Helminth-released EVs are considered one of the strategies employed to deliver signals to the host immune system which are crucial to maintain the long-term parasite-host interaction [1]. Previous reports have evidenced the properties of sEVs released by the larval stage of E. granulosus and their interaction with host cells such as hepatocytes, macrophages and DCs [13, 15, 35]. In this work, we isolated and characterized sEVs from control and drug-treated E. granulosus-protoscoleces and studied the immune response profile induced on murine DCs.
The production, release and uptake of EVs are regulated by intracellular and extracellular stimuli [36]. Likewise, therapeutic interventions represent both types of stimuli, in which the drugs can inhibit or activate the biogenesis and release of exosomes [37] and alter their morphological characteristics and cargo [38–39]. Thus, in this context, we aimed to identify potential differences in the protein pattern and initial immune response of sEVs coming from E. granulosus protoscolex cultures under pharmacological treatment as a potential strategy for monitoring drug response. Consistently with several reports, the size of sEVs determined by DLS and corroborated by TEM was between 100–200 nm (Fig. 1A-B). This diameter was in the upper range of sEVs and was slightly larger than our previous determination for the exosome-like vesicles of the E. granulosus larval stage [13, 40]. Also, we have previously shown that an increment in intracellular calcium in presence of loperamide, was able to increase the E. granulosus-EVs formation and secretion similarly to other reports as well as a greater abundance in their protein cargo [13, 41–42]. Here, our hypothesis was partially verified since sEVs purified from all conditions showed a similar protein pattern (Fig. 1C). However, although in control samples or in presence of ABZSO the total amount of proteins was similar, in presence of metformin, it was greater (Fig. 1C). This fact could be associated with an enhanced release of EVs under metformin treatment as it has been reported in mesenchymal stem cell-derived EVs [43]. It is known that metformin through the activation of AMPK, induces indirect inhibition of TORC1 in the parasite, and as has been described in animal cell models, sustained inhibition of TORC1 activates the exosome release concomitant with activation of autophagy [44–45].
Particularly, we detected 107 and 8 exclusive proteins in the sEV-cargo from metformin and ABZSO-treated parasites respectively, which probably are associated with the drug-induced cellular alterations [29, 44, 46]. Since that metformin is capable of causing lysosomal damage, also in AMPK-dependent manner, our findings suggest that these perturbations could be contributing to the higher protein cargo into sEVs obtained under metformin treatment [47]. Beyond the proteins involved in exosome biogenesis and the characteristic sEV markers, we exclusively identify a variety of enzymes in metformin-treated protoscoleces-derived EVs, which suggest that they could be associated with a role in the parasite-parasite interaction and/or in the host-parasite interface. Among them, we found enzymes that could modulate the proliferation as estradiol 17β-dehydrogenase (W6U9Y5, a steroidogenic enzyme that controls the last step in the formation of estrogens) which catalyzes the conversion of estrone to a more potent estrogen, 17β-estradiol -E2-, with potential binding capacity to parasite nuclear receptors to promote transcription [20]. In the same way, hypoxanthine-guanine phospho-ribosyltransferase (W6ULW2) and GMP-synthase (W6UEZ1) are involved in the purine biosynthesis, while lethal (2) giant larvae protein (W6UD39, which plays an important role in regulating cell polarity and asymmetric division) functions as tumor suppressor controlling proliferation by Notch signaling as has been previously reported in exosomes [48]. Moreover, we detected other enzymes such as mannose-1P guanyltransferase-α (W6U7H3) involved in GDP-mannose biosynthesis required for protein N-glycosylation which was also identified in colon and ovarian cancer exosomes, and αN-acetyl-galactosaminidase (W6UG73) and endoglycoceramidase (W6UMJ6) which remove carbohydrate residues from glycopeptides and glycolipids being able to induce changes in parasite antigenicity [49–50]. Interestingly, all these enzyme-cargo could modify the glycan moiety of parasite and/or host glycoproteins during the sEVs interactions with their environment, which also possess highly glycosylated antigens (Table S4).
