Unraveling new players in helminth pathology: extracellular vesicles from Fasciola hepatica and Dicrocoelium dendriticum exert different effects on hepatic stellate cells and hepatocytes.

Fasciola hepatica and Dicrocoelium dendriticum are parasitic trematodes residing in the bile ducts of mammalian hosts, causing, in some cases, impairment of liver function and hepatic fibrosis. Previous studies have shown that extracellular vesicles released by F. hepatica (FhEVs) and D. dendriticum (DdEVs) induce a distinct phenotype in human macrophages, but there is limited information on the effect of parasitic EVs on liver cells, which interact directly with the worms in natural infections. In this study, we isolated FhEVs and DdEVs by size exclusion chromatography and labeled them with a lipophilic fluorescent dye to analyze their uptake by human hepatic stellate cells (HSC) and hepatocytes, important cell types in liver pathology, using synthetic liposomes as internal labeling and uptake control. We analyzed EV uptake and the proteome profiles after the treatment with EVs for both cell types. Our results reveal that EVs establish unique and specific interactions with stellate cells and hepatocytes, suggesting a different role of EVs derived from each parasite, depending on the migration route to reach their final niche. FhEVs have a cytostatic effect on HSCs, but induce the extracellular matrix secretion and elicit anti-inflammatory responses in hepatocytes. DdEVs have a more potent anti-proliferative effect than FhEVs and trigger a global inflammatory response, increasing the levels of NF-κB and other inflammatory mediators in both cell types. These interactions may have a major influence on the progression of the disease, serving to generate conditions that may favor the establishment of the helminths in the host.

Extracellular vesicles from the trematodes Fasciola hepatica and Dicrocoelium dendriticum trigger different responses in human THP-1 macrophages.

Extracellular vesicles (EVs) released by the helminths Dicrocoelium dendriticum and Fasciola hepatica are important modulators of the host immune response, contributing to the establishment of the infection. Monocytes and, in particular, macrophages are major regulators of the inflammatory response and are likely responsible for the phagocytosis of most of the parasite EVs. In this study, we isolated EVs from F. hepatica (FhEVs) and D. dendriticum (DdEVs) by size exclusion chromatography (SEC) and characterized them by nanoparticle tracking analysis, transmission electron microscopy and LC-MS/MS, and analyzed the cohort of proteins. The treatment of monocytes/macrophages with FhEVs, DdEVs or EV-depleted fractions from SEC, demonstrated species-specific effects of the EVs. In particular, FhEVs reduce the migratory capacity of monocytes and the analysis of the cytokine profile showed that they induce a mixed M1/M2 response, exerting anti-inflammatory properties in Lipopolysaccharide-activated macrophages. In contrast, DdEVs do not affect monocyte migration and seem to have pro-inflammatory properties. These results correlate with the differences in the life cycle of both parasites, suggesting different host immune responses. Only F. hepatica migrates to the bile duct through the liver parenchyma, driving the host immune response to heal deep erosions. Furthermore, the proteomic analysis of the macrophages upon FhEV treatment identified several proteins that might be involved in FhEV-macrophage interactions.

Proteomic Analysis of Extracellular Vesicles From Fasciola hepatica Hatching Eggs and Juveniles in Culture.

The identification of extracellular vesicles (EVs) in Fasciola hepatica has provided a new way to understand parasite-host communication. Most of the studies on EVs have focused on the adult stage of F. hepatica, but recently, the presence of EVs from different developmental stages has been reported. To better understand the potential role of EVs in the biology of the parasite and in the infection process, the protein cargo of EVs from embryonated eggs and newly-excysted juvenile (NEJs) flukes cultured up to 28 days, has been analyzed. EVs were isolated by size exclusion chromatography and evaluated by nanoparticle tracking analysis and transmission electron microscopy. LC-MS/MS proteomic analysis of EVs revealed the presence of 23 different proteins from embryonated egg-derived EVs and 29 different proteins from NEJ-derived EVs. Most of the identified proteins had been previously described in EVs from F. hepatica adults, including cytoskeletal proteins, glycolytic enzymes, stress-related proteins and tetraspanins. Nevertheless, EVs from hatching eggs and NEJs exhibited qualitative differences in composition, when compared to EVs form adults, including the absence of cathepsin cysteine peptidases. The differential content of the EVs released by the different developmental stages of the parasite reflect the intense activity of NEJs at this early stage, with several proteins involved in membrane traffic and cell physiology. This new set of identified proteins could help to understand key metabolic, biochemical and molecular mechanisms mediated by EVs that take place upon egg hatching and after parasite excystment.