Introduction: The E. multilocularis laminated layer (LL) is a heavily glycosylated parasitic structure that plays an important role in protecting the larval stage (metacestode) of this parasite from physiological and immunological host reactions. We elaborated an experimental design with the idea to modify the (glycan) surface of the LL by a targeted digestion. This should allow the host defense to more easily recognize and attack (or kill) the parasite by immune-mediated effects. Methods: Experimentally, E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or β1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry. Results: Our experiments demonstrated that the parasite vesicular structure can be directly destroyed by adding galactosidases into the in vitro culture system, resulting in the fact that the parasite metacestode vesicles could not anymore infect and develop in mice after this glycan digestion. Moreover, when compared to the mice inoculated with E. multilocularis metacestode without galactosidases, PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with β1-3-galactosidase, with a lower IL-10 secretion from CD4+ Teffs; there was no difference of PD1 and IL-10 expression levels regarding CD4+ Teff from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galac­tosidase. Discussion: We raised our hypothesis that this “aborting” effect may be linked to an altered PD1 and IL-10 response fine-tuning between immunopathology and immune protection. These findings justify a continuation of these experiments upon therapeutical in vivo administration of the enzymes.

Alveolar echinococcosis (AE) is one of the clinically most severe zoonotic helminthic diseases in humans, characterized by a chronic progressive hepatic damage caused by the continuous proliferation of the larval (metacestode) stage of Echinococcus multilocularis. The treatment options of AE in a given patient depend on the metacestode tissue size and its location: first choice is a radical (curative) resection of the parasite by surgery [1, 2]. If not feasible, long-term to life-long daily medication with albendazole (ABZ) provides a good health improvement in many of the patients; however, some patients experience side effects such as hepatotoxicity and drug interactions [3]. Consequently, alternative treatment options are urgently required.

The E. multilocularis cestode is outwardly protected by a massive layer represented by a carbohydrate-rich extracellular matrix, termed the laminated layer (LL). Biochemical analysis of the E. multilocularis LL showed that the non-decorated cores of the matrix, together with Galpβ1-3 (Galpα1-4Galpβ1-4GlcNAcpβ1-6) GalNAc, comprise over 96% of the glycans in molar terms [4]. This simple LL glycome is synthesized by E. multilocularis both under in vitro and in vivo conditions [4]. Our experimental aim was to biochemically modify the surface of LL in vitro such as to render it more easily accessible for an attack by immune cells, or even to directly harm the parasite by a targeted digestion of the glycan from the E. multilocularis LL. To achieve this, the parasites were first cultured in vitro with α1-3,4,6-galactosidase or β1-3-galactosidase, and after a 2-day recovery phase in vitro, they were reinjected into mice for assessing their in vivo viability. Control was a non-galactosidase-treated culture otherwise processed and maintained under identical conditions. Parasite weight as well as PD1 and IL-10 were measured as selective key parameters in necropsied mice.

Parasite Preparation, in vitro Cell Culture, and Experimental Treatment

E. multilocularis (H95) metacestodes were cultured as described by Spiliotis el al. [5]. In brief, metacestodes were isolated and continuously maintained by serial passages (vegetative transfer) in C57BL/6 mice. For the present experiments, the metacestode tissue was obtained from previously infected animals by aseptic removal from the peritoneal cavity. After grinding the tissue through a sterile 50-μm sieve, vesicular cysts were collected, washed, and subsequently incubated with 100 U/mL penicillin, 100 μg/mL streptomycin, and 5 μg/mL tetracycline at +4°C overnight (∼1 mL vesicles per well suspended in 40 mL medium), then put into DMEM culture medium (Gibco, Basel, Switzerland) containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5 μg/mL tetracycline, with 5 × 106 rat Reuber hepatocytes as feeder cells for 2 weeks (vesicle and culture medium volumes per 50-mL flask as above). After this preincubation period, they were treated for 11 days under the following conditions: (a) with α1-3,4,6-galactosidase (New England Biolabs, cat. P0747, purity ≥95%) at 10 units/mL and without hepatocytes in the culture, (b) with β1-3-galactosidase (New England Biolabs, cat. P0726, purity ≥95%), at 10 units/mL and without hepatocytes in the culture, (c) with 1% Triton X-100 as a positive inactivating control and without hepatocytes in the culture [6], and (d) parasites without any treatment (galactosidases and Triton X-100) as a negative control, without hepatocytes in the culture. Morphological changes were examined under the microscope at days 1, 3, 5, 7, 9, and 11 (shown in Fig. 1).

