Examination of IpLITR-mediated signal transduction events: the cross-talk regulation of phagocytosis

• Author / Creator

  Zwozdesky, Myron A

• Cells perceive their environment through cell surface-expressed transmembrane (TM) receptors. TM receptors transduce extracellular cues to highly sophisticated intracellular signaling pathways. Transduction of multiple signaling inputs fine-tunes the regulation of vital effector responses, like phagocytosis. All metazoan species possess an innate immune system, which is critical for host protection and homeostasis. Multiple innate immune cell-types sense the extracellular microenvironment through immunoregulatory receptors and several immunoregulatory receptor families have been identified in vertebrates. Members of the immunoglobulin superfamily (IgSF) are conserved throughout mammalian, avian, amphibian and fish species. Although some families contain highly diversified members in teleost fish, they operate via evolutionarily conserved principles of signaling for regulating effector responses. This has allowed the study of conserved and novel immunoregulatory receptor-mediated signal transduction capabilities using fish receptor models.
  Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a polygenic and polymorphic immunoregulatory receptor family that share structural and phylogenetic relationships with vertebrate IgSF receptor-types. This family contains stimulatory and inhibitory forms that regulate several innate immune cell effector responses via classical as well as novel cytoplasmic tail (CYT)-dependent signaling capabilities. My thesis is focused on using an IpLITR model to better understand how receptor CYT signaling versatility affects effector response regulation. Previous functional and biochemical characterizations were performed in innate immune cell-types. The stimulatory IpLITR 2.6b associated with the ITAM-containing adaptor molecule IpFcRγ-L and activated phagocytosis, cytokine secretion and degranulation in rat mast cells through conserved pathways. Conversely, the inhibitory IpLITR 1.1b abrogated cytotoxicity via predictable ITIM-dependent SHP pathways in mouse natural killer (NK) cells. Uniquely, IpLITR 1.1b also used an ITIM-independent Csk pathway to abrogate cytotoxicity. Surprisingly, IpLITR 1.1b activated a unique ITAM-independent phagocytic phenotype in rat mast cells that occurs in two distinct actin polymerization-dependent stages to capture and then internalize targets. Importantly, IpLITR 1.1b evokes different functional outcomes by switching its signaling potential between cell-types (i.e. NK cell vs. mast cell), however, detailed mechanistic studies of this potential were lacking.
  Non-immune epithelial cell-types do not express IgSF immune receptors, however, they express sub-membrane proximal signaling molecules and downstream effectors of actin polymerization. This offers a cellular background for isolating receptor CYT-specific signaling events away from co-activated pathways. Therefore, the overall objective of my thesis is to establish a non-immune epithelioid cell system to examine details of IpLITR CYT-dependent signaling versatility. My specific research aims were to: (1) characterize signaling molecule recruitment to the IpLITR 1.1b CYT; (2) examine IpLITR 1.1b CYT-dependent phagocytic signaling potential; (3) establish an assay for measuring IpLITR CYT-dependent cross-talk signaling potential; and (4) examine IpLITR 1.1b CYT requirements for down-regulating ITAM-driven phagocytosis.
  My research has demonstrated that although IpLITR CYTs recruit various stimulatory signaling molecules, the functional outcome is predicted by the presence of canonical CYT motifs when expressed in epithelioid cells. Specifically, IpLITR 1.1b recruited Nck and Syk to the proximal and distal CYT segments, respectively, in GST pulldown assays. However, while IpFcRγ-L activated phagocytosis, IpLITR 1.1b CYT did not. Furthermore, co-immunoprecipitation showed that the IpLITR 1.1b CYT recruited the inhibitory molecules Csk and SHP2 in cells activated with pervanadate. This facilitated a shift in my objectives based on previous IpLITR characterizations to assess a novel aspect of IpLITR signaling potential. IpLITRs contain highly conserved extracellular domains that may bind common targets when co-expressed in catfish immune cells. Since IpFcRγ-L orchestrated signaling events that activated phagocytosis in epithelioid cells, I examined IpLITR 1.1b CYT-dependent cross-talk signaling potential. Using a novel imaging flow cytometry-based phagocytosis assay, I demonstrated IpLITR 1.1b CYT-dependent cross-talk down-regulation of ITAM-driven phagocytic signaling upon co-cross-linking with bead targets. To examine the details of this down-regulation, I used site-directed mutagenesis, dominant-negative signaling molecule constructs and shRNA-mediated knockdown techniques. I showed that IpLITR 1.1b CYT-dependent down-regulation of phagocytosis requires both Y453 and Y477 for maximum inhibition, however, neither SHP2 nor Csk were critically involved. These results indicate that IpLITR 1.1b uses a novel proximal and distal CYT-dependent co-operative inhibitory signaling mechanism to down-regulate ITAM-driven phagocytosis. Overall, I have demonstrated novel inhibitory signaling capabilities for IpLITR 1.1b, including the first report of cross-talk signaling potential within a teleost immunoregulatory receptor family. My work shows that teleost immunoregulatory receptors are excellent models to advance the understanding of innate immunoregulatory processes in vertebrates.

• Subjects / Keywords

  • innate
  • LITR
  • regulation
  • cross-talk
  • signal
  • immunity
  • transduction
  • phagocytosis

• Graduation date

  Fall 2019

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-cszb-gz20

• License

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