Characterization of exosome-like vesicles derived from Taenia pisiformis cysticercus and their immunoregulatory role on macrophages

Abstract

Background: Taenia pisiformis is one of the most common intestinal parasites in canines, and leads to serious economic losses in the rabbit breeding industry. Exosome-like vesicles from parasites play crucial roles in host-parasite interactions by transferring cargo from parasites to host cells and by modulating host immunological response through inducing production of host-derived cytokines. Nevertheless, the mechanism by which exosome-like vesicles from T. pisiformis cysticercus regulate the macrophage immune response remains unknown. Methods: Using ultracentrifugation, we isolated exosome-like vesicles from excretory/secretory products (ESP) of T. pisiformis cysticercus. The morphology and size of purified vesicles were confirmed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The components of proteins and miRNAs within these vesicles were identified by proteomic analysis and high-throughput small RNA sequencing. The biological function of targets of exosomal miRNAs was predicted by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Moreover, the expression of Th1-and Th2-type immune response associated cytokines in RAW264.7 macrophages were evaluated by qPCR and ELISA. We found that exosome-like vesicles were typical cup-shaped vesicles with diameters from 30 to 150 nm. A total of 87 proteins were identified by proteomic analysis, including proteins prominently associated with exosome-like vesicles biogenesis and vesicle trafficking. 41 known miRNAs and 18 novel miRNAs were identified in the exosome-like vesicles. Eleven selected miRNAs, including 7 known miRNAs (miR-71-5p, miR-10a-5p, miR-let-7-5p, miR-745-3p, miR-219-5p, miR-124-3p and miR-4989-3p) and 4 novel miRNAs (novel-mir-3, novel-mir-7, novelmir-8 and novel-mir-11) were validated to exist in metacestiodes and exosome-like vesicles of T. pisiformis cysticercus by qPCR. The functions of most targets of exosomal miRNAs were mainly associated with signal transduction and the immune system. Additionally, T. pisiformis cysticercus-derived vesicles induced the production of IL-4, IL-6, IL-10, IL-13 and Arg-1, but downregulated the expression of IL-12, IFN-γ and iNOS in RAW264.7 macrophages. Conclusions: We demonstrated that proteins and miRNAs enclosed within exosome-like vesicles from T. pisiformis cysticercus have immunomodulatory functions. Furthermore, exosome-like vesicles were shown to induce the

Exosomal microRNA let-7-5p from Taenia pisiformis Cysticercus Prompted Macrophage to M2 Polarization through Inhibiting the Expression of C/EBP δ

“…Further, ITEM analysis indicated that the isolated vesicles contained two exosomal markers CD9 and CD63 ( Figure 1 B). The size distribution of the particles was shown to range predominantly from 50 nm to 200 nm in diameter ( Figure 1 C), consistent with our previous study [ 14 ]. Taken together, the TEM, ITEM and NTA analyses confirmed that exosome-like vesicles were successfully isolated from ESPs of T. pisiformis cysticercus.…”

“…Culture media were refreshed after 12 h to remove contaminants from host components. The excretory/secretory products (ESPs) of T. pisiformis cysticerci were collected at 24 h and 48 h. Isolation of exosome-like vesicles was carried out from pooled ESPs as described in our previous study [ 14 ]. The size of isolated exosome-like vesicles was determined using a NanoSight LM10 instrument (Nanosight, Wiltshire, UK).…”

“…In the gastrointestinal nematode Heligmosomoides polygyrus , Buck et al [ 13 ] identified secreted exosomes that contained miRNAs and Y RNA that suppressed the expression of Il33r and Dusp1, suggesting exosomal miRNAs could regulate target gene expression in host cells. Although our previous results showed that Th2 immune response inhibition can be induced by exosome-like vesicles derived from T. pisiformis metacestodes both in immunized rabbits in vivo and macrophages in vitro [ 14 ], the exact molecular mechanisms regarding the exosome-like vesicles in modulating of Th2 immune response/M2 macrophage polarization requires further investigation.…”

“…The let-7 miRNA family is involved in developmental transitions, cell signaling, and the regulation of immune response [ 17 , 18 ]. In addition, vertebrate miRNA let-7c can polarize macrophages toward the M2 phenotype by regulating the expression of C/EBP-δ [ 19 ], and our previous study demonstrated miRNA let-7 is one of the most abundant miRNAs in exosome-like vesicles of T. pisiformis metacestodes [ 14 ]. Thus, we speculated that T. pisiformis metacesode exosome-like vesicles were also involved in macrophage polarization through exosomal let-7 mediated gene regulation, thereby inhibiting the host inflammatory response.…”

Parasitic Helminth-Derived microRNAs and Extracellular Vesicle Cargos as Biomarkers for Helminthic Infections

“…The composition of EVs, such as small RNA and/or protein profiles is available for some cestode species (Wang et al, 2020b). In E. granulosus, studies were carried out on EVs isolated from hydatid cyst fluid (HCF) directly from hosts with CE (Siles-Lucas et al, 2017; Zhou et al, 2019) and cultured protoscoleces (Nicolao et al, 2019), leading to the identification of highly immunogenic antigens, such as antigen 5, Antigen B, P29 and endophilin-1.…”