Hemocyte siRNA uptake is increased by 5′ cholesterol-TEG addition in Biomphalaria glabrata, snail vector of schistosome

Biological materials

B. glabrata originated from Guadeloupe (BgGUA2) and its sympatric strain of S. mansoni (SmGH2) were used for experimental approaches. BgGUA2 was provided from M. Blouin (Oregon State University) in 2013. These snails were collected in 2005 from Dans Fond on the island of Guadeloupe (DFO; N:16°18.500′, W:061°30.720′). For infection experiments, each snail was individually exposed for 12 h to 10 miracidia in 5 mL of pond water. Breeding conditions and maintenance of parasite cycle have been conducted as previously described (Portet et al., 2019).

The laboratory and experimenters possessed an official certificate from the French Ministry of National Education, Research, and Technology, CNRS and DRAAF Languedoc Roussillon for experiments on animals, animal housing, and animal breeding (#A66040; decree # 87–848, October 19, 1987; and authorization # 007083).

siRNA uptake and interference

Small interfering RNA duplexes against BgTEP1 and Green Fluorescent Protein (GFP used as control) were synthetized and modified by Eurogentec Company and purified by standard SePOP desalting. In this study, two siRNA targeting BgTEP1 transcripts mRNA (GenBank accession No. FJ480411) which are 5′ GAC-AGA-UUC- UCA-UCA-AAC-A and 5′ GAG-UAU-GAU-UUA-CCA-AGA-U and one against the cnidarian GFP (GenBank accession No. EU430082): 5′ CAA-GCU-GAC-CCU-GAA-GUU-C were synthetized. The GFP siRNA was designed with and without a Cholesteryl-TEG added in 5′ to assess if this modification can increase siRNA cellular uptake. A Cy3 dye was linked to the 3′ termini of each RNA molecule for siRNA duplex in order to follow cellular uptake by flow cytometry approach. To investigate the silencing effect on the BgTEP1 mRNA level and on snail susceptibility phenotype to S. mansoni, the different siRNAs (BgTEP and GFP) were designed only with a Cholesteryl-TEG added in 5′ end. Two siRNA targeting BgTEP1 transcripts suppress mRNA were thus injected in equimolar quantity in the same snails. Each duplex siRNA was annealed and shipped dried by the manufacture (Eurogentec). siRNA was suspended in sterile Chernin’s balanced salt solution (CBSS). Concentrations of siRNA were adjusted so injected volumes do not exceed 5 µl. Each siRNA was injected into the cardiac sinus of B. glabrata snails (7–8 mm in diameter), using a 50 µl Hamilton syringe with a 26s needle (Hamilton) (Baeza Garcia et al., 2010).

siRNA delivery into hemocytes ex vivo

500 µL of hemolymph were recovered by head-foot retraction from pool of 3 adult snails, transferred to a 1.5 ml microcentrifuge low DNA binding tube and then incubated with 2 µg of Cy3-labeled GFP siRNA with and without Chol-TEG at 24 °C after mixed by gentle inversion. siRNA delivery was monitored from 30 min to 1, 2, 4, 6 and 8 h post-incubation by flow cytometry analysis (see FACS analysis section). For each experimental point, 4 biological replicates were performed. For comparing the percentage of fluorescent cells following the different treatments a Fisher Exact test was used, results were considered significant for P value ≤ 0.05. For comparison of siRNA incorporation over the kinetic after injection, a Kruskall–Wallis with Dunn post-hoc tests were used to test significant differences (P value ≤ 0.05) between experimental conditions.

siRNA delivery on hemocytes in vivo

2 µg of Cy3-labeled Chol-TEG siRNA targeted GFP (500 ng/µl) were injected in the snail pericardial cavity. Cellular uptake of siRNA was evaluated at 2, 4, 6, 12 and 24 h after injection (n = 4 snails for each condition). The hemolymph was recovered as described above. A negative control consisting in analysing the hemocyte fluorescence from uninjected snails was also performed. Then, a similar approach was conducted by varying the amount of siRNA injected to snails (2, 5 and 10 µg). Hemolymph was recovered 2 h after injection from 4 individual snails used as biological replicates. Significant differences in kinetic of siRNA cellular uptake following injection were tested using Kruskall-Wallis followed by Dunn post-hoc test (P value ≤ 0.05). Significant differences between doses of siRNA were tested using a Mann–Whitney U test. Results were considered significant when P value ≤ 0.05.

