Early host-parasite interaction models reveal a key role for fibrinolysis in Fasciola hepatica intestinal migration

Early host-parasite interaction models reveal a key role for fibrinolysis in Fasciola hepatica intestinal migration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Early host-parasite interaction models reveal a key role for fibrinolysis in Fasciola hepatica intestinal migration Judit Serrat, Marta López-García, María Torres-Valle, Verónica Molina-Hernández, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6822677/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Parasites & Vectors → Version 1 posted 9 You are reading this latest preprint version Abstract Background Fasciola hepatica is the most common etiologic agent of fasciolosis, a parasitic disease that affects millions of ruminants worldwide and a zoonotic human infection of public health concern. Upon ingestion of infective metacercariae, F. hepatica newly excysted juveniles (FhNEJ) emerge in the duodenum and cross the intestinal wall to initiate a migration route that culminates with their establishment within the hepatic bile ducts. The ability of FhNEJ to exploit the broad-spectrum activities of host plasmin, the central protease of the fibrinolytic system, has been proposed as a strategy employed by these parasites to migrate across the intestinal wall while minimising energy expenditure. Methods Mouse intestinal epithelial cells (mPSIEC) were stimulated with FhNEJ and plasminogen (PLG), the zymogen of plasmin, to understand whether FhNEJ-stimulated plasmin generation modulates processes relevant to parasite migration through the intestinal wall, including extracellular matrix (ECM) degradation and the secretion of ECM-degrading enzymes. Plasmin-mediated cellular responses were further examined by proteomic analysis of mPSIEC whole-cell lysates. In parallel, the contribution of the fibrinolytic system in FhNEJ migration was studied in vivo by infecting mice with F. hepatica metacercariae following pharmacological inhibition of fibrinolysis. Results Co-stimulation of mPSIEC with FhNEJ and PLG led to increased plasmin generation in the intestinal pericellular space, which was associated with enhanced collagen degradation and secretion of the urokinase-type plasminogen activator (u-PA). In addition, using independent cell culture replicates and a stringent statistical pipeline, we identified a robust set of differentially expressed proteins in mPSIEC following stimulation with FhNEJ and PLG. These proteins were involved in cell adhesion, migration, ECM remodelling, immune evasion, and fibrinolysis. Despite inter-experimental variability, FhNEJ migration in mice was reduced upon pharmacological inhibition of fibrinolysis, supporting the contribution of host fibrinolysis to parasite invasion in vivo . Conclusions Altogether, this work provides unprecedented insights into the role of the host fibrinolytic system to FhNEJ migration across mammalian host tissues, thereby advancing our understanding of host-parasite relationships during early-stage fasciolosis and highlighting interesting directions for future research in this area. Fasciola hepatica fibrinolysis plasminogen plasmin migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction A paradigmatic example of host-parasite relationship is the ability of parasites to engage with the fibrinolytic system of their definitive hosts, a strategy that is considered a key aspect of parasite dissemination and survival within the mammalian organism [1–3]. The most common mechanism of interaction between parasites and host fibrinolysis is through the expression of lysine-rich proteins at the host-parasite interface—either on the parasite surface or secreted into the tissue microenvironment surrounding the parasite—that interact with plasminogen (PLG), the main zymogen of the fibrinolytic route. Upon binding to its receptors (PLG-Rs), PLG undergoes a conformational change that exposes the target sites of its activators, the proteases tissue-type and urokinase-type PLG activators (t-PA and u-PA, respectively), which convert PLG into its catalytically active form, the serine protease plasmin [4]. The classical function of the fibrinolytic system is to degrade the fibrin network that stabilises blood clots in the vascular endothelium [4]; however, due to the broad spectrum of plasmin substrates, this system also participates in multiple biological processes that are both unrelated and independent of fibrin degradation and blood clot removal, including cell migration [5,6]. Plasmin facilitates cell migration by degrading different components of the extracellular matrix (ECM), including collagens, laminin, fibronectin, and proteoglycans [7–11], as well as by cleaving and activating the zymogens of different metalloproteinases (MMPs), which are considered master regulators of ECM remodelling during homeostasis, development, and disease [12]. Additionally, fibrinolysis-unrelated substrates of plasmin also include complement molecules and immunoglobulins [13]. Based on this, the stimulation of plasmin generation from host PLG within the tissue microenvironment surrounding parasites has been postulated as a mechanism facilitating immune evasion and their migration through host tissues [2,3,13]. Fasciola hepatica is one of many parasites whose interaction with the host fibrinolytic system has been experimentally shown [14–18]. F. hepatica is a helminth trematode with an indirect life cycle that includes freshwater snails as intermediate hosts, where the asexual phases of the parasites develop, and a mammalian definitive host, where adult flukes mature. Definitive hosts, typically ruminants and humans, become infected by ingestion of water plants contaminated with infective metacercariae, which excyst in the duodenum and release the newly excysted juveniles (FhNEJ). Shortly after excystment, FhNEJ cross the intestinal wall, and the juvenile flukes subsequently migrate through the peritoneum and liver parenchyma before entering the major hepatic bile ducts, where they mature into adults and produce fertilised eggs that are shed with the faeces [19]. F. hepatica is the most widespread etiologic agent of fasciolosis, a chronic and debilitating disease that affects millions of ruminants worldwide, thereby representing an economically important affection of domestic livestock that poses major threats to animal welfare and global food security [20,21]. Since cases of human infection are predominantly concentrated in tropical areas of the globe, human fasciolosis is categorized by the Word Health Organization a food-borne neglected tropical disease of growing public health concern requiring targeted intervention for its elimination [22,23]. Using in vitro assays, we and others have previously shown that FhNEJ interact with the host fibrinolytic system by multiple mechanisms. These include the expression of proteins that bind to PLG, the precursor zymogen of u-PA (pro-u-PA), or both at the host-parasite interface [14–16,18], as well as the expression of proteases that can stimulate pro-u-PA conversion into its catalytically active form, which further potentiates plasmin generation from host PLG [15]. Notably, FhNEJ-stimulated plasmin generation contributes to the endogenous capacity of these parasites to degrade laminin [24], one of the major components of the intestinal basement membrane [25], indicating that the interaction between FhNEJ and host fibrinolysis may facilitate their migration across the intestinal wall. Since FhNEJ trans-intestinal migration is considered a crucial step for infection success, a deeper understanding of the mechanisms governing this process, including the contribution of the host fibrinolytic system, may offer potential avenues for developing novel therapeutic and control strategies against this widespread parasite [26]. Although the interaction between parasites and host fibrinolysis system is well-established [1–3], only a minority of studies have performed functional assays using physiologically-relevant experimental models to assess whether it serves any physiological roles [1]. The abovementioned interplay between F. hepatica and the host fibrinolytic system exemplifies this gap. In the present study, we addressed this limitation by exploring the functional relevance of the interaction between FhNEJ and host fibrinolysis using a co-culture system of epithelial cells derived from the mouse small intestine, which simulates the first contact between FhNEJ and host tissues [27,28], and a mouse model of early-stage fasciolosis [29]. Materials and Methods Culture of mouse primary small intestinal epithelial cells C57BL/6 mouse primary small intestinal epithelial cells (mPSIEC; Cell Biologics) were cultured as previously described [27,28] and following the manufacturer’s instructions. Briefly, cells were plated in 6 cm 2 dishes (Corning) pre-coated with a gelatin-based coating solution (Cell Biologics) and grown in complete epithelial cell medium (Cell Biologics) in a humidified, 5% CO 2 atmosphere at 37 ºC. Cell medium was replaced every 48 h and cells were tripsinised and split at a 1:3 ratio when confluence was reached following standard cell culture procedures. Excystment of F. hepatica metacercariae F. hepatica metacercariae (Italian strain, Ridgeway Research Ltd) were excysted as previously described [14]. Briefly, metacercariae were incubated for 1 h at 37 ºC in a solution containing CO 2 and 0.02 M sodium dithionite (Sigma), followed by three washes with distilled water and incubation in excystment medium [Hank’s balanced salt solution (Sigma) supplemented with 10% lamb bile (obtained from a local abattoir) and 30 mM HEPES (Sigma) at pH 7.4] at 37 ºC. FhNEJ were manually recovered under a stereomicroscope using a 20 μL pipette every hour after addition of excystment medium, immediately transferred to a clean plate containing complete epithelial cell medium, and incubated in a humidified, 5% CO 2 atmosphere at 37 ºC to induce recovery for 1 h prior to transfer to mPSIEC cultures. In vitro interaction model FhNEJ were co-cultured with mPSIEC as previously described [27,28], with some modifications. At passage 5, mPSIEC were cultured in 6-well plates until confluence was reached. At that point, 200 FhNEJ were added to the corresponding wells in 2 mL of complete epithelial cell medium in the presence or absence of 10 µg/mL of human PLG (Origene). A condition where cells were co-incubated with 200 FhNEJ, PLG (10 µg/mL), and 50 mM of the lysine analogue 6-aminocaproic acid (ε-ACA; Sigma) was included to assess whether the mechanism of FhNEJ-induced plasmin generation is lysine-dependent. Cells left unstimulated, cells stimulated only with 200 FhNEJ, and cells stimulated only with PLG (10 µg/mL) were used as controls. The parasites were separated from the cells 24 h after stimulation, and whole-cell lysates and cell culture supernatants were harvested for downstream analyses. First, 1 mL of each culture supernatant was carefully aspirated and transferred to 1.5 mL tubes, followed by centrifugation for 5 min at 13,000 x g and 4 ºC. The pellets containing cell debris were discarded and clean supernatants were frozen at -80 ºC until use. Whole-cell lysates were obtained by washing the cells in pre-warmed PBS followed by scraping in 200 µL of RIPA buffer (Sigma). Whole-cell lysates were transferred to 1.5 mL tubes, vigorously vortexed for 30 s and centrifuged for 5 min at 13,000 x g and 4 ºC. Pellets containing the cell debris were discarded and supernatants were transferred to clean 1.5 mL tubes and stored at –80 ºC until use. Protein concentrations were determined using the Pierce BCA Protein Assay kit (Thermo Fisher) and ranged between 1,03–1,37 mg/mL protein, depending on the sample. Each