Abstract
In the last two decades, extracellular vesicles (EVs) from the three domains of life, Archaea, Bacteria and Eukaryotes, have gained increasing scientific attention. As such, the role of EVs in host-pathogen communication and immune modulation are being intensely investigated. Pivotal to EV research is the determination of how and where EVs are taken up by recipient cells and organs in vivo, which requires suitable tracking strategies including labelling. Labelling of EVs is often performed post-isolation which increases risks of non-specific labelling and the introduction of labelling artefacts. Here we exploited the inability of helminths to de novo synthesise fatty acids to enable labelling of EVs by whole organism uptake of fluorescent lipid analogues and the subsequent incorporation in EVs. We showed uptake of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rho) in Anisakis spp. and Trichuris suis larvae. EVs isolated from the supernatant of Anisakis spp. labelled with DOPE-Rho were characterised to assess the effects of labelling on size, structure and fluorescence of EVs. Fluorescent EVs were successfully taken up by the human macrophage cell line THP-1. This study, therefore, presents a novel staining method that can be utilized by the EV field in parasitology and potentially across multiple species.
Highlights
Extracellular vesicles, small membranous vesicles that contain a cargo of bioactive molecules are released from organisms spanning all three domains of life [1,2], including parasitic helminths and their hosts [3,4]
extracellular vesicles (EVs) isolated from the supernatant of Anisakis spp. labelled with DOPE-Rho were characterised to assess the effects of labelling on size, structure and fluorescence of EVs
Theaddition additionofof fluorescent lipid analogue, DOPE-Rho, to culture was actively in andand subsequently incorporated by larvae of nematodes from from two differing classes of upvivo in vivo subsequently incorporated by larvae of nematodes two differing classes of nematodes
Summary
Extracellular vesicles, small membranous vesicles that contain a cargo of bioactive molecules are released from organisms spanning all three domains of life [1,2], including parasitic helminths and their hosts [3,4]. An alternative method for pre-labelling of EVs was demonstrated by the culture of human bladder cancer cell lines in the presence of the fluorescent lipid analogue, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl [15]. Helminths present a highly suitable organism for the assessment of fluorescent lipid analogue labelling strategies of EVs in vivo. This proof-of-concept study aimed to validate the in vivo uptake of 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rho) in nematodes of two different classes; the larval stage of Trichuris suis (L1), a whipworm belonging to Enoplea, and Anisakis spp. This study assessed the applicability of this technique for labelling of EVs from Anisakis spp. through in vivo uptake of the fluorescent lipid analogue, DOPE-Rho and the functional application of this method in human THP-1 cell uptake studies
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