Flow cytometry analysis

After both in vivo and in vitro siRNA delivery, 100 µL of hemolymph were fixed with 100 µL of 4% paraformaldehyde solution in PBS for 5 min. Then, hemocytes were collected after centrifugation at 1,000 rpm, 10 min and re-suspended in PBS snail as previously described (Pinaud et al., 2016). Hemocyte preparations were stored at 4 °C and analysed one day after storage. The Flow Cytometry was performed using a FACS Canto BD Biosciences (RIO Imaging Platform, Montpellier, France). For each sample, around 10,000 events were counted. The results were analysed and visualised using the FlowJo V 10.0.8 software. To demonstrate internalization of cholesteryl-TEG siRNA into hemocytes, 500 µL of hemolymph from pool of 3 adult snails were plated for 1 h on polystyrene chamber slides. Then, hemocytes were incubated 2 h with 2 µg of Cy3-labeled siRNA. Hemocytes were washed twice in PBS and fixed in 4% paraformaldehyde for 5 min. After rinsing with PBS, actin filaments were stained with phalloidin  conjugated with Alexa 488 (Thermo Fisher) for 15 min and the cell nucleus with DAPI (Biotum) for 30 seconds as previously described (Portet et al., 2018). After rinsing, slides were mounted in Dako fluorescent mounting medium (Dako) and examined using a fluorescence confocal laser-scanning microscope (Zeiss LSM 700, Bio-Environment platform). For presentation, images were imported into ImageJ software.

RNA extraction and Quantitative RT-PCR analysis

Using HPLC syringe, 10 µg of Chol-TEG siRNAs targeting BgTEP1 (quantity ratio 1:1) or GFP were injected in snail pericardial cavity (2µg/µl). Hemolymph was recovered at 1, 2, 3 and 4 days after injection from 4 independent pools of 3 snails. Hemocytes were recovered by centrifugation and hemocyte total RNA was then extracted using the Total RNA Purification Micro Kit (Norgen Biotek) according to the manufacturer’s instructions. Reverse transcription was performed from 100 ng of RNA with oligo dT using Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase (Thermo Scientific, Whaltham, Massachussetts, USA) according to manufacturer’s instructions. Quantitative PCR (Q-PCR) was performed using NO ROX SYBR® Master Mix blue dTTP (Takyon) and run on LightCycler 480 thermocycler (Roche) according to the manufacturer’s instructions. Sequence of primers used for BgTEP1 amplification (forward: 5′ CGT-ACT-TAC-CCT-CGC-TC, reverse: 5′ ACC-ATT-AGA-TCC-ACT-GGA-AGA-TA) and ribosomal protein S19 (accession number: XM_013211381) (forward: 5′ TTC-TGT-TGC-TCG-CCA-C, reverse: 5′ CCT-GTA-TTT-GCA-TCC-TGT-T) were designed using LightCycler Probe Design software version 1.0 (Roche Diagnostics). 2 µl of cDNA diluted to 1/20 in ultrapure water was used in a reaction mixture containing 0.1 µM of each primer. The cycling program was as follows: denaturation step at 95 °C for 2 min, 40 cycles of amplification (denaturation at 95 °C for 10 s, annealing and elongation at 60 °C for 20 s), with a final step at 60 °C for 5 min. QPCR was ended by a melting curve step from 65 to 97 °C with a heating rate of 0.11 °C/s and continuous fluorescence measurement. The cycle threshold (C_t) was determined using the second derivative method of the LightCycler 480 Software release 1.5 (Roche). Sequence of PCR amplicon was checked by Sanger sequencing. The relative expression of BgTEP1 was calculated with the ΔΔCt method as the efficiency of both couple of primers presented the same PCR amplification efficiency. Briefly, BgTEP1 expression was normalised to the endogenous control, the ribosomal protein S19, for each condition (ΔCt, Ct_BgTEP1-Ct_S19). Then, the relative expression of BgTEP1 was determined by the ΔΔCt method based on this expression of the nonsilencing control group (ΔΔCt, ΔCt_{(siRNA injected)} − ΔCt_{(untreated snails)}). For graphical representation, relative expression data presented are determined as the ratio between relative expression of BgTEP1 with respect to S19 level under BgTEP and GFP interference condition (Pfaffl, 2001; Knight et al., 2011). Kruskall-Wallis with Dunn post-hoc tests were used to test significant differences (P value ≤ 0.05) between experimental conditions.

