Advertisement

Metabolic reprogramming of hepatocytes by Schistosoma mansoni eggs

Open AccessPublished:November 06, 2022DOI:https://doi.org/10.1016/j.jhepr.2022.100625

      Highlights

      • -
        S. mansoni soluble egg products induce hepatocellular metabolic reprogramming
      • -
        S. mansoni eggs mobilize, incorporate, and store host lipids
      • -
        S. mansoni induced parenchymal reprogramming causes metabolic exhaustion in the liver
      • -
        the parasite induced metabolic crisis associated redox imbalance provokes DNA damage
      • -
        the egg induced processes can harm hepatocytes independent of the host´s immune response

      Abstract

      Background & Aims

      Schistosomiasis, is a parasitic infection, which globally affects more than 200 million people. Schistosome eggs, but not the adult worms, are mainly responsible for schistosomiasis-specific morbidity in the liver. It is unclear if S. mansoni eggs consume host metabolites, and how this compromises the host parenchyma.

      Methods

      Metabolic reprogramming was analyzed by MALDI-MSI imaging, LC- and HPTLC-HRMS, metabolite quantification, confocal laser scanning microscopy, live cell imaging, quantitative real-time PCR, western blotting, assessment of DNA damage, and immunohistology in hamster models and functional experiments in human cell lines. Major results were validated in human biopsies.

      Results

      Notably, eggs mobilize, incorporate, and store host lipids, while the metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes. Administration of ROS scavengers ameliorates these deleterious effects.

      Conclusions

      Our findings indicate that S. mansoni eggs completely reprogram lipid and carbohydrate metabolism by soluble factors, which results in oxidative stress-induced cell damage in the host parenchyma.

      Lay summary

      The authors demonstrate that soluble egg products of the parasite S. mansoni induce hepatocellular reprogramming causing metabolic exhaustion and a strong redox imbalance. Notably, eggs mobilize, incorporate, and store host lipids, while the metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes, independent of the host´s immune response.

      Graphical abstract

      Keywords

      Abbreviations:

      AKT (Protein kinase B), AMPK (AMP-activated protein kinase), DAG (diacylglyceride), DMPE (dimethyl-phosphatidylethanolamine), ERK (extracellular-signal regulated kinases), G6PDH (glucose-6-phosphate dehydrogenase), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), GCK (glucokinase), GSH (reduced L-glutathione), GS (glycogen synthase), H2A.x (H2a histone family member x), HCC (hepatocellular carcinoma), hRF (retention factor * 100), IPSE (IL-4-inducing principle from Schistosoma mansoni eggs), JNK (c-JUN N-terminal kinase), MALDI (matrix assisted laser desorption ionization), MDA (malondialdehyde), MSI (MALDI-MS imaging), OA (oleic acid), PARP (poly(ADP-ribose) polymerase), PC (phosphatidylcholine), PDH (pyruvate dehydrogenase), PE (phosphatidylethanolamine), PI (phosphatidylinositol), PKM (pyruvate kinase in mammals), PLIN2 (perilipin 2), PPP (pentose phosphate pathway), PYGL (hepatic glycogen phosphorylase), ROS (reactive oxygen species), SEA (soluble egg antigens), S. (japonicum Schistosoma japonicum), SM (sphingomyelin), S. (mansoni Schistosoma mansoni), SP600125 (JNK inhibitor), STAT3 (signal transducers and activators of transcription 3), TAG (triacylglyceride), Th1 (T-helper type 1), TORC1 (target of rapamycin kinase complex I), WNT (wingless and Int-1)

      Conflict of interest

      B.S. and C.G.G. are consultants of TransMIT GmbH, Giessen, Germany. The other authors declare that they have no conflicts of interest.

      Financial support

      This work was supported by grants from Deutsche Forschungsgemeinschaft DFG RO3714/4-1 and Sp314-13-1, INST 162/500-1 FUGG, INST 162/471-1 FUGG; INST 162/536-1 FUGG, GILEAD (support program Infectiology 2017), from Hessian Ministry of Science, Higher Education and Art (HMWK), LOEWE Center DRUID, in part by the Wellcome Trust [CGG, TQ; FUGI, Grant number 107475/Z/15/Z], and the University Hospital Giessen and Marburg (UKGM).

      Authors contributions

      MR and ER conceived the project and directed the study. V.vB., M.R., C.G.G., R.A.W., and E.R. were involved in writing of the manuscript. V.vB., L.H., A.B., N.B., S.G., V.W., L.R., S.W., K.T., T.Q., S.H., P.K., S.G., K.W., A.M., G.S., and M.R. performed experiments. T.Q., S.H., B.S., G.S., F.H.F., G.M., K.B., P.W., and C.G.G. supervised experiments and contributed samples, materials, methods and instrumentation. A.B., L.H., S.G., V.W., L.R., S.W., A.M., J.P.-K., and M.R. were involved in statistical analysis. All authors analyzed, interpreted, discussed the data, and reviewed the manuscript.

      Introduction

      Schistosomiasis, one of the most important parasitic infections worldwide, is caused by trematodes of the genus Schistosoma. It is accompanied by severe clinical symptoms as well as socioeconomic problems, and more than 200,000 deaths per year. According to the World Health Organization (WHO), schistosomiasis is a neglected tropical disease, and at least 236.6 million people required preventive treatment in 2019

      WHO: Fact-Sheets Schistosomiasis 2021; Available from: https://www.who.int/news-room/fact-sheets/detail/schistosomiasis.

