Special considerations for studies of extracellular vesicles from parasitic helminths: A community‐led roadmap to increase rigour and reproducibility

Abstract

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross‐species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well‐recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth‐derived EVs in these processes and highlights EVs as an important participant in cross‐phylum communication. While the mammalian EV field is guided by a community‐agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan‐helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth‐derived EVs to complement the MISEV guidelines. We summarise community‐agreed standards for studying EVs derived from this broad set of non‐model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

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

“…To verify their presence and characterize the EVs (size and morphology), both NTA and TEM were used (EVtrack ID EV220006), as recommended by White et al. ( 2023 ). The isolated EVs were visualized by TEM, confirming the presence of typical round‐shaped vesicles within the size range of 30–200 nm (data not shown).…”

“…The isolation of EVs from parasites was performed as described previously (Sánchez‐López et al., 2020 ) and following the recent recommendations issued by White et al. ( 2023 ). Briefly, ESP were centrifuged at 16,000 × g/20 min at 4°C and filtered through 0.22 μm membrane filters to exclude large EVs.…”

Monoclonal antibody-based localization of major diagnostic antigens in metacestode tissue, excretory/secretory products, and extracellular vesicles of Echinococcus species

“…specific epitopes. The EV fractions of the mentioned extravesicular ESP and intravesicular fluid fractions were isolated and purified with a protocol from Eichenberger et al ( Eichenberger, 2021 ), according to MISEV guidelines ( Théry et al., 2018 ; White et al., 2023 ). In brief, a combination of differential ultracentrifugation followed by Opti-prep ® discontinuous gradient (ODG) was used to purify EV fractions (fractions 3 to 8).…”

“…In recent years, substantial research was focused on the description of extracellular vesicles (EVs) in multiple helminth species. Helminths release EVs into their environment, which have various effects that are relevant in parasite-host interactions ( White et al., 2023 ). So far, this work was mainly focused on the characterization of EV-molecules and their specific interactions with host cells and tissues.…”

Proteomic characterization of extracellular vesicles released by third stage larvae of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae)

“…Nevertheless, studies focused to investigate molecules shaping the adaptation of these parasites to their natural hosts are rather limited ( Mehrdana and Buchmann, 2017 ; Palomba et al., 2020 ; Trumbić et al., 2021 ; Harbar et al., 2022 ). Recent studies have revealed that extracellular vesicles (EVs) represent a new paradigm in the “cross-talk” between parasites and their hosts, playing a crucial role in pathogenesis’s mechanisms including the parasite’s capacity for immune evasion ( Marcilla et al., 2012 ; Barteneva et al., 2013 ; Marcilla et al., 2014 ; Coakley et al., 2015 ; Sánchez-López et al., 2021 ; White et al, 2022a) . In addition, helminth-derived EVs are recently proposed as key players in helminth-microbiota crosstalk ( Rooney et al., 2022 ).…”