experimental condition was conducted in triplicate. Plasmin activity in cell culture supernatants Plasmin activity in cell culture supernatants collected from mPSIEC in the in vitro interaction model was assayed by measuring the amidolytic activity of generated plasmin on a plasmin-specific chromogenic substrate, as previously described [14], with minor modifications. In every well of a transparent, flat-bottom, 96-well microtiter plate, equal volumes of samples were mixed with 1 mM of D-Val-Leu-Lys 4-nitroanilide dihydrochloride chromogenic substrate (S-2251, Sigma) in a total volume of 100 µL of PBS. The microplates were incubated for up to 24 h at 37 ºC and substrate cleavage was assessed by measuring absorbance at 405 nm every hour in a Multiskan GO spectrophotometer (Thermo Fisher). Wells containing 0.02 µM of plasmin (Origene) instead of cell culture supernatant were used to control for substrate specificity. All the reactions were performed in technical triplicate. Analysis of u-PA levels in cell culture supernatants The levels of u-PA in cell culture supernatants (100 µL) were measured by enzyme-linked immunosorbent assay (ELISA) using the Mouse PLAU/Upa (Urokinase-Type Plasminogen Activator) ELISA kit (FineTest) and following the manufacturer’s instructions. The reaction was stopped 11 min after addition of the 3,3′,5,5′-tetramethylbenzidine substrate, and the amount of u-PA in the samples was quantified by fitting a regression line to the linear portion of a standard curve with known u-PA concentrations. All the reactions were performed in technical duplicate. Analysis of total collagen in cell culture supernatants The levels of total collagen in cell culture supernatants (20 µL) were measured by enzymatic digestion of collagen into N-Gly terminal peptides that react with a dye reagent to form a fluorescent complex, using the Collagen Assay Kit (Assay Genie) and following the manufacturer’s instructions. The amount of collagen in the samples was quantified by fitting a regression line to the linear portion of a standard curve with known collagen concentrations. All the reactions were performed in technical duplicate. Zymography The ability of plasmin to degrade the mPSIEC coating solution was measured by preparing 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels using standard protocols but replacing water with the mPSIEC coating solution (Cell Biologics). Next, different amounts of plasmin (Origene) were mixed with non-reducing sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromophenol blue) in a total volume of 18 µL and samples were loaded onto the gel in duplicate. Electrophoresis was performed at constant 15 mA for 20 min followed by 30 mA. After electrophoresis, the gel was soaked twice in wash buffer (5 mM CaCl 2 , 2% Triton-X, pH 7.4) for 30 min at room temperature, followed by overnight incubation at 37 ºC in digestion buffer (5 mM CaCl 2 , 0.1 M glycine, pH 8.4) [30]. All the incubations were performed with mild shaking. After incubation, the gels were stained with Coomassie brilliant blue staining, following standard procedures, and imaged in a Chemidoc MP Imaging System (BioRad). Analysis of MMP levels in cell culture supernatants The levels of MMP-2, MMP-3, MMP-8, pro-MMP-9 and MMP-12 in mPSIEC culture supernatants (25 µL) were analysed on a Luminex xMAP platform using a 5-plex mouse MMP magnetic bead panel (Millipore) and following the manufacturer’s instructions. Multiplex assays were analysed on a Liquichip Luminex 100 Liquid Array Multiplexing System XYP Lab (Luminex Corporation, Qiagen) with Luminex Xponent 3.1 software (Luminex Corporation), following the manufacturer’s technical guidelines. Data analysis was performed with the Milliplex Analyst 5.1 Software (Merck). All the reactions were performed in technical duplicate. The experiment and analysis were conducted at the Flow Cytometry Facility of the National Centre for Biotechnology (Spanish National Research Council; CNB-CSIC, Madrid, Spain). Proteomic analysis of mPSIEC whole-cell lysates Sample preparation. Protein concentrations were determined using the Macherey-Nagel Protein Quantification Assay (Macherey-Nagel) and equal amounts of protein were reduced with 2 mM dithiothreitol at 60 ºC for 20 min, and alkylated with 5 mM iodoacetamide dilutes in 50 mM ammonium bicarbonate at room temperature for 30 min. Next, proteins were purified using the SP3 protocol [31,32] to eliminate detergents prior to in solution protein digestion. Purified proteins were digested overnight at 37 ºC with 100 ng of sequencing-grade trypsin (Promega) diluted in 50 mM ammonium bicarbonate and the reaction was stopped by addition of 0.1% trifluoroacetic acid. LC-MS/MS analysis. Liquid chromatography (LC) was performed by loading 200 ng of digested peptides (diluted in 20 µL of 0.1% formic acid) onto an Evotip Pure tip (EvoSep) following the manufacturer’s instructions, followed by separation using the Evosep One system on an analytical column Performance 15 cm × 150 μm, 1.5 μm (Evosep). The eluted peptides were ionized in a captive Spray with 1700 V at 200 ºC and tandem mass spectrometry analysis (MS/MS) was performed in a TimsTOF fleX mass spectrometer (Bruker) in data-independent acquisition Parallel Accumulation-Serial Fragmentation (diaPASEF) mode. Data processing and protein quantification. The PASER system (Bruker) was used to send the raw data for subsequent analysis and quantification with DIA-NN v1.8 software (https://github.com/vdemichev/DiaNN). First, an in silico -predicted spectral library was built from the Uniprot Mus musculus database using DIA-NN v1.8, using QuantUMS (high precision) as the quantification strategy. After raw data normalization and quantification, proteins were filtered at 1% false discovery rate (FDR) to ensure confidence in protein identification. The proteomic analysis was carried out at the Proteomics Unit of the Central Support Service for Experimental Research (SCSIE) of the University of Valencia (Spain), a member of the Instituto de Salud Carlos III (ISCIII) ProteoRed Proteomics Platform. Statistical analysis. Peak intensities of all the conditions were transformed to the logarithm with base e using the log1p function in R. Next, a stringent statistical pipeline employing three distinct methodologies was applied to identify differentially expressed proteins (DEPs) with high robustness, as previously described [33]. First, the glmnet package in R was employed to fit an elastic-net regularised regression model to select those variables (proteins) that better explain each experimental condition [34] ( Fig. S1 ). To refine the model, the nearZeroVar function from the caret package in R was applied to identify variables with near-zero variance, indicating very low variability across observations. These proteins were discarded from subsequent analysis. The values of the regularisation parameters (alpha and lambda) were optimized with the train function of the caret package through cross-validation re-sampling to obtain the regularised model that best fit our data. The log-transformed expression values of the selected proteins were standardized through Z-score normalization to ensure direct comparability of protein expression levels across samples , followed by representation in heatmaps. Second, feature selection by elastic-net was validated through Partial Least Squares Discriminant Analysis (PLS-DA) using the mixOmics package in R [35] ( Fig. S2 ). This package contains the vip function, which estimates the importance of each variable in the projection. When vip>1.5, the influence of the variable on the response is very high [33] . Third, differential expression analysis between selected pairs of experimental conditions was performed using the limma package in R to get fold-change (FC) differences in protein expression along with the corresponding p-values [36] . Only proteins that were selected by both elastic-net and PLS-DA (vip>1.5) and exhibited a significant fold-change (p-value<0.05), as calculated by limma, were considered differentially expressed . This analysis was performed by the Statistics and Omics Data Analysis Unit of the Central Support Service for Experimental Research (SCSIE) of the University of Valencia, Spain. Analysis of interaction effects. To identify proteins whose differential expression was specifically driven by the interaction of FhNEJ and PLG, rather than by either factor alone, an interaction effect analysis was conducted on the list of DEPs identified using the abovementioned statistical pipeline in the Untreated vs. FhNEJ+PLG comparison. To ensure that only proteins influenced by the interaction between FhNEJ and PLG were retained, proteins that were also differentially expressed in either the Untreated vs. FhNEJ or Untreated vs. PLG comparisons were excluded unless their expression in the combined treatment deviated significantly from the expected additive effect. This approach is conceptually similar to established methodologies in gene interaction studies aimed at detecting synergistic genetic perturbations, where interaction effects are assessed by comparing the observed combined effects to the expected additive effects of individual perturbations [37,38]. To this end, an expected additive FC was computed as the sum of the individual effects of FhNEJ and PLG, based on FC values obtained from the Untreated vs. FhNEJ and Untreated vs. PLG comparisons. An interaction score was then calculated as the difference between the observed FC in the Untreated vs. FhNEJ+PLG comparison and the expected additive FC. Proteins common to all three comparisons were considered to be differentially expressed as a result of the interaction between FhNEJ and PLG only if their absolute interaction score exceeded a threshold. To avoid arbitrary thresholding, interaction scores were filtered using the 1.5 × IQR rule, where IQR is the interquartile range, a standard method for outlier detection based on Tukey’s definition of extreme values [39]. Therefore, the interaction threshold was defined as: median interaction score + (1.5 × IQR); where the median interaction score is the median of all the interaction scores calculated across proteins in the Untreated vs. FhNEJ+PLG condition. This analysis ensured that only proteins exhibiting substantial interaction effects—whether synergistic or antagonistic—were retained for further investigation. In addition to this analysis, a complementary approach was performed to identify proteins whose differential expression in the FhNEJ+PLG condition, when compared to FhNEJ-alone, could not be explained solely by the effect of PLG. In this case, proteins were filtered based on their FC values in the Untreated vs. PLG comparison. A threshold was defined as the median absolute FC ± (1.5 × IQR) in this comparison, capturing the typical range of PLG-induced expression changes. Only proteins in the FhNEJ vs. FhNEJ+PLG comparison with an absolute FC exceeding this threshold were retained. Animal ethics protocol All animals received human care in accordance with the guidelines for the protection of animals used for scientific purposes (Directive 2010/63EU, Decision 2012/707/UE, and RD 53/2013). All procedures were approved by the Ethical Animal Experimentation Committee of the University of Córdoba and by the Junta de Andalucía (project nr. 2021P|/22). In vivo model 20 wild-type, 49–55-day-old female C57BL/6 mice (Charles River Laboratories) were divided in two groups: one group (n=10) was injected intraperitoneally with mouse PLG activator inhibitor 1 (PAI-1) recombinant stable mutant (50 µg per mouse; Innovative Research) to inhibit the fibrinolytic route, as previously described [40]; and the control group (n=10) was injected intraperitoneally with sterile phosphate-buffered saline (PBS). All mice were challenged with F. hepatica metacercariae 30 min after injection, following a protocol recently described by our lab [29]. First, mice were orally infected with 125 F. hepatica metacercariae as follows: metacercariae were resuspended in a total volume of 50 µL of PBS and oral infection was performed using a sterile 20–200 µL pipette tip trimmed 0.5 cm and pre-washed with PBS containing 0.01% Triton X-100 to prevent the metacercariae from sticking to the plastic tip. Second, 8 days after challenge with F. hepatica metacercariae, mice were euthanised after 24 h fasting through CO 2 overdose followed by cervical dislocation. Prior to dissection, 2 mL of sterile PBS were injected into the peritoneal cavity, taking care to avoid puncturing any organs. Peritoneal fluid was then collected by making a small abdominal incision, followed by careful aspiration with a pipette tip. The liquid was transferred to 1.5 mL tubes and immediately frozen at –80 ºC. For the recovery of juvenile worms from the hepatic parenchyma, livers were carefully dissected with tweezers under a stereomicroscope and the number of juvenile parasites was manually counted by independent, trained, and experimental group-blinded researchers that were unfamiliar with the objective of the experiment. The experiment was conducted over two consecutive days, using 5 mice per group per day, with the same reagents and personnel. Animal handling was performed by qualified staff at the Experimental Animal Facility of the University of Córdoba (Spain). Mice were housed in groups of 4 per cage, with aspen wood bedding, and provided food and water ad libitum on a standard dry pellet diet for rodents. The experiment was conducted after 1 week of acclimatization at 22 ± 3 ºC, 50-60% relative humidity, and a 12 h light/dark cycle. PAI-1 and t-PA activity assays The activities of PAI-1 and t-PA in peritoneal fluids of mice were measured using the commercially available Mouse active PAI1 ELISA Kit (Innovative Research) and Mouse Active tPA ELISA Kit (Innovative Research), following the manufacturer’s instructions. The amounts of active PAI-1 and t-PA in the samples were quantified by fitting a regression line to the linear portion of a standard curve with known active PAI-1/t-PA concentrations. All the reactions were performed in technical duplicate. Statistical analysis Plots were created, and statistical analyses were performed using Prism 10 software (GraphPad Software). Unless otherwise stated, graph bars represent the mean of three cell culture replicates, each calculated as the average of two or three technical replicates. Error bars represent either the standard error of the mean (SEM) or the standard deviation (SD) to reflect the precision of the sample mean as an estimate of the population mean or the variability among individual technical measurements, respectively. The data from the in vivo experiment was analysed using a two-way analysis of variance (ANOVA), considering both treatment groups and the day on which the experiment was conducted as factors. Based on the result of this analysis, the data was plotted separately for each day and presented as mean ± SEM. Since normality in data distribution cannot be confidently determined with small sample sizes [41], non-parametric methods were chosen to determine statistically significant differences between experimental conditions across biological replicates. Specifically, the Mann-Whitney U test was used to assess differences between two experimental conditions; and comparisons between more than two experimental conditions were performed via Kruskal-Wallis test followed by Dunn’s post hoc analysis of pairwise comparisons. Unless otherwise stated, the threshold for significance was set at α ≤ 0.1. Results FhNEJ stimulate plasmin generation in the pericellular space of mouse intestinal epithelial cells In order to study whether FhNEJ stimulate plasmin generation from host PLG in an in vitro system of host-parasite interactions [27,28], mPSIEC were co-incubated for 24 hours with FhNEJ in the presence of host PLG and without the exogenous addition of PLG activators. A condition where cells were co-incubated with FhNEJ, PLG, and the lysine analogue ε-ACA was included to assess whether FhNEJ-induced plasmin generation is mediated by lysine residues present in FhNEJ-derived PLG-interacting proteins, as previously described [14]. Plasmin generation in cell culture supernatants of mPSIEC was assessed using a plasmin-specific chromogenic substrate, which revealed that cells incubated with PLG alone showed a small degree of plasmin generation that was increased in the presence of FhNEJ. This effect was observed across three independent cell culture replicates and was completely abrogated in the presence of ε-ACA, indicating that the observed increased plasmin generation is mediated by lysine residues (Fig. 1 ). FhNEJ increase u-PA levels in mPSIEC culture supernatants and promote collagen degradation in a PLG-dependent manner We next addressed how mPSIEC respond to the presence of FhNEJ in terms of u-PA release and collagen degradation, and whether these responses would be modulated by the interaction between FhNEJ and PLG. The levels of u-PA in cell culture supernatants of mPSIEC left unstimulated, stimulated with FhNEJ and PLG, or simulated with either component alone were assessed through ELISA, and total collagen in these samples was quantified by measuring the amount of N-Gly terminal peptides generated upon treatment of the samples with a collagenolytic enzyme. These experiments revealed that, in contrast to cell culture supernatants from mPSIEC single-treated with either FhNEJ or PLG, cell culture supernatants from mPSIEC stimulated with both FhNEJ and PLG exhibited a statistically significant increase in u-PA levels (Fig. 2 A) and a decrease in collagen (Fig. 2 B, S3A) compared to supernatants from untreated mPSIEC. In line with this, standard zymography performed with increasing amounts of recombinant plasmin revealed that this protease degrades the ECM coating solution used to culture mPSIEC ( Fig. S3 B ). Altogether, these results show that the stimulation of mPSIEC with both FhNEJ and PLG results in increased u-PA release and ECM degradation in this in vitro cell system. Regulation of MMP levels in mPSIEC culture supernatants by FhNEJ We went on to evaluate whether FhNEJ influence the secretion of different ECM-degrading MMPs by mPSIEC, and whether this would be influenced by PLG. To that end, the levels of MMP-2, -3, -8, -12, and pro-MMP-9 were analysed through multiplexed assays in cell culture supernatants of mPSIEC left unstimulated, stimulated with FhNEJ and PLG, or stimulated with either component alone (Fig. 3 ). This experiment revealed that MMP-2 and pro-MMP-9 are the MMPs showing greater concentrations in the cell culture supernatants of mPSIEC. Specifically, the levels of MMP-2 were similar across the different experimental conditions, whereas those of pro-MMP-9 were only detectable in the conditions incubated with FhNEJ (FhNEJ-only and FhNEJ + PLG). Similar results were obtained for MMP-3, -8, and − 12, despite their levels in mPSIEC culture supernatants being much lower than those observed for MMP-2 and pro-MMP-9. Overall, although some experimental conditions could not be quantified due to values falling below the detection limits of the technique, these results indicate that MMP levels in cell culture supernatants substantially increase upon incubation of mPSIEC with FhNEJ, regardless of PLG availability. Proteomic profiling reveals mPSIEC responses driven by the interaction between FhNEJ and host PLG To characterise in greater detail how mPSIEC respond to FhNEJ and whether these responses are modulated by their interaction with host PLG, a proteomic analysis was conducted in whole-cell lysates from mPSIEC left unstimulated, stimulated with FhNEJ and PLG, or stimulated with either component alone. This analysis identified a total of 8136 proteins with FDR > 1% that were included in the differential expression analysis. We compared the protein expression profiles of untreated cells to cells stimulated with both FhNEJ and PLG to identify proteins that are differentially regulated by the interaction between both factors, which results in increased plasmin activity in cell culture supernatants (Fig. 1 ). In order to keep only proteins regulated by the combination of FhNEJ and PLG, and not by either component alone, DEPs identified in both the untreated vs. FhNEJ and the untreated vs. PLG comparisons that overlapped with DEPs identified in the untreated vs. FhNEJ + PLG comparison were not considered as differentially regulated by FhNEJ + PLG unless their FC values showed an interaction effect. As a result of this interaction analysis, we identified a total of 84 DEPs in mPSIEC treated with FhNEJ and PLG that are specifically attributable to the combined effect of both components (Table 1 ). Table 1 DEPs in untreated vs. FhNEJ + PLG-treated mPSIEC. A negative fold-change indicates that proteins were overexpressed in FhNEJ + PLG-treated vs. untreated mPSIEC. The opposite is true when the fold-change is positive. Genes logFC adjPVal Genes logFC adjPVal Cap1 -11.5641 1.16E-08 Mrgbp 0.315304 0.041192 Eif1ad11 -10.8284 0.044884 Sgo2 0.321909 0.044884 Zfp606 -9.98762 1.16E-08 Tmem184b 0.337123 0.042419 Serpinb5 -9.09 1.29E-05 Cdca2 0.337796 0.044267 A130006I12Rik -8.8194 6.9E-06 Aurka 0.337847 0.037707 S1pr2 -8.79117 6.89E-05 Glul 0.353169 0.029492 Pptc7 -7.5837 3.26E-07 Blm 0.365228 0.032615 Tmem14c -0.70435 0.044884 Actn4 0.36718 0.046035 Rab3b -0.67455 0.031743 Rab38 0.367389 0.026216 Mmp11 -0.67003 0.046035 Aurkb 0.370126 0.047626 Dennd3 -0.4902 0.004343 Sash1 0.377697 0.044267 Desi2 -0.44875 0.044884 Rpl35 0.398069 0.012174 Dhx38 -0.44049 0.035042 Acsl4 0.398548 0.007476 Srsf2 -0.43185 0.041835 Cox6a1 0.405347 0.028322 Gorasp1 -0.38835 0.011505 Esco2 0.419492 0.018064 Ccn2 -0.37626 0.046035 0610010K14Rik 0.476796 0.044884 Atrx -0.32326 0.04661 Arhgap20 0.48969 0.044267 Slc25a30 -0.31699 0.046035 Cmip 0.490543 0.046035 P4ha2 -0.26881 0.044267 Aven 0.490844 0.041192 Slc12a7 -0.2512 0.026216 Gmnn 0.495586 0.047626 Scel -0.24909 0.044267 Cdc34 0.522914 0.040503 Gpam -0.23204 0.046035 Suco 0.539981 0.018465 Sppl2a 0.191843 0.042419 Cetn2 0.553036 0.040283 Ska3 0.199186 0.0426 Nifk 0.619508 0.046035 Lama5 0.223652 0.040283 Fnip1 0.677284 0.006195 Slc9a3r2 0.224492 0.046404 Lrig1 0.745655 0.00283 Dvl2 0.239475 0.046035 Sema3a 0.831001 0.031743 Cdv3 0.244815 0.035681 Serpind1 0.858629 0.032615 Notch2 0.247477 0.022438 Fn1 0.888985 0.031603 Lamb1 0.250854 0.018889 Zfhx4 1.130508 0.010738 Prkd2 0.254831 0.048562 Thbs1 1.239887 0.035681 Diaph3 0.255139 0.018889 Serpinb7 1.278409 0.003469 Rnf4 0.25624 0.044267 A2m 1.423682 0.016865 Prr11 0.265523 0.04991 Itih4 1.425836 0.026216 Spdl1 0.271143 0.031743 Tmc5 1.469909 0.002405 Melk 0.27314 0.048562 C7 1.471351 0.003517 Calu 0.28254 0.022262 Dnah14 1.608509 0.026216 Fam50a 0.286497 0.011574 Mettl25 7.646163 4.28E-05 Mphosph10 0.292922 0.028712 Pigr 8.709757 4.38E-06 Msmo1 0.302965 0.031743 Sema3d 10.34484 3.39E-07 Ror1 0.311029 0.021179 Lamtor3-ps 12.05803 0.00283 Tns2 0.313294 0.017259 Alb 12.61229 2.51E-07 In addition to proteins shown in Table 1 , we also considered as differentially regulated as a result of the interaction between FhNEJ and PLG those DEPs identified by comparing mPSIEC stimulated with FhNEJ + PLG to cells stimulated with only FhNEJ. Following a similar rationale as described above, we excluded proteins regulated by PLG alone by discarding those DEPs in the FhNEJ vs. FhNEJ + PLG comparison that were common to those identified in the Untreated vs. PLG comparison unless their fold change under the FhNEJ + PLG condition exceeded the typical