      . Climate change and globalization will inevitably influence both, the distribution of the parasites and the incidence of schistosomiasis, even in areas where it is currently not endemic. Reports about an outbreak of urogenital schistosomiasis in Corsica (France), demonstrate the potential risk for schistosomiasis to spread into other areas
      • Boissier J.
      • Grech-Angelini S.
      • Webster B.L.
      • Allienne J.-F.
      • Huyse T.
      • Mas-Coma S.
      • et al.
      Outbreak of urogenital schistosomiasis in Corsica (France): An epidemiological case study.
      .
      In intestinal schistosomiasis schistosome eggs cause inflammatory responses leading to granuloma formation
      • Costain A.H.
      • MacDonald A.S.
      • Smits H.H.
      Schistosome egg migration: mechanisms, pathogenesis and host immune responses.
      . The process of egg excretion is driven by an immune-dependent formation of granulomatous inflammation in the gut. A similar immunopathology is caused in the hepatic dead-end
      • Schwartz C.
      • Fallon P.G.
      Schistosoma "eggs-iting" the host: granuloma formation and egg excretion.
      . In contrast to intestinal granulomas, liver granulomas become fibrotic over time, which often leads to obstructive portal lesions and portal hypertension, resulting in gastrointestinal bleeding, hepatic encephalopathy, or liver failure
      • Schwartz C.
      • Fallon P.G.
      Schistosoma "eggs-iting" the host: granuloma formation and egg excretion.
      .
      There is compelling evidence that schistosome eggs, but not the adult worms, are mainly responsible for schistosomiasis-specific morbidity
      • Olveda D.U.
      • Olveda R.M.
      • McManus D.P.
      • Cai P.
      • Chau T.N.
      • Lam A.K.
      • et al.
      The chronic enteropathogenic disease schistosomiasis.
      . Unusually for trematodes, S. mansoni eggs are not self-contained. As central part of the eggs, the supporting vitelline cells are unable to drive embryonic development, which also depends on host factors. Taking into account density and volume changes between deposition and maturation, it has been estimated that an egg increases in mass > threefold
      • Ashton P.D.
      • Harrop R.
      • Shah B.
      • Wilson R.A.
      The schistosome egg: development and secretions.
      . This means that more than two-thirds of tissue constituents in a mature egg are externally derived, while even neglecting energy requirements during embryogenesis
      • Ashton P.D.
      • Harrop R.
      • Shah B.
      • Wilson R.A.
      The schistosome egg: development and secretions.
      .
      In endemic areas, S. mansoni infection is associated with anemia and chronic malnutrition
      • Kabongo M.M.
      • Linsuke S.
      • Baloji S.
      • Mukunda F.
      • Raquel IdL.
      • Stauber C.
      • et al.
      Schistosoma mansoni infection and its association with nutrition and health outcomes: a household survey in school-aged children living in Kasansa, Democratic Republic of the Congo.
      . It is yet unclear if schistosomiasis is a cause of anemia and malnutrition or if both are threatening consequences of the poverty-related living conditions in endemic areas. Nevertheless, malnutrition is one of the major factors driving schistosomiasis [
      • Maciel P.S.
      • Gonçalves R.
      • Antonelli LRdV.
      • Fonseca C.T.
      Schistosoma mansoni infection is impacted by malnutrition.
      ]. The current study illuminates the physiologic consequences of an S. mansoni infection, thus improving our understanding of the connections between malnutrition and schistosomiasis.
      In contrast to S. mansoni-associated malnutrition in endemic areas, also positive metabolic effects of infection have been shown in humans suffering from the metabolic syndrome
      • Wolde M.
      • Berhe N.
      • Medhin G.
      • Chala F.
      • van Die I.
      • Tsegaye A.
      Inverse associations of Schistosoma mansoni infection and metabolic syndromes in humans: a cross-sectional study in northeast Ethiopia.
      . Furthermore, the injection of S. mansoni eggs into mice indicated that the cholesterol-lowering effect observed in the serum during infection is mediated by factors released from eggs, while adult worms seemed to have little or no effects
      • Stanley R.G.
      • Jackson C.L.
      • Griffiths K.
      • Doenhoff M.J.
      Effects of Schistosoma mansoni worms and eggs on circulating cholesterol and liver lipids in mice.
      . S. japonicum infection induced host genes involved in catabolism including glucose uptake, glycolysis, fatty acid oxidation, and suppression of anabolism (glycogen synthesis) in the liver. This might be regulated by macrophage metabolic states involving AMPK (AMP-activated protein kinase), AKT (Protein kinase B), and TORC1 (target of rapamycin kinase complex I) pathways, as shown by in vitro stimulation with SEA (representing the total soluble complement of homogenized eggs)
      • Xu Z.-P.
      • Chang H.
      • Ni Y.-Y.
      • Li C.
      • Chen L.
      • Hou M.
      • et al.
      Schistosoma japonicum infection causes a reprogramming of glycolipid metabolism in the liver.
      . A recently published study demonstrated reprogramming of the metabolic signature of macrophages after S. mansoni infection
      • Cortes-Selva D.
      • Gibbs L.
      • Maschek J.A.
      • Nascimento M.
      • van Ry T.
      • Cox J.E.
      • et al.
      Metabolic reprogramming of the myeloid lineage by Schistosoma mansoni infection persists independently of antigen exposure.
      . Of note, this reprogramming paralleled the establishment of a longevity memory-like phenotype in myeloid cells of infected mice
      • Cortes-Selva D.
      • Gibbs L.
      • Maschek J.A.
      • Nascimento M.
      • van Ry T.
      • Cox J.E.
      • et al.
      Metabolic reprogramming of the myeloid lineage by Schistosoma mansoni infection persists independently of antigen exposure.
      .
      Imbalances of nutrition supply, i.e. undernutrition as well as overnutrition, have been associated with enhanced hepatic oxidative stress that promotes liver disease
      • Risi R.
      • Tuccinardi D.
      • Mariani S.
      • Lubrano C.
      • Manfrini S.
      • Donini L.M.
      • et al.
      Liver disease in obesity and underweight: the two sides of the coin.
      . Increased oxidative stress during S. mansoni infection has been attributed to the inflammatory response of granulomatous immune cells within the liver
      • Oliveira RB de
      • Senger M.R.
      • Vasques L.M.
      • Gasparotto J.
      • dos Santos J.P.A.
      • Pasquali MAdB.
      • et al.
      Schistosoma mansoni infection causes oxidative stress and alters receptor for advanced glycation endproduct (RAGE) and tau levels in multiple organs in mice.
      . High ROS levels induce oncogenic signaling and may promote cancer by increasing DNA mutations
      • Srinivas U.S.
      • Tan B.W.Q.
      • Vellayappan B.A.
      • Jeyasekharan A.D.
      ROS and the DNA damage response in cancer.
      . Previously, we discovered that SEA, including IPSE/alpha‐1 of S. mansoni, activates oncogenic signaling in liver and colon
      • Weglage J.
      • Wolters F.
      • Hehr L.
      • Lichtenberger J.
      • Wulz C.
      • Hempel F.
      • et al.
      Schistosoma mansoni eggs induce Wnt/β-catenin signaling and activate the protooncogene c-Jun in human and hamster colon.
      ,
      • Roderfeld M.
      • Padem S.
      • Lichtenberger J.
      • Quack T.
      • Weiskirchen R.
      • Longerich T.
      • et al.
      Schistosoma mansoni egg secreted antigens activate HCC-associated transcription factors c-Jun and STAT3 in hamster and human hepatocytes.
      .
      In this study we provide first evidence that S. mansoni eggs are capable of inducing metabolic reprogramming of the host parenchyma, in order to take up host metabolites. These processes involve hepatic exhaustion, finally leading to hepatic DNA damage.

      Methods (Details in supplementary CTAT Table)

      Human Material

      Pseudonymized human colon samples were kindly provided by Dr. Senckenberg Institute of Pathology, University Hospital Frankfurt. The use of pseudonymized human residual samples that were routinely taken for pathologic assessment was approved by the local ethics committee (AZ 05/19). According to the ethics vote, an informed consent was not required for our retrospective analyses of archived tissues.

      Animal Experimentation

      Biomphalaria glabrata snails served as intermediate hosts and Syrian hamsters (Mesocricetus auratus) as final hosts for maintaining the life-cycle of a Liberian strain of S. mansoni
      • Grevelding C.G.
      The female-specific W1 sequence of the Puerto Rican strain of Schistosoma mansoni occurs in both genders of a Liberian strain.
      . Bs (= mixed sex) and ms (= monosex) worm populations were generated by polymiracidial and monomiracidial intermediate host infections, respectively
      • Grevelding C.G.
      Genomic instability in Schistosoma mansoni.
      . Bs infections(n=36 ♀) were carried out at the age of 8 weeks and were maintained for 46 days, and ms infections (n=17 ♀) 67 days
      • Lu Z.
      • Sessler F.
      • Holroyd N.
      • Hahnel S.
      • Quack T.
      • Berriman M.
      • et al.
      Schistosome sex matters: A deep view into gonad-specific and pairing-dependent transcriptomes reveals a complex gender interplay.
      to ensure a complete maturation of the worms; females need longer to grow and develop in the absence of male partners. Untreated hamsters (n=6 ♀) were used as supercontrols. All animal experiments were performed in accordance with the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes (ETS No 123; revised Appendix A) and were approved by the Regional Council Giessen (V54-19 c 20/15 c GI 18/10 Nr. A26/2018).

      MALDI MSI analysis and identification by LC-MS/MS

      MALDI MSI and LC-MS/MS experiments were performed as described elsewhere
      • Wiedemann K.R.
      • Peter Ventura A.
      • Gerbig S.
      • Roderfeld M.
      • Quack T.
      • Grevelding C.G.
      • et al.
      Changes in the lipid profile of hamster liver after Schistosoma mansoni infection, characterized by mass spectrometry imaging and LC-MS/MS analysis.
      . For data analysis, ”Lipid Match Flow”
      • Koelmel J.P.
      • Kroeger N.M.
      • Ulmer C.Z.
      • Bowden J.A.
      • Patterson R.E.
      • Cochran J.A.
      • et al.
      LipidMatch: an automated workflow for rule-based lipid identification using untargeted high-resolution tandem mass spectrometry data.
      was used for identification, ”Perseus”
      • Tyanova S.
      • Temu T.
      • Sinitcyn P.
      • Carlson A.
      • Hein M.Y.
      • Geiger T.
      • et al.
      The Perseus computational platform for comprehensive analysis of (prote)omics data.
      for statistical analysis and ”Mirion”
      • Paschke C.
      • Leisner A.
      • Hester A.
      • Maass K.
      • Guenther S.
      • Bouschen W.
      • et al.
      Mirion—a software package for automatic processing of mass spectrometric images.
      for image generation. Detailed description of the method: Supplemental material.

      Isolation of soluble egg antigens

      S. mansoni eggs were obtained from livers of bs-infected hamsters at day 46 post infection, and SEA was isolated as described earlier
      • Schramm G.
      • Falcone F.H.
      • Gronow A.
      • Haisch K.
      • Mamat U.
      • Doenhoff M.J.
      • et al.
      Molecular characterization of an interleukin-4-inducing factor from Schistosoma mansoni eggs.
      .

      Isolation of S. mansoni eggs

      Liver eggs and in vitro-laid eggs of S. mansoni-infected hamsters were isolated as described elsewhere with minor modifications
      • Schramm G.
      • Falcone F.H.
      • Gronow A.
      • Haisch K.
      • Mamat U.
      • Doenhoff M.J.
      • et al.
      Molecular characterization of an interleukin-4-inducing factor from Schistosoma mansoni eggs.
      ,
      • Dalton J.P.
      • Day S.R.
      • Drew A.C.
      • Brindley P.J.
      A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.
      .

