124 | Immunomodulatory effects of extracellular vesicles from invertebrate species on human cells

Kristian Riolo¹, Francesco Di Pietro¹, Sabrina Oliva¹, Claudia Mosca¹, Valeria Stefanizzi¹, Giovanna Montana², Annalisa Pinsino², Alessia Giannetto¹ | ¹Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; ²Institute of Translational Pharmacology IFT, National Research Council CNR, Palermo, Italy.

Extracellular vesicles (EVs) are heterogeneous membrane-bound particles actively released by cells and represent key mediators of intercellular communication through the transfer of bioactive cargo, including proteins, lipids, and microRNAs. Invertebrates, particularly echinoderms and arthropods—the most abundant organisms on Earth—have evolved efficient molecular strategies to survive in highly dynamic and hostile environments, such as marine ecosystems or decaying organic matter. As a result, these organisms constitute a largely unexplored reservoir of bioactive compounds with potential antimicrobial, antiviral, anticancer, antioxidant, and immunomodulatory properties. H. illucens, a saprophagous insect at the larval stage, and Paracentrotus lividus inhabit highly challenging environments characterized by intense microbial exposure and dynamic environmental stress conditions, making them suitable models to investigate the potential release of extracellular vesicle in their circulating fluids. In this study, we isolated and characterized, for the first time, extracellular vesicles from both H. illucens larvae (Hi-EVs) and P. lividus (Pl-EVs) model organisms, before evaluating their biological effects on human cells for their potential therapeutic applications. Based on the capability of the EV content to be influenced by the physiological state of the originating cells, EVs were isolated from both organisms under basal and challenging conditions to assess stimulus-dependent biological activities on human cells. In particular, Hi-EVs were isolated from larvae maintained under physiological conditions or subjected to immune stimulation (with Lactobacillus casei, Salmonella enterica, lipopolysaccharide, or sterile puncture injury), and Pl-EVs from sea urchin immune cells cultured in vitro at physiological or challenging conditions (LPS and titanium dioxide nanoparticles). Proteomic analysis revealed distinct protein cargo profiles among EV populations, indicating that different stimuli modulate EV cargo in both animal models. The immunomodulatory potential of EVs was evaluated in the human U937 monocyte-like cell line by assessing inflammatory responses through ELISA and gene expression analysis of key cytokines. Results showed that EVs from both species were able to modulate cytokine expression (including IL-6 and IL-8) in a species- and stimulus-dependent manner. Overall, this study provides the first evidence of EV production in H. illucens and P. lividus and offers preliminary insights into their molecular content and biological activity, those encouraging future investigations into invertebrate-derived EVs as a novel and promising source of therapeutically relevant immunomodulatory biomolecules.
Studies were supported by the MUR-PNRR PRIN 2022 Project SURPRISE (GA P2022LASKT).