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Complement-mediated phagocytosis in neutrophils

Periodic Reporting for period 1 - CR-PHAGOCYTOSIS (Complement-mediated phagocytosis in neutrophils)

Reporting period: 2015-04-01 to 2017-03-31

The goal of this project is to understand how the complement system directs neutrophil phagocytosis of bacteria. The complement system is part of innate immunity that quickly becomes activated by pathogens, resulting in the production of “opsonin” molecules that label these target cells for recognition by phagocytic cells. During infection, neutrophils rapidly migrate toward target cells, and subsequently ingest and kill them. This process is critical for clearance of bacterial infections, but the underlying molecular mechanisms are not well understood, since these phenomena are often studied in complex physiological environments. Due to rapid rise in antibiotic-resistant bacteria, it is critical to explore alternative treatment strategies to handle bacterial infections in the near future. By understanding how immune cells (i.e. neutrophils) recognize and destroy bacteria, we could “guide” the complement system to tag these bacteria for destruction. Further, since complement-mediated phagocytosis is an important component of many immunotherapies, this information can be exploited to improve these treatments. The main objectives in this project include the development of tools to study complement opsonin-receptor interactions at a molecular level, to determine the role of complement opsonins in neutrophil uptake, activation and killing, and to study the collaborative action of antibodies and complement in neutrophil functioning.
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.
This project has advanced our understanding of how complement opsonization drives phagocytosis. We now understand the functional roles of opsonins and antibodies in neutrophil recognition and uptake, which was not previously characterized in a purified environment. Our understanding of how distribution and density of complement opsonin and antibody mixtures drive phagocytosis can be used to improve the efficacy of immunotherapies against cancer and infectious diseases. By targeting complement activation and opsonization in particular locations on cell surfaces relative to antibody binding sites, recognition and uptake (and in turn, killing) by immune cells will be enhanced.

Furthermore, we have demonstrated the broad applicability of our tools, which can be used to study various physiological processes in which complement plays a role. Specifically, we used opsonized beads to study the role of complement inhibitors on surface-specific complement convertase enzymes. These studies have guided the discovery of new complement inhibitors, which can serve as a foundation for new therapeutics.

We have also used opsonized beads (and complement receptor cell lines) to evaluate the dynamics of opsonin cleavage in the presence of whole blood, which is critical in understanding how opsonins direct the effector functions of complement in a physiological context. In addition, we have used opsonized beads and bacteria to investigate the role of opsonins in driving complement-mediated adaptive immunity. Using this information, we can “direct” complement opsonization on bacterial surfaces to efficiently evoke the desired response (i.e. adaptive immune response, bacterial killing, etc.). This is a promising strategy in combating the rise in antibiotic-resistant bacterial infections.

Overall, the work from this project provides the tools to study a wide variety of complement-dependent processes at a molecular level, guides the development of therapeutic molecules against highly-specific active targets of the complement cascade, and provides strategies for improved cancer immunotherapies and treatment of antibiotic-resistant bacterial infections.