BgTEP1 protein expression level analysis

Using the procedure described previously, 10 µg of Chol-TEG BgTEP1 siRNA were injected in 4 replicates of 3 snails for each condition. Cell free hemolymph was collected 1, 2, 3 and 4 days post injection and protein content was determined using 2-D Quant Kit (GE Healthcare). Five microliters (15 µg total protein/µL of hemolymph) of hemolymph were denatured 5 min at 99 °C in 1X Laemmli buffer plus β-mercaptoethanol, before proteins separation on 7.5% SDS-polyacrylamide gel and transferred onto a 0.2 µm Hybond ECL membrane (GE Healthcare) using a Transblot transfer system (Bio-Rad). After saturation over a period of 3 h at room temperature in TBSTM [1 × TBS (500 mM Tris-HCl, 1.5 M NaCl, pH 7.5), 0.05% Tween-20, 5% non-fat milk], the protein blots were incubated overnight at 4 °C in TBSTM, with a 1:5,000 dilution of the rat polyclonal anti-BgTEP1-N-ter antibody (Agro-Bio), raised against the recombinant BgTEP1 N-terminal part (amino acid 204 to 521 from BgTEP1 protein sequence (GenBank ID QEQ12617)). The blots were washed three times with TBST, and further incubated with 1:5,000 dilution of the commercial horseradish peroxidase-conjugated rabbit anti-rat IgG (H + L) antibody (CliniSciences #6180-05). Blots were finally washed three times with TBST, and one time with TBS, and then revealed in the presence of an enhanced chemiluminescent substrate (SuperSignal West Pico Plus Chemiluminescent Substrate, Thermo Scientific). Image acquisition was performed using ChemiDoc MP Imaging System (Bio-Rad) for 3 s. Blot picture and relative quantitative analysis of bands were performed using Image Lab Software version 4.0.1.

Biomphalysin was used as internal control for protein loading and to exclude a protein degradation. A western blot on the same samples was performed according to the same procedure used for BgTEP1 protein detection. To reveal Biomphalysin in the cell-free hemolymph, a polyclonal antibody raised against this toxin (Agro-Bio) and an HRP-conjugated goat anti-rabbit IgG secondary antibody (ImmunoPure Antibody, Pierce) were diluted at 1:1,000 and 1:7000 respectively in TBST.

Effect of BgTEP1 mRNA silencing on S. mansoni infection phenotype

BgGUA2 snails were injected with 10 µg of Chol-TEG siRNA duplexes specific for BgTEP1 (43 snails) and GFP (34 snails) and individually exposed for 12 h to 10 S. mansoni GH2 miracidia in 5 ml of pond water at 3 days after siRNA injection. Then, 16 days after parasite exposure, the snails were fixed in Raillet-Henry solution. The prevalence (P: % of snail infected) and the intensity (I: number of parasites per infected snail) were determined by counting the primary sporocysts present in each snail tissue as described previously (Portela et al., 2013). Briefly, the snails were relaxed in water containing an excess of crystalline menthol for 6 h. The snail shell was removed and the body was fixed in modified Raillet-Henry’s solution (930 ml distilled water, 6g sodium chloride, 50ml formol 40%, 20 ml 95 acetic acid). The presence of Sp1 in each snail was numbered by visual inspection and meticulous dissection of the snail tissues. Following Raillet-Henry’s fixation the Sp1 were readily observable as opaque white bodies within a yellow snail tissue background. Significant changes in prevalence and intensity were tested by Fisher exact test and Mann–Whitney U test.