      Cell culture experiments

      HepG2 cells (stock ordered in 2019, CLS # 330198, expanded and stored as cryostocks for consistent quality in culture for up to 10 passages per cryostock) were stimulated with 15 μg/mL SEA and/or 10 mM reduced L-gluthatione (GSH) for the indicated time points. For co-culture assays, HepG2 cells were treated with 400 μM fluorescently labeled oleic acid (Avanti Polar Lipids, Alabaster, AL, USA, #810259C) for 24 h and washed three times with PBS prior to co-culturing with 100 S. mansoni eggs per well (24-well plate) for further 24 h. In vitro-laid or liver-extracted eggs were co-cultured in the same compartment or in transwells with a permeable polycarbonate membrane (Corning, New York, USA), as indicated in the figure legends. Subsequently, eggs were washed three times and analyzed directly or after fixation with 1% formaldehyde by confocal laser scanning microscopy (CLSM).

      Confocal laser scanning microscopy and live cell imaging

      For lipid staining of eggs, fluorescently-labeled oleic acid (flOA; TopFlour Oleic Acid, Avanti SKU 810259C) was applied and uptake analyzed by CLSM. Liver eggs or in vitro-laid eggs were cultured up to 24 h with 50 μg/mL flOA (mixed with 200 μM oleic acid) or with HepG2 cells pretreated with 50 μg/mL flOA (mixed with 200 μM oleic acid) 24 h prior to co-culture with eggs. Negative controls received oleic acid only. Image acquisition was done on a Leica TCS SP5 VIS using a 488 nm argon laser. Autofluorescence of eggs was excited with a DPSS laser at 561 nm to visualize egg contours. Live imaging of flOA uptake was conducted by image acquisition every 20 sec over a period of 30 min. Confocal z-stacks were composed of 100-122 images with a step size of 0.5 μm and further processed in IMARIS imaging software (Bitplane). Quantification of flOA fluorescence per egg was achieved by acquiring 15-25 z-stacks and averaging the mean grey values of the egg in all images using Image J.

      Histochemistry and immunohistochemistry

      Periodic acid-Schiff (PAS) reaction was applied for visualization of glycogen distribution in hepatic tissue. For analyzing the hepatic distribution of neutral triacylglycerides (TAGs) and lipids, liver tissue was stained by the fat-soluble Oil red O dye (Serva, Heidelberg, Germany, #31170). Immunohistochemical detections were performed as described previously
      • Helmrich N.
      • Roderfeld M.
      • Baier A.
      • Windhorst A.
      • Herebian D.
      • Mayatepek E.
      • et al.
      Pharmacological antagonization of cannabinoid receptor 1 improves cholestasis in Abcb4-/- mice.
      .

      TAG Assay

      The TAG concentration of homogenized hamster liver lysates was determined as recommended by the manufacturer (Triacylglyceride Assay Kit – Quantification, Abcam, Cambridge, UK, #ab65336).

      High-performance thin-layer chromatography (HPTLC)

      Liver tissue extracts were prepared ten times more concentrated than described
      • Irungbam K.
      • Roderfeld M.
      • Glimm H.
      • Hempel F.
      • Schneider F.
      • Hehr L.
      • et al.
      Cholestasis impairs hepatic lipid storage via AMPK and CREB signaling in hepatitis B virus surface protein transgenic mice.
      . Instrumentation used was from CAMAG, Muttenz, Switzerland. Samples and calibration standards (1 mg/mL in chloroform/methanol 3:1, V/V) were applied as 8-mm bands (ATS4) on HPTLC silica gel 60 F254 MS grade plates (Merck, Darmstadt, Germany) and dried (40 °C, 5 min). Separation of polar lipids was performed with chloroform/methanol/ammonia (25%)/water 60:30:3:1 (V/V/V/V) up to 50 mm, and of non-polar lipids with n-hexane/diethyl ether/acetic acid 40:10:1 (V/V/V) up to 65 mm migration distance after 20 min pre-saturation (via filter paper, Twin-Trough Chamber). The dried plate (5 min, cold air stream) was immersed into the primuline reagent (0.05 % in acetone/water 4:1, V/V). Fluorescence detection (FLD) was at 366/>400 nm (TLC Scanner 4). For visualization of saccharides and amino acids per reagent sequence, the primuline-treated polar lipid plate was immersed into the ninhydrin and then aniline-diphenylamine-o-phosphoric acid reagents
      • Mehl A.
      • Schmidt L.J.
      • Schmidt L.
      • Morlock G.E.
      High-throughput planar solid-phase extraction coupled to orbitrap high-resolution mass spectrometry via the autoTLC-MS interface for screening of 66 multi-class antibiotic residues in food of animal origin.
      . The primuline-treated non-polar lipid plate was sprayed with phosphomolybdic acid reagent (20 mg/mL solution in ethanol), heated (110°C, 3 min) and detected at white light illumination. For high-resolution mass spectrometry (HRMS), samples and standards were applied in duplicate on prewashed plates
      • Morlock G.E.
      Background mass signals in TLC/HPTLC–ESI-MS and practical advises for use of the TLC-MS interface.
      , which were cut after development. One plate part was used for detection via the primuline reagent, and the other via HPTLC–HRMS as described
      • Irungbam K.
      • Roderfeld M.
      • Glimm H.
      • Hempel F.
      • Schneider F.
      • Hehr L.
      • et al.
      Cholestasis impairs hepatic lipid storage via AMPK and CREB signaling in hepatitis B virus surface protein transgenic mice.
      .

      Reporter gene assay

      Analysis of AP-1 promotor-activity in SW620 cells has been described previously
      • Weglage J.
      • Wolters F.
      • Hehr L.
      • Lichtenberger J.
      • Wulz C.
      • Hempel F.
      • et al.
      Schistosoma mansoni eggs induce Wnt/β-catenin signaling and activate the protooncogene c-Jun in human and hamster colon.
      .

      Western blot analysis

      Western blotting was performed as described before
      • Roderfeld M.
      • Rath T.
      • Pasupuleti S.
      • Zimmermann M.
      • Neumann C.
      • Churin Y.
      • et al.
      Bone marrow transplantation improves hepatic fibrosis in Abcb4-/- mice via Th1 response and matrix metalloproteinase activity.
      .

      Glycogen assay

      Liver tissue and glycogen standard samples were dissolved in 2 M hydrochloric acid, boiled for 1 h and neutralized with 2 M sodium hydroxide. After centrifugation, the supernatant was analyzed with the Glucose Assay Kit (Merck) according to the manufacturer´s protocol.

      Malondialdehyde (MDA) assay

      Lipid-peroxidation as a marker for oxidative stress was measured in liver samples and HepG2 cells by the MDA Assay Kit (Merck), according to the manufacturer´s protocol.

      Pyruvate assay

      The pyruvate content of HepG2 cells was determined by the Pyruvate Assay Kit (Merck) according to the manufacturer´s protocol.

      Quantitative realtime polymerase chain reaction (qRT-PCR)

      mRNA isolation, transcription, quantitative real-time PCR, and data analysis was performed as described before
      • Irungbam K.
      • Churin Y.
      • Matono T.
      • Weglage J.
      • Ocker M.
      • Glebe D.
      • et al.
      Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2.
      .

      Quantification of S. mansoni eggs

      Liver tissue (100 mg) of bs-infected hamsters (n=36) was digested in 5 % potassium hydroxide at 37°C for 16 h
      • Cheever A.W.
      Conditions affecting the accuracy of potassium hydroxide digestion techniques for counting Schistosoma mansoni eggs in tissues.
      . Subsequently, eggs were counted independently by two persons three times per sample. The number of eggs was calculated per mg of liver tissue.

      Comet assay

      The alkaline comet assay (Abcam #ab238544) for assessing DNA damage in HepG2 cells was performed according to the manufacturer´s protocol.

      Statistic analysis

      The present study is of an exploratory nature. Therefore the group size were not estimated in advance by pre-specified effect size. The study was started with an existing number of cryopreserved organs that were not required for the maintenance of the parasite life cycle. Statistic analysis was performed using SPSS version 26.0 (SPSS Inc., IBM corporation, Armonk, NY, RRID:SCR_002865). Subsequent comparison of groups within each Kruskal-Wallis test were Bonferroni-corrected. Because of the exploratory nature of the study, no further adjustment for p-values was performed. Densitometrically assessed data from western blots (hamster colon) were depicted as mean or median ± 95% confidence intervals.