range of PLG-induced expression changes. By doing so, we identified a total of 15 proteins that were differentially regulated in mPSIEC as a result of the stimulation with both FhNEJ and PLG (Table 2 ). Notably, this analysis identified PLG (Plg) as significantly upregulated in FhNEJ + PLG-treated cells compared to cells treated with FhNEJ alone, highlighting the accuracy of the approach. By combining both analyses, we identified 94 unique DEPs in double-treated mPSIEC, which are involved in cell adhesion and migration, ECM remodelling, mucosal immunity, and fibrinolysis. These included cyclase-associated protein 1 (Cap1), serpin b5, the Ras-related protein Rab3b, actinin-4 (Actn4), MMP-11 (Mmp11), the tissue inhibitor of metalloproteinase 3 (Timp3), fibronectin 1 (Fn1), quiescin sulfhydryl oxidase 1 (Qsox1), semaphorin 3D (Sema3d), the polymeric immunoglobulin receptor (Pigr), carbohydrate sulfotransferase 2 (Chst2), the complement molecule C7, the sphingosine 1-phosphate receptor 2 (S1pr2), alpha-2 macroglobulin (A2m), and carboxipeptidase 2 (Cpb2), also known as the thrombin activatable fibrinolysis inhibitor (TAFI). Table 2 DEPs in FhNEJ-treated vs. FhNEJ + PLG-treated mPSIEC. A negative fold-change indicates that proteins were overexpressed in FhNEJ + PLG-treated vs. FhNEJ-treated mPSIEC. The opposite is true when the fold-change is positive. Genes logFC adjPVal Cap1 -11.5928 2.45E-08 Cpb2 -8.97008 9.76E-07 Ube2l6 -8.30553 9.76E-07 Qsox1 -9.00044 9.76E-07 Sema3d 10.18779 9.76E-07 Klhl30 10.2297 4.84E-06 Chst2 7.69985 5.86E-06 Timp3 -0.59491 0.004089 Rab3b -0.50349 0.010792 Plg -1.34697 0.014915 Dennd3 -0.42075 0.022473 Phip -0.55656 0.025486 Ccn1 -0.45733 0.043756 Scel -0.36049 0.043756 Slco2a1 -0.33878 0.044832 The contribution of the fibrinolytic system to FhNEJ migration in a mouse model of fasciolosis Finally, in an effort to understand the contribution of the host fibrinolytic system in the migration of F. hepatica juveniles during the earliest stages of infection in a physiologically-relevant experimental system, we employed a mouse model of acute fasciolosis [29] to quantify the amount of juvenile flukes that successfully migrate to the livers of control mice and mice whose fibrinolytic route had been pharmacologically inhibited prior to challenge with F. hepatica metacercariae (Fig. 4 A). Due to technical constraints, the experiment was performed across two consecutive days, employing five mice per group per day. Statistical analysis using two-way ANOVA revealed that the day of the experiment gave statistically significant differences in FhNEJ counts ( Table S2 ), as PAI-1-treated mice from day 1—but not control mice—exhibited significantly different amounts of liver-migrating flukes than their counterparts from day 2 ( Fig. S4 ). Therefore, PAI-1-treated mice could not be treated as absolute replicates and data was subsequently plotted and analysed separately for each day. This analysis revealed that PAI-1 treatment prior to F. hepatica infection resulted in a significant decrease in the number of juvenile flukes recovered from the livers of infected mice eight days after challenge with F. hepatica metacercariae, but only in the experiment conducted on day 1 (Fig. 4 B). To assess potential differences in PAI-1 treatment efficacy across different experimental days, we measured the levels of fibrinolysis in the peritoneal fluids of all mice, which were collected immediately after euthanasia. However, no statistically significant differences were observed in either PAI-1 or t-PA activities between control and PAI-1-treated mice on any experimental day ( Fig. S5 ). Discussion Aiming to understand the contribution of the fibrinolytic system in biological processes that might facilitate FhNEJ migration, commercially-available primary intestinal epithelial cells derived from the mouse small intestine (mPSIEC) were stimulated with FhNEJ [27,28] and PLG for 24 hours, after which cell culture supernatants and mPSIEC whole-cell lysates were harvested for downstream analyses. Additionally, mPSIEC left unstimulated or stimulated with either FhNEJ or PLG alone served as negative controls, and an additional subset of mPSIEC stimulated with FhNEJ, PLG, and the lysine analogue ε-ACA was used to assess whether plasmin generation depends on the presence of lysine residues in FhNEJ-derived PLG-interacting proteins, as previously described [14]. Although FhNEJ take only a few hours to cross the intestinal wall [19], we extended the stimulation period to up to 24 hours, as previously described [28], to account for potential delays in cellular responses due to the artificial and reductionist nature of the co-culture system. First, our enzymatic assays revealed that cell culture supernatants from mPSIEC stimulated with both FhNEJ and PLG showed increased plasmin activity compared to those from control or single-treated cells, indicating that FhNEJ stimulate plasmin generation in the pericellular space of the mouse small intestine in the presence of host PLG. Since FhNEJ cannot directly cleave and activate PLG [14], and mPSIEC were stimulated in the absence of exogenous addition of either t-PA or u-PA, these results suggest that mPSIEC express the PLG activator(s) required to convert PLG into plasmin, which might have important physiological and pathological implications. Notably, FhNEJ-induced plasmin generation was completely abrogated in the presence of ε-ACA, indicating that this phenomenon depends on the interaction between lysine residues present in PLG-interacting proteins expressed by FhNEJ and the PLG kringle domains, replicating the mechanism of PLG activation utilised by physiologic PLG-Rs. Since u-PA is expressed by epithelial cells of the small intestine, presumably to loosen intercellular junctions and facilitate epithelial cell turnover at the top of the villi [42], we studied whether the combination of FhNEJ and PLG would increase the availability of this PLG activator in the mPSIEC extracellular space. Results revealed that cell culture supernatants of mPSIEC stimulated with both FhNEJ and PLG contained significantly more u-PA than untreated or single-treated cells, suggesting that plasmin generation in the pericellular space may initiate a positive feedback loop that enhances u-PA release by double-treated cells. These findings indicate that plasmin may enhance fibrinolysis within the small intestinal micro-environment through two complementary mechanisms: the well-established ability of this protease to cleave and activate the zymogen form of u-PA [43,44] and the stimulation of u-PA secretion by intestinal cells, as demonstrated in the present study. Plasmin is considered a master regulator of cell migration through the degradation of different ECM components [5,6], including collagen type IV and laminin [11,45], two major structural proteins of the basement membrane underlying the intestinal epithelium [25]. To determine whether FhNEJ-mediated plasmin generation enhances the degradation of intestinal ECM components, we quantified the levels of total collagen in cell culture supernatants of mPSIEC incubated with FhNEJ, PLG, or both. Decreased collagen levels in cell culture supernatants was interpreted as increased degradation of this ECM protein, as previously described [46]. Our results revealed that collagen levels slightly decrease in mPSIEC supernatants treated with either FhNEJ or PLG, but the reduction becomes significant only when both are combined, indicating that FhNEJ-induced plasmin generation promotes collagen degradation. This notion is supported by the observation that plasmin is capable of degrading the ECM coating solution employed for mPSIEC culture. Although the interaction between helminth parasites and the host fibrinolytic system is well-established and has long been proposed to facilitate tissue migration through enhanced ECM degradation [1–3], to our knowledge, this is the first experimental demonstration of increased ECM degradation resulting from parasite-induced plasmin generation in a trematode, as this phenomenon had only been shown in the nematode Dirofilaria immitis [46]. Considering the potent collagenolytic properties of FhCL3 [47,48], which is highly expressed and secreted by FhNEJ [49], the residual, non-statistically significant collagen degradation that we detected in cell culture supernatants from mPSIEC only treated with FhNEJ is somewhat unexpected. However, this could be explained by the fact that the commercial kit that we used in our experiments fails to detect the specific collagen isoforms that are degraded by FhCL3, that such isoforms are not present in the ECM coating employed here to culture mPSIEC, or that the extent of collagen degradation by FhNEJ under physiologic conditions is less pronounced than that observed in in vitro experiments performed in the absence of host cells [47,48]. In addition to directly degrading ECM components, plasmin activates the precursor zymogens of MMP-1, -2, -3, -9, -10, -12, and − 13, either via direct cleavage or by activating other MMPs that, in turn, cleave and activate the MMP precursor [50–57]. MMPs are highly proficient at degrading ECM components [12], and given their close relationship with the fibrinolytic system [5], we addressed whether FhNEJ-mediated plasmin generation would simulate MMP release by mPSIEC [58], which could facilitate FhNEJ trans-intestinal migration by expanding the arsenal of enzymes that are available in the extracellular space for ECM degradation. Our results showed that FhNEJ substantially stimulate the secretion of MMP-3, -8, -12, and pro-MMP-9 by mPSIEC, a response that was not further stimulated in the presence of PLG, indicating that FhNEJ-induced plasmin generation does not affect the secretion of these enzymes by host cells. Since MMP-like enzymes in the FhNEJ secretome constitute only 0.04% of the total protein content [59], we believe that their contribution to the measured MMP levels in cell culture supernatants is likely negligible. Regardless of MMP secretion, it remains to be addressed whether FhNEJ-induced plasmin generation in the intestinal epithelium facilitates the activation of MMP precursors that could contribute to the observed collagen degradation and ultimately facilitate the migration of these parasites across the host intestine. Since plasmin influences biological processes not only by regulating the activity of proteolytic enzymes but also by modulating intracellular signalling pathways that culminate in changes in gene expression [60–63], we conducted a proteomic analysis on whole-cell lysates of mPSIEC treated with both FhNEJ and PLG to investigate how these cells respond to elevated plasmin levels in the pericellular space. To this end, we applied a stringent statistical pipeline [33] combined with an analysis of interaction effects to identify proteins with statistically different abundances in FhNEJ + PLG-treated mPSIEC compared to untreated cells that are specific to the combination of FhNEJ and PLG. This analysis included elastic-net penalised regression, which has emerged as a powerful and validated approach for robust feature selection, enabling the reliable identification of condition-specific gene and protein expression signatures in diverse biomedical fields [64–67]. Additionally, differential expression was performed with the limma package, which applies a combination of statistical principles that outperform ordinary t -tests in proteomics data analysis when sample sizes are small [36,68]. In parallel to this, we also compared the proteomes of FhNEJ-treated and FhNEJ + PLG-treated mPSIEC to reduce the overall variability and enhance the detection of subtle but biologically meaningful proteomic changes specifically induced by the combined action of FhNEJ and PLG that