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

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 Cysticercus pisiformis regulate macrophage immune response remains unknown. Methods: Using ultracentrifugation, we isolated exosome-like vesicles from excretory/secretory products (ESP) of C. pisiformis. The morphology and size of puried vesicles were conrmed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The components of proteins and miRNA within these vesicles were identied 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 qRT-PCR 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 identied by proteomic analysis, including proteins prominently associated with exosome-like vesicles biogenesis and vesicle tracking. 41 known miRNAs and 18 novel miRNAs were identied in the exosome-like vesicles. 11 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, novel-mir-8, and novel-mir-11) were validated to exist in metacestiodes and exosome-like vesicles of C. pisiformis by qRT-PCR. The functions of most targets of exosomal miRNAs were mainly associated with signal transduction and the immune system. Additionally, C. pisiformis-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 C. pisiformis have immunomodulatory functions. Furthermore, exosome-like vesicles were shown to induce the macrophage Th2-type immune response in vitro. Our study suggests that exosome-like vesicles play an important role in the interaction between cysticerci and their hosts. qRT-PCR SuperMix (TransGen Biotech) on an ABI7500. The qRT-PCR reaction system consisted of 10 μl of 2×TransStart Tip Green qPCR SuperMix, 0.4 μl of TransScript One-Step RT Enzyme Mix, 0.4 μl of Passive Reference Dye, 0.8 μl of Forward primer, 0.8 μl of Reverse primer, 2 μl of RNA template and 5.6 μl of ddH 2 O into a total volume of 20 μl. qRT-PCR reaction procedures and data analysis were performed as previously described.


Background
T. pisiformis, a common intestinal tapeworm in the canines and felines, is widely distributed around the world [1,2]. Cysticercus pisiformis, the larval stage of T. pisiformis, causes considerable economic losses to the rabbit breeding industry [3]. Infection in the de nitive host may occur when they consume lagomorph internal organs infected by C. pisiformis. Oryctolagus cuniculus become infected through ingestion of water or forage contaminated with T. pisiformis eggs. C. pisiformis usually parasitizes the liver capsule, peritoneum, greater omentum and mesentery, and occasionally in other organs such as the pelvis or lungs [3,4]. Rabbits infected with T. pisiformis have a weakened immunologic resistance and RPMI-1640 culture medium (Invitrogen, Carlsbad, CA, USA) and maintained in T-25 asks in RPMI-1640 medium supplemented with 10% exosome-depleted fetal bovine serum (FBS), 100 μg/ml streptomycin and 100 IU/ml penicillin at 37 °C under 5% CO 2 . Each ask contained 50 cysts in 15 ml culture medium.
To ensure host components were expelled thoroughly from larvae, the medium was changed after 12 h [30]. ESP from C. pisiformis were obtained at 24 h and 48 h and stored at 4 °C prior to centrifugation.
Exosome-like vesicles from the ESP of C. pisiformis were puri ed by serialcentrifugation as previously described [31]. 100 ml pooled ESP from C. pisiformis were subjected to successive centrifugations at 300×g for 10 min and 10,000×g for 30 min to remove cellular debris and dead cells. The supernatant was harvested and centrifuged at 75,000×g at 4 °C for 90 min to remove large vesicles. This supernatant was collected and centrifuged at 110,000×g for 90 min at 4 °C. The resultant pellet was obtained and centrifuged at 110,000×g for 90 min to remove remaining protein contaminants, and re-suspended in 50 μl PBS puri ed with a 0.22 μm lter. The concentration of puri ed exosomal proteins was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scienti c, Waltham, MA, USA). All aliquots were stored at -80°C until further use.

