As mentioned in the previous section, complement opsonin molecules play a pivotal role in phagocytosis. In addition, these opsonins are involved in many other effector functions of complement, including clearance of immune complexes, stimulation of adaptive immunity, and inflammation, and direct cell lysis. The most critical opsonins for these functions are derived from complement component C3. These opsonins are unique in that they contain a thioester bond that can covalently attach to cell surface, and act as a long-lived complement “tag” for the numerous functions described above. However, the surface specificity of these complement effector functions poses a major challenge to studying the underlying molecular mechanisms. In this project, we developed several new tools to study complement-mediated phagocytosis in a purified system. Most importantly, we generated purified complement opsonin molecules, and chemically linked these to small bacteria-sized beads in their natural orientation (via the thioester bond). In this way, we can mimic complement opsonization of bacteria in the absence of other confounding factors. We utilized this model to develop a functional assay for surface-specific complement activation by convertase enzymes, which we have used to study mechanisms of complement activation (and inhibition) and subsequent cellular responses. This work was published in BMC Biology (Berends, Gorham, et. al., BMC Biology, 2015). Furthermore, we generated additional tools to enhance our understanding of neutrophil phagocytosis. We created cell lines expressing single complement receptors, in order to determine which opsonins and receptors are involved in neutrophil phagocytosis. Using freshly isolated human neutrophils, we studied the binding and uptake of beads opsonized with complement opsonins under different conditions. Furthermore, we also examined the collaboration between antibodies and complement in driving phagocytosis. We showed that antibodies and certain complement opsonins can induce phagocytosis of beads on their own in a concentration-dependent manner. We also successfully developed methods to artificially opsonize various bacterial strains with complement opsonins, in order to study the roles of individual opsonins in a purified manner, but in the context of other (non-complement) immunostimulatory molecules on the bacterial surface. A manuscript describing this work is in preparation and will be published within the remaining six months of the fellow’s tenure in the lab.
