Expression analysis of Carassius auratus-leukocyte-immune-type receptors (CaLITRs) during goldfish kidney macrophage development and in activated kidney leukocyte cultures.

Carassius auratus leukocyte immune-type receptors (CaLITRs) were recently discovered immunoregulatory receptors in goldfish that have diverse immunoglobulin-like (Ig-like) ectodomains and intracellular signaling motifs. Genomic analysis shows that CaLITR-types are also located as distinct gene clusters across multiple goldfish chromosomes. For example, CaLITR1 (unplaced) is a functionally ambiguous receptor having two Ig-like domains, a transmembrane domain (TM), and a short cytoplasmic tail (CYT) devoid of any recognizable signaling motifs. CaLITR2 (Chr47) is a putative inhibitory receptor containing four Ig-like domains, a TM, and a long CYT with an immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). A putative activating receptor-type, CaLITR3 (Chr3), has four Ig-like domains, a TM, and a short CYT containing a positively charged histidine residue and CaLITR4 (ChrLG28B) is a receptor with putative multifunctional signaling potential as well as five Ig-like domains, a TM, and a long tyrosine-motif containing CYT region. The variable genomic locations of the CaLITRs suggest that they are likely under the influence of different cis- and/or trans-regulatory elements. To better understand the transcriptional activities of select CaLITRs from variable genomic regions, we used an RT-qPCR-based approach to examine the expression of CaLITR1, CaLITR2, CaLITR3, and CaLITR4 during goldfish primary kidney macrophage (PKM) development and in mixed leukocyte reaction cultures (MLRs) of the goldfish. Our results showed that the select CaLITRs are differentially expressed during PKM development and in goldfish MLRs exposed to T-cell mitogens/immunosuppressive drugs, supporting that the transcription of these CaLITRs is likely regulated by distinct cis- and/or trans-regulatory elements.

A subset of leukocyte immune-type receptors (LITRs) regulates phagocytosis in channel catfish (Ictalurus punctatus) leukocytes.

Channel catfish, Ictalurus punctatus, leukocyte immune-type receptors (LITRs) constitute a large family of paired, immunoregulatory receptors unique to teleosts. A role for LITRs in phagocytosis has been proposed based on studies in mammalian cell lines; however, LITR-mediated phagocytosis has not been examined in the catfish model. In this study, we use two anti-LITR monoclonal antibodies, CC41 and 125.2, to contrast the effects of crosslinking subsets of inhibitory and activating LITRs. Briefly, LITRs expressed by catfish γδ T cells, αß T cells, and macrophage cell lines were crosslinked using mAb-conjugated fluorescent microbeads, and bead uptake was evaluated by flow cytometry and confirmed by confocal microscopy. A clear difference in the uptake of 125.2- and CC41-conjugated beads was observed. Crosslinking LITRs with mAb 125.2 resulted in efficient bead internalization, while mAb CC41 crosslinking of inhibitory LITRs resulted predominantly in a capturing phenotype. Pretreating catfish macrophages with mAb CC41 resulted in a marked decrease in LITR-mediated phagocytosis of 125.2-conjugated beads. Overall, these findings provide insight into fish immunobiology and validate LITRs as regulators of phagocytosis in catfish macrophages and γδ T cells.

Identification of distinct LRC- and Fc receptor complex-like chromosomal regions in fish supports that teleost leukocyte immune-type receptors are distant relatives of mammalian Fc receptor-like molecules.

Leukocyte immune-type receptors (LITRs) are a large family of immunoregulatory receptor-types originally identified in the channel catfish (Ictalurus punctatus (Ip)LITRs). Phylogenetic analyses of LITRs show that they share distant evolutionary relationships with important mammalian immunoregulatory receptors belonging to the Fc receptors family and the leukocyte receptor complex (LRC), but their syntenic relationships with these immunoglobulin superfamily members have not been investigated. To further examine the possible evolutionary connections between teleost LITRs and various mammalian immunoregulatory receptor-types, we surveyed the genomic databases of representative vertebrate taxa and our 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 shows that these receptors share related Ig-like domains indicative of their common ancestry. However, contrary to our previous reports, no supportive evidence for phylogenetic relationships between the Ig-like domains of LITRs with the Ig-like domains of LRC-encoded mammalian immunoregulatory receptors was found. We also identified an LRC-like region in the zebrafish genome, but no expanded litr-related genes were located in this region. Similarly, no lilr-related genes were found in spotted gar, a representative basal ray-finned fish. Finally, two distantly related fcrls and an LRC-like gene were identified in the elephant shark genome, suggesting that the loss of an immunoregulatory receptor-containing LRC region may be unique to ray-finned fish.