may be overlooked in the broader untreated vs. FhNEJ + PLG-treated contrast. Supporting the accuracy of this approach, only the comparison between FhNEJ-treated and FhNEJ + PLG-treated cells captured PLG as a significantly abundant protein in whole-cell lysates of double-treated mPSIEC (Table 2 ). Given that mPSIEC were thoroughly washed before harvesting, we assume that the amount of residual PLG in these samples was negligible, reinforcing that comparing the protein expression profiles of FhNEJ-treated and FhNEJ + PLG-treated cells effectively fine-tunes the analysis to detect subtle proteomic shifts that are undetectable in the untreated vs. FhNEJ + PLG differential expression analysis. Some of the DEPs resulting from the interaction between FhNEJ and PLG are involved in biological processes that could be beneficial to FhNEJ trans-intestinal migration and survival within the mammalian organism, including cell adhesion and migration, cytoskeleton dynamics, ECM remodelling, mucosal immunity, and fibrinolysis. For instance, we identified Cap1, serpin b5, Rab3b, and Actn4 as differentially regulated upon mPSIEC stimulation with both FhNEJ and PLG. Cap1 is involved in cell adhesion and motility through the regulation of actin cytoskeleton dynamics [69], serpin b5 is a non-inhibitory serpin that regulates u-PA/u-PAR-mediated cell adhesion [70], and Rab3b is required for the distribution of the major tight junction (TJ) adaptor protein zonula occludens 1 to the apical membrane of epithelial cells [71,72]. Similarly, depletion of Actn4 inhibits the recruitment of occludin to TJs, thereby disrupting the formation of functional intercellular junctions [73]. Differential expression of these proteins in mPSIEC stimulated with both FhNEJ and PLG indicates that plasmin might regulate parasite adhesion to the intestinal epithelium and modulate epithelial junction dynamics, potentially compromising epithelial barrier integrity and facilitating FhNEJ migration across the host intestine. Additionally, some of the DEPs identified in mPSIEC incubated with both FhNEJ and PLG play important roles in ECM remodelling. Among others, we found differential expression of Mmp11 and Timp3 in double-treated cells, suggesting that FhNEJ-induced plasmin generation alters the protease/anti-protease balance of the MMP system to ultimately regulate ECM degradation. Overexpression of Mmp-11 is particularly compelling as this metalloproteinase degrades collagen type IV [74], a major structural component of the intestinal basement membrane [25]. In the context of ECM remodelling, it is noteworthy that we also detected downregulation of the ECM protein fibronectin in whole-cell lysates of mPSIEC incubated with both FhNEJ and PLG. Decreased Fn1 expression in double-treated mPSIEC aligns with Mmp-11 overexpression in these cells, as MMP-11 was shown to supress fibronectin gene expression in an in vivo zebrafish model of ECM remodelling [75]. Moreover, the overexpression of Qsox1, which is required for the incorporation of laminin into the ECM [76], provides further evidence that FhNEJ regulate tissue integrity through the modulation of ECM dynamics/deposition in a plasmin(ogen)-dependent manner. Another notable functional aspect of the interaction between FhNEJ and PLG, highlighted by our proteomic analysis, is the identification of proteins linked to host immune responses and, potentially, to FhNEJ immune evasion mechanisms. Among others, we found downregulation of Sema3d, Pigr, Chst2, and the complement molecule C7 in mPSIEC treated with both FhNEJ and PLG. Sema3d has been recently shown to promote pancreatic cancer progression by stimulating macrophage polarization into a protumourigenic M2 phenotype [77]. Since M2 polarization is a hallmark of anti- Fasciola immune responses [78], this finding offers novel insights into how these responses might be regulated at the intestinal level during infection with FhNEJ. In the context of FhNEJ trans-intestinal migration, the downregulation of Pigr in mPSIEC upon stimulation with FhNEJ and PLG is particularly noteworthy, as this protein is a key regulator of mucosal immunity by facilitating the translocation of immunoglobulin A (IgA) to the apical compartment of the intestinal epithelium [79]. In fact, mice lacking Pigr expression show increased bacterial infiltration into the liver due to impaired IgA-mediated antimicrobial defence in the gut [80]. In line with this, the downregulation of Chst2 upon stimulation with FhNEJ and PLG may also represent a mechanism to evade mucosal immunity, as this protein is involved in lymphocyte homing to peripheral lymphoid organs and presumably to inflamed sites by modulating the synthesis of carbohydrate ligands that interact with L-selectin molecules expressed on the lymphocyte surface [81,82]. The complement molecule C7 is essential for the assembly of the lethal, pore-forming membrane attack complex, a key effector in antibacterial defence. Previous studies had shown that F. hepatica expresses molecules that inhibit complement proteases, thereby evading complement-mediated responses [83]. To the best of our knowledge, the present study provides the first evidence that FhNEJ modulate the expression of C7 in mouse intestinal cells, an effect observed only upon co-stimulation with both FhNEJ and PLG. Intriguingly, we also observed a downregulation of the complement protein C3 in FhNEJ-treated mPSIEC compared to untreated controls ( Table S1 ), suggesting that FhNEJ modulate the expression of complement molecules through plasmin(ogen)-dependent and independent mechanisms. The S1pr2 is another immune-related protein that we identified as differentially regulated in double-treated mPSIEC. This protein regulates the onset of type 2 immune responses in the lung by inhibiting the secretion of interleukin 33 (IL-33) by lung macrophages [84]. IL-33 is also secreted by intestinal epithelial cells and has recently been shown to represent an indispensable trigger of intestinal type 2 immune responses that lead to successful gastrointestinal nematode expulsion [85]. Based on this, the observed S1pr2 overexpression in mPSIEC upon stimulation with both FhNEJ and PLG may serve to reduce IL-33 release by intestinal epithelial cells and suppress the onset of anti-helminth immunity in the intestine, providing an intriguing direction for future investigation. Moreover, our proteomic analysis indicated that plasmin itself might also be regulating fibrinolysis in the small intestine tissue microenvironment, as double-treated mPSIEC differentially regulate the expression of two major fibrinolysis inhibitors: A2m and TAFI, which were downregulated and overexpressed, respectively, in mPSIEC upon stimulation with FhNEJ and PLG. This finding suggests that fine-tuning fibrinolysis may benefit the parasite by balancing proteolytic activity such that it facilitates tissue penetration while preventing excessive ECM degradation, which could inadvertently lead to severe tissue damage and exacerbated inflammatory responses in the intestine. Finally, we employed a mouse model of acute fasciolosis [29] to conclusively determine whether the fibrinolytic system is involved in the intra-mammalian migration of F. hepatica juveniles during the early infection. To that end, mice were injected with either PBS or PAI-1 to inhibit the fibrinolytic route prior to infection with F. hepatica metacercariae. We utilised a recombinant and stable-mutant version of PAI-1, as previously described [40], because the wild-type inhibitor has a very short half-life of approximately two hours [86]. Eight days post-infection was selected as the time point for quantifying infection success based on previous studies indicating that juvenile recovery from the livers of infected mice is highest at this time [29]. Results showed that the number of liver-migrating flukes in PAI-1-treated mice was significantly lower than that observed in control mice, but only in the experiment conducted on day 1. Since differences in juvenile counts across experimental days were observed only in PAI-1-treated mice, but not in their control counterparts, we suspected that variations in PAI-1 treatment efficacy between days might explain these results. To investigate this possibility, fibrinolysis was evaluated by measuring the levels of active PAI-1 and t-PA in peritoneal fluids collected immediately after euthanasia. Although the differences in active PAI-1 and t-PA levels between PAI-1-treated and control mice on day 1 were more consistent with the expected inhibition of fibrinolysis than those observed in mice from day 2, these differences were small and did not reach statistical significance. This is likely due to the fact that peritoneal fluids were collected eight days after PAI-1 treatment, exceeding the maximum half-life of six days achieved for this factor through random mutagenesis [86]. Altogether, these results highlight the complexity of working with genetically diverse organisms such as F. hepatica , which likely constitutes one of several factors contributing to the inconsistent and variable outcomes reported in vaccine trials over the past decades [87,88]. This variability not only hinders data interpretation but also raises ethical concerns regarding the use of large animal cohorts to overcome inter-individual heterogeneity and meet significance thresholds that are inherently arbitrary. Notwithstanding these points, these findings provide unprecedented insights into the role of the fibrinolytic system in FhNEJ migration in vivo , supporting the long-standing hypothesis that FhNEJ-stimulated plasmin generation from host PLG contributes to the dissemination of F. hepatica juveniles through mammalian tissues. Conclusions In summary, this study demonstrates that plasmin is generated in the pericellular space of mouse intestinal epithelial cells in the presence of FhNEJ and host-derived PLG. This process enhances collagen degradation and u-PA release into the extracellular environment, potentially facilitating FhNEJ migration across the intestinal wall. Additionally, FhNEJ-induced plasmin generation triggers changes in the expression levels or abundance of proteins involved in biological processes that could be critical during the earliest stages of infection, including cell adhesion and migration, ECM remodelling, immune evasion, and fibrinolysis. Finally, despite the inherent challenges associated to in vivo models of F. hepatica infection, our findings provide the first functional evidence supporting the long-proposed role of the fibrinolytic system in facilitating the migration of F. hepatica juveniles through mammalian tissues. Collectively, these results underscore the contribution of the fibrinolytic system in parasite dissemination during early-stage fasciolosis, providing interesting insights into host-parasite relationships established in the initial stages of infection and highlighting potential avenues for future research in this area. Abbreviations Actn4: actinin-4; ANOVA: analysis of variance; A2m: alpha-2 macroglobulin; Cbp2: carboxipeptidase; Chst2: carbohydrate sulfotransferase 2; C7: complement molecule 7; DEP: differentially expressed protein; ECM: extracellular matrix; ELISA: enzyme-linked immunosorbent assay; FC: fold change; FDR: false discovery rate; FhNEJ: F. hepatica newly excysted juvenile; Fn1: fibronectin 1; IgA: immunoglobulin A; IL-33: interleukin 33; IQR: interquartile range; MMPs: matrix metalloproteinases; MMP11: matrix metalloproteinase 11; mPSIEC: mouse primary small intestinal epithelial cells; MS/MS: tandem mass spectrometry; PBS: phosphate-buffered saline; Pigr: polymeric immunoglobulin receptor; PLG: plasminogen; PLG-R: plasminogen receptor; PLS-DA: partial least squares discriminant analysis; pro-u-PA: precursor of