      Results

      S. mansoni eggs disrupt hepatic lipid metabolism

      To study the role of S. mansoni eggs in the liver in vivo, we used a hamster model and infections with bisex (bs)- and monosex (ms)-cercariae. This allowed to discriminate between worm-induced and egg-mediated effects in vivo (Fig. 1A). For analyzing the hepatic distribution of certain lipid species in livers of bs-infected hamsters, we performed MALDI-MS imaging
      • Spengler B.
      Mass spectrometry imaging of biomolecular information.
      , revealing the relative abundance and locations of hepatic lipids (Fig. 1A). MS imaging experiments revealed that all detected triacylglycerides accumulated in the granuloma area with highest levels inside the eggs (Fig. 1B arrowheads) while they were depleted in the surrounding liver tissue compared to ni controls (Fig. 1B, SFig. 1 and 2). Please note, that the color encodes for the concentration of the target molecule shown in Fig. 1B and SFig. 1 and 2.
      Figure thumbnail gr1
      Fig. 1Infection with S. mansoni induces alterations in the composition and distribution of hepatic lipids. (A) Infection of hamster with S. mansoni cercariae of both sexes (bisex, bs infection) or with clonal cercariae of one sex (monosex, ms infection) in order to compare egg-induced vs worm-only effects. The distribution of lipids was analyzed by MALDI-MS imaging in three biological replicates of bs-infected- and ms-infected hamster, and ni (non infected) control cryosections, respectively. (B) S. mansoni infection causes an accumulation of triacylglycerides in the granuloma area with highest levels inside the eggs (arrowheads). Triacylglycerides were depleted in the surrounding liver tissue compared to ni controls. The panels depict the distribution of TG(16:0_18:1_18:2)+K, m/z 895.714876 in the liver of bs-infected-hamster and ni controls. (C) The distribution of lipid species differs characteristically in eggs, granuloma, and the surrounding of granulomas. Upper left: Brightfield image. Middle left: Dimethylphosphatidylethanolamine DMPE (18:2/22:6) was found depleted in granulomas and enriched in eggs. Lower left: Dimethylphosphatidylethanolamine DMPE (18:0/22:5) was also detected with higher intensity in the eggs, but no depletion was observed for the granuloma regions. Upper right: DMPE (18:0/22:4) was mainly found in granulomas with slight enrichments in eggs. Middle right: DMPE (15:0/18:2) was found depleted in some part of granulomatous regions. Lower right: phosphatidylcholine m/z 810.532050 PC(17:2_18:3) [M+HCO2]- was found enriched in the outer borders of granulomas. A second set of MSI-pictures demonstrating altered lipid distribution in S. mansoni-infected hamsters is depicted in . These experiments were performed at least three times independently. (D) The quantification of selected lipid species revealed enhanced hepatic levels of SM and oleic acid (OA) in bs infected animals. TO triolein, Chol cholesterol, ni non-infected, ms monosex-infected, bs bisex-infected hamsters. These experiments were performed at least three times independently. Levels of significance are indicated in the figure (Kruskal-Wallis-test).
      In total, we detected 77 triacylglycerides with altered concentrations - 33 were depleted and 43 enriched in livers of bs-infected hamsters compared to the controls (SFig. 3). Adult schistosomes modify fatty acids from their host for biosynthetic purposes and incorporate those in phospholipids and neutral lipids
      • Bexkens M.L.
      • Mebius M.M.
      • Houweling M.
      • Brouwers J.F.
      • Tielens A.G.
      • van Hellemond J.J.
      Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead.
      . Here, we demonstrate that distinct isoforms of dimethyl-phosphatidylethanolamine (DMPE) were enriched in the eggs while they were depleted in the granuloma and in the surrounding liver parenchyma (Fig. 1C). Representative MS images of different lipid classes are shown for the three animal groups (SFig. 4, Fig. 1C focuses on the distribution of the lipids in and around the granuloma). We detected characteristic distributions of lipids in the liver of bs-infected versus ms-infected and ni animals. DMPE (18:2/22:6) was evenly distributed in the parenchyma of bs-infected hamster liver but depleted in granuloma and enriched in eggs (SFig. 4B). On the other hand, DMPE (18:0/22:5) was found in parenchyma and enriched in granuloma and eggs (SFig. 4C). DMPE (18:0/22:4) was not detected in hepatic parenchyma but in granulomas, with enrichment in eggs (SFig. 4D). DMPE (15:0/18:2) was found evenly distributed in all samples despite the granulomatous regions of bs-infected samples (SFig. 4E). A different lipid species, phosphatidylcholine (PC) (17:2/18:3) was enriched in the outer borders of granulomas but evenly distributed in samples from both controls (SFig. 4F). The distribution of other lipid species, like lysophosphatidylethanolamines (LPE), phosphatidylethanolamines (PE), and Ceramides (Cer) are shown in SFig. 5 and here exemplarily demonstrated as overlay of different lipid species in SFigs. 6 and 7 (please note, that the colors depicts different lipid species in SFigs. 6 and 7, while the color encodes for the concentration of one target molecule in Fig. 1B and SFigs. 1-2 and SFigs. 4-5). While the dimethyl-phosphatidylethanolamine DMPE(18:3_20:4) (SFig. 6B and in SFig. 6E in blue) was depleted in the granulomatous area (dashed lines), the phosphatidylcholine lipid PC(15:0_22:6) was enriched (in red in SFig. 6C and E, red arrowheads). Granulomas and surrounding tissues were differentiated, and a substructure was detected within the granuloma. Plasmenyl-PE(O-18:0_20:4) (green arrows) was only found in the outer regions of the granulomas. All lipids detected in this region were identified as plasmalogens. Further phosphatidylinositol lipid PI(18:0_22:4) (SFig. 7A, red arrowheads) was enriched in distict parts of granulomas while plasmenyl-PE (P-16:0_18:1) (green arrows) was found in the center and around the periphery of the granulomas (SFig. 7).
      Next, we quantified spatioregionally occurring lipids by HPTLC–FLD using the primuline reagent. The chromatogram and the respective densitogram of the non-polar lipids (SFig. 8A/B) showed well-separated blue fluorescence of cholesterol (hRF 13), oleic acid (hRF 22), triolein (hRF 45) and cholesteryl oleate (hRF 79). A reagent sequence of: first of primuline and then phosphomolybdic acid on the same plate resulted in dark green lipid zones (SFig 9). The tentative assignment of the different lipid species (based on hRF and reference substances) was confirmed by HPTLC–HRMS, exemplarily shown for the liver samples of S. mansoni ms- and bs-infected hamsters (SFig. 10, track 5 versus 15). Here, we unambiguously confirmed the presence of oleic acid (m/z 281.2486), triolein (m/z 907.7727) and cholesteryl oleate (m/z 673.5896) (SFig. 10A/B and D–G). Oleic acid, stearic acid (m/z 283.2642), linoleic acid (m/z 279.2329), and linolenic acid (m/z 277.2171) were present in liver samples of ms-infected hamsters (SFig. 10D). Cholesterol (SFig. 10C) showed the mass signals of two oxidation products at m/z 425.3391 ([M+O+Na]+) and 441.3340 ([M+2O+Na]+, and only a very weak signal at m/z 409.3443 [M+Na]+, all in accordance with the mass signals of liver samples of ms-infected hamsters. In ms-infected hamsters, triolein or similar triacylglycerides (TAGs) were absent (SFig. 10A/E). However, we confirmed a strongly fluorescing zone of cholesteryl oleate (SFig. 10A/F/G), which was absent in liver samples of bs-infected hamsters. The comparatively more complex mass spectra of phosphatidic acid, sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine were assigned to each respective lipid class (SFig. 11), whose presence we confirmed in the samples, except for phosphatidic acid. The low phosphatidic acid content (SFig. 8B, weakly blue fluorescent zone at hRF of 9) was most likely masked by the mass signals of saccharides (glucose) and amino acids (SFig. 9C), whose coelution was proven by derivatization (SFig. 9B, red zone at hRF 5 and SFig. 9C, blue-grey zone at hRF 6). The quantitative analysis of the eight lipid compounds in the 15 samples (STable 1) showed good reproducibility (mean %RSD 7.9%, n = 3, ranged 0−29%). The performance of the linear calibration lines was sufficient (coefficients of determination >0.997 and reproducibilities %RSD <10%, n = 3). The quantitative results revealed enhanced hepatic levels of sphingomyelins and oleic acid in bs-infected animals, while global hepatic levels of PE, PC, triolein, and cholesterol showed no alteration (Fig. 1D and SFigs. 8−11).