Although it is well known that exosomes are naturally antigen transport carriers, this is the first work that highlights the antigenic content, including several known and potential uncharacterized antigens, of E. granulosus-sEVs obtained both in presence or absence of drug treatment (Fig. 2 and Tables S4-S5). This considerable antigenic cargo can be transferred to DCs to promote their maturation and cytokine transcription activation (Fig. 4) that allows the initiation of T-cell-mediated immune responses to favor the host as has been described for cancer cell exosomes [51]. The maturation and modulation of DCs by E. granulosus E/S products, including soluble antigens affect the release of cytokines that regulate the profile of immune responses to mainly induce immune tolerance [52–54]. Similarly, a previous study demonstrated that EVs derived from this cestode possess an immunosuppressive effect on murine CD4+ and CD8+ T cells proliferation and significantly inhibit IL-10, IFN-γ, IL-6, IL-17A and TNF-α secretion and promote IL-2 and IL-4 secretion [14]. Comparably, sEVs derived from Taenia pisiformis cysticercus induced the M2 macrophage polarization [55]. Conversely, here the sEVs obtained from all conditions studied carried similar antigenic characteristics that allowed induction of IL-12 in DCs favoring a Th1 response (Fig. 4) as was observed in M1-derived macrophages after exposure to exosome-like vesicles from Schistosoma japonicum [56]. Remarkably, the parasite sEVs obtained under metformin treatment increased the expression of pro-inflammatory cytokines such as IL-6, TNF-α and IL-10 even at higher levels than the LPS control used in our assays (Fig. 4). In this line of evidence, a recent report showed that E. granulosus exosome-like vesicles induced maturation and differentiation of BMDCs towards to a pro-inflammatory profile with the production of IL-6, IL-12, IL-β, TNF-α and IFN-γ promoted by egr-miR-277a-3p and the regulation of the NF-kB p65/p50 ratio [57]. On the other hand, we also observed a decrease in the gene transcription of IL-6 and TGF-β in BMDCs exposed to control and ABZSO-derived sEVs. These data, added to the lack of IL-23 induction, correlate to that reported in murine lymphocytes where the Th17 profile was not induced after the exposure to EVs from E. granulosus [14]. Interestingly, the EVs from Trichinella spiralis were reported to generate a Th1/Th2 mixed immune response characterized by the release of IL-12, IFN-γ, IL-4 and IL-10 in the serum of immunized mice [58]. Therefore, the observed heterogeneous responses probably depend on the composition of the EVs including the antigenic protein and other biomolecules and especially on the context they were generated.
The description and characterization of sEVs antigens and immunomodulatory molecules are crucial to elucidate the potential role of these vesicles as tools for the identification of new biomarkers for the echinococcosis, in immunotherapy for autoimmune diseases and for the development of a new generation of vaccines. We found several unknown putative antigens that possess a high number of epitopes (Table S5). These proteins, like the known antigens present in these worm-derived sEVs (Table S4), may be O-glycosylated, which may result in recognition by C-type lectins and contribute to the immunoregulatory activities of EVs [59]. Likewise, the N-glycosylation could mediate vesicle internalization and consequent immunomodulation as has been reported for S. mansoni EVs in monocyte-derived dendritic cells through DC-SIGN [60]. Therefore, extensive protein glycosylation could have a key role in the conformational integrity, antigenicity, and immunogenicity of E. granulosus sEVs-antigens. Among the proteins detected in our samples, antigen 5, which corresponds to one of the most immunogenic and abundant hydatid fluid antigens, contains at least 10 and 2 O- and N-glycosylation sites, respectively. Considering that antigen 5 is detected at higher ratios in albendazole-treated patients [61], an expected possibility was to observe a higher abundance of this antigen in sEVs from ABZSO-treated parasites as we previously showed for EVs obtained under treatment with loperamide [13]. However, in the present study antigen 5 shows a similar abundance in all studied conditions indicating that probably the higher ratio observed in albendazole-treated patients corresponds to an increment in cyst permeability rather than to a greater antigen production and release either in a soluble or sEVs-linked manner. As we mentioned before, the increment in cyst permeability by albendazole favors the leakage of antigens which promote antibody production [62] and the pro-inflammatory response which is beneficial to limit parasitic progression in both cystic or alveolar echinococcosis [22, 63–64].
