Characterization of extracellular vesicles released by S. stercoralis infective larvae isolated from clinical samples

Extracellular vesicles (EVs) represent an important mechanism of inter-cellular and inter-kingdom communication, thus mediating host-pathogen interactions. Numerous helminth parasites have already been reported to shed EV-like structures displaying functional effects on target cells. However, to the best of our knowledge, EVs from S. stercoralis are yet to be investigated. Here we hypothesized that S. stercoralis iL3 might release EVs involved in the interaction with the host immune system. S. stercoralis iL3 were isolated from fecal samples obtained from patients attended at the IRCCS Sacro Cuore Don Calabria Hospital. The samples were maintained in medium supplemented with EXO-free FBS in 1µm trans-well inserts. The conditioned medium was collected and replaced every day, and EVs prepared by differential ultracentrifugation. EVs were analyzed by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and untargeted proteomics to identify their cargo. Moreover, THP-1 (monocytic cell line) were exposed to iL3 EVs over-time and the release of cytokines in the conditioned medium was assessed. Our results indicate that S. stercoralis iL3 release EVs-like structures of 80-115nm in diameter, as determined by NTA and TEM. These EVs contain a cargo of more than 100 different proteins, including some important markers of helminth-derived EVs. When used to stimulate THP-1, iL3-EVs induced a strong cell activation with release of pro-inflammatory cytokines and alarmins in the conditioned medium as soon as 6h post-exposure. These results demonstrate the ability of S. stercoralis to release EVs, which are able to stimulate the host immune response. These results pave the way for more in-depth investigations on the specific role of iL3-derived EVs in the host-pathogen interaction in human strongyloidiasis.