Animals and parasites
Animals used in this study were purchased from the Laboratory Animal Center of Lanzhou Veterinary Research Institute. New Zealand white rabbits were housed individually in wire cages equipped with a plastic nest and had access to food and water ad libitum. Fresh T. pisiformis cysticerci were harvested from New Zealand white rabbits 50 days after infection with 500 eggs of T. pisiformis.
Exosome-like vesicles isolation
To prepare exosome-like vesicles derived from T. pisiformis cysticercus, rabbits infected with T. pisiformis cysticerci were sedated with xylazine (5 mg/kg) and ketamine (25 mg/kg), and euthanized with a lethal dose of sodium pentobarbital (100 mg/kg). Metacestodes collected from the peritoneum and greater omentum of rabbits were washed thoroughly in sterile 0.9% sodium chloride containing 100 μg/ml streptomycin and 100 IU/ml penicillin (Life Technologies, Grand Island, NY, USA). The larvae were washed three times with RPMI-1640 culture medium (Invitrogen, Carlsbad, CA, USA) and maintained in T25 flasks 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% CO2. Each flask contained 50 cysts in 15 ml culture medium. To ensure host components were expelled thoroughly from larvae, the medium was changed after 12 h . ESP from T. pisiformis cysticerci were obtained at 24 h and 48 h and stored at 4 °C prior to centrifugation.
Exosome-like vesicles from the ESP of T. pisiformis cysticerci were purified by serial centrifugation as previously described . 100 ml pooled ESP from cysticercus pisiformis were subjected to two 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. The supernatant was collected and centrifuged at 110,000× g for 90 min at 4 °C. The resultant pellet was re-suspended in 12 ml filtered PBS, then centrifuged at 110,000× g for 90 min to remove remaining protein contaminants, and re-suspended in 50 μl PBS. The concentration of purified exosomal proteins was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 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 cysticercus pisiformis were visualized by TEM. Briefly, 10 μl of exosomes from cysticercus pisiformis were loaded onto a 200-mesh formvar-coated copper grid (Agar Scientific Ltd, Stansted, UK) for 10 min and the excess stain was removed by blotting with filter 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, Wiltshire, 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 . The measurement procedure was performed as previously described . Each sample was measured in triplicate and the NTA analytical software (version 2.3; Nanosight, Wiltshire, UK) was used to capture and analyze the data.
Mass spectrometry analysis
To identify the proteins of exosome-like vesicles from T. pisiformis cysticercus, three biological replicates were prepared as described above. Each 10 μg pelleted exosome was lysed with 150 μl RIPA lysis buffer (Beyotime, Shanghai, China) and separated by 12% polyacrylamide gel electrophoresis (PAGE), respectively. All bands were cut into 1 mm3 cubes and washed thrice with 25 mM NH4CO3 in 50% acetonitrile (ACN) for 1 h, and subjected to dehydration with ACN and reduction with 10 mM dithiothreitol (DTT) at 56 °C for 1 h. Alkylation was carried by the addition of 55 mM iodoacetamide (IAM) at room temperature for 45 min. In-gel digestion was performed using 2.5 μg trypsin at a ratio (w/w) of 1:40 (enzyme:substrate) at 37 °C overnight and was stopped with 5% formic acid (FA). Peptides were desalted with a Waters Oasis HLB column (Waters, Milford, MA, USA) and eluted in 2% ACN and 0.1% FA before drying with a Benchtop Centrifugal Vacuum Concentrator (Labconco, Kansas City, MO, USA). Peptides were subjected to LC-20AD nano-HPLC (Shimadzu, Kyoto, Japan) spectrometry for peptide separation and data analysis. Briefly, samples were loaded onto a C18 trap column at 15 μl/min in solvent A (2% ACN, 0.1% FA) and the peptides were eluted and loaded onto an analytical column using a 44 min gradient, from 2% to 35% solvent B (98% ACN, 0.1% FA), at a flow rate of 400 nl/min. The eluate was subjected to nanoelectrospray ionization followed by tandem mass spectrometry in a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Relative parameters were set as a positive ion mode and data dependent mode with full MS scans from 200 to 1800 m/z, resolution at 70,000, normalized collision energy at 27, charge state at 2 +, 3 +, 4 + and 5 +, and resolution at 17,500. After the survey scans, the top 15 most abundant precursor ions were fragmented by high-energy collision dissociation (HCD). Since genome information on T. pisiformis was not yet obtained, the MS data analysis was carried out by Mascot software (version 2.3.02; Matrix Science, London, UK) using genomes from three parasites that have high kinship with T. pisiformis, including T. solium (http://www.genedb.org/Homepage/T.solium), E. granulosus (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/524/195/GCA_000524195.1_ASM52419v1/) and E. multilocularis (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/469/725/GCA_000469725.3_EMULTI002/). Additionally, common Repository of Adventitious Proteins (cRAP) (http://www.thegpm.org/crap/) was used to analyze proteins commonly found in proteomic experiments and Oryctolagus cuniculus genome database (https://www.uniprot.org/uniprot/?query=taxonomy:9986) was used to remove the protein from 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 fixed modifications and oxidation (M), Gln- > pyro-Glu (N-term Q), deamidated (NQ) as variable modifications. False discovery rate (FDR) lower than 0.01 was used as a screening condition [34, 35].