Transmission electron microscopy (TEM)
The morphology and size of exosome-like vesicles from C. pisiformis were visualized by TEM. Brie y, 10 μL of exosomes fromC. pisiformis were loaded onto a 200-mesh formvar-coated copper grid (Agar Scienti c, UK) for 10 min and the excess stain was removed by blotting with lter paper. Exosome-like vesicle pellets were negatively stained with a 3% solution of phosphotungstic acid (pH 7.0) for 1 min at room temperature. Grids were air dried and imaged using a Hitachi TEM at a voltage of 80 kV.
Nanoparticle tracking analysis (NTA) The size distribution and number of exosome-like vesicles were analyzed by measuring the rate of Brownian motion of each particle using a NanoSight LM10 instrument (Nanosight, UK). The LM10 uses digital cameras to directly track the movement of individual particles in solution, thereby enabling the determination of particle size distribution as well as the number of nanoparticles [32]. The measurement procedure was performed as previously described [33]. Each sample was measured in triplicate and the NTA analytical software (version 2.3) was utilized to capture and analyze the data.

Mass spectrometry analysis
To identify the proteins of exosome-like vesicles from C. pisiformis, three biological replicates samples were prepared as described above. Each 10 μg pelleted exosomes were lysed with 150μl RIPA lysis buffer and separated by 12% polyacrylamide gel electrophoresis (PAGE), respectively. All bands were cut into 1 hosts. Database search parameters were set as follows: trypsin as enzyme; peptide mass tolerance of 20 ppm and fragment mass tolerance of 0.05 Da; + 1, + 2, + 3 as peptide charge; a maximum of one missed cleavage; carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K) as xed modi cations and oxidation (M), Gln-> pyro-Glu (N-term Q), deamidated (NQ) as variable modi cations. False discovery rate (FDR) lower than 0.01 was used as screening condition [34][35].
Gene ontology (GO) analysis of the identi ed proteins was conducted using the Gene ontology database (http://www.geneontology.org). Functional annotations of the proteins were performed using Blast2GO program (https://www.blast2go.com) against the non-redundant protein database (NCBInr). Additionally, the Clusters of Orthologous Groups (COG) database (http://www.ncbi.nlm.nih.gov/COG/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) were used to classify and group these identi ed proteins.

Western blot analysis
The protein concentration of exosome-like vesicles, soluble worm antigens (SAg) and ESP from C. pisiformis were measured using a BCA protein assay kit. 15 μg of total protein was denatured at 100 °C for 10 min and separated by 12% SDS-PAGE. The proteins were transferred to polyvinylidene uoride membranes (Millipore, Burlington, MA, USA) for 13 min and blocked with 5% non-fat milk in PBST for 2 h at room temperature. Two antibodies of anti-14-3-3 and anti-enolase (both from T. solium produced in rabbits were prepared in our lab, 1:200) [36] were separately added to the membrane and incubated at 4°C overnight. The membranes were washed three times with PBST and incubated with HRP-goat-anti rabbit IgG (H + L) (1:1000, Beyotime, China). The bands were developed using an ECL chemiluminescence working solution (Beyotime, China) following the manufacture's instruction.
RNA extraction and high-throughput small RNA sequencing Exosome-like vesicles derived from C. pisiformis and fresh metacestodes of T. pisiformis (served as positive control) in three biological replicates were prepared as described above, and total RNA was extracted using TRIzol reagent (Invitrogen). RNA sample integrity and quality were determined by Agilent  (18-30 nt). Afterwards, the mapped reads were aligned to miRBase database (http://mirbase.org) and Echinococcus spp. metacestode miRNA dataset to annotate known miRNAs (E-value < 0.05). Small RNA expression pro les, including miRNA, snRNA, snoRNA, tRNA, rRNA, and piRNA were annotated by RFam database (http://rfam.janelia.org). RepBase database (http://www.girinst.org/repbase) and pre-setting reference genome database were also used to identify small RNAs. In addition, the unannotated sequences were used to predict potential novel miRNA candidates through searching the characteristic hairpin structure of the miRNA precursor [37]. The prediction of targets of exosomal miRNAs was conducted using RNAhybrid, miRanda and TargetScan software. The potential biological functions of target genes were predicted using the KEGG database. Quantitative real-time PCR (qRT-PCR) for miRNAs and mRNAs miRNAs from 50 μl exosome-like vesicles of C. pisiformis and 20 mg C. pisiformis tissue were extracted using an miRNeasy kit (Qiagen, Germantown, MD, USA) following the manufacturer's instructions. The rst-strand cDNA was synthesized using 2 μg of total miRNA using Mir-X™ miRNA First-Strand Synthesis Kit (Takara, Japan) according to the manufacturer's protocols. The qPCR reaction system consisted of 12.5 μl of 2×TB Green Advantage Premix, 0.5 μl of 50×ROX Dye, 0.5 μl of miRNA-speci c forward primer, 0.5 of universal miRNA reverse primer, 2 μl of cDNA and 9 μl of ddH 2 O into a total volume of 25 μl. qPCR reactions were performed on an ABI7500 instrument according to the following parameters: initial activation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Data was evaluated using online software (http://pcrdataanalysis.sabiosciences.com/mirna). All miRNA primers were purchased from Guangzhou RiboBio Co., Ltd (Additional le 1: Table S1). As a reference, cel-miR-39-3p was added to each sample to monitor miRNA extraction e ciency and normalize sample-to-sample variation. The relative abundance of miRNAs was calculated and normalized using the 2 −ΔΔCt method.
Enzyme-linked immunosorbent assay (ELISA) Following RAW264.7 cell stimulation with LPS, IL-4, exosome-like vesicles from C. pisiformis, PBS, or a combination for 12 h, 24 h and 36 h, the cell-free supernatants were harvested and frozen at -80 °C until the assay was performed. The levels of Th1 and Th2 cytokines in the supernatant was assessed using commercially available mouse cytokine (IL-4, IL-6, IL-10, IL-13, IL-12 and IFN-γ) ELISA kits (RayBiotech, Peachtree Corners, GA, USA) according to the manufacturers' protocols. Each experiment was performed in triplicate.