Fig. 1.

Experimental design E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or β1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry.

Fig. 1.

Experimental design E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or β1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry.

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Viability Assays

In vivo Reinoculation into Mice from the Cultures with Galactosidases

In vitro-cultured and experimentally treated as well as untreated controls were used to infect mice for assessing the viability status of the vesicles. Methodically, the 40-mL cultured volume per 50-mL flask (including all vesicles) was aspirated with a syringe (1.2 mm Ø needle) and injected into the peritoneal cavity of mice (200 μL/mouse) (shown in Fig. 1).

Mice

Sixteen female 8-week-old wild-type C57/BL6 mice were purchased from Charles River GmbH (Sulzfeld, Germany) and randomly divided into 4 groups (4 mice per group) as follows: (1) noninfected control; (2) E. multilocularis vesicles from the culture treated with α1-3,4,6-galactosidase; (3) E. multilocularis vesicles from the culture treated with β1-3-galactosidase; (4) E. multilocularis vesicles from the cultures without α1-3,4,6-galactosidase or β1-3-galactosidase treatment. There were no parasite vesicles left after having been treated with 1% Triton X-100, thus this material could not anymore be included into viability assays.

Sampling

At the end of the experiment (10 months after the infection/inoculation), mice were sacrificed by CO2 euthanasia, and parasite tissues were searched for within the whole peritoneal cavity and the liver. If present, the parasite tissue was dissected such as to remove fat and connective tissues for subsequent determination of the parasite mass. Peritoneal exudate cells were collected by peritoneal rinsing with 5 mL RPMI 1640. Cells were subsequently washed twice with, and then resuspended in, PBS containing 3% FBS for cell staining.

Flow Cytometry

Aliquots of 106 cells/100 μL of staining buffer per well were incubated each with 1 μg of purified anti-CD16/CD32 for 20 min in the dark, in order to block nonspecific binding of antibodies to the FcγIII and FcγII receptors. Cell suspensions were incubated with a cell viability dye eFluorTM 506 at 4°C in the dark (Thermo Fisher Scientific) to exclude dead cells. Subsequently, these cells were separately stained with 1 μg of anti-CD4-APC (RM4-4, Biolegend) and anti-PD1-eFluor 450 (J43, eBioscience) for 20 min. For cytokines and transcription factors, the cells were subsequently fixed, permeabilized, and stained with anti Foxp3-Percp cy5.5 (FJK-16 s, eBioscience) and anti-IL-10 PE (JES3-9D7, eBioscience) for 15 min, according to the manufacturer’s instructions (Foxp3/Transcription Factor Staining Buffer Set; eBioscience). Corresponding fluorochrome-labeled isotype control antibodies were used for staining controls. Cells resuspended in 250 μL of buffer (0.15 m NaCl, 1 mm NaH2PO4·H2O, 10 mm Na2HPO4·2H2O, and 3 mm NaN3) were analyzed in a flow cytometer BD LSR II (BD Pharmingen Inc., San Diego, CA, USA) using the corresponding BD FACSDiva software. Flow cytometric analysis was done using FlowJo software (Treestar, Inc., Ashland, OR, USA).

Statistical Analyses

All data were analyzed by Prism GraphPad. The results are presented as means ± SD. Normality of data was assessed by D’Agostino & Pearson and Shapiro-Wilk tests. For normally distributed groups of data, one-way ANOVA with Bonferroni’s correction was used to compare the differences between groups. Significance was defined as p < 0.05 for all tests.

Parasite Cultures in vitro-Treated with Galactosidase-α1-3,4,6 or Galactosidase-β1-3

E. multilocularis metacestode vesicles, having been initially cultured for 2 weeks in vitro with hepatocytes as feeder cells, were subsequently treated for 11 days with galactosidases in their respective working concentration without hepatocytes in the cultures. Microscopical analysis showed that all the cultures of the vesicles started to collapse by day 1 post-treatment (identical effect for both enzymes), and this effect increased till day 5 post-treatment. The parasites maintained a collapsed status after day 5 till day 11 (the end of investigation). After day 5 of treatment, the collapse phenomenon affected both the surface and the inside of the vesicle, including all enzyme-treated cysts (shown in Fig. 2). Non-treated vesicles remained fully intact for the whole experimental period, whereas Triton X-100 treatment destroyed the vesicles within 1 day of treatment (shown in Fig. 2). Overall, the results showed that the parasite structures were destroyed by adding the glycan-affecting galactosidases in the in vitro culture system.