the urokinase-type plasminogen activator; Qsox1: quiescin sulfhydryl oxidase 1; Sema3d: semaphorin 3D; SDS: sodium dodecyl sulphate; SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SD: standard deviation; SEM: standard error of the mean; S1pr2: sphingosine 1-phosphate receptor 2; u-PA: urokinase-type plasminogen activator; TAFI: thrombin activatable fibrinolysis inhibitor; Timp3: tissue inhibitor of metalloproteinase 3; TJ: tight junction; ε-ACA: 6-aminocaproic acid. Declarations Author Contribution JS planned and carried out all the experiments, analysed the data, and wrote the main manuscript text. MT provided technical assistance with the experiments. MLG, VMH, and MTRC contributed to the conception and implementation of the in vivo mouse model. MSL interpreted the results and was involved in their planning. JGM conceived the study, secured funding, planned the experiments, and contributed to their analysis and interpretation. All authors participated in revising the manuscript and have approved the submitted version. Acknowledgement JGM acknowledges funding received from projects ULYSSES (RTI2018-093463-J-100) and PERSEUS (PID2023-152150OB-C21) funded by MICIU/AEI/10.13039/501100011033 and FEDER, EU, and URANUS (CNS2022-135561) funded by MICIU/AEI/10.13039/501100011033 and NextGenerationEU/PRTR. JS acknowledges the support of the Junta de Castilla y León for her Predoctoral contract. MLG acknowledges the support of the Spanish Ministry of Science and Innovation for her FPU Predoctoral contract. We thank Miguel Ángel Sánchez Luengo and Teresa Osteso Ibáñez (CNB-CSIC, Madrid, Spain) for their support with the multiplexed experiment, Raquel Gavidia (SCSIE, Valencia, Spain) for the statistical analysis of proteomics data, and Laura Barrios (Department of Applied Statistics, CSIC, Madrid, Spain) for her valuable advice on how to analyse the in vivo data. Data Availability Data is provided within the manuscript or supplementary information files. References Diosdado A, Simón F, Serrat J, González-Miguel J. Interaction of helminth parasites with the haemostatic system of their vertebrate hosts: a scoping review. Parasite. 2022;29:35. González-Miguel J, Siles-Lucas M, Kartashev V, Morchón R, Simón F. Plasmin in parasitic chronic infections: friend or foe? Trends Parasitol. 2016 Apr;32(4):325–35. Figuera L, Gómez-Arreaza A, Avilán L. 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Feature selection by elastic-net penalized regression improves sample clustering. An elastic-net penalised regression model was performed to select for proteins that are specifically expressed in each experimental condition. The figures show heatmap representations of protein expression levels (normalized to Z-score) of all the proteins identified (left panels) or proteins selected as specific to each experimental condition after feature selection through elastic-net regularised regression (right panels), for selected, biologically-relevant pairwise comparisons: untreated mPSIEC vs. mPSIEC treated with FhNEJ (A); untreated mPSIEC vs. mPSIEC treated with both FhNEJ and PLG (B); and FhNEJ-treated mPSIEC vs. mPSIEC treated with both FhNEJ and PLG (C). Heatmaps created with R software. FigureS2.pdf Additional File 2: Figure S2. PLS-DA analysis of selected pairwise comparisons. PLS-DA was performed to identify the proteins most relevant for distinguishing between experimental conditions. Only those elastic-net pre-selected proteins that contributed substantially to the newly projected bidimensional space (as determined by vip>1.5) were considered as specific to each experimental condition. Plots are shown for selected, biologically-relevant pairwise comparisons: untreated mPSIEC vs. FhNEJ-treated mPSIEC (top left); untreated mPSIEC vs. mPSIEC treated with both FhNEJ and PLG (top right); and FhNEJ-treated mPSIEC vs. mPSIEC treated with both FhNEJ and PLG (bottom). Plots created with R software. FigureS3.pdf Additional File 3: Figure S3. A) The effect of plasmin on the detection of total collagen was determined using the same commercial kit as in Figure 2B by pre-incubating total collagen with 2 µM plasmin for 24 hours at 37 ºC followed by enzymatic digestion and detection of N-Gly terminal peptides, as specified by the manufacturer’s instructions. Total collagen left undigested (“Undigested”) or digested without plasmin pre-incubation (“+ Enzyme”) served as controls. Bars indicate the mean of three technical replicates ± SD (***p≤0.001, ****p≤0.0001; one-way ANOVA followed by Tukey post hoc analysis of pairwise comparisons). RFU, relative fluorescent units (excitation 380 nm, emission 460 nm). B) Zymography showing plasmin-mediated degradation of the ECM coating used for mPSIEC culture. Different amounts of plasmin (2 µM, 0.5 µM, and 0.1 µM) were loaded onto 10% SDS-PAGE gels prepared using mPSIEC coating solution instead of water. Light bands (arrowhead) corresponding to degradation of the mPSIEC coating solution by plasmin were revealed by Coomassie brilliant blue staining. FigureS4.pdf Additional File 4: Figure S4. Fluke recovery in control and PAI-1-treated mice across experimental days. The number of flukes recovered from control (A) and PAI-1-treated (B) mice was plotted separately to assess possible variation in intra-group responses across days . Data points represent the number of juveniles recovered in the liver of each mouse, and bars indicate the mean ± SEM. Asterisks indicate significant differences between experimental conditions (ns, not significant; * *p≤0.1; Mann-Whitney U test). FigureS5.pdf Additional File 5: Figure S5. Analysis of PAI-1 and t-PA activities in peritoneal fluids of F. hepatica-infected mice. Eight days after challenge with F. hepatica metacercariae, mice were euthanized and the levels of active PAI-1 (A) and t-PA (B) in peritoneal fluids were quantified by ELISA using commercially-available kits. Data points represent the levels of active PAI-1 (A) or t-PA (B) in the peritoneal fluid of each mouse, and bars indicate the mean ± SEM. None of the differences were significant ( Mann-Whitney U test). GraphicalAbstract.png AdditionalFile67.docx Cite Share Download PDF Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 17 Jul, 2025 Reviews received at journal 17 Jul, 2025 Reviews received at journal 15 Jul, 2025 Reviewers agreed at journal 26 Jun, 2025 Reviewers agreed at journal 25 Jun, 2025 Reviewers invited by journal 24 Jun, 2025 Editor assigned by journal 07 Jun, 2025 Submission checks completed at journal 07 Jun, 2025 First submitted to journal 04 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6822677","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466587022,"identity":"5b548c28-e230-4d9c-a8d8-a10439b3a5cb","order_by":0,"name":"Judit Serrat","email":"","orcid":"","institution":"Institute of Natural Resources and Agrobiology of Salamanca (IRNASA-CSIC)","correspondingAuthor":false,"prefix":"","firstName":"Judit","middleName":"","lastName":"Serrat","suffix":""},{"id":466587023,"identity":"a1559db8-35ed-4a81-bcdb-a156bcb13689","order_by":1,"name":"Marta López-García","email":"","orcid":"","institution":"Institute of Natural Resources and Agrobiology of Salamanca (IRNASA-CSIC)","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"López-García","suffix":""},{"id":466587024,"identity":"15d71270-d208-46ae-827a-fbc6966faf5c","order_by":2,"name":"María Torres-Valle","email":"","orcid":"","institution":"Institute of Natural Resources and Agrobiology of Salamanca (IRNASA-CSIC)","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Torres-Valle","suffix":""},{"id":466587025,"identity":"96771315-6d8a-49a4-b9a4-ad9ef16dcd01","order_by":3,"name":"Verónica Molina-Hernández","email":"","orcid":"","institution":"Universidad de Córdoba","correspondingAuthor":false,"prefix":"","firstName":"Verónica","middleName":"","lastName":"Molina-Hernández","suffix":""},{"id":466587026,"identity":"76b64484-2b12-4f4b-9842-5b73870bd08b","order_by":4,"name":"María Teresa Ruiz-Campillo","email":"","orcid":"","institution":"Universidad de Córdoba","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Teresa","lastName":"Ruiz-Campillo","suffix":""},{"id":466587027,"identity":"60c9a101-88b8-4733-ba03-103b44484326","order_by":5,"name":"Mar Siles-Lucas","email":"","orcid":"","institution":"Institute of Natural Resources and Agrobiology of Salamanca (IRNASA-CSIC)","correspondingAuthor":false,"prefix":"","firstName":"Mar","middleName":"","lastName":"Siles-Lucas","suffix":""},{"id":466587028,"identity":"73e02a92-fd2a-460c-9f90-7156c426e5e7","order_by":6,"name":"Javier González-Miguel","email":"data:image/png;base64,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","orcid":"","institution":"Institute of Natural Resources and Agrobiology of Salamanca (IRNASA-CSIC)","correspondingAuthor":true,"prefix":"","firstName":"Javier","middleName":"","lastName":"González-Miguel","suffix":""}],"badges":[],"createdAt":"2025-06-04 17:09:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6822677/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6822677/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06992-9","type":"published","date":"2025-09-24T15:58:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83974428,"identity":"114cfa8d-1950-48ef-8cfd-5d65c45d8c62","added_by":"auto","created_at":"2025-06-05 08:41:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMouse intestinal epithelial cells facilitate FhNEJ-induced plasmin generation from PLG.\u003c/strong\u003e mPSIEC cultures were incubated with FhNEJ and PLG and plasmin generation on cell culture supernatants (OD at 405 nm) was assessed 24 hours after stimulation in an enzymatic assay by mixing equal amounts of cell culture supernatants with a plasmin-specific chromogenic substrate. Cells left untreated (Unt) or single-treated with either FhNEJ or PLG served as negative controls for FhNEJ-induced plasmin generation, and cells incubated with FhNEJ and PLG in the presence of ε-ACA were used to assess whether FhNEJ-induced plasmin generation is mediated by lysine residues. A condition was included in where the plasmin-specific chromogenic substrate was incubated with recombinant plasmin (Plm) instead of cell culture supernatant to control for substrate specificity. The results from three independent cell culture replicates are shown. Lines indicate the mean of three technical replicates ± SD. Error is displayed only for the PLG-only and FhNEJ+PLG conditions for simplification purposes.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/10142b83e95d62aff1af5f5d.png"},{"id":83974750,"identity":"41db464d-ae7c-493a-83aa-d9c401ee975c","added_by":"auto","created_at":"2025-06-05 08:49:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFhNEJ stimulate u-PA release and collagen degradation by mPSIEC in the presence of PLG. \u003c/strong\u003emPSIEC cultures were stimulated with FhNEJ and PLG and the levels of u-PA (A) and total collagen (B) were analysed in cell culture supernatants 24 hours after stimulation by ELISA (A) or by measuring the amount of N-Gly terminal peptides generated after enzymatic digestion with a collagenolytic enzyme (B). In both panels, cells left untreated or single-treated with either FhNEJ or PLG serve as negative controls. Data points represent the value obtained for each independent cell culture replicate, calculated as the average of two technical replicates, and bars represent the mean ± SEM. Only statistically significant differences are shown (*p≤0.1; Kruskal-Wallis test followed by Dunn’s post hoc test for pairwise comparisons).