Examining the Effects of Cytoplasmic Tail Splicing Event in Goldfish (Carassius auratus) Leukocyte Immune-type receptors
Creators: Al-Rikabi, Hussain Isam
Publisher: name: University of Alberta Library
Year: 2024

<p>The innate immune system is important in clearing infections and maintaining homeostasis. Across vertebrates, innate immunity is capable of executing potent effector responses through the actions of highly specialized cells. Effector responses, such as phagocytosis and cytokine secretion, are mediated by the actions of conserved and specialized immunoregulatory receptors. The transduction of extracellular events across the cell membrane by immune receptors allows for the fine-tuning of innate effector responses. The evolutionary conserved nature of these receptors across vertebrates bridges the gap to understanding vertebrate immunity by providing an opportunity to study such processes within a range of model systems of teleost fishes. <br>
In 2006, a novel and diverse family of leukocyte immune-type receptors (LITRs) in channel catfish (Ictalurus punctatus) was identified (Stafford et al. 2006). Phylogenetic studies and sequence analyses suggest that LITRs belong to the immunoglobulin superfamily (IgSF) and that they are related to mammalian Fc receptors (FcRs), FcRLs and other Ig-type proteins (Wang et al. 2020). IpLITRs contain both stimulatory and inhibitory types of receptors that influence the functions of innate immune cells through their cytoplasmic tail (CYT) regions. This results in both classical and distinct intracellular signalling pathways. Past research on different types of IpLITRs in our lab has enabled us to investigate how immune receptors interact with adaptor signalling molecules, form both homo- and heterodimers, and regulate cell cytotoxicity using both classical and unconventional inhibitory components. Additionally, we have studied how these receptors differ in capturing and engulfing targets, and how they use unique mechanisms to interfere with the phagocytic response. <br>
Although the catfish model has provided valuable insights into immunoregulatory receptor types in teleosts, our lab has recently focused on exploring other teleost models that present a variety of LITR proteins to study the intricacies of immunoregulation in early vertebrates. Utilizing a transcriptome developed from goldfish spleen tissue, our lab identified 37 unique CaLITRs, each characterized by their own specific extracellular and intracellular domain configurations. Two LITRs of interest are the goldfish (Carassius auratus) CaLITR 2.0 and CaLITR 2.1 forms. These receptors are identical except for a 29-amino acid deletion in the CaLITR2.1 CYT region that contains a tyrosine (Y) motif potentially involved in cell signalling. This deletion is presumably due to alternative splicing of the goldfish LITR 2 transcript, and it may play functional roles in the fine-tuning of immune effector functions. To investigate this, CaLITR 2.0 and 2.1 were individually cloned into pDisplay vector, then expressed on the cell surface of mammalian AD293 cells with an N-terminal hemagglutinin (HA)-epitope tag. Latex yellow-green (YG) beads opsonized with anti-HA mAb were then used to stimulate the transfected cells. Finally, their phagocytic activities are examined using an ImageStream X flow cytometer. <br>
Due to the inability to express native CaLITR constructs in AD293 cells, chimeric constructs (C-CaLITR) composed of the extracellular and transmembrane domains of the positive control, IpLITR 2.6b, and the CYT region of CaLITR 2.0 or CaLITR 2.1, were created and used in the phagocytosis assays. C-CaLITR 2.0 showed significantly lower phagocytic activity than the splice variant, C-CaLITR 2.1. This observation was consistent when cells were stimulated with 4.5 µm or 2.0 µm beads, as well as increasing antibody concentration to opsonize bead targets. The CYT region-splicing event removes an immunoreceptor tyrosine-based inhibitory motif (ITIM) motif, which generally inhibits immune responses, leaving only an immunoreceptor tyrosine-based switch motif (ITSM) in the CYT region of CaLITR 2.1. ITSMs are known to have either inhibitory or stimulatory effects on cell signalling, depending on factors such as the cell type, the intracellular adaptor molecules available, and the co-stimulation of other receptors expressed by the same cell. My results suggest that i) removal of the ITIM amplifies the phagocytic response of C-CaLITR 2.1, and 2) an ITSM-containing receptor can uniquely mediate phagocytosis. The underlying signalling components of C-CaLITR 2.1-mediated phagocytosis involve actin, PI3K, SFK and Syk signalling pathways, which were preliminarily investigated using pharmacological inhibitors. Overall, my results show the first evidence of how CYT region-targeted splicing events can fine-tune immunoregulatory receptor functional potentials.</p>