Statistical analyses
Statistical analyses were conducted using GraphPad Prism5.0. Comparisons between groups were assessed using the unpaired Student′s t-test. Differences among multiple groups were analyzed by one way analysis of variance (ANOVA) using SPSS 24.0 (SPSS Inc., Chicago, IL, USA). Data were presented as the means ± standard error of the mean (SEM). Statistical signi cance was indicated as * for P < 0.05, ** for P < 0.01, and *** for P < 0.001.

Results
Size and morphological analysis of C. pisiformis-derived exosome-like vesicles To con rm the presence of exosome-like vesicles isolated from the culture medium of C. pisiformis, the pellets obtained from sequential centrifugation were subjected to TEM and NTA analysis. TEM images showed that these vesicles were spherical, approximately 30-150 nm in diameter and with lipid bilayerbound membrane structures (Fig. 1a). The particle size distribution of the vesicles was distributed around 50-150 nm and peaked at a mean diameter of 98.47 nm (Fig. 1b), which had the prototypical size characteristic of exosome-like vesicles and was consistent with exosomes from other parasites [10,23,27,[38][39][40]. This data indicated that we successfully isolated and puri ed exosome-like vesicles from C. pisiformis.

Characterization of C. pisiformis exosomal protein cargo
The MS/MS analysis identi ed 87 parasite-unique proteins in C. pisiformis-derived exosome-like vesicles (Additional le 2: Table S3). GO analysis showed that these proteins were classi ed to 40 categories by cellular component, biological process, and molecular function (Fig. 2, Additional le 2: Table S4). In terms of cellular component, the proteins were mostly related to membrane (16.06%), cell part (16.06%) and membrane part (11.92%). Biological process studies suggested that these exosomal proteins were involved in cellular process (18.39%), biological regulation (11.49%) and regulation of biological process (10.34%). In addition, most of molecular functions were classi ed into three categories: binding (40.43%), catalytic activity (34.04%) and transporter activity (9.57%) in the identi ed proteins. The top 50 parasiteorigin proteins with unique spectra numbers ≥ 3 are presented in Table 1, and some of these were identi ed as the most common exosomal proteins in Exocarta, mainly including chaperones (Heat shock protein and Beta-soluble NSF attachment protein), cytoskeletal proteins (Actin, Rab and Tubulin), metabolic enzymes (enolase, phosphoenolpyruvate carboxykinase and fructose 1, 6 bisphosphate aldolase ), molecules associated with signal transduction (annexin, 14-3-3 proteins, programmed cell death 6 interacting protein), elongation factor 1-alpha, and phosphoglycerate kinase. Most of these parasite-origin proteins have been described in exosome-like vesicles from Echinococcus and other atworm parasites [27,41,42].