Fig. 2.

Morphological changes of E. multilocularis metacestodes cultured with galactosidases at different time points. Negative: E. multilocularis metacestode culture without any treatment in the culture (galactosidase and Triton X-100); TX-100: E. multilocularis metacestode with 1% Triton X-100 in the culture; α-Gal: E. multilocularis metacestode with 10 Units/mL α1-3,4,6-galactosidase in the culture; β-Gal: E. multilocularis metacestode with 10 Units/mL β1-3-galactosidase in the culture. Magnification, ×200.

Fig. 2.

Morphological changes of E. multilocularis metacestodes cultured with galactosidases at different time points. Negative: E. multilocularis metacestode culture without any treatment in the culture (galactosidase and Triton X-100); TX-100: E. multilocularis metacestode with 1% Triton X-100 in the culture; α-Gal: E. multilocularis metacestode with 10 Units/mL α1-3,4,6-galactosidase in the culture; β-Gal: E. multilocularis metacestode with 10 Units/mL β1-3-galactosidase in the culture. Magnification, ×200.

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Viability Assays

Parasite vesicles that had been in vitro-treated with α1-3,4,6-galactosidase or β1-3-galactosidase were intraperitoneally inoculated into mice to assess their viability status. Since there were no parasite vesicles left after being treated with 1% Triton X-100, this test condition was not included into viability assays.

We found only very small calcified lesions in mice inoculated with E. multilocularis metacestode pretreated with α or β-galactosidase, whereas the metacestode tissue recovered from mice infected with non-treated vesicles exhibited a conventional multivesiculated tumor-like structure. Besides the morphologically evident differences, the parasite weight was significantly lower in mice inoculated with parasites treated either with α1-3,4,6-galactosidase (0.017 ± 0.006 g) or β1-3-galactosidase (0.003 ± 0.003), when compared to non-treated controls (13.460 ± 3.087 g) (shown in Fig. 3a). Overall, these experiments showed that the parasite lost its ability to develop in mice upon pretreatment with galactosidases.

Fig. 3.

Parasite treated with galactosidases viability by reinoculation into mice, and PD1 and IL-10 expression in CD4 Teffs. a Parasite weight from the mice reinoculated with parasites pretreated with galactosidases. Frequency of PD1+ cells (b) and IL-10+ cells (c) in CD4 Teffs from the liver of mice reinoculated with parasites pretreated with galactosidases, corresponding noninfected animals as negative controls. Data represent mean ± SD of 4 mice in each group. Comparison between groups was performed using one-way ANOVA for statistical analysis. *p < 0.05. Control: noninfected wild type mice; AE: E. multilocularis metacestode without pretreatment of galactosidases reinoculated into wild-type mice; α-Gal: E. multilocularis metacestode with pretreatment of α1-3,4,6-galactosidase reinoculated into wild-type mice; β-Gal: E. multilocularis metacestode with pretreatment of β1-3-galactosidase reinoculated into wild-type mice.

Fig. 3.

Parasite treated with galactosidases viability by reinoculation into mice, and PD1 and IL-10 expression in CD4 Teffs. a Parasite weight from the mice reinoculated with parasites pretreated with galactosidases. Frequency of PD1+ cells (b) and IL-10+ cells (c) in CD4 Teffs from the liver of mice reinoculated with parasites pretreated with galactosidases, corresponding noninfected animals as negative controls. Data represent mean ± SD of 4 mice in each group. Comparison between groups was performed using one-way ANOVA for statistical analysis. *p < 0.05. Control: noninfected wild type mice; AE: E. multilocularis metacestode without pretreatment of galactosidases reinoculated into wild-type mice; α-Gal: E. multilocularis metacestode with pretreatment of α1-3,4,6-galactosidase reinoculated into wild-type mice; β-Gal: E. multilocularis metacestode with pretreatment of β1-3-galactosidase reinoculated into wild-type mice.

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PD1 Expression in Mice Inoculated with E. multilocularis Metacestode Pretreated with Galactosidases

Flow cytometrical analyses showed that PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with β1-3-galactosidase (shown in Fig. 3b). However, there was no difference in PD1 expression on CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galactosidase, when compared to the mice inoculated with E. multilocularis metacestode without galactosidase pretreatment (shown in Fig. 3b).

