All animal experiments were performed under the section of Environmental Parasitology, Tokyo Med. & Dent. Univ (TMDU). All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (A2021-131A).
Animal and Parasite
To maintain the life cycle of Schistosoma japonicum, ICR mice (5weeks old) from SLC (Hamamatsu, Japan) and the intermediate host, Oncomelania hupensis nosophora (Yamanashi strain), were infected as previously described 55. Snails collected from the field were infected with five to seven miracidia each. They were kept in an thermostatic chamber at 28°C for over four months. The infected shells were placed between glass slides to crush the shells and release the cercariae inside. The mice anesthetized by peritoneal administration of sodium pentobarbital in saline (40mg/kg) have their abdomens shaved and were infected with a cover glass to which 50 cercariae were attached.
The infected animals were kept in a controlled temperature and humidity environment with a 12:12 h light: dark cycle. The mice had free access to water and food in the facilities approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (2010002C2). Humane endpoints were applied when an evidence of severe pain, excessive distress, suffering or an impending death was observable in any of the animals, which were then euthanized. All mice were euthanized at the end of the experiment by intraperitoneal injection of sodium pentobarbital in saline solution (100 mg/kg). The status of all mice was checked daily using a composite score including vitality, secretions, fur quality, mobility, dyspnea, ascites, neurological signs, and ability to ingest water or food. All methods were carried out in accordance with relevant guidelines and regulations. All animal handling and methods complied with the ARRIVE guidelines.
Adult worms were collected by perfusion from euthanized mice at seven weeks post-infection. They were placed in a conditioned medium consisting of RPMI1640 containing 10% exosome-depleted FBS (Thermo Fisher Science, USA), penicillin, streptomycin, L-glutamine (Thermo Fisher Science). After 24 h, all worms were washed with the conditional medium three times, and the surviving worms were used for experiments.
Extracellular vesicles (EVs) secreted from parasites
To collect EVs, adult worms (three pairs) were cultured in a conditional medium for a week at 37°C in a CO2 incubator. For TEM analysis, the supernatant was subjected to ultracentrifugation at 100,000 × g for two hours and washed twice with PBS. Alternatively, for RNA extraction, the supernatant was treated with the Total Exosome Isolation Reagent (Thermo Fisher Scientific). The pellet was dissolved in TRI Reagent (MRC, USA). Furthermore, to check whether the EVs were vesicles containing phosphatidylserine in the membrane, the MagCapture™ Exosome Isolation Kit PS (WAKO, Japan) was used to purify the vesicles. In brief, the supernatant from the worms (three pairs/well) cultured for three days was used as a sample for purification using this kit. RNA extraction was performed by adding TRI Reagent directly to the beads binding to the EVs after washing. On the other hand, Total Exosome Isolation Reagent was added to the flow-through fractions and RNA not contained in EVs was extracted using the same procedure as above.
Cultured paired worms and treatment with RBCs and inhibitory experiment of EV production by inhibitors
The paired adult worms were directly picked up, and single-sex worms detached naturally through incubation for 24 h were also used as a single sex worm. All worms were placed into a 12 well-culture plastic plate in conditional medium. For treatment with erythrocytes, RBCs were prepared from the whole blood of euthanized ICR mice (5 weeks old). The cells were collected from the RBC fraction after centrifugation at 3,000 × g and washed with the conditional medium three times. Furthermore, ghost RBCs were prepared after puncturing and reconstruction of RBCs as a standard method. Adult worms (three pairs of worms or three worms per well; 5 wells/group) were cultured with or without RBCs or ghost RBCs (108 cells) in a 2 ml conditional medium for a week at 37°C in a CO2 incubator. After culture, all worms were confirmed to survive, and the supernatant was collected for EV preparation. All worms were collected for RNA extraction and the female worms cultured with RBC were collected for transmission electron microscopy (TEM). For the inhibitor test, three paired worms were treated with EV inhibitors suspended in DMSO, and then mouse RBCs were added after an hour. The appropriate concentration of each inhibitor was based on data from previous studies (10 μM Y27632, 5 μM Manumycin A, 80 μM Calpeptin, 10 μM GW4869) 33. After three days, the supernatants were collected from each well for RNA extraction from EVs, and all worms were removed for egg counting. The eggs were treated with 4% KOH at 37°C overnight, and all eggs were counted under a microscope.
