EXAMINATION OF CHANNEL CATFISH (ICTALURUS PUNCTATUS) LEUKOCYTE IMMUNE-TYPE RECEPTOR (IPLITR)-MEDIATED CROSS-TALK REGULATION OF PHAGOCYTOSIS
Chenjie Fei, University of Alberta
Chenjie Fei, Myron Zwozdesky, Dustin Lillico, Luke Gerla, and James L. Stafford
Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
Channel catfish leukocyte immune-type receptors regulate various innate immune cell responses via classical and unique intracellular signaling networks. We have shown IpLITR 2.6b/IpFcRγ-L activates immune cell effector responses using a canonical ITAM-dependent signaling pathway whereas IpLITR 1.1b is an inhibitory receptor due to ITIM-mediated phosphatase recruitment. Since both stimulatory and inhibitory IpLITR-types are co-expressed by catfish immune cell-types, integrated signaling between these proteins (i.e. cross-talk) are likely important for their overall immunoregulatory functions. Known IpLITRs also share similar extracellular regions suggesting that they may engage common ligands, however detailed mechanisms regarding potential cross-talk between different IpLITR-types remain unknown. Here we generated AD293 cell lines co-expressing the N-terminal HA-tagged IpLITR 2.6b/IpFcRγ-L proteins and a FLAG-tagged IpLITR 1.1b construct. This allowed co-engagement of the receptors with mAb-coated microbeads and development of an imaging flow cytometry-based phagocytosis crosstalk assay. Our results show that IpLITR 1.1b inhibits IpLITR 2.6b/IpFcRγ L-mediated phagocytic response. Induction of phosphotyrosine signals downstream of IpLITR 2.6b/IpFcRγ-L activation was significantly reduced when co-engaged with IpLITR 1.1b, likely through the activation of Src homology region 2 domain containing phosphatase-2 (SHP-2). Additionally, two tyrosine motifs (i.e. a Csk-binding motif and an ITIM) in the cytoplasmic region of IpLITR 1.1b were shown to be critical for sustaining the inhibition of phagocytosis, providing new information about coordinated recruitment of effector molecules (e.g. Csk and SHP-2) in regulating phagocytosis. Overall, this work reveals that co-engagement of IpLITRs can fine-tune the balance between activating and inhibitory signals, which governs the overall control of a vital innate cell effector response.