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.