      S. mansoni eggs exhaust hepatocellular neutral lipids

      Next, we analyzed the concentration of TAGs in whole liver lysates of bs-, ms- and ni-hamsters (Fig. 2A). In line with a significant reduction of whole hepatic TAGs in bs-samples compared to ms and ni, we detected the corresponding downregulation of hepatic mRNA and protein expression levels of rate-limiting enzymes for lipid synthesis, namely fatty acid synthase (FAS) and acetyl-CoA-carboxylase (ACC1), in liver lysates of bs-infected hamsters (Fig. 2B and C). Notably, the differences between ms-infected- and bs-infected hamsters underline that the bisexual infection with egg-production caused pronounced effects (Fig. 2A-C). Immunohistochemistry revealed that infection with S. mansoni reduced parenchymal FAS expression with the exception of perigranulomatous hepatocytes (red arrow SFig. 12).
      Figure thumbnail gr2
      Fig. 2S. mansoni infection exhausts the host´s hepatic neutral lipid depots while parasite eggs accumulate lipids. (A) Hepatic TAG content was reduced in bs-infected hamsters. (B and C) Hepatic mRNA and protein levels of fatty acid synthase (Fas) and acetyl-CoA-carboxylase 1 (Acc1), both rate limiting enzymes for lipid synthesis, were reduced in bs-infected hamsters. Representative western blots are depicted. ni non-infected, ms monosex-infected, bs bisex-infected hamsters. (D) PLIN2-staining visualized the reduction of neutral lipids in liver parenchyma (p) of bisex-infected (bs) hamsters and the accumulation of neutral lipids in S. mansoni eggs (red arrowheads). Black arrowheads depict S. mansoni-infection-specific hepatic hemosiderin deposits, *central vein, #portal tract, 200 x, bar 100 μm, black dotted line indicates granuloma. (E and F) Confocal microscopy clearly demonstrated the sites of lipid accumulation (white arrowheads) inside the eggs (liver) cultured with fluorescently labeled oleic acid (flOA, in green), bars 20 μm (E) and 10 μm (F). A live video showing the active uptake of flOA by an egg (liver) was deposited online (suppl. Film 1) and also a 3D video demonstrating the lipid distribution inside liver-extracted eggs can be found online (suppl. Film 2). (G) To prove if S. mansoni eggs mobilize and take up lipids from hepatocytes, HepG2 cells were fed with flOA, washed three times and subsequently co-cultivated with eggs in a transwell system. Around 3% of the eggs freshly isolated from the host liver took up flOA from HepG2 cells after 24 h of co-culture. The workflow of the experiment depicted schematically (left panel) and representative pictures of a liver-derived egg that took up fluorescently labeled oleic acid (bar 25 μm, right panel). Remarkably, nearly 100% of pre-matured in vitro laid eggs took up flOA from HepG2 cells. These experiments were performed at least three times independently. (H) CLSM-based quantification of fluorescence intensity of individual eggs from coculture performed at 4 °C (white bar) or with 4% PFA fixed HepG2 cells (grey bar) in comparison to the conventional experiment as a control (con, green bar). At least 10 eggs per condition were analyzed in each of two independent experiments. Levels of significance are indicated in the figure (Kruskal-Wallis-test).

      S. mansoni eggs mobilize and take up lipids from hepatocytes

      Furthermore, we observed a strong reduction of PLIN2, a marker for neutral lipid storage, in the liver parenchyma compared to ni- and ms-infected liver specimens (Fig 2D). Notably, eggs were positively stained for PLIN2 (red arrowheads, Fig. 2D). The accumulation of neutral lipids in the eggs was confirmed by Oil-Red staining (red arrowhead, SFig. 13). Please note that some perigranulomatuos hepatocytes were positive for Oil-Red staining (red arrow, SFig. 13).
      Next, we investigated whether the eggs of S. mansoni were able to actively take up lipids. We found flOA uptake by freshly isolated eggs using confocal live-cell imaging (suppl. Films 1 and 2) and confocal fluorescence microscopy (Fig. 2E-F, SFig. 14A-C). The uptake occurred rapidly in some living eggs, starting after 10 min (SFig. 14A/B). We also observed flOA in fully developed miracidia (SFig. 14C). Next, we analyzed the uptake of flOA into in vitro-laid eggs of different maturity over 18 hours (SFig. 15). Eggs incorporated lipid independent of their maturity stage. Depending on their maturity, however, lipids accumulated in distinct structures of the egg. Young eggs (SFig. 15A) incorporated flOA into their vitelline cells. The oocyte remained negative. During embryo development, flOA-positive vitelline-cell content was found inside the embryo as small granular structures as well as in the sub-shell area (SFig. 15B). When the miracidium matured, the sub-shell envelopes (Reynolds‘ layer and van-Lichtenberg‘s envelope) took up flOA (SFig. 15C). Additionally, we questioned whether eggs mobilize and take up lipids from human hepatocytes. As shown schematically in Fig. 2G, we co-cultured in vitro-laid eggs with HepG2 cells, which had been pretreated with flOA (SFig. 16) 24 h prior to the coculture. Nearly all pre-matured in vitro-laid eggs took up flOA from HepG2 in transwell cocultures (Fig. 2G) but also in direct cocultures (SFig. 14D-E). Based on a previous study design
      • Toribio V.
      • Morales S.
      • López-Martín S.
      • Cardeñes B.
      • Cabañas C.
      • Yáñez-Mó M.
      Development of a quantitative method to measure EV uptake.
      , we performed two additional control experiments and demonstrated that the fixation of flOA-fed HepG2 cells or co-culturing of flOA-fed HepG2 cells with in vitro-laid eggs at 4°C impaired flOA uptake by the eggs, suggesting that we were measuring a process that requires an active cellular metabolism (Fig. 2H).
      The following is/are the supplementary data related to this article:

      S. mansoni eggs dysregulate the carbohydrate-metabolism

      Next, we analyzed in detail how S. mansoni infection affects the hepatic carbohydrate metabolism of the host. As depicted in Fig. 3A, we found hepatic glycogen to be significantly reduced in livers of bs- compared to ms-infected and ni-hamsters. Furthermore, the amount of hepatic glycogen inversely correlated with the number of eggs per mg hamster liver (Fig. 3B). In line with glycogen exhaustion, we found a significant downregulation of rate-limiting enzymes responsible for the glycogen turnover such as glycogen synthase (GS) and glycogen phosphorylase (PYGL) in liver lysates of bs-infected hamsters (SFig. 17). Remarkably, hepatic protein levels of the rate-limiting glycolysis-initiating enzyme glucokinase (GCK) were significantly increased in the bs group (Fig. 3C). PAS staining visualized a deprivation of intracellular glycogen in parenchymal cells, while the eggs were strongly stained and thus appeared to be rich in glycogen (Fig. 3D). Western blot and immunohistochemistry demonstrated a significant increase of the hepatic protein levels of the glycolytic enzymes pyruvate kinase 1 and 2 (PKM 1 and 2), both in bs-infected animals (Fig. 3E/F). Notably, PKM1 and PKM2 occurred in different areas and cells in the livers of bs-infected hamsters, i.e. PKM1 in the hepatic parenchyma (SFig. 18A) and PKM2 in the granulomas (SFig. 18B). The opposite regulation of elevated glycolysis and reduced glycogenesis rate-limiting enzymes was confirmed by stimulating human hepatoma cell lines with SEA (SFig. 19). Pyruvate dehydrogenase (PDH) catalyzes the conversion of pyruvate, the end-product of glycolysis, into acetyl-CoA, which links glycolysis and the citric acid cycle. Hepatic PDH was induced by S. mansoni infection (SFig. 20). In addition, we observed an elevation of glucose-6-phosphate dehydrogenase (G6PDH), which catalyzes the transition of glucose-6-phosphate into 6-phosphogluconate, the first step in the PPP (Fig. 3G, SFig. 21).
      Figure thumbnail gr3
      Fig. 3S. mansoni infection exhausts the host´s hepatic carbohydrate storage. (A) The hepatic glycogen content was diminished approximately 6-fold in livers of bs-infected hamsters. (B) Hepatic glycogen content inversely correlated with the number of eggs per mg liver tissue. (C) Glucokinase, the first rate limiting enzyme of glycolysis, was upregulated in livers of bs-infected hamsters. A representative western blot is shown. (D) PAS staining of histologic liver sections revealed homogenous hepatic glycogen storage in ni animals (left panel), zoned glycogen storage in livers of ms-infected hamsters with a reduction of glycogen deposits around the central veins* (dotted red line indicates zonation), as well as a complete absence of glycogen in the liver parenchyma (p) of bs-infected hamsters, while eggs were strongly positive stained (red arrowheads). *central vein, #portal tract, 200 x, bar 100 μm, black dotted line indicates granuloma. (E and F) Western blot analysis demonstrated the induction of rate-limiting enzymes of glycolysis like PKM1 (E) and PKM2 (F) in livers of bs-infected hamsters. (G) G6PDH, the rate-limiting enzyme of the PPP of glycolysis, was also upregulated in livers of bs-infected hamsters. n=3-5, ni non-infected, ms monosex-infected, bs bisex-infected hamsters. These experiments were performed at least three times independently. Levels of significance are indicated in the figure (Kruskal-Wallis-test and non-linear regression).
      Finally, we wondered which evolutionary mechanisms might have promoted the ability of eggs to reprogram the host´s metabolism in order to utilize the host´s lipid and glucose reserves. This question led to the idea, that the hepatic effects might have their evolutionary origin in the egg´s translocation through the bowel wall into the gut lumen. Remarkably, we found a similar regulation of PKM2, GK2, and PEPCK2 expression in the colon of bs-infected hamsters (SFig. 22 A-D).