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/503fed501e0c67958ce73b5b.png"},{"id":83974432,"identity":"02f03cca-b67c-4533-af15-21da63642689","added_by":"auto","created_at":"2025-06-05 08:41:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased secretion of MMP-3, MMP-8, MMP-12, and pro-MMP-9 by mPSIEC upon stimulation with FhNEJ. \u003c/strong\u003emPSIEC cultures were stimulated with FhNEJ and PLG, with either component alone, or left unstimulated, and the levels of MMP-2, -3, -8, -12, and pro-MMP-9 were simultaneously assayed using multiplexed assays. In all graphs, data points represent the value obtained for each independent cell culture replicate, calculated as the average of two technical replicates, and bars represent the mean ± SEM. Only statistically significant differences are shown (*p≤0.1; Kruskal-Wallis test followed by Dunn’s post hoc test for pairwise comparisons). Note that statistical analysis could not be performed for MMP-8, MMP-12, and pro-MMP-9 due to some MMP levels being below the detection limit of the technique (PLG-only in MMP-8 analysis; PLG-only in MMP-12 analysis; and Untreated and PLG-only in pro-MMP-9 analysis).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/d94f820fb30c22fc405bbbc5.png"},{"id":83974433,"identity":"47879f9a-5e7c-4cd9-8b5d-57205305fb3d","added_by":"auto","created_at":"2025-06-05 08:41:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":108350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy of the influence of the fibrinolytic system in FhNEJ migration in a mouse model of fasciolosis. \u003c/strong\u003eA) Schematic representation of the experimental design. Thirty minutes prior to infection with 125 \u003cem\u003eF. hepatica\u003c/em\u003emetacercariae, mice were injected with either PBS (control) or a recombinant, stable-mutant version of PAI-1 to inhibit the fibrinolytic route, as previously described [40]. Eight days after challenge with \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae, mice were euthanized and the number of juvenile flukes in the livers were manually counted under a stereomicroscope. Created with Biorender.com. B) Counts of juvenile flukes recovered from the livers of PAI-1-treated and control mice eight days after challenge with \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae. \u003cem\u003eData points represent the number of juveniles recovered in the liver of each mouse, and bars indicate the mean ± SEM. Asterisks indicate significant differences between experimental conditions (ns, not significant; \u003c/em\u003e*p≤0.1; Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/a7819f3800d135278361c025.png"},{"id":92430586,"identity":"b719d91e-f55c-4e20-af2a-fca5b9dde5ba","added_by":"auto","created_at":"2025-09-29 16:06:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1639932,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/8b13bc05-e0bf-4d43-92fc-335c2c68e50c.pdf"},{"id":83974751,"identity":"3ed63096-04de-44b8-ba34-003681862857","added_by":"auto","created_at":"2025-06-05 08:49:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1295249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional File 1: Figure S1. \u003c/strong\u003eFeature selection by elastic-net penalized regression improves sample clustering.\u003cstrong\u003e \u003c/strong\u003eAn elastic-net penalised regression model was performed to select for proteins that are specifically expressed in each experimental condition. The figures show heatmap representations of protein expression levels (normalized to Z-score) of all the proteins identified (left panels) or proteins selected as specific to each experimental condition after feature selection through elastic-net regularised regression (right panels), for selected, biologically-relevant pairwise comparisons: untreated mPSIEC vs. mPSIEC treated with FhNEJ (A); untreated mPSIEC vs. mPSIEC treated with both FhNEJ and PLG (B); and FhNEJ-treated mPSIEC vs. mPSIEC treated with both FhNEJ and PLG (C). Heatmaps created with R software.\u003c/p\u003e","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/dfbc383c0b574aed844acdb3.pdf"},{"id":83974752,"identity":"30d79cbc-f158-452a-8961-99e3db6b6459","added_by":"auto","created_at":"2025-06-05 08:49:35","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":88158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 2: Figure S2.\u003c/strong\u003e PLS-DA analysis of selected pairwise comparisons. PLS-DA was performed to identify the proteins most relevant for distinguishing between experimental conditions. Only those elastic-net pre-selected proteins that contributed substantially to the newly projected bidimensional space (as determined by \u003cem\u003evip\u0026gt;1.5) were considered as specific to each experimental condition. Plots are shown for \u003c/em\u003eselected, biologically-relevant pairwise comparisons: untreated mPSIEC vs. FhNEJ-treated mPSIEC (top left); untreated mPSIEC vs. mPSIEC treated with both FhNEJ and PLG (top right); and FhNEJ-treated mPSIEC vs. mPSIEC treated with both FhNEJ and PLG (bottom). \u003cem\u003ePlots created with R software.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/b3a08249da110fdb05f73c7a.pdf"},{"id":83974754,"identity":"d879b603-a0d0-4d23-bd49-6e3e18c79295","added_by":"auto","created_at":"2025-06-05 08:49:35","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":663511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional File 3: Figure S3. \u003c/strong\u003eA)\u003cstrong\u003e \u003c/strong\u003eThe effect of plasmin on the detection of total collagen was determined using the same commercial kit as in Figure 2B by pre-incubating total collagen with 2 µM plasmin for 24 hours at 37 ºC followed by enzymatic digestion and detection of N-Gly terminal peptides, as specified by the manufacturer’s instructions. Total collagen left undigested (“Undigested”) or digested without plasmin pre-incubation (“+ Enzyme”) served as controls. Bars indicate the mean of three technical replicates ± SD (***p≤0.001, ****p≤0.0001; one-way ANOVA followed by Tukey post hoc analysis of pairwise comparisons). RFU, relative fluorescent units (excitation 380 nm, emission 460 nm). B) Zymography showing plasmin-mediated degradation of the ECM coating used for mPSIEC culture. Different amounts of plasmin (2 µM, 0.5 µM, and 0.1 µM) were loaded onto 10% SDS-PAGE gels prepared using mPSIEC coating solution instead of water. Light bands (arrowhead) corresponding to degradation of the mPSIEC coating solution by plasmin were revealed by Coomassie brilliant blue staining.\u003c/p\u003e","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/feccb69a4bf71d8cb3cd6868.pdf"},{"id":83974431,"identity":"c1968059-5fbb-4168-8cdb-6c136de5521c","added_by":"auto","created_at":"2025-06-05 08:41:35","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":25396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAdditional File 4: Figure S4. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eFluke recovery in control and PAI-1-treated mice across experimental days. \u003c/em\u003eThe number of flukes recovered from control (A) and PAI-1-treated (B) mice was plotted separately to assess possible variation in intra-group responses across days\u003cem\u003e. Data points represent the number of juveniles recovered in the liver of each mouse, and bars indicate the mean ± SEM. Asterisks indicate significant differences between experimental conditions (ns, not significant; *\u003c/em\u003e*p≤0.1; Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"FigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/6ff1728c63322ec0b0fc1c64.pdf"},{"id":83974437,"identity":"3ba5cf17-74e9-4056-986c-979a0f1ad1fc","added_by":"auto","created_at":"2025-06-05 08:41:35","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":28250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAdditional File 5: Figure S5. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eAnalysis of PAI-1 and t-PA activities in peritoneal fluids of F. hepatica-infected mice. Eight days after challenge with F. hepatica metacercariae, mice were euthanized and the levels of active PAI-1 (A) and t-PA (B) in peritoneal fluids were quantified by ELISA using commercially-available kits. Data points represent the levels of active PAI-1 (A) or t-PA (B) in the peritoneal fluid of each mouse, and bars indicate the mean ± SEM. None of the differences were significant (\u003c/em\u003eMann-Whitney U test).\u003c/p\u003e","description":"","filename":"FigureS5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/1f34e777c0dfd49e2cb1a9c9.pdf"},{"id":83974440,"identity":"74bba61a-8ff3-4185-8493-ab7312271781","added_by":"auto","created_at":"2025-06-05 08:41:35","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1880589,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/678226f6b5bbda45c40b8a0f.png"},{"id":83974436,"identity":"3a2f513f-c441-489f-825a-f57bf3beccbd","added_by":"auto","created_at":"2025-06-05 08:41:35","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":19521,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile67.docx","url":"https://assets-eu.researchsquare.com/files/rs-6822677/v1/25b34f0d4910bc3c6af625ae.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Early host-parasite interaction models reveal a key role for fibrinolysis in Fasciola hepatica intestinal migration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA paradigmatic example of host-parasite relationship is the ability of parasites to engage with the fibrinolytic system of their definitive hosts, a strategy that is considered a key aspect of parasite dissemination and survival within the mammalian organism [1\u0026ndash;3]. The most common mechanism of interaction between parasites and host fibrinolysis is through the expression of lysine-rich proteins at the host-parasite interface\u0026mdash;either on the parasite surface or secreted into the tissue microenvironment surrounding the parasite\u0026mdash;that interact with plasminogen (PLG), the main zymogen of the fibrinolytic route. Upon binding to its receptors (PLG-Rs), PLG undergoes a conformational change that exposes the target sites of its activators, the proteases tissue-type and urokinase-type PLG activators (t-PA and u-PA, respectively), which convert PLG into its catalytically active form, the serine protease plasmin [4].\u003c/p\u003e \u003cp\u003eThe classical function of the fibrinolytic system is to degrade the fibrin network that stabilises blood clots in the vascular endothelium [4]; however, due to the broad spectrum of plasmin substrates, this system also participates in multiple biological processes that are both unrelated and independent of fibrin degradation and blood clot removal, including cell migration [5,6]. Plasmin facilitates cell migration by degrading different components of the extracellular matrix (ECM), including collagens, laminin, fibronectin, and proteoglycans [7\u0026ndash;11], as well as by cleaving and activating the zymogens of different metalloproteinases (MMPs), which are considered master regulators of ECM remodelling during homeostasis, development, and disease [12]. Additionally, fibrinolysis-unrelated substrates of plasmin also include complement molecules and immunoglobulins [13]. Based on this, the stimulation of plasmin generation from host PLG within the tissue microenvironment surrounding parasites has been postulated as a mechanism facilitating immune evasion and their migration through host tissues [2,3,13].