Exploring molecular signaling mechanisms in invertebrate immunity: Leaping back and forth through evolution

Phagocytosis is a highly destructive cellular response, lying at the heart of innate immunity. Phagocytosis is often accompanied of additional destructive cell responses, such as the production of reactive oxygen intermediates (ROI) and nitric oxide (NO). Phagocytosis is triggered by the interaction of phagocyte membrane receptors with microbial surface molecules, or host molecules deposited on the pathogen surface. This interaction activates various intracellular signaling enzymes, which regulate pathogen internalization and destruction. In mammals, signaling enzymes of the PI-3K, PCK and ERK families are involved in the regulation of phagocytosis, and ROI or NO production. In addition, phagocytosis can be enhanced when cells are exposed soluble microbial derivatives, recognized by non-phagocytic receptors. In mammals, the bacterial tri-peptide fMLP interacts with a membrane receptor coupled to heterotrimeric G signaling proteins. This interaction enhances phagocytosis (via PI-3K, PKC, and ERK) and activates chemotaxis. We used microscopic techniques, flow cytometry, and in vitro phagocytosis, chemotaxis, ROI and NO production assays to functionally characterize mussel (Mytilus galloprovincialis), roach (Gomphadorhina portentosa), cricket (Acheta domesticus), beetle (Zophobas morio), and moth (Galleria mellonella) hemocytes. We report the existence of conserved signaling mechanisms regulating phagocytosis, chemotaxis, ROI and NO production in these organisms, similar to those present in mammalian phagocytes. These conserved mechanisms include PI-3K, PKC, and ERK utilization for immune functions, as well as phagocytosis and chemotaxis activation by fMLP. Interestingly, these conserved mechanisms presented differences that were hemocyte type- or species- specific, and also depended on the phagocytic target being internalized. The data presented in this work show that the information, reagents, and techniques available for the study of mammalian immunity can be successfully used as stepping stones for the exploration of invertebrate immunity, unveiling not only conserved molecular mechanisms, but also novel aspects of innate immunity.