Gene ontology (GO) analysis of the identified proteins was conducted using the Gene ontology database (http://www.geneontology.org). Functional annotations of the proteins were performed using the 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 identified proteins.
Western blot analysis
The protein concentration of exosome-like vesicles, soluble worm antigens (SAg) and ESP from T. pisiformis cysticercus were measured using a BCA protein assay kit. Fifteen μg of total protein was denatured at 100 °C for 10 min and separated by 12% SDS-PAGE. The proteins were transferred to polyvinylidene fluoride 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 (1:200; both from T. solium produced in rabbits and were prepared in our laboratory)  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, Shanghai, China). The bands were developed using an ECL chemiluminescence working solution (Beyotime) following the manufacturer’s instructions.
RNA extraction and high-throughput small RNA sequencing
Exosome-like vesicles derived from cysticerci and fresh T. pisiformis cysticerci (served as a 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 using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). High-throughput small RNA sequencing was carried out by BGI (Shenzhen, China). Briefly, RNA fragments (18–30 nt) were separated by PAGE. After ligation of 3' and 5' adaptors to both ends of small RNAs, the ligation products were used for reverse transcription PCR. The PCR products (100–120 bp) were further separated on a PAGE gel and small RNA sequencing libraries were generated using a TruSeq Small RNA Library Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. Library sequencing was carried out on an Illumina HiSeq 2500 system (Illumina), and small RNA clean reads were obtained after removing adaptor reads, low quality reads and contaminants. Because of the unavailability of T. pisiformis genome, we used the T. solium genome (https://parasite.wormbase.org/Taenia_solium_prjna170813/Info/Index/), E. granulosus (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/524/195/GCA_000524195.1_ASM52419v1/) and E. multilocularis (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/469/725/GCA_000469725.3_EMULTI002/) as references to align screened small RNA sequences (18–30 nt). Afterwards, the mapped reads were aligned to the miRBase database (http://mirbase.org) and Echinococcus spp. metacestode miRNA dataset to annotate known miRNAs (E-value < 0.05). Small RNA expression profiles, including miRNA, snRNA, snoRNA, tRNA, rRNA, and piRNA, were annotated using the RFam database (http://rfam.janelia.org). The RepBase database (http://www.girinst.org/repbase) and the 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 . 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.
Macrophage cell culture and treatment
RAW264.7 murine macrophage cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and cultured in a 37 °C incubator with 5% CO2. The cells were plated in 6-well plates (1 × 106 cells/ml) and incubated with exosome-depleted DMEM conditioned media containing either 200 ng/μl LPS (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/ml IL-4 (Sigma-Aldrich), 50 μg/ml T. pisiformis cysticercus derived exosome-like vesicles, sterile PBS, or a combination with LPS + exosome-like vesicles, or IL-4 + exosome-like vesicles. All treatments were conducted in triplicate.
Quantitative real-time PCR (qPCR) for miRNAs and mRNAs
miRNAs from 50 μl exosome-like vesicles of T. pisiformis cysticercus and 20 mg T. pisiformis cysticerci tissue were extracted using an miRNeasy kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. The first-strand cDNA was synthesized using 2 μg of total miRNA using Mir-X™ miRNA First-Strand Synthesis Kit (Takara, Shiga, 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-specific forward primer, 0.5 μl of universal miRNA reverse primer, 2 μl of cDNA and 9 μl of ddH2O. qPCR reactions were performed on an ABI7500 instrument ( Thermo Fisher Scientific, Waltham, MA, USA) 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 (Guangzhou, China) (Additional file 1: Table S1). As a reference, cel-miR-39-3p was added to each sample to monitor miRNA extraction efficiency and normalize sample-to-sample variation. The relative abundance of miRNAs was calculated and normalized using the 2−ΔΔCq method.
To determine the relative expression of cytokines in RAW264.7 macrophages during T. pisiformis cysticercus exosome-like vesicles stimulation, total RNA from differently treated cells were isolated and probed by qPCR using mouse-specific primers (IL-4, IL-6, IL-10, IL-12, IL-13, Arg-1, iNOS, and the housekeeping gene GAPDH) (Genecopoeia, Guangzhou, China) (Additional file 1: Table S2). The qPCR was conducted using the TransScript Green One-Step qPCR SuperMix (TransGen Biotech, Beijjing, China) on an ABI7500 instrument. The qPCR 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 ddH2O. qPCR reaction procedures and data analysis were performed as previously described.
Enzyme-linked immunosorbent assay (ELISA)
Following RAW264.7 cell stimulation with LPS, IL-4, exosome-like vesicles from T. pisiformis cysticercus, 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 were conducted using GraphPad Prism 5.0. Comparisons between groups were assessed using the unpaired Student’s t-test. Differences among multiple groups were analyzed by a one-way analysis of variance (ANOVA) using SPSS 24.0 (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean (SE). Statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.