Identification of goldfish (Carassius auratus L.) leukocyte immune-type receptors shows alternative splicing as a potential mechanism for receptor diversification.

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family.

Catfish lymphocytes expressing CC41-reactive leukocyte immune-type receptors (LITRs) proliferate in response to Edwardsiella ictaluri infection in vitro.

Monoclonal antibodies (mAbs) CC34 and CC41 recognize overlapping subsets of leukocyte immune-type receptors (LITRs). The mAb CC34 was raised against the clonal TS32.15 cytotoxic T cell line and the mAb CC41 was raised against the clonal NK cell line TS10.1. In this study, an in vitro model was developed to monitor CC34- and CC41-reactive cells in response to Edwardsiella ictaluri infection. Briefly, head kidney leukocytes and peripheral blood lymphocytes (PBL) were isolated from individual catfish and labeled with CellTrace Violet and CellTrace FarRed dye, respectively. Head kidney-derived macrophages were infected with E. ictaluri and then cocultured with autologous PBL. The combined cell cultures were then analyzed using flow cytometry. A significant increase in CC41 staining was observed in the PBL population at 2, 5 and 7 days after culture, which suggest that LITRs are involved in cell-mediated immunity to E. ictaluri.

Imaging flow cytometry and confocal microscopy-based examination of F-actin and phosphoinositide dynamics during leukocyte immune-type receptor-mediated phagocytic events.

Cells of the innate immune system rapidly detect and eliminate invading microbes using surface-expressed immunoregulatory receptors that translate extracellular binding events into potent effector responses. Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins that have been shown to regulate several innate immune cell effector responses including the phagocytic process. The mechanisms by which these receptors regulate phagocytosis are not entirely understood but we have previously shown that different IpLITR-types use ITAM-dependent as well as ITAM-independent pathways for controlling target engulfment. The main objective of this study was to develop and use imaging flow cytometry and confocal microscopy-based assays to further examine both F-actin and phosphoinositide dynamics that occur during the different IpLITR-mediated phagocytic pathways. Results show that the ITAM-dependent IpLITR-induced phagocytic response promotes canonical changes in F-actin polymerization and PI(4,5)P2 redistributions. However, the ITAM-independent IpLITR phagocytic response induced unique patterns of F-actin and PI(4,5)P2 redistributions, which are likely due to its ability to regulate alternative signaling pathways. Additionally, both IpLITR-induced phagocytic pathways induced target internalization into PI(3)P-enriched phagosomes indicative of a maturing phagosome compartment. Overall, this imaging-based platform can be further applied to monitor the recruitment and distribution of signaling molecules during IpLITR-mediated phagocytic processes and may serve as a useful strategy for functional examinations of other immunoregulatory receptor-types in fish.

Imaging flow cytometry and GST pulldown assays provide new insights into channel catfish leukocyte immune-type receptor-mediated phagocytic pathways.

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) control various innate immune cell effector responses including the phagocytic process. This large immunoregulatory receptor family also consists of multiple receptor-types with variable signaling abilities that is dependent on their inherent or acquired tyrosine-containing cytoplasmic tail (CYT) regions. For example, IpLITR 2.6b associates with the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor molecule IpFcRγ-L, and when expressed in mammalian cells it activates phagocytosis using a similar profile of intracellular signaling mediators that also regulate the prototypical mammalian Fc receptor (FcR) phagocytic pathway. Alternatively, IpLITR 1.1b contains a long tyrosine-containing CYT with multifunctional capabilities including both inhibitory and stimulatory actions. Recently, we demonstrated that IpLITR 1.1b activates a unique phagocytic pathway involving the generation of multiple plasma membrane extensions that rapidly capture extracellular targets and secure them on the cell surface in phagocytic cup-like structures. Occasionally, these captured targets are completely engulfed albeit at a significantly lower rate than what was observed for IpLITR 2.6b. While this novel IpLITR 1.1b phagocytic activity is insensitive to classical blockers of phagocytosis, its distinct target capture and engulfment actions depend on the engagement of the actin polymerization machinery. However, it is not known how this protein translates target recognition into intracellular signaling events during this atypical mode of phagocytosis. Using imaging flow cytometry and GST pulldown assays, the aims of this study were to specifically examine what regions of the IpLITR 1.1b CYT trigger phagocytosis and to establish what profile of intracellular signaling molecules likely participate in its actions. Our results show that in stably transfected AD293 cells, the membrane proximal and distal CYT segments of IpLITR 1.1b independently regulate its phagocytic activities. These CYT regions were also shown to differentially recruit various SH2 domain-containing intracellular mediators, which provides new information about the dynamic immunoregulatory abilities of IpLITR 1.1b. Overall, this work further advances our understanding of how certain immunoregulatory receptor-types link extracellular target binding events to the actin polymerization machinery during a non-classical mode of phagocytosis.