Transmission electron microscopy (TEM) analysis
The isolated EVs were loaded and negatively stained with 1% (w/v) uranyl acetate solution on copper grids (400 mesh) covered with formvar film. These specimens were observed using TEM at 75 kV (H-7100, Hitachi, Tokyo, Japan). TEM using adult worms was performed as previously described 56. In brief, the adult single female worms after ingestion of the erythrocytes were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 24 h. The fixed worms were successively immersed in 0.5% osmium tetroxide (OsO4) in 0.1 M phosphate buffer for an hour, 2% low-molecular-weight tannic acid (LMW-TA, Electron Microscopy Sciences, Hatfield, PA) in 0.05 M maleate buffer for four hours, and 1% uranyl acetate in 0.05 M maleate buffer for three hours. The worms were then dehydrated with a graded series of ethanol and embedded in Oken Epok 812 epoxy resin (Oken-shoji, Tokyo, Japan). Ultrathin sections (90–100 nm thickness) were produced using an ultra 45° diamond knife (Diatome, Biel, Switzerland) and transferred to 50-mesh copper grids coated with a Formvar membrane. Ultrathin sections stained with lead citrate and uranyl acetate were digitally photographed using an H-7100 transmission electron microscope (Hitachi High-Technologies, Tokyo) equipped with a C4742-95 CCD camera (Hamamatsu Photonics, Shizuoka, Japan).
RNA extractions and cDNA synthesis of miRNA
RNA was extracted from EVs using Direct-zol™ RNA Kits (Zymo Research, USA). The quality of the extracted RNA was evaluated using a Bioanalyzer (Agilent, USA), and the concentration of total RNA was also measured. cDNA synthesis for miRNA detection was performed using a Simple miRNA Detection Kit (Biodynamics, Japan) or Mir-X™ miRNA First-Strand Synthesis Kit (Takara Bio Inc., Japan).
miRNA-seq library and sequencing
The miRNA-seq was performed using total RNA samples by contact analysis (GNAS, Japan). In brief, miRNA-seq libraries were prepared from 50 ng of total RNA using the TruSeq RNA Library Preparation Kit (Illumina, UK). All samples were processed with three biological replicates from five worm groups representing one biological replicate. Libraries were multiplexed and sequenced on an Illumina HiSeq 2500 with 50 bp paired-5’end reads. The sequence counts were detected as expression values and annotations were determined by inputting them into the miRBase of the S. japonicum. The value of each expression value when the total count was set to one million was used as the data in each sample. The top 20 annotated miRNAs were enumerated by averaging each of the three types of data.
Quantitative real-time PCR (qRT–PCR) analysis
cDNA prepared from miRNAs from EVs was used for qRT–PCR. Briefly, miRNA expression of EVs was analyzed using universal primers and miRNA-specific forward primers (Sup. S1 Table) in a mixture of the KAPA SYBR Fast qPCR kit (Kapa Biosystems, South Africa). The reaction was performed using the LightCycler480 system (Roche, USA) at 95°C for 2 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 1 s. After amplification, the reaction was performed at 95°C for 5 s, 60°C for 1 min, and then gradually heated to 95°C for melting curve analysis. In the expression analysis of miRNAs, the concentration of EVs RNAs recovered in another experiment was measured by the bioanalyzer, and a standard line for analysis was drawn using this as the standard concentration. each miRNA in EVs was measured by the absolute quantification method against the standard line. In each graph, the absolute quantitative value of each miRNA was expressed as a relative value to the value of the control group to be compared.
Statistical differences between the two groups were assessed using an unpaired two-tailed t-test and were considered significant at p < 0.05. In the miRNA expression analysis, statistical analysis was performed based on the miRNA absolute quantitative values in EVs obtained from three to five groups of cultured adult worms and compared between two groups. In the inhibitor experiment, statistical analysis was performed by comparing between the DMSO-treated groups and each treated group.