Identification and functional characterization of a subset of goldfish (Carassius auratus) leukocyte immune-type receptors (CaLITRs) expressed by myeloid cell-types
Creators: Wang, Jiahui
Publisher: name: University of Alberta Library
Year: 2024

<p>Originally discovered in the channel catfish, Ictalurus punctatus leukocyte immune-type receptors (IpLITRs) are members of the immunoglobulin superfamily (IgSF) and several of their transcripts are known to be expressed in a variety of catfish innate and adaptive immune cells. Phylogenetically, IpLITRs have a unique “chimeric” composition of Ig-like domains that are distantly related to mammalian immunoregulatory receptors of the Fc receptor (FcR) family and leukocyte receptor complex (LRC). Using heterologous overexpression systems, our lab has shown that select IpLITRs likely use dynamic tyrosine-based signaling mechanisms to control various innate immune effector responses. Although LITRs have been studied for almost two decades, their immunoregulatory roles in teleost immunity remain poorly understood. The goldfish, with its published genome, well-characterized primary myeloid cells such as primary kidney macrophages (PKMs) and neutrophils (PKNs), and large molecular toolkit, represent an ideal model system for studying the native functions of LITRs in teleost immune cells. <br>
The overall aim of my thesis was to set up the goldfish as a new model system for investigating the native immunoregulatory roles of LITRs in teleost immunity. I have identified and cloned a total of seventeen calitr transcripts. The mRNAs of several CaLITRs were found to be present in an assortment of goldfish tissues and isolated PKMs and PKNs. Sequence analyses of these receptors have shown that they have diverse configurations of Ig-like domains and cytoplasmic tails (CYTs) containing various tyrosine-based signaling motifs such as immunoreceptor tyrosine-based activation motifs (ITAMs), immunoreceptor tyrosine-based switch motifs (ITSMs), and immunoreceptor tyrosine-based inhibition motifs (ITIMs). Additionally, I have also identified several putative CaLITR ectodomain and CYT splice forms. These results indicate that alternative splicing may play a significant role in diversifying the ligand binding and/or signaling potentials of CaLITRs. Overall, the identification and cloning of the various calitr sequences open up new avenues for exploring how these sequences may contribute to the regulation of the goldfish immune system.<br>
Using a comparative genomics approach, I further examined the possible evolutionary connections between teleost LITRs and various vertebrate immunoregulatory receptor types. My results show that teleost litrs generally exist in large genomic clusters, which are linked to vangl2, arhgef11, and slam family genes, features that are also shared by amphibian and mammalian Fc receptor-like molecules (fcrls). Moreover, detailed phylogenetic comparisons between the individual Ig-like domains of LITRs and mammalian FCRLs show that these receptors share related Ig-like domains indicative of their common ancestry. These data support that LITRs are likely to be distant homologs of vertebrate FCRLs. <br>
    Using qPCR, I investigated the transcriptional activities of several calitrs from distinct goldfish chromosomes. My findings demonstrate that these calitrs are differentially expressed during PKM development and in activated goldfish leukocyte cultures. Specifically, I showed that calitr1 exhibited transcriptional kinetics similar to several goldfish proinflammatory markers in mitogen-simulated goldfish leukocytes, while the expression of the other calitrs examined (calitr2, calitr3, and calitr4) remained unchanged under the same conditions. Furthermore, immunosuppressive drugs, including dimethyl sulfoxide (DMSO), and to a much less extent, cyclosporin A (CsA), were shown to downregulate the mRNA expressions of several goldfish proinflammatory genes and calitr1, with minimal to no effects observed on the expression of the other calitrs examined. Altogether, these results suggest that the select calitrs from distinct goldfish chromosomes are likely under the influence of different cis- and trans-regulatory elements. <br>
    To further study the native roles of CaLITRs in goldfish myeloid cells, I developed and characterized several CaLITR pAbs specific for CaLITR3, a predicted stimulatory receptor-type with a transmembrane domain (TM) containing a positively charged histidine. I demonstrate that the anti-CaL3.D1 pAb stains various hematopoietic cell types within the goldfish kidney, as well as in PKNs and PKMs. Moreover, cross-linking of the anti-CaL3.D1-pAb on PKN membranes induces phosphorylation of p38 and ERK1/2, critical components of the MAPK pathway involved in controlling a wide variety of innate immune effector responses such as NETosis, respiratory burst, and cytokine release. These findings support the stimulatory potential of CaLITR3 proteins as activators of fish granulocytes and pave the way for a more in-depth examination of the immunoregulatory functions of CaLITRs in goldfish myeloid cells. </p>