate-elicited) peritoneal macrophages (PMacs) and in vitro differentiated bone marrow-derived macrophages (BMDMs). The E. coli and zymosan used were labeled with the pH-sensitive dye pHrodo™, which fluoresces in the acidic environment of the phagosome but not in the neutral pH of the extracellular buffer, allowing us to specifically detect internalized particles. By utilizing flow cytometry (FACS) as a sensitive and objective measure of pathogen uptake, we determined not only the percentage of cells that internalized at least one target, but also the level of fluorescence/cell as a relative readout of the number of particles ingested. By plotting the mean fluorescence intensity (MFI) against time we were able to calculate the rate of internalization (slope of the MFI vs time line) and compare the results from two or more macrophage populations. We found that PKC-ε knockout macrophages had significantly lower rates of internalization for both unopsonized and IgG-opsonized pHrodo® E. coli particles, as well as a decrease in the rate of internalization of unopsonized zymosan. This suggests that the phagocytic defect in PKC-ε null macrophages is not restricted to one or a small subset of receptors, as the defect was seen across organism and target size.
Pierce, Rebekah Marie, "Use of a pH-Sensitive Probe to Study the Role of PKC-epsilon in Phagocytosis of Pathogens" (2015). Biological Sciences. 29.