      S. mansoni eggs induce oxidative stress

      G6PDH is a key regulator of the oxidative branch of the PPP and indispensable for maintaining the cytosolic pool of NADPH, thus being essential for the cellular redox balance
      • Stincone A.
      • Prigione A.
      • Cramer T.
      • Wamelink M.M.C.
      • Campbell K.
      • Cheung E.
      • et al.
      The return of metabolism: biochemistry and physiology of the pentose phosphate pathway.
      . We observed a significant augmentation of MDA (malondialdehyde), a secondary reaction product of lipid peroxidation, in livers of S. mansoni-infected hamsters (Fig. 4A). GSH (reduced L-glutathione) significantly decreased the SEA-induced MDA in human hepatoma cells (Fig. 4B). Catalase is one of the crucial antioxidant enzymes that mitigates oxidative stress by destroying cellular hydrogen peroxide to produce water and oxygen. We detected both, mRNA and protein levels of catalase significantly downregulated in hamster livers (Fig. 4C/D). The downregulated mRNA level of the host´s catalase in the bs-group correlated with an increasing number of eggs in the liver (Fig. 4E). Notably, catalase mRNA levels also decreased by stimulation with soluble egg products in human hepatoma cells (SFig. 23).
      Figure thumbnail gr4
      Fig. 4S. mansoni egg-induced hepatocellular oxidative stress. (A) Hepatic MDA levels were increased in hamster liver upon bs infection. (B) SEA stimulation induced MDA in HepG2 cells. The induction was abolished by the addition of reduction equivalents in form of GSH (n=6). (C and D) Hepatic catalase expression is reduced in hamster liver of the bs group (C, mRNA, D, protein level). (E) Hepatic catalase mRNA levels and the number of eggs per mg liver tissue inversely correlated with exponential trend. Data were normalized to the control group. (F) Pyruvate quantification demonstrated SEA-induced glycolysis in HepG2 cells, which was reduced to unstimulated levels by the addition of reduction equivalents in form of GSH. The experiment was repeated three times. Data were normalized to the control group. Kruskall-Wallis test was performed to asses group differences. ni non-infected, ms monosex-infected, bs bisex-infected hamsters. These experiments were performed at least three times independently. Levels of significance are indicated in the figure (Kruskal-Wallis-test).
      Surprisingly, the infection with S. manoni increased hepatic mRNA levels of Mn2+-dependent superoxide dismutase (MnSod), while CuZnSod mRNA levels decreased (SFig. 24A/B). In the bs group, glutathione peroxidase, Gsh-Px, was not regulated (SFig. 24C). SEA treatment induced the accumulation of pyruvate in HepG2 cells, which was reversed by GSH (Fig. 4F). Strikingly, also flOA transfer from flOA-fed HepG2 cells to eggs was reduced in the presence of GSH in co-cultures (SFig. 25). Accordingly, we found a similar regulation of Gsh-Px expression in the colon of bs-infected hamsters (SFig. 26).

      S. mansoni egg-induced oxidative stress activates the protooncogen c-Jun and DNA damage

      We investigated whether the exhaustion of hepatocytes and the associated oxidative stress might trigger the activation of oncogenic signaling and DNA damage. In human hepatoma cells, GSH inhibited the egg product-induced activation of ERK, c-JUN, and STAT3 (Fig. 5 A-C). In parallel, we observed significantly elevated protein expression levels of the DNA damage marker γ-H2A.x in bs-infected hamsters and in HepG2 cells following SEA treatment, which could be reversed in vitro by the ROS scavenger GSH (Fig. 5D/E).
      Figure thumbnail gr5
      Fig. 5S. mansoni eggs induce malignant signaling and oxidative stress-dependent DNA damage in HepG2 cells. (A-C) Western blot analyses revealed the activation of ERK, c-JUN, and STAT3 in SEA-stimulated HepG2 cells. The addition of GSH diminished SEA-induced HCC-associated signaling to control levels. Representative western blots are depicted. ni non-infected, ms monosex-infected, bs bisex-infected hamsters. (D) γH2A.x, a marker for DNA double strand breaks, is induced in the bs group. (E) Stimulation with SEA induced γH2A.x, while the addition of GSH abolished this effect. (F) Comet assay demonstrated that SEA-induced DNA damage in HepG2 cells, and that DNA damage was reduced by addition of GSH. Kruskall-Wallis test was performed to asses group differences. These experiments were performed at least three times independently. Levels of significance are indicated in the figure (Kruskal-Wallis-test).
      In line with these findings, we demonstrated an SEA-induced DNA damage in vitro by comet assay. This effect was reversed by reduced GSH (Fig. 5F). As the antagonization of oxidative stress abolishes the SEA-induced c-Jun activation (Fig. 5B), we further analyzed whether the functional activation of AP1 promotor activity is influenced by GSH. The SEA induced functional activation of AP1 promotor activity was also reduced by GSH in the human colon cell line SW620 (SFig. 27).

      Proof of clinical relevance in human biopsies

      In order to obtain a model-independent proof of the most important results from hamster and human cell lines, some major targets were visualized in histological slices colon biopsies that were routinely taken for diagnosis from a 23 years old male patient suffering from schistosomiasis (Fig. 6). S. mansoni eggs were positively stained for PLIN2 (Fig. 6A) and PAS (Fig. 6B) indicating enhanced accumulation of neutral lipids (A) and glycogen (B) in eggs passing the rectal wall. The staining for PKM2 demonstrated enhanced glycolysis in inflammatory cells around the eggs (Fig. 6C). A visual comparison of PKM2 in infected vs non-infected human tissue is depicted in SFig. 28.
      Figure thumbnail gr6
      Fig. 6Validation of metabolic implications with tissue samples of a patient infected with S. mansoni. (A) Histologic specimen of a colon biopsy from a 23 year old patient with schistosomiasis was stained for PLIN2. The signal for PLIN2 indicated an accumulation of neutral lipids inside the eggs (red arrowheads). (B) The positive PAS reaction demonstrated an accumulation of glycogen in most of the eggs passing the bowel wall (purple arrowheads). (C) Enhanced staining for PKM2 (red arrowheads) in granulomatous infiltrates around the eggs (black arrowhead). Representative stainings are depicted, bars 100 (right) and 200 μm (left).

