Examination of channel catfish (Ictalurus punctatus) leukocyte immune-type receptor-mediated crosstalk regulation of phagocytosis

• Author / Creator

  Fei,Chenjie

• Across vertebrates, innate immune cells are capable of initiating a range of potent effector responses that are designed to destroy or contain foreign microbial invaders. The execution and regulation of various innate cellular responses is mediated by a dedicated repertoire of cell surface-expressed immune receptor proteins. These specialized receptors, known as immunoregulatory receptors, sense extracellular stimuli and activate intracellular signaling cascades that elicit appropriate effector responses.
  Channel catfish (Ictalurus punctatus) leukocyte immune type receptors (IpLITRs) belong to a polygenic and polymorphic immunoregulatory receptor family. These teleost proteins are structurally and phylogenetically related to several mammalian immunoregulatory proteins within the immunoglobulin super family (IgSF). The IpLITR family consists of multiple members that exist as generally as putative inhibitory or stimulatory types. Functional characterization of representative IpLITR-types using heterologous overexpression approaches has shown that these teleost proteins are capable of mediating various innate effector responses via classical as well as unique intracellular signaling networks. Previously, it was shown that IpLITR 2.6b activates phagocytosis and degranulation in rat basophil leukocytes (RBL-2H3) using a canonical ITAM-dependent signaling pathway; whereas a putative inhibitory IpLITR-type (i.e. IpLITR 1.1b) functioned as a potent inhibitor of NK cell-mediated cytotoxicity via an ITIM-mediated phosphatases recruitment as well as a distinct ITIM-independent signaling pathway. Moreover, specific triggering of IpLITR 1.1b in myeloid cells activate the phagocytic responses, likely through activation of an alternative ITAM-independent signaling cascade. This unique ability of IpLITR 1.1b to exert both inhibitory and stimulatory effects is likely dependent on selective recruitments of intracellular effectors. To further understand mechanistic details underlying this functional plasticity, the overall objectives of my thesis study was to use IpLITR 1.1b as an alternative vertebrate immunoregulatory receptor model to further explore the signaling potential of this unique teleost protein. Specifically, my research aims were; i) to examine IpLITR 1.1b-mediated integrated control of phagocytic responses using a new flow cytometry-based platform; ii) to investigate the potential IpLITR-mediated receptor crosstalk regulation of the phagocytic response; and iii) to dissect the molecular details underlying the inhibitory actions of IpLITR 1.1b on IpLITR 2.6b activated phagocytosis
  Overall, my thesis research demonstrates the utility of imaging flow cytometry as a valuable platform to investigate the dynamic signaling potential of IpLITR-types during their regulation of immune cell effector responses. My results further support the notion that engagement of IpLITR 1.1b results in selective recruitments of intracellular effectors for the intricate tuning of effector processes in a context-dependent manner. I also show for the first time that IpLITR types can not only function independently, but that they also integrate proximal signaling events downstream via receptor crosstalk to modulate innate effector responses. Specifically, I have shown that IpLITR 1.1b is capable of cross-inhibiting IpLITR 2.6b-mediated phagocytosis when co-expressed in AD293 cells and this inhibitory effect is likely dependent on down-regulating phosphotyrosine signaling cascades. Furthermore, I show that two tyrosine-based motifs (ITIMs and Csk-binding motif) in the cytoplasmic tail of IpLITR 1.1b are minimally required for sustaining the inhibitory effects on IpLITR 2.6b-mediated phagocytosis. My biochemical studies revealed the coordinated recruitments of endogenous Csk and SHP2 molecules to IpLITR 1.1b during receptor crosstalk, which act in concert to sustain the inhibitory effects on IpLITR 2.6b-mediated phagocytosis. Overall, this research represents the first report of IpLITR-mediated integrated control of phagocytosis via receptor crosstalk and provides the basis for further understanding the mechanistic details underlying this important regulatory process.

• Subjects / Keywords

  • Receptor crosstalk
  • Phagocytosis
  • Innate immunity
  • Immunoregulatory receptors
  • Comparative immunology
  • Intracellular signaling
  • Teleost fish

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-axw3-mg52

• License

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