In vitro Host-Parasite Models Combined With Proteomics Reveal Cell Type-Specific Responses of Early Migrating...

The trematode Fasciola hepatica has a complex life cycle involving two hosts. Infection of the definitive host, mainly livestock and humans, occurs through ingestion of the infective stage, the metacercariae, which excyst in the duodenum and release newly excysted juveniles (FhNEJ). These FhNEJ first interact with small intestinal epithelial cells (SIECs), penetrate across the intestinal wall, through the peritoneum, migrating over mesothelial cells (MCs) toward the liver and the biliary tree, where they develop into adult worms and establish chronically. We recently described the in vitro interactions of FhNEJ with SIEC at the proteomic level. Here, we extend this analysis to FhNEJ interactions with MC and hepatic stellate cells using a single cell type in vitro model combined with SWATH-MS-based proteomics. However, a weak proteomic response was observed in both the parasite and MCs or hepatic stellate cells upon direct coincubation, with no clear signatures of targeted immune or structural activation. Based on these findings, we developed a sequential cell type in vitro model in which FhNEJ were first incubated with SIECs and then with MCs, to assess whether a specific sequence of host cell contacts is required to elicit detectable proteomic responses. This approach revealed a higher number of differentially expressed proteins upon interaction than in the single cell type in vitro model, particularly in the parasite, reflecting increased activity during cell-to-cell transitions, including processes such as proteolysis, which likely support tissue migration and host interaction. The results indicate that FhNEJ exhibit a dynamic response to host cell-specific interactions, with early activation following SIEC interaction and subsequent attenuation after contact with MCs, notably affecting focal adhesion pathways in the latter cell type. These findings support the idea that sequential host cell encounters are necessary for effective recognition and interaction by the FhNEJ.