Moreover, we also identified several proteins associated with immunomodulation and interaction with the host (Figs. 3 and S1). A well represented protein was the T-cell immunomodulatory protein (W6V8B8 ortholog of Em-TIP), which has been previously characterized as an E/S product in E. multilocularis primary cell cultures involved in metacestode development and in promotion of interferon IFN-γ release by murine Th1-cells during the early stages of infection [65]. Besides, it has been reported that potential orthologs of this protein are involved in regulating inflammatory cytokines, in acute graft-versus-host disease model, in Plasmodium berghei infection and in Cryptosporidium parvum invasion [66–69]. Therefore, considering their adhesion and immunomodulatory characteristics, the Eg-TIP present in sEVs could have a role in parasite establishment and in the modulation of the early Th1 response. Besides, the LTA4 hydrolase (W6UMY4) and Prostaglandin-E(2)-9-ketoreductase (W6U2E2) detected in our samples could also be involved in the pro-inflammatory profile that generate the E. granulosus sEVs. In this sense, these enzymes mediate the synthesis of pro-inflammatory mediators which represent key factors in type-2 inflammation [70–71] which, among other functions, can induce granulocytes recruitment as has been reported in Caenorhabditis elegans and Nippostrongylus brasiliensis [72].
Additionally, in this study, we also detected several proteins related to protein–ligand or protein–protein interactions such as leucine-rich repeat (LRR) proteins (W6UG49 and W6UFQ5, Figs. 3 and S1). As an evolutionary conserved strategy, plants, invertebrates and vertebrates, use LRR-containing domains to sense pathogen patterns as a first line of defense [73]. Parasite LRR proteins could play a critical role in mimicking and desensitizing host sensors [74–75]. Moreover, in pathogens such as Leishmania and Leptospira interrogans, proteins with LRR domains have been shown to be involved in mediating pathogenicity, host cell attachment and invasion [76–77]. On the other hand, we also identified a scavenger receptor class B member (W6V978) homolog to a transmembrane glycoprotein found in macrophages, microglia, microvascular endothelium, cardiac and skeletal muscle, adipocytes, and platelets implicated in angiogenesis, atherosclerosis, phagocytosis, inflammation, lipid metabolism, and removal of apoptotic cells [78]. The expression of a CD36-like class B scavenger receptor in S. mansoni and Opisthorchis viverrini has been associated with the acquisition of host lipids (LDL, IDL and fatty acid) probably for nutritional, developmental and/or immune evasion purposes [79–80]. Therefore, the presence of this homologous protein in the E. granulosus sEVs could be associated with the amelioration in lipid uptake in the target organ for improving parasite growth. Finally, two basement membrane specific heparan sulfate proteoglycan core proteins (W6V2K4 and W6UKD6) were detected in Echinococcus sEVs, which also have been found in E/S products of Hymenolepis diminuta and associated with transport [81]. Interestingly in the same line evidence with our results, homologs for these proteins present in mast cell-derived EVs can be associated with inactive TGFβ-1 which mediates signaling in endosomes of the recipient cell allowing regulation of its phenotype and function [82].
Overall, this work revealed that exosome-like vesicles obtained from E. granulosus larvae could contribute in the process of parasite-host communication and in the initial type 1 immune response in the host, which is enhanced by drug treatment with ABZSO and metformin. We determine that the sEVs of this cestode induced production of pro-inflammatory cytokines from BMDC, which could promote a Th1 profile in T cells, mainly with those vesicles derived from metformin treatment. This effect could be associated with the antimicrobial and antitumor potential of metformin which strengthens cellular immunity in the host and that is related to the interference with the endoplasmic reticulum to Golgi apparatus trafficking [83–84]. These results in part contribute to understand the high pharmacological efficacies of these drugs in in vivo echinococcosis experimental models, where the sEVs could participate in the potentiation of the host immune response for parasite growth control [28, 85]. Thus, based on the prominent stability, antigenic properties, long-circulating half-life, and favorable safety profile that possess the sEVs, they or their derived engineered nanovesicles filled with selected antigenic proteins, could be considered good alternatives for the screening of new vaccine models in patients with established echinococcosis as a strategy for secondary disease protection.