Validation of C. pisiformis exosome-like vesicles proteomics results
To verify the proteomic data, two exosome-like vesicle enriched proteins (14-3-3 and enolase) were used for con rmation by western blotting. The results showed that these proteins were detected in the exosome-like vesicles, ESP and SAg of C. pisiformis (Fig. 3), consistent with the MS result that 14-3-3 and enolase were enriched in exosome-like vesicles from C. pisiformis.
Small RNA sequencing data analysis and qRT-PCR validation To identify the small RNA components in exosome-like vesicles from C. pisiformis, total RNA extracted from exosome-like vesicles and larvae of T. pisiformis were analyzed using small RNA high-throughput sequencing. The results revealed that a total of 36,961,810 and 31,592,685 raw reads were identi ed in exosome-like vesicles and metacestodes from the small RNA sequencing library, respectively. After read ltering and processing, a total of 24,329,002 (65.82%) and 24,897,949 (77.81%) clean reads were obtained. Among these reads, approximately 14,568,998 (59.88%) and 6,804,366 (27.66%) reads were mapped to the reference genome database. All mapped reads were used for small RNA classi cation, including miRNA, tRNA, rRNA, snRNA and snoRNA ( Fig. 4a and Fig. 4b).
A total of 41 and 59 known miRNAs were identi ed in two libraries. Among them, all of miRNAs identi ed in exosome-like vesicles were found in larvae libraries (Fig. 4c, Additional le 3: Table S5 and Table S6).
The miRNA length distribution of the two libraries showed that almost all of miRNAs were 20-24 nt (Fig.  4d) and the predominant species was 22 nt, a typical length of Dicer-processed products, which was consistent with the previous reports in other cestodes [30,[43][44][45]. Among identi ed exosomal miRNAs, the most abundant miRNAs were miR-277, followed by miR-10 and miR-71 (Additional le 3: Table S5). Furthermore, 18 novel miRNAs were successfully predicted in the exosome-like vesicles and metacestodes using Mireap (Additional le3: Table S7).