Ex vivo siRNA delivery into hemocyte is enhanced by lipid-conjugation

To assess the delivery of siRNA conjugated to cholesterol in snail hemocytes, naked and modified (Chol-TEG) GFP-siRNA were coupled with the fluorochrome Cy3. Two micrograms of labelled GFP-siRNA were added to 500 µL of freshly collected hemolymph from a pool of adult snails. Monitoring of cell uptake by FACS revealed that siRNA were quickly internalized by hemocytes in both conditions and showed a high number of fluorescent cells after 30 min of exposition (Fig. 1 and Fig. S1). More siRNA were uptaken by cells when cholesterol was conjugated (Figs. 1A and 1B), 47% of cells were counted with a cytosolic fluorescence compared to 5% of cells when using unmodified siRNA (Fisher exact test, p = 0.00001, n = 4 per group). No significant differences were observed when comparing unmodified siRNA to untreated snails hemocyte auto-fluorescence (Fisher exact test, p = 0.3687, n = 4 for unmodified siRNA and n = 8 for control). A closing parenthesis has been deleted here: [(Fisher exact test,...for control)]. Please check, and correct if necessary (Fig. 1C). Cholesterol conjugation enhances significantly the cellular uptake of siRNA. Also, internalization of siRNA did not increase over time in vitro condition; the number of fluorescent hemocytes remains similar after 30 min or 8 h exposure time for both conditions (GFP-cy3 Kruskal-Wallis test p = 0.145552, df = 5, n = 24, H = 8.200 ; Chol-TEG-GFP-cy3 Kruskal-Wallis test p = 0.643170, df = 5, n = 24, H = 3.369826, n = 4 per group) (Fig. 1C). Therefore, Chol-TEG siRNA was used for subsequent experiments.

In vivo siRNA delivery into hemocyte

To further validate our GFP-siRNA delivery condition in vivo, 2 µg of Chol-TEG-siRNA against GFP mRNA were injected in snail pericardial cavity. Flow cytometry analysis was performed on hemolymph recovered between 2 h and 24 h post-injection to follow hemocyte uptake efficiency in vivo. Overall, the percentage of internalized Chol-TEG-siRNA is much lower than that obtained in vitro. Indeed, only 1.9 to 3.3% of hemocytes have incorporated Chol-TEG-siRNA regardless of exposure time (no significant differences over time: Kruskal-Wallis test p = 0.062544, df = 4, n = 20, H = 8.9428) (Fig. 2) compared to around 50% for in-vitro condition after 30 min (Fig. 1C). Therefore, to optimize cellular uptake, increasing amounts of siRNA have been injected into snails and hemocytes were recovered 2 h post-injection. The proportion of fluorescent cells after 2 h post-injection increased with higher Chol-TEG siRNA concentrations (Fig. 3). Hemocytes have internalized significantly more Chol-TEG-siRNA from 11% or 55% after the injection of 5 µg or 10 µg respectively (Mann–Whitney U- test, U = 0 ; z-score = −2.50672 ; P = 0.00604, n = 4 per condition) (Fig. 3). Consequently, we decided to use a dose of 10 µg of siRNA coupled with Cholesteryl-TEG for further analysis.

Knockdown of BgTEP expression in guadeloupean snails

Once we have demonstrated that cholesterol conjugation promotes siRNA delivery into hemocytes, its efficiency for BgTEP1 gene silencing was tested. BgTEP1 is a thioester-containing protein playing the role in innate immunity as complement-like factor secreted in snail hemolymph (Li et al., 2020a; Mone et al., 2010; Wu et al., 2017). Furthermore, a previous report showed that BgTEP1 could act as an opsonin by binding to the surface of diverse intruders (Portet et al., 2018). Whole snails were microinjected with 10 µg of Chol-TEG siRNA against BgTEP1 or GFP (control). Transcriptional expression analysis of BgTEP1 was focused on circulating hemocytes, normalised with the S19 housekeeping gene and compared with Chol-TEG siGFP injection. Thus, we found a significant reduction in BgTEP1 expression of 4-, 8.5-, and 5.5-fold at day 2, 3 and 4, respectively, when compared to day 1 after siRNA injection (Kruskall-Wallis test: P = 0.000054, df = 4, n = 45, H = 24.848) (Fig. 4A). The efficiency of knockdown was also confirmed at the protein level. Indeed, we observed a decrease in plasmatic BgTEP1 protein in hemolymph from nearly 50% at day 1 to 80% at day 2 and 3 and to 90% at 4 days after siRNA injection, while the amount of Biomphalysin used as control for protein loading did not vary significantly (Fig. 4B). Since mRNA level of BgTEP1 was decreased 2 to 4 days after siRNA injection, BgGUA2 snails were exposed to 10 miracidia of S. mansoni GH2 at the third day (GFP and BgTEP1). No differences in prevalence and intensity of infection were observed between siBgTEP1- (P = 14.7%, I = 1, n = 34) and siGFP (P = 18.6%, I = 1.12, n = 43) - control injected snails at 16 days following infection (Fisher exact test, P = 0.3687).