      Discussion

      Genes encoding the beta-oxidation pathway are lacking in S. mansoni at any stage of its life cycle
      • Bexkens M.L.
      • Mebius M.M.
      • Houweling M.
      • Brouwers J.F.
      • Tielens A.G.
      • van Hellemond J.J.
      Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead.
      . Therefore, S. mansoni may not be capable of catabolizing fatty acids for energy production. We demonstrated that eggs mobilized and took-up flOA from well-differentiated human hepatoma cells. The development of larvae requires energy resources but also structural components for the synthesis of cell membranes like lipids that cannot be synthesized by the parasite itself. A single ovum in a newly laid egg does not have high nutritional requirements. After 2-3 days, however, the developing embryo will take more nutrients from its host environment. Moreover, we identified distinct phospholipid species that were enriched in hepatic eggs as previously proposed
      • Bexkens M.L.
      • Mebius M.M.
      • Houweling M.
      • Brouwers J.F.
      • Tielens A.G.
      • van Hellemond J.J.
      Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead.
      but depleted in granulomas and the surrounding liver parenchyma. Furthermore, lipid mobilization from host cells requires an active cellular metabolism and can be suppressed by the addition of a ROS scavenger GSH. During the migration of eggs through host tissues the uptake of lipids and other molecules might be facilitated by cribriform-like pores inside the eggshell
      • Neill P.J.
      • Smith J.H.
      • Doughty B.L.
      • Kemp M.
      The ultrastructure of the Schistosoma mansoni egg.
      ,
      • Stjernholm R.L.
      • Warren K.S.
      Schistosoma mansoni: Utilization of exogenous metabolites by eggs in vitro.
      .
      Our data strengthen the idea that nutrient uptake from the host is a basic prerequisite for the increase of the eggs´ biomass and dimensions during development
      • Ashton P.D.
      • Harrop R.
      • Shah B.
      • Wilson R.A.
      The schistosome egg: development and secretions.
      ,
      • Jurberg A.D.
      • Gonçalves T.
      • Costa T.A.
      • Mattos ACA de
      • Pascarelli B.M.
      • Manso PPA de
      • et al.
      The embryonic development of Schistosoma mansoni eggs: proposal for a new staging system.
      . This may explain why we found exhausted hepatic glycogen stores in the presence of eggs. However, the glycogen content of eggs increases with the embryonic development and is highest at the miracidium stage
      • Jurberg A.D.
      • Gonçalves T.
      • Costa T.A.
      • Mattos ACA de
      • Pascarelli B.M.
      • Manso PPA de
      • et al.
      The embryonic development of Schistosoma mansoni eggs: proposal for a new staging system.
      . It has been observed that S. mansoni eggs directly absorb glucose in vitro
      • Bexkens M.L.
      • Mebius M.M.
      • Houweling M.
      • Brouwers J.F.
      • Tielens A.G.
      • van Hellemond J.J.
      Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead.
      . Acetyl-CoA and glycerol-3-phosphate, both products of the glycolytic breakdown, serve as components for the de novo fatty-acid synthesis
      • Bexkens M.L.
      • Mebius M.M.
      • Houweling M.
      • Brouwers J.F.
      • Tielens A.G.
      • van Hellemond J.J.
      Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead.
      . It has been hypothesized that S. mansoni eggs store neutral lipids among others for developmental processes of miracidia, which require phospholipids e.g., for synthesizing new membranes
      • Bexkens M.L.
      • Mebius M.M.
      • Houweling M.
      • Brouwers J.F.
      • Tielens A.G.
      • van Hellemond J.J.
      Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead.
      .
      In S. japonicum-infected mice, changes in gene expression of catabolism and anabolism in the liver were affected or could occur via the macrophage M2 phenotype state
      • Xu Z.-P.
      • Chang H.
      • Ni Y.-Y.
      • Li C.
      • Chen L.
      • Hou M.
      • et al.
      Schistosoma japonicum infection causes a reprogramming of glycolipid metabolism in the liver.
      . Our data however, clearly demonstrate that soluble egg products modulate the metabolic reprogramming of the host´s hepatic carbohydrate and lipid metabolism, independently of any immune response. In line with our findings, maturation of S. japonicum eggs depends on cholesteryl ester uptake from HDL and occurs via a CD36-related protein from the egg developmental stages
      • Okumura-Noji K.
      • Miura Y.
      • Lu R.
      • Asai K.
      • Ohta N.
      • Brindley P.J.
      • et al.
      CD36-related protein in Schistosoma japonicum: candidate mediator of selective cholesteryl ester uptake from high-density lipoprotein for egg maturation.
      . Thus, depletion of host lipids/glycogen storage during S. mansoni infection is likely effected by the eggs itself.
      Most of the factors we analyzed indicate that the hepatic metabolism of ms-infected hamsters is different to that of bs-infected hamsters, which underlines the role of eggs for these processes. Remarkably, some parameters like PDH or catalase expression are also different between ni- and ms-infected animals, which presumably reflects the effects on hepatic metabolism and oxidative stress that are even caused by adult worms alone. Nevertheless, biomolecular insights into reprogramming of the host’s metabolism by S. mansoni might also be important when considering the two sides of the same coin – detrimental malnutrition of infected patients in endemic countries versus beneficial anti-obesity effects that have been explored in the context of obesity.
      Metabolic dysregulation as well as DNA damage induced by S. mansoni SEA was restored by the addition of a ROS scavenger in vitro. We demonstrated by in vitro assays that SEA is capable of inducing oxidative stress, subsequently resulting in the activation of oncogenic signaling and DNA damage. In addition, we found a metabolism-associated dysregulation of the antioxidant system, which is in line with previous observations
      • Oliveira RB de
      • Senger M.R.
      • Vasques L.M.
      • Gasparotto J.
      • dos Santos J.P.A.
      • Pasquali MAdB.
      • et al.
      Schistosoma mansoni infection causes oxidative stress and alters receptor for advanced glycation endproduct (RAGE) and tau levels in multiple organs in mice.
      .
      The phenomenon of exploitation the host´s energy reserves constitutes a parasitic principle. In the case of schistosome eggs, however, for the first time we describe metabolic deprivation and effects on carbohydrate as well as lipid regulation. Nutrient deficiency resulted in oxidative stress in the livers of bs-infected hamsters as well as in SEA-stimulated human hepatoma cells in vitro. SEA significantly inhibited the expression of catalase in vitro, which inversely correlated with elevated egg load in vivo. Furthermore, we demonstrated that oncogenic signaling and DNA damage are consequences of egg-mediated metabolic deprivation, and that the ensuing oxidative stress can be neutralized by the addition of the ROS scavenger GSH. Our data suggest metabolic reprogramming of the host in order to acquire the hosts lipid and carbohydrate reserves, absolutely essential for miracidial development within the egg.
      In summary, our results shed new light on the schistosome parasite – host parenchyma interaction, at the level of the egg stage. We demonstrate that the S. mansoni egg take advantage of its host environment through metabolic reprogramming of hepatocytes and enterocytes. Eggs ending up in the liver cause oxidative stress-induced- and malignancy-associated signaling as well as DNA damage, which in combination might precondition or promote hepatocellular damage.

      Acknowledgements

      The authors thank Heike Mueller, Annette Tschuschner, Christina Scheld, Georgette Stovall, and Bianca Kulik for excellent technical assistance. Technical support by TransMIT GmbH, Giessen, Germany, is gratefully acknowledged.

      Appendix A. Supplementary data

      The following is/are the supplementary data to this article:

      1 References

      Author names in bold designate shared co-first authorship
      1. WHO: Fact-Sheets Schistosomiasis 2021; Available from: https://www.who.int/news-room/fact-sheets/detail/schistosomiasis.