Bioinformatics analysis of known miRNAs in exosome-like vesicles
To determine the potential biological functions of the targets of the exosome-like vesicle miRNAs of C. pisiformis , the targets of the exosomal miRNAs were predicted using RNAhybrid and miRanda software packages. A total of 99,278 targets of 41 miRNAs were identi ed (Additional le 4: Table S8). KEGG analysis revealed that the potential biological functions of most targets were involved mainly in signal transduction and immune system, except for cancer, global and overview maps (Fig. 6). Moreover, some of well-known immune-related miRNAs were identi ed in exosome-like vesicles, including miR-2a, miR-9, miR-10a, miR-71 and let-7-5p [23,28,[46][47][48]. Therefore, we speculated that the C. pisiformis derived exosome-like vesicles might be involved in modulating host immune response by delivering immunerelated miRNA content.
T. pisiformis exosome-like vesicles stimulated secretion of molecules related to the Th2-type immune response in macrophages To investigate the potential function of exosome-like vesicles as determined by bioinformatics, we examined the in vitro effects of exosome-like vesicles on the release of Th1-and Th2-associated bioactive molecules in RAW264.7 macrophages. The qRT-PCR results showed that the mRNA levels of IFN-γ and iNOS at 12 h, 24 h and 36 h increased signi cantly in LPS-activated macrophages (Fig. 7a, 7b and 7g). The expression of IL-6, IL-10, IL-13 and Arg-1 increased signi cantly in IL-4-activated macrophages (Fig. 7d, 7e, 7f and 7h). The mRNA levels of IFN-γ and iNOS at 12 h, 24 h and 36 h and IL-12 at 24 h decreased signi cantly in the LPS + EXO group compared to the LPS group (Fig. 7a, 7g and  7b), while expression of Arg-1 at 12 h, 24 h and 36 h, IL-4 at 12 h and 24 h, IL-6 at 24 h, IL-10 at 24 h and IL-13 at 36 h increased signi cantly in LPS + EXO group compared with LPS group (Fig. 7c, 7d, 7e and   7f). These data showed that C. pisiformis exosome-like vesicles primed macrophages to secrete Th2 related bioactive molecules.
To further validate the qRT-PCR results, the supernatant from those groups were collected and used to detect the expression of IL-12, IFN-γ, IL-4, IL-6, IL-10, and IL-13. The results of ELISA assay showed that both the expression levels of IFN-γ and IL-12 at 12 h, 24 h and 36 h were signi cantly increased in macrophages after treatment with LPS, while the production of IFN-γ and IL-12 at 12 h and 36 h decreased signi cantly in macrophages stimulated with EXO or LPS + EXO compared with LPS treated cells ( Fig. 8a and 8b). Macrophages treated with EXO obviously increased the level of IL-4 secretion at 12 h, 24 h and 36 h. However, cells treated with LPS + EXO produced remarkably lower levels of IL-4 after 24 h than did cells treated with EXO alone (Fig. 8c). The secretion levels of IL-6, IL-10 and IL-13 at 12 h and 24 h were signi cantly increased in macrophages treated with IL-4 (Fig. 8d, 8e and 8f), and the levels of IL-6 and IL-10 at 12 h and 36 h were also increased by treatment with EXO compared to the PBS control ( Fig. 8d and 8e).The expression of IL-6 and IL-10 at 24 h were increased by treatment with IL-4 + EXO compared to the IL-4 control ( Fig. 8d and 8e). The expression of IL-13 at 12 h had no obvious change after treatment with EXO. However, combination treatment with IL-4 + EXO tended to produce more IL-13 (P < 0.001) at 24 h and 36 h than did IL-4 treatment alone (Fig. 8f). Taken together, these data suggest that macrophages stimulated by exosome-like vesicles from C. pisiformis produced mainly Th2 cytokines.

