Teleost leukocyte immune-type receptors activate distinct phagocytic modes for target acquisition and engulfment

Abstract

Channel catfish (Ictalurus punctatus) IpLITRs belong to the Ig superfamily and regulate innate immune cell effector responses. This study tested the hypothesis that ITAM-dependent and ITAM-independent phagocytic pathways are engaged by different subtypes of the IpLITR family. When stably expressed in RBL-2H3 cells, the ITAM-containing fusion-construct IpLITR 2.6b/IpFcRγ-L stimulated phagocytic responses that were abrogated at suboptimal incubation temperatures and by pharmacological inhibitors of the classic signaling components of the mammalian FcR-dependent phagocytic pathway. Interestingly, the ITIM-containing receptor IpLITR 1.1b also induced phagocytosis through an actin-dependent mechanism, but this process was insensitive to the pharmacological inhibitors tested and remained functional at temperatures as low as 22°C. The IpLITR 1.1b also displayed a unique target-acquisition phenotype that consisted of complex, membranous protrusions, which captured targets in phagocytic cup-like structures but often failed to completely engulf targets. Taken together, these findings suggest that teleost immunoregulatory receptors that associate with ITAM-containing adaptors can engage conserved components of the phagocytic machinery to engulf extracellular targets akin to the classic FcR-mediated response in mammals. Alternatively, IpLITR 1.1b displays a stalled phagocytic phenotype that is likely dependent on the selective recruitment of the minimal molecular machinery required for target capture but results in incomplete target engulfment. Overall, this study demonstrates that IpLITRs can selectively engage distinct components of the phagocytic process and provides important new information regarding the target acquisition as well as internalization mechanisms involved in controlling phagocytic responses across vertebrates.

A Leukocyte Immune-Type Receptor Subset Is a Marker of Antiviral Cytotoxic Cells in Channel Catfish, Ictalurus punctatus

“…More recently, it was demonstrated that the inhibitory TS32.17 LITR 1.1b, which in addition to two ITIMs contains an atypical ITAM-like (ExxYx 2 Vx 16 Yx 2 V) sequence and an ITSM in its CYT, also mediated phagocytosis when expressed in rat basophilic leukemia-2H3 cells (39). Although both TS32.17 LITR1.1b and the chimeric activating LITR2.6b/IpFcRg-L receptors were shown to induce ERK1/2 and protein kinase B (Akt)-dependent signal transduction, there were differences in their signaling kinetics and sensitivity to pharmacological inhibitors (64). Combined, these experiments demonstrated a dual signal capability of TS32.17 LITR1.1b and further support the notion that different LITR isoforms may be involved in fine-tuning various immune response(s).…”

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

“…Classically, the mediators recruited to FcR-FcRγ complexes include isoforms of PI3Ks, Vav, and Rho family GTPases [ 51 , 52 , 53 , 54 ]; in particular, Rac1 and Cdc42 dynamically control actin polymerization and are required for phagocytic cup formation as well as pseudopod extension [ 55 , 56 ]. Selective pharmacological inhibitors of these mediators were profiled in phagocytic assays in order to obtain detailed information regarding the biochemical pathways that facilitate IpLITR 2.6b/IpFcRγ-mediated phagocytosis and to compare these mechanisms with the classical mammalian FcR phagocytic mode [ 57 ]. Phagocytic activity was measured using a flow cytometric-based assay and 4.5 µm fluorescent polystyrene microsphere targets.…”

“…IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis relied on a similar subset of intracellular effectors for target engulfment and was comparable to the ITAM- and SFK-dependent mode of phagocytosis utilized by mammalian FcRs [ 49 , 50 , 51 ]. Specifically, the IpLITR 2.6b/IpFcRγ-L phagocytic response was inhibited using small molecule inhibitors that targeted SFKs, Syk, PI3Ks, Cdc42, Rac1/2/3, phosphoinositide-dependent kinase 1 (PDK1), Akt, PKC, MEK1/2, and F-actin polymerization [ 57 ]. These data provide further biochemical details describing the phagocytic pathway engaged by a teleost immunoregulatory receptor.…”

“…Details of an alternative ITAM-independent IpLITR-induced phagocytic pathway were revealed in [ 57 ]. These studies indicated that despite convergence on the control of actin polymerization dynamics, IpLITR 1.1b clearly operates independently of several of the key components of the ITAM-dependent signaling machinery.…”

“…Incomplete target engulfment is indicative of a stalled phagocytic phenotype that was often observed during IpLITR 1.1b-mediated phagocytosis (~44% of cells examined), but was rarely observed for the IpLITR 2.6b/IpFcRγ-L-expressing cells (~3% of cells) that readily internalized extracellular targets ( i.e. , ~60% of the cells analyzed had completely engulfed two or more beads) [ 57 ]. Although the capture and partial engulfment phenotype was common during IpLITR 1.1b-mediated phagocytosis, ~30% of the analyzed cells internalized at least one 4.5 µm bead.…”