Enolase and 16.5-kDa Tegument-Associated Protein in Fasciola hepatica Extracellular Vesicles: Clues to Their Role in Pathogenesis

Fasciolosis, caused by Fasciola hepatica, is a parasitic zoonosis that induces liver fibrosis in infected hosts, including ruminants and humans. Extracellular vesicles secreted by F. hepatica (FhEVs) play a crucial role in modulating host immune responses and promoting tissue re-modelling. This work explores the effects of two proteins found in FhEVs, enolase (Fhenolase), enriched in the vesicular lumen, as well as the 16.5-kDa tegument-associated protein (Fh16.5TP), highly abundant in the EV membrane, on hepatic and liver-associated immune cells. Recombinant proteins (r-Fhenolase and r-Fh16.5TP) were produced to evaluate their impact on cell viability, inflammatory responses, proteomic profiles and EV secretion in THP1-XBlue CD14 macrophages, HepG2 hepatocytes and LX-2 hepatic stellate cells (HSCs). Interestingly, r-Fhenolase, but not r-Fh16.5TP, showed anti-inflammatory properties in lipopolysaccharide (LPS)–activated macrophages, by reducing NF-κB activation and inducing significant changes in the protein cargo of macrophage-derived EVs, which contained lower levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6. Proteomic analysis of cells treated with r-Fhenolase revealed distinct alterations in proteins related to fibrotic and inflammatory pathways, including a reduction in extracellular matrix (ECM) proteins and suggesting a potential role in mitigating liver fibrosis. Furthermore, r-Fhenolase reduced EV production and fibrotic markers in hepatic cells, but not in macrophages. In contrast, r-Fh16.5TP increased pro-fibrotic proteins in both, cells and EVs, and increased EV production specifically in LX-2 cells, indicating its possible contribution to fibrosis progression in fasciolosis. These findings represent a first approach to analyse EV-associated proteins and study their potential role in the molecular mechanisms of F. hepatica–host interactions.