Trypsin differentially modulates the surface expression and function of channel catfish leukocyte immune-type receptors.

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are immunoregulatory proteins that control innate immune cellular responses. Previously, we demonstrated that two representative IpLITR forms, IpLITR 2.6b and IpLITR 1.1b, engage distinct components of the phagocytic machinery resulting in unique target capture and engulfment phenotypes. IpLITR-induced phagocytic mechanisms were also differentially susceptible to temperature and pharmacological inhibitors of canonical signaling mediators. In the present study, we examined the sensitivity of IpLITR-mediated phagocytosis to the endogenous serine-protease trypsin, a well-known mediator of immunoregulatory receptor functions. Trypsin selectively reduced IpLITR 1.1b cell surface expression and phagocytic activity in a dose-dependent manner. We also observed a significant alteration of the IpLITR 1.1b phagocytic phenotype post-trypsin exposure; whereas, the IpLITR 2.6b-mediated target engulfment phenotype was unchanged. Recovery experiments suggested that trypsin-induced inhibition of IpLITR 1.1b-dependent phagocytosis was reversible and that the re-establishment of phagocytic function was associated with a recovery of receptor surface expression. Cell-surface biotinylation and immunoprecipitation studies demonstrated that IpLITR 1.1b normally exists as a mature (∼70 kDa) protein on the cell surface. However, trypsin treatment reduced expression of the mature receptor and processed IpLITR 1.1b into an ∼60 kDa form. The trypsin-generated and putative immature IpLITR 1.1b form was not present on the cell surface; suggesting that the cleaved receptor may have been internalized, post-processing, by regulated endocytosis. Taken together, these results reveal a unique role for trypsin as a selective modulator of IpLITR-mediated phagocytosis and highlight a conserved role for serine proteases as potent immunomodulatory factors.

Biochemical and Functional Insights into the Integrated Regulation of Innate Immune Cell Responses by Teleost Leukocyte Immune-Type Receptors.

Across vertebrates, innate immunity consists of a complex assortment of highly specialized cells capable of unleashing potent effector responses designed to destroy or mitigate foreign pathogens. The execution of various innate cellular behaviors such as phagocytosis, degranulation, or cell-mediated cytotoxicity are functionally indistinguishable when being performed by immune cells isolated from humans or teleost fishes; vertebrates that diverged from one another more than 450 million years ago. This suggests that vital components of the vertebrate innate defense machinery are conserved and investigating such processes in a range of model systems provides an important opportunity to identify fundamental features of vertebrate immunity. One characteristic that is highly conserved across vertebrate systems is that cellular immune responses are dependent on specialized immunoregulatory receptors that sense environmental stimuli and initiate intracellular cascades that can elicit appropriate effector responses. A wide variety of immunoregulatory receptor families have been extensively studied in mammals, and many have been identified as cell- and function-specific regulators of a range of innate responses. Although much less is known in fish, the growing database of genomic information has recently allowed for the identification of several immunoregulatory receptor gene families in teleosts. Many of these putative immunoregulatory receptors have yet to be assigned any specific role(s), and much of what is known has been based solely on structural and/or phylogenetic relationships with mammalian receptor families. As an attempt to address some of these shortcomings, this review will focus on our growing understanding of the functional roles played by specific members of the channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs), which appear to be important regulators of several innate cellular responses via classical as well as unique biochemical signaling networks.

Multigene families of immunoglobulin domain-containing innate immune receptors in zebrafish: deciphering the differences.

Five large multigene families encoding innate-type immune receptors that are comprised of immunoglobulin domains have been identified in bony fish, of which four do not possess definable mammalian orthologs. The members of some of the multigene families exhibit unusually extensive patterns of divergence and the individual family members demonstrate marked variation in interspecific comparisons. As a group, the gene families reveal striking differences in domain type and content, mechanisms of intracellular signaling, basic structural features, haplotype and allelic variation and ligand binding. The potential functional roles of these innate immune receptors, their relationships to immune genes in higher vertebrate species and the basis for their adaptive evolution are of broad interest. Ongoing investigations are expected to provide new insight into alternative mechanisms of immunity.