        • Boissier J.
        • Grech-Angelini S.
        • Webster B.L.
        • Allienne J.-F.
        • Huyse T.
        • Mas-Coma S.
        • et al.
        Outbreak of urogenital schistosomiasis in Corsica (France): An epidemiological case study.
        Lancet Infect. Dis. 2016; 16: 971-979
        • Costain A.H.
        • MacDonald A.S.
        • Smits H.H.
        Schistosome egg migration: mechanisms, pathogenesis and host immune responses.
        Front. Immunol. 2018; 9: 3042
        • Schwartz C.
        • Fallon P.G.
        Schistosoma "eggs-iting" the host: granuloma formation and egg excretion.
        Front. Immunol. 2018; 9: 2492
        • Olveda D.U.
        • Olveda R.M.
        • McManus D.P.
        • Cai P.
        • Chau T.N.
        • Lam A.K.
        • et al.
        The chronic enteropathogenic disease schistosomiasis.
        Int. J. Inf. Dis. 2014; 28: 193-203
        • Ashton P.D.
        • Harrop R.
        • Shah B.
        • Wilson R.A.
        The schistosome egg: development and secretions.
        Parasitology. 2001; 122: 329-338
        • Kabongo M.M.
        • Linsuke S.
        • Baloji S.
        • Mukunda F.
        • Raquel IdL.
        • Stauber C.
        • et al.
        Schistosoma mansoni infection and its association with nutrition and health outcomes: a household survey in school-aged children living in Kasansa, Democratic Republic of the Congo.
        Pan Afr. med. J. 2018; 31: 197
        • Maciel P.S.
        • Gonçalves R.
        • Antonelli LRdV.
        • Fonseca C.T.
        Schistosoma mansoni infection is impacted by malnutrition.
        Front. Microbiol. 2021; 12635843
        • Wolde M.
        • Berhe N.
        • Medhin G.
        • Chala F.
        • van Die I.
        • Tsegaye A.
        Inverse associations of Schistosoma mansoni infection and metabolic syndromes in humans: a cross-sectional study in northeast Ethiopia.
        Microbiol. Insights. 2019; 121178636119849934
        • Stanley R.G.
        • Jackson C.L.
        • Griffiths K.
        • Doenhoff M.J.
        Effects of Schistosoma mansoni worms and eggs on circulating cholesterol and liver lipids in mice.
        Atherosclerosis. 2009; 207: 131-138
        • Xu Z.-P.
        • Chang H.
        • Ni Y.-Y.
        • Li C.
        • Chen L.
        • Hou M.
        • et al.
        Schistosoma japonicum infection causes a reprogramming of glycolipid metabolism in the liver.
        Parasites & vectors. 2019; 12: 388
        • Cortes-Selva D.
        • Gibbs L.
        • Maschek J.A.
        • Nascimento M.
        • van Ry T.
        • Cox J.E.
        • et al.
        Metabolic reprogramming of the myeloid lineage by Schistosoma mansoni infection persists independently of antigen exposure.
        PLoS Pathogens. 2021; 17e1009198
        • Risi R.
        • Tuccinardi D.
        • Mariani S.
        • Lubrano C.
        • Manfrini S.
        • Donini L.M.
        • et al.
        Liver disease in obesity and underweight: the two sides of the coin.
        A narrative review. Eating and weight disorders EWD. 2021; 26: 2097-2107
        • Oliveira RB de
        • Senger M.R.
        • Vasques L.M.
        • Gasparotto J.
        • dos Santos J.P.A.
        • Pasquali MAdB.
        • et al.
        Schistosoma mansoni infection causes oxidative stress and alters receptor for advanced glycation endproduct (RAGE) and tau levels in multiple organs in mice.
        Int. J. Parasitol. 2013; 43: 371-379
        • Srinivas U.S.
        • Tan B.W.Q.
        • Vellayappan B.A.
        • Jeyasekharan A.D.
        ROS and the DNA damage response in cancer.
        Redox Biol. 2019; 25101084
        • Weglage J.
        • Wolters F.
        • Hehr L.
        • Lichtenberger J.
        • Wulz C.
        • Hempel F.
        • et al.
        Schistosoma mansoni eggs induce Wnt/β-catenin signaling and activate the protooncogene c-Jun in human and hamster colon.
        Sci. Rep. 2020; 10: 2492
        • Roderfeld M.
        • Padem S.
        • Lichtenberger J.
        • Quack T.
        • Weiskirchen R.
        • Longerich T.
        • et al.
        Schistosoma mansoni egg secreted antigens activate HCC-associated transcription factors c-Jun and STAT3 in hamster and human hepatocytes.
        Hepatology. 2020; 72: 626-641
        • Grevelding C.G.
        The female-specific W1 sequence of the Puerto Rican strain of Schistosoma mansoni occurs in both genders of a Liberian strain.
        Mol. Biochem. Parasitol. 1995; 71: 269-272
        • Grevelding C.G.
        Genomic instability in Schistosoma mansoni.
        Mol. Biochem. Parasitol. 1999; 101: 207-216
        • Lu Z.
        • Sessler F.
        • Holroyd N.
        • Hahnel S.
        • Quack T.
        • Berriman M.
        • et al.
        Schistosome sex matters: A deep view into gonad-specific and pairing-dependent transcriptomes reveals a complex gender interplay.
        Sci. Rep. 2016; 631150
        • Wiedemann K.R.
        • Peter Ventura A.
        • Gerbig S.
        • Roderfeld M.
        • Quack T.
        • Grevelding C.G.
        • et al.
        Changes in the lipid profile of hamster liver after Schistosoma mansoni infection, characterized by mass spectrometry imaging and LC-MS/MS analysis.
        Analytical and bioanalytical chemistry. 2022; https://doi.org/10.1007/s00216-022-04006-6
        • Koelmel J.P.
        • Kroeger N.M.
        • Ulmer C.Z.
        • Bowden J.A.
        • Patterson R.E.
        • Cochran J.A.
        • et al.
        LipidMatch: an automated workflow for rule-based lipid identification using untargeted high-resolution tandem mass spectrometry data.
        BMC Bioinform. 2017; 18: 331
        • Tyanova S.
        • Temu T.
        • Sinitcyn P.
        • Carlson A.
        • Hein M.Y.
        • Geiger T.
        • et al.
        The Perseus computational platform for comprehensive analysis of (prote)omics data.
        Nat. Methods. 2016; 13: 731-740
        • Paschke C.
        • Leisner A.
        • Hester A.
        • Maass K.
        • Guenther S.
        • Bouschen W.
        • et al.
        Mirion—a software package for automatic processing of mass spectrometric images.
        J. Am. Soc. Mass Spectrom. 2013; 24: 1296-1306
        • Schramm G.
        • Falcone F.H.
        • Gronow A.
        • Haisch K.
        • Mamat U.
        • Doenhoff M.J.
        • et al.
        Molecular characterization of an interleukin-4-inducing factor from Schistosoma mansoni eggs.
        J. Biol. Chem. 2003; 278: 18384-18392
        • Dalton J.P.
        • Day S.R.
        • Drew A.C.
        • Brindley P.J.
        A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.
        Parasitology. 1997; 115: 29-32
        • Helmrich N.
        • Roderfeld M.
        • Baier A.
        • Windhorst A.
        • Herebian D.
        • Mayatepek E.
        • et al.
        Pharmacological antagonization of cannabinoid receptor 1 improves cholestasis in Abcb4-/- mice.
        CMGH. 2021; 13: 1041-1055
        • Irungbam K.
        • Roderfeld M.
        • Glimm H.
        • Hempel F.
        • Schneider F.
        • Hehr L.
        • et al.
        Cholestasis impairs hepatic lipid storage via AMPK and CREB signaling in hepatitis B virus surface protein transgenic mice.
        Lab. Invest. 2020; 100: 1411-1424
        • Mehl A.
        • Schmidt L.J.
        • Schmidt L.
        • Morlock G.E.
        High-throughput planar solid-phase extraction coupled to orbitrap high-resolution mass spectrometry via the autoTLC-MS interface for screening of 66 multi-class antibiotic residues in food of animal origin.
        Food chemistry. 2021; 351129211
        • Morlock G.E.
        Background mass signals in TLC/HPTLC–ESI-MS and practical advises for use of the TLC-MS interface.
        J. Liq. Chromatogr. 2014; 37: 2892-2914
        • Roderfeld M.
        • Rath T.
        • Pasupuleti S.
        • Zimmermann M.
        • Neumann C.
        • Churin Y.
        • et al.
        Bone marrow transplantation improves hepatic fibrosis in Abcb4-/- mice via Th1 response and matrix metalloproteinase activity.
        Gut. 2012; 61: 907-916
        • Irungbam K.
        • Churin Y.
        • Matono T.
        • Weglage J.
        • Ocker M.
        • Glebe D.
        • et al.
        Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2.
        Lab. Invest. 2020; 100: 454-465
        • Cheever A.W.
        Conditions affecting the accuracy of potassium hydroxide digestion techniques for counting Schistosoma mansoni eggs in tissues.
        Bull. World Health Organ. 1968; 39: 328-331
        • Spengler B.
        Mass spectrometry imaging of biomolecular information.
        Anal. Chem. 2015; 87: 64-82
        • Bexkens M.L.
        • Mebius M.M.
        • Houweling M.
        • Brouwers J.F.
        • Tielens A.G.
        • van Hellemond J.J.
        Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead.
        Int. J. Parasitol. 2019; 49: 647-656
        • Toribio V.
        • Morales S.
        • López-Martín S.
        • Cardeñes B.
        • Cabañas C.
        • Yáñez-Mó M.
        Development of a quantitative method to measure EV uptake.
        Sci. Rep. 2019; 910522
        • Stincone A.
        • Prigione A.
        • Cramer T.
        • Wamelink M.M.C.
        • Campbell K.
        • Cheung E.
        • et al.
        The return of metabolism: biochemistry and physiology of the pentose phosphate pathway.
        Biol. rev. biol. proc. Camb. Philos. Soc. 2015; 90: 927-963
        • Neill P.J.
        • Smith J.H.
        • Doughty B.L.
        • Kemp M.
        The ultrastructure of the Schistosoma mansoni egg.
        Am. J. Trop. Med. Hyg. 1988; 39: 52-65
        • Stjernholm R.L.
        • Warren K.S.
        Schistosoma mansoni: Utilization of exogenous metabolites by eggs in vitro.
        Exp. Parasitol. 1974; 36: 222-232
        • Jurberg A.D.
        • Gonçalves T.
        • Costa T.A.
        • Mattos ACA de
        • Pascarelli B.M.
        • Manso PPA de
        • et al.
        The embryonic development of Schistosoma mansoni eggs: proposal for a new staging system.
        Dev. Genes Evol. 2009; 219: 219-234
        • Okumura-Noji K.
        • Miura Y.
        • Lu R.
        • Asai K.
        • Ohta N.
        • Brindley P.J.
        • et al.
        CD36-related protein in Schistosoma japonicum: candidate mediator of selective cholesteryl ester uptake from high-density lipoprotein for egg maturation.
        FASEB J. 2013; 27: 1236-1244