Year: 2020
Type: dissertation
Abstract: Phagocytosis is an evolutionarily conserved mechanism that serves as the first line of host defense in multicellular organisms. Phagocytes are exposed to a variety of ligands on heterogeneous target particles in vivo, and this recognition is driven by an extensive repertoire of receptors with distinct ligand specificity. However, our current view of phagocytosis has been shaped by studying individual receptor-ligand interactions and their downstream signaling pathways. This makes it physiologically relevant to observe the simultaneous engagement of multiple phagocytic receptors and the underlying coordination of downstream signaling pathways for efficient phagocytosis. Potential crosstalk between the two well-characterized opsonic receptors, FcγR and CR3, was briefly explored in the early 1970s, where macrophages were challenged with dual-opsonized target particles. However, subsequent studies on receptor crosstalk have been primarily restricted to the use of single opsonin on different target particles and typically presented at relatively high opsonin concentrations to induce maximal receptor engagement and signaling. Beyond validating these initial explorations on receptor crosstalk, we attempted to identify the early signaling mechanisms that underlie the binding and phagocytosis of dual-opsonized target particles (IgG and IgM/C3bi) during the simultaneous activation of both opsonic receptors, compared to single receptor activation. For this purpose, we used signaling protein inhibitor studies as well as live-cell brightfield and fluorescence imaging to fully understand the role of cytoskeletal elements and tyrosine kinases, F-actin dynamics, internalization kinetics and phagosome maturation rates for FcγRs and CR3. Importantly, opsonic receptors were studied together and in isolation, in the context of relatively low opsonin concentrations on target particles deemed optimal through flow cytometry analysis and particle binding assays. We saw enhanced particle binding and a synergistic effect on particle internalization when FcγRs and CR3 were simultaneously engaged with relatively low opsonin concentrations on target particles. Next, we investigated the early signaling requirements for the binding and uptake of single and dual-opsonized particles. Inhibition of early signaling and cytoskeletal molecules revealed a differential involvement of Src kinase for FcγRs- versus CR3- and dual receptor-mediated phagocytosis. Src activity recruits Syk kinase, and we observed intermediate levels of Syk phosphorylation in dual-opsonized particles compared to those opsonized with IgG or IgM/C3bi alone. These results likely correlate to the intermediate levels of F-actin that is recruited to sites of dual-opsonized particle uptake and underlie the notoriously delayed internalization of IgM/C3bi-opsonized target particles by macrophages.
Source: TSpace (University of Toronto)
Author: Urja Naik
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3159601251