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Examination of IpLITR-mediated control of the phagocytic process

  • Author / Creator
    Lillico, Dustin
  • The ability of immune cells to perform a range of potent antimicrobial effector responses is tightly regulated by intracellular signaling events. These signals are transduced via distinct sets of membrane expressed proteins termed immunoregulatory receptors that translate extracellular cues (e.g. ligand binding) into immunological responses. Such effectors are vital for the elimination of invading microbes and the removal of cellular debris and dying cells from the body. One such effector response is the engulfment and destruction of extracellular targets such as microbes, cellular debris, and necrotic or apoptotic cells by the process known as phagocytosis. Phagocytosis evolved from a nutrient acquisition process in primitive unicellular organisms into a dynamic, complex and fundamental component of innate immunity. The ability of phagocytic cells to recognize and internalize large extracellular particulates is dependent on the expression of specialized immunoregulatory receptors on the cell surface. Phagocytic receptors relay their engagements with extracellular targets to promote filamentous (F)-actin (F-actin) polymerization events that induce dynamic remodelling and reshaping of the plasma membrane through specialized intracellular signaling events. Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a polygenic and polymorphic immunoregulatory receptor family that share basic structural and distant phylogenetic relationships with several immunoregulatory proteins within the mammalian immunoglobulin super family (IgSF). IpLITRs exist as both stimulatory and inhibitory sub-types, which regulate several innate effector responses via classical as well as unique biochemical signaling networks. The focus of my thesis was to utilize IpLITRs as a receptor model system for better understanding the control of innate effector responses in teleost. Previously, it was demonstrated that the stimulatory IpLITR 2.6b directly associated with ITAM-encoding adaptors (e.g IpFcRγ-L) and induced cellular degranulation and phagocytosis when expressed in mammalian myeloid cell-line. Alternatively, the inhibitory receptor IpLITR 1.1b abrogated NK cell-mediated killing via SHP-dependent and SHP-independent mechanisms, which was revealed after transfection and expression in primary mouse NK cells. Since catfish myeloid cells express both stimulatory and inhibitory IpLITR types, the signaling and functional potential of IpLITR 1.1b in myeloid cells was examined and activities were compared to that of the stimulatory IpLITR 2.6b/IpFcRγ-L receptor. Surprisingly, IpLITR 1.1b when expressed in myeloid cells exhibited phagocytic activities. However, the mechanism surrounding IpLITR 1.1b’s stimulatory capabilities remained unclear. Therefore, the overall objective of my thesis was on the examination of the regulatory capabilities of IpLITRs. Specifically, my research aims were; (1) to characterize the ITAM-independent phagocytic pathway facilitated by IpLITR 1.1b; (2) to confirm that the surface expression of IpLITR 1.1b was necessary for the unique target acquisition and engulfment phenotype; (3) to examine IpLITR 1.1b selective induction of F-actin dynamics; (4) to examine the recruitment of Syk and Nck during IpLITR 1.1b-induced filopodia and the capture and internalization of targets. My research demonstrates IpLITRs can selectively activate distinct components of the phagocytic process. Specifically, I show that IpLITR 1.1b uses an alternative phagocytic pathway that is functionally distinct from the classical ITAM-mediated response. I further show trypsin selectively reduced IpLITR 1.1b cell surface expression levels and phagocytic activity. I also observed a significant alteration of the IpLITR 1.1b phagocytic phenotype post-trypsin exposure while IpLITR 2.6b//IpFcRγ-L-mediated target engulfment phenotype was unchanged, indicating the presence of IpLITR 1.1b on the cellular surface effects the way in which cells engage in target-cell interactions through the formation of membranous protrusions. In addition, I show that during the early stages of the IpLITR 1.1b-mediated phagocytic process, filopodia-like structures retract after target contacts to secure captured microspheres to the cell surface. I identified IpLITR 1.1b endogenously colocalizes with Nck in filopodia-like plasma membrane structures and suggesting IpLITR 1.1b-Nck interactions play a pivotal role in the receptor-specific formation of filopodia. Lastly, I demonstrate IpLITR 1.1b recruits Syk and Nck to sites of bead-cell interfaces providing new mechanistic details regarding ITAM-independent phagocytosis. Overall, these results offer new insights regarding the capability of immunoregulatory receptors to initiate filopodia formation and provide new understandings into the cellular events surrounding alternative transduction dynamics that regulate F-actin polymerization and membrane remodelling events. Therefore, IpLITRs as a receptor model system serve to extend our understanding of how other vertebrate immunoregulatory receptor-types may link with intracellular signaling components to regulate effector responses such as phagocytosis.

  • Subjects / Keywords
  • Graduation date
    Fall 2018
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3057D79P
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.