IL-10 Expression in Mice Inoculated with E. multilocularis Metacestode Pretreated with Galactosidases

Flow cytometrical analyses showed that IL-10 expression levels were higher in CD4+ Teffs from mice inoculated with E. multilocularis metacestode without galactosidase pretreatment, but much lower in those cell types from mice inoculated with E. multilocularis metacestode pretreated with β1-3-galactosidase, when compared to the mice inoculated with E. multilocularis metacestode without galactosidase pretreatment (shown in Fig. 3c).

The E. multilocularis LL is a heavily glycosylated structure that plays an important role in protecting the parasite from physiological and immunological host reactions. We developed an explorative preliminary experimental treatment study based upon a new concept to enzymatically digest in vitro the E. multilocularis LL with appropriate galactosidases. We showed for the first time that this parasite structure can not only be directly affected and destroyed by adding galactosidases in vitro, but that these resulting vesicles also do not develop in mice anymore, leading to an “abortive” or “dying-out” course of infection. This confirms that intact LL is very critical in parasite survival and might thus represent a curative target by digesting its “sugar” and directly killing the parasite. However, because of the limited knowledge about the detailed sugar components of E. multilocularis LL, other glycan-degrading enzymes against a sugar not present on the LL or with very low density, a non-relevant enzyme as a control for alpha- and beta-galactosidases was missing in the in vitro assay. This effect needs to be further studied.

In one of our previous studies [7], blockade of the programmed cell death-1 (PD1)/PD-L1 pathway significantly decreased parasite growth, which was associated with increased Th1 response in the secondary AE model (intraperitoneal infection with metacestodes) and decreased Treg/Th2 responses in the natural primary AE model (oral infection with eggs). Therefore, we included a few crucial immunological parameters in this study, such as to find putative correlations between an immunologically induced impairment of the metacestode growth, and, as presently, a biochemically impaired vesicle growth potential. PD1/PD-L1 signaling has been demonstrated to be involved in the modulation of both central and peripheral tolerance and plays a vital role in immune tolerance and T-cell exhaustion and has emerged as a key target for immunotherapy [8]. In our previous work, PD-1/PD-L1 immune check point blockade showed to exhibit a therapeutic effect against murine AE in both primary and secondary infection models [7], which was most likely associated with an elevated IL-10 concentration [data submitted to PLOS NTD]. However, this differs from the results found in a majority of patients among various malignant tumor types, who did not benefit from PD-1/PD-L1 blockade. Findings showed that IL-10 increased the expression of PD-1 and its ligand PD-Ll on ovarian myeloid dendritic cells (TIDCs) and PD1 blockade resulted in compensatory release of IL-10 by TIDCs [8]. In the present study, we found that PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with β1-3-galactosidase, with a lower IL-10 secretion from CD4 Teffs; there was no difference of PD1 and IL-10 expression levels regarding CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galactosidase. This might indicate that different mechanisms are used to keep the balance between PD1 and IL-10, thus putatively fine-tuning between immunopathological and immune-protective effects. However, it remains still unclear if the immune-related expression patterns (IL-10 and PD1) directly and causatively contributed to controlling the parasite burden, or if the biochemical alteration of the metacestode surface already was responsible for hampering metacestode viability. In order to elucidate the active contribution of immunity to reduce the parasite load or to kill the parasite, appropriate further experimentally studies will be needed.

Furthermore, in a next experimental step, we will characterize glycan profiles, or expression of structures of interest if possible, after enzymatic treatment, thus to show how stable the enzyme effect is, by comparing different time points and at the final harvest from mice. Based on this, glycan digestion could be an attractive alternative (or additional) approach for treating AE in vivo, a procedure very different from the presently available tools such as medication with benzimidazoles [9]. Overall, the present results justify to follow-up subsequent experiments aiming at an appropriate in vivo experiment.

The animal studies were performed in accordance with the recommendations of the Swiss Guidelines for the Care and Use of Laboratory Animals. The protocol was approved by the governmental Commission for Animal Experimentation of the Canton of Bern (Approval No. BE112/17).

The authors declare no commercial or financial conflict of interests.

This work was supported by the Swiss National Science Foundation 160108 to BG, 184757 to SG, and 189136 to SLL.

J.W., S.G., G.B., B.G. conceived the presented idea and planned the experiments; J.W. carried out the experiment and wrote the paper; and D.G. and S.L.L. contributed to the interpretation of the results. All authors provided critical feedback and helped shape the research, analysis, and manuscript.

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