Discussion
Exosomes, nano-sized endosome derived membrane vesicles, play vital roles in intercellular communication [49]. An increasing body of studies has revealed exosomes as a ubiquitous molecular mechanism that can transfer bioactive molecules from pathogens to host cells in order to regulate host immune response or promote parasite survival [50]. In the present study, we isolated the exosome-like vesicles derived from C. pisiformis, pro led their proteins and miRNAs, and further evaluated their immunomodulatory roles in RAW264.7 macrophages treated with exosome-like vesicles.
We identi ed seven enzymes involved in energy metabolism (glycolysis, gluconeogenesis, and tricarboxylic acid cycle). Among them, enolase, fructose 1, 6 bisphosphate aldolase and phosphoenolpyruvate carboxykinase are commonly observed in the context of parasite-derived exosome-like vesicles [30,42]. It is well-known that, in addition to participating in the glycolysis and gluconeogenesis pathways, enolase can act as a plasminogen receptor as well, which prevents blood clots and facilitates parasite migration within hosts [36], suggesting that enolases within exosome-like vesicles could be important for C. pisiformis survival in hosts. Through proteomic analysis, we were able to identify multiple proteins related to exosome biogenesis, including Rabs (Rab-2A, Rab-4A, Rab-6A, Rab-10 and Rab-14), vesicular fusion protein, Vps4, transforming protein RhoA and Rab effectors otoferlin.
Most of these molecules have been reported in exosome-like vesicles from Echinococcus and other atworm parasites [27,41]. In light of the fact that these proteins are typical molecules associated with the ESCRT-dependent pathway, we hypothesized that ESCRT is likely the major route involved in the formation of exosome-like vesicles and the sorting of cargo into exosome-like vesicles, consistent with a previous study of adult F. hepatica [41]. Moreover, the proteomics analysis identi ed tegument-speci c proteins from C. pisiformis exosome-like vesicles, providing evidence for the possible roles of eoxsomelike vesicles in parasite survival through modi cation of host immune response.
The most signi cant nding of this study was that exosome-like vesicles from C. pisiformis induced the macrophages toward the M2 phenotype and produced a Th2-type immune response. When RAW264.7 macrophages were treated with C. pisiformis exosome-like vesicles, the production of Arg-1, IL-4, IL-6, IL-10 and IL-13 was signi cantly increased. In contrast, the expression of iNOS, IFN-γ and IL-12 was signi cantly decreased, revealing that C. pisiformis exosome-like vesicles participate in promoting macrophages to M2 polarization. There is evidence that injection of exosomes from the intestinal uke Echinostoma caproni in BALB/c mice primes balanced Th2/Treg immune responses, which alleviates intestinal symptom severity in subsequent challenge infections and bene ts parasite survival [56]. The immunomodulatory capacity of exosomes from the murine gastrointestinal nematode, Heligmosomoides polygyrus, has been demonstrated by suppressing innate type 2 lymphoid cell responses. Furthermore, H.
polygyrus EVs have been shown to suppress the expression of IL1RL1/ST2, the IL-33 receptor, and type 2 innate lymphoid cell responses [57,58]. Similarly, our previous studies showed that rabbits immunized with exosomes from C. pisiformis displayed a higher production of IL-10, which results in decreasing signi cantly in worm reduction after challenging tapeworm eggs (unpublished). These studies suggest that exosome-like vesicles could play important roles in host Th2-type immune response induced by C. pisiformis.
In summary, although the presence of exosome-like vesicles has been demonstrated in several parasites, this is the rst systemic study on exosome-like vesicles derived from ESP of C. pisiformis in terms of morphology, size, content and immune regulation. The present work revealed that exosome-like vesicles participated in the process of parasite-host communication and the modulation of host Th2-type immune response induced by Cysticercus. The present investigation provides new insights into a deep understanding of molecular cargo in exosome-like vesicles of C. pisiformis and the pathogenesis of exosome-like vesicle-mediated metacestodiasis.

Conclusions
We successfully puri ed exosome-like vesicles from C. pisiformis and pro led their protein and miRNA components, demonstrating the potential biological functions of exosome-like vesicles in host immune response. Interestingly, the present study reveals the upregulation of molecules associated with Th2-type immune response in RAW264.7 macrophages after stimulation with exosome-like vesicles from C .pisiformis, which might facilitate survival of T. pisiformis metacestodes in rabbits. Further exploration of exosomal miRNA targets will be bene cial to elucidate the immunodulatory mechanism and the important roles of exosome-like vesicles in the interaction between the host and C. pisiformis.  Both unique peptide number and unique spectra number ≥ 3 were listed. Proteins listed in bold font represent the most common proteins of the "top 30" exosomes-like vesicles in ExoCarta.       LPS and IL-4 served as positive controls for M1 phenotype and M2 phenotype molecules, respectively.

Figures
PBS served as the negative control for cytokine stimulation. a and b Induction of M1 markers in RAW264.7 macrophages treated with C. pisiformis exosome-like vesicles. c, d, e, and f Induction of M2 markers in RAW264.7 macrophages by C. pisiformis exosome-like vesicles. Data for the nal analysis are from three independent experiments and are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 and *** P < 0.001 were considered statistically signi cant compared to PBS-treated RAW264.7 macrophages. # P < 0.05, ## P < 0.01 and ### P < 0.001 were considered statistically signi cant compared to LPS-treated RAW264.7 macrophages.