Zebrafish (Danio rerio) Leukocyte Immune-Type Receptor (DrLITR) Variable Control of the Phagocytic Response
Creators: Niemand, Rikus R
Publisher: name: University of Alberta Library
Year: 2023

<p>To combat microbial invaders, immune cells elicit a range of potent antimicrobial effector responses coordinated by complex intracellular signalling events. These events are initiated by the engagement of cell surface-expressed proteins, termed immunoregulatory receptors, that bind to and recognize foreign bodies. The transduction of extracellular events across the cell membrane by immune receptors allows for fine-tuning of immunological responses against microbes and for the resolution of tissue injury.<br>
Leukocyte immune-type receptors (LITRs) are a polymorphic and polygenic group of immunoregulatory proteins originally discovered in channel catfish (Ictalurus punctatus; IpLITRs) that share structural and phylogenetic similarities with mammalian members of the immunoglobulin superfamily (IgSF). IpLITRs contain both putative stimulatory and inhibitory receptor-types that regulate innate immune cellular functions through their cytoplasmic tail (CYT) regions, inducing both classical and unique intracellular signalling networks. Previous characterization of IpLITR-types has allowed our lab to examine how immune receptors associate with adaptor signalling molecules and form homo- and heterodimers, inhibit cellular cytotoxicity through classical and non-classical inhibitory components, induce differing modes of target capture and engulfment through an inhibitory receptor-type, as well as cross-inhibiting the phagocytic response using novel CYT signalling network mechanisms.<br>
Our lab has identified new LITR-types within the zebrafish genome (Danio rerio; DrLITRs) that have been shown to be ubiquitously expressed during embryogenic development and zebrafish adulthood. While some of these DrLITR-types are indicative of classical stimulatory or inhibitory receptors, DrLITR 1.2 was discovered to contain both activating and inhibitory motifs (i.e., immunoreceptor tyrosine-based activation motif; ITAM and immunoreceptor tyrosine-based inhibitory motif; ITIM) within the same receptor CYT. This arrangement is unusual as these motifs typically exist on separate stimulatory (i.e., ITAM-containing) or inhibitory (i.e., ITIM-containing) immunoregulatory receptors that co-engage to fine-tune cellular signalling and effector responses. My overall objective in this thesis was to examine the role both an ITAM and an ITIM play in controlling DrLITR-mediated signalling. My research aims were, i) to create DrLITR 1.2 as well as motif dysfunctional receptor constructs to generate DrLITR-expressing AD-293 cells, ii) to use these newly created construct-expressing cells to examine the phagocytic capacity of DrLITR 1.2 using a flow cytometric phagocytosis assay, and iii) to examine the novel ITIM-dependent augmentation of DrLITR 1.2-mediated phagocytosis.<br>
My results show that engagement of DrLITR 1.2-expressing cells with phagocytic targets resulted in a robust phagocytic response dependent on the presence of a functional ITAM within the receptor CYT. In addition, I also show that the ITIM motif surprisingly enhances the overall phagocytic response of the receptor while simultaneously decreasing the receptor’s ability to bind to targets. Utilizing confocal fluorescence microscopy, I show that the ITIM-associated inhibitory signalling molecule SHP-2 is localized to the bead-cell interface during the phagocytic engulfment of targets relying on the presence of a functional ITIM within the receptor CYT. Similarly, the ITAM-associated stimulatory signalling molecule Syk was also shown to be recruited to the phagocytic synapse and was dependent on a functional ITAM. Using pharmacological inhibition profiling, I also show that DrLITR 1.2 uses signalling molecules indicative of other ITAM-containing receptors and that the ITIM may play a role in protecting DrLITR 1.2 from crosstalk inhibition. Overall, the data presented in this thesis provides the first functional characterization of teleost immune receptors containing both an ITAM as well as an ITIM within the same receptor CYT. This thesis uncovers new information of how immunoreceptor tyrosine-based motifs fine-tune immunoregulatory receptor-mediated signalling responses.</p>