\u003c/p\u003e \u003cp\u003e \u003cem\u003eFasciola hepatica\u003c/em\u003e is one of many parasites whose interaction with the host fibrinolytic system has been experimentally shown [14\u0026ndash;18]. \u003cem\u003eF. hepatica\u003c/em\u003e is a helminth trematode with an indirect life cycle that includes freshwater snails as intermediate hosts, where the asexual phases of the parasites develop, and a mammalian definitive host, where adult flukes mature. Definitive hosts, typically ruminants and humans, become infected by ingestion of water plants contaminated with infective metacercariae, which excyst in the duodenum and release the newly excysted juveniles (FhNEJ). Shortly after excystment, FhNEJ cross the intestinal wall, and the juvenile flukes subsequently migrate through the peritoneum and liver parenchyma before entering the major hepatic bile ducts, where they mature into adults and produce fertilised eggs that are shed with the faeces [19]. \u003cem\u003eF. hepatica\u003c/em\u003e is the most widespread etiologic agent of fasciolosis, a chronic and debilitating disease that affects millions of ruminants worldwide, thereby representing an economically important affection of domestic livestock that poses major threats to animal welfare and global food security [20,21]. Since cases of human infection are predominantly concentrated in tropical areas of the globe, human fasciolosis is categorized by the Word Health Organization a food-borne neglected tropical disease of growing public health concern requiring targeted intervention for its elimination [22,23].\u003c/p\u003e \u003cp\u003eUsing \u003cem\u003ein vitro\u003c/em\u003e assays, we and others have previously shown that FhNEJ interact with the host fibrinolytic system by multiple mechanisms. These include the expression of proteins that bind to PLG, the precursor zymogen of u-PA (pro-u-PA), or both at the host-parasite interface [14\u0026ndash;16,18], as well as the expression of proteases that can stimulate pro-u-PA conversion into its catalytically active form, which further potentiates plasmin generation from host PLG [15]. Notably, FhNEJ-stimulated plasmin generation contributes to the endogenous capacity of these parasites to degrade laminin [24], one of the major components of the intestinal basement membrane [25], indicating that the interaction between FhNEJ and host fibrinolysis may facilitate their migration across the intestinal wall. Since FhNEJ trans-intestinal migration is considered a crucial step for infection success, a deeper understanding of the mechanisms governing this process, including the contribution of the host fibrinolytic system, may offer potential avenues for developing novel therapeutic and control strategies against this widespread parasite [26].\u003c/p\u003e \u003cp\u003eAlthough the interaction between parasites and host fibrinolysis system is well-established [1\u0026ndash;3], only a minority of studies have performed functional assays using physiologically-relevant experimental models to assess whether it serves any physiological roles [1]. The abovementioned interplay between \u003cem\u003eF. hepatica\u003c/em\u003e and the host fibrinolytic system exemplifies this gap. In the present study, we addressed this limitation by exploring the functional relevance of the interaction between FhNEJ and host fibrinolysis using a co-culture system of epithelial cells derived from the mouse small intestine, which simulates the first contact between FhNEJ and host tissues [27,28], and a mouse model of early-stage fasciolosis [29].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eCulture of mouse primary small intestinal epithelial cells\u003c/h2\u003e\n\u003cp\u003eC57BL/6 mouse primary small intestinal epithelial cells (mPSIEC; Cell Biologics) were cultured as previously described\u0026nbsp;[27,28]\u0026nbsp;and following the manufacturer’s instructions. Briefly, cells were plated in 6 cm\u003csup\u003e2\u003c/sup\u003e dishes (Corning) pre-coated with a gelatin-based coating solution (Cell Biologics) and grown in complete epithelial cell medium (Cell Biologics) in a humidified, 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37 ºC. Cell medium was replaced every 48 h and cells were tripsinised and split at a 1:3 ratio when confluence was reached following standard cell culture procedures.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eExcystment of \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eF. hepatica\u003c/em\u003e metacercariae (Italian strain, Ridgeway Research Ltd) were excysted as previously described\u0026nbsp;[14]. Briefly, metacercariae were incubated for 1 h at 37 ºC in a solution containing CO\u003csub\u003e2\u003c/sub\u003e and 0.02 M sodium dithionite (Sigma), followed by three washes with distilled water and incubation in excystment medium [Hank’s balanced salt solution (Sigma) supplemented with 10% lamb bile (obtained from a local abattoir) and 30 mM HEPES (Sigma) at pH 7.4] at 37 ºC. FhNEJ were manually recovered under a stereomicroscope using a 20 μL pipette every hour after addition of excystment medium, immediately transferred to a clean plate containing complete epithelial cell medium, and incubated in a humidified, 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37 ºC to induce recovery for 1 h prior to transfer to mPSIEC cultures.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eIn vitro\u003c/em\u003e interaction model\u003c/h2\u003e\n\u003cp\u003eFhNEJ were co-cultured with mPSIEC as previously described\u0026nbsp;[27,28], with some modifications. At passage 5, mPSIEC were cultured in 6-well plates until confluence was reached. At that point, 200 FhNEJ were added to the corresponding wells in 2 mL of complete epithelial cell medium in the presence or absence of 10\u0026nbsp;µg/mL of human PLG (Origene). A condition where cells were co-incubated with 200 FhNEJ, PLG (10\u0026nbsp;µg/mL), and 50 mM of the lysine analogue 6-aminocaproic acid (ε-ACA; Sigma) was included to assess whether the mechanism of FhNEJ-induced plasmin generation is lysine-dependent. Cells left unstimulated, cells stimulated only with 200 FhNEJ, and cells stimulated only with PLG (10\u0026nbsp;µg/mL) were used as controls. The parasites were separated from the cells 24 h after stimulation, and whole-cell lysates and cell culture supernatants were harvested for downstream analyses. First, 1 mL of each culture supernatant was carefully aspirated and transferred to 1.5 mL tubes, followed by centrifugation for 5 min at 13,000 x\u003cem\u003eg\u003c/em\u003e and 4 ºC. The pellets containing cell debris were discarded and clean supernatants were frozen at -80 ºC until use. Whole-cell lysates were obtained by washing the cells in pre-warmed PBS followed by scraping in 200\u0026nbsp;µL of RIPA buffer (Sigma). Whole-cell lysates were transferred to 1.5 mL tubes, vigorously vortexed for 30 s and centrifuged for 5 min at 13,000 x\u003cem\u003eg\u003c/em\u003e and 4 ºC. Pellets containing the cell debris were discarded and supernatants were transferred to clean 1.5 mL tubes and stored at\u0026nbsp;–80 ºC until use. Protein concentrations were determined using the Pierce BCA Protein Assay kit (Thermo Fisher) and ranged between 1,03–1,37 mg/mL protein, depending on the sample. Each experimental condition was conducted in triplicate.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePlasmin activity in cell culture supernatants\u003c/h2\u003e\n\u003cp\u003ePlasmin activity in cell culture supernatants collected from mPSIEC in the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003einteraction model was assayed by measuring the amidolytic activity of generated plasmin on a plasmin-specific chromogenic substrate, as previously described\u0026nbsp;[14], with minor modifications. In every well of a transparent, flat-bottom, 96-well microtiter plate, equal volumes of samples were mixed with 1 mM of D-Val-Leu-Lys 4-nitroanilide dihydrochloride chromogenic substrate (S-2251, Sigma) in a total volume of 100\u0026nbsp;µL of PBS. The microplates were incubated for up to 24 h at 37 ºC and substrate cleavage was assessed by measuring absorbance at 405 nm every hour in a Multiskan GO spectrophotometer (Thermo Fisher). Wells containing 0.02\u0026nbsp;µM of plasmin (Origene) instead of cell culture supernatant were used to control for substrate specificity. All the reactions were performed in technical triplicate.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAnalysis of u-PA levels in cell culture supernatants\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe levels of u-PA in cell culture supernatants (100\u0026nbsp;µL) were measured by enzyme-linked immunosorbent assay (ELISA) using the Mouse PLAU/Upa (Urokinase-Type Plasminogen Activator) ELISA kit (FineTest) and following the manufacturer’s instructions. The reaction was stopped 11 min after addition of the 3,3′,5,5′-tetramethylbenzidine substrate, and the amount of u-PA in the samples was quantified by fitting a regression line to the linear portion of a standard curve with known u-PA concentrations. All the reactions were performed in technical duplicate.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAnalysis of total collagen in cell culture supernatants\u003c/h2\u003e\n\u003cp\u003eThe levels of total collagen in cell culture supernatants (20\u0026nbsp;µL) were measured by enzymatic digestion of collagen into N-Gly terminal peptides that react with a dye reagent to form a fluorescent complex, using the Collagen Assay Kit (Assay Genie) and following the manufacturer’s instructions. The amount of collagen in the samples was quantified by fitting a regression line to the linear portion of a standard curve with known collagen concentrations. All the reactions were performed in technical duplicate.\u003c/p\u003e\n\u003ch2\u003eZymography\u003c/h2\u003e\n\u003cp\u003eThe ability of plasmin to degrade the mPSIEC coating solution was measured by preparing 10%\u0026nbsp;sodium dodecyl sulfate (SDS)-polyacrylamide gels using standard protocols but replacing water with the mPSIEC coating solution (Cell Biologics). Next, different amounts of plasmin (Origene) were mixed with non-reducing sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromophenol blue) in a total volume of 18 µL and samples were loaded onto the gel in duplicate. Electrophoresis was performed at constant 15 mA for 20 min followed by 30 mA. After electrophoresis, the gel was soaked twice in wash buffer (5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 2% Triton-X, pH 7.4) for 30 min at room temperature, followed by overnight incubation at 37 ºC in digestion buffer (5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.1 M glycine, pH 8.4) [30]. All the incubations were performed with mild shaking. After incubation, the gels were stained with Coomassie brilliant blue staining, following standard procedures, and imaged in a Chemidoc MP Imaging System (BioRad).\u003c/p\u003e\n\u003ch2\u003eAnalysis of MMP levels in cell culture supernatants\u003c/h2\u003e\n\u003cp\u003eThe levels of MMP-2, MMP-3, MMP-8, pro-MMP-9 and MMP-12 in mPSIEC culture supernatants (25\u0026nbsp;µL) were analysed on a Luminex xMAP platform using a 5-plex mouse MMP magnetic bead panel (Millipore) and following the manufacturer’s instructions. Multiplex assays were analysed on a Liquichip Luminex 100 Liquid Array Multiplexing System XYP Lab (Luminex Corporation, Qiagen) with Luminex Xponent 3.1 software (Luminex Corporation), following the manufacturer’s technical guidelines. Data analysis was performed with the Milliplex Analyst 5.1 Software (Merck). All the reactions were performed in technical duplicate. The experiment and analysis were conducted at the Flow Cytometry Facility of the National Centre for Biotechnology (Spanish National Research Council; CNB-CSIC, Madrid, Spain).\u003c/p\u003e\n\u003ch2\u003eProteomic analysis of mPSIEC whole-cell lysates\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eSample preparation.\u003c/strong\u003e Protein concentrations were determined using the Macherey-Nagel Protein Quantification Assay (Macherey-Nagel) and equal amounts of protein were reduced with 2 mM dithiothreitol at 60 ºC for 20 min, and alkylated with 5 mM iodoacetamide dilutes in 50 mM ammonium bicarbonate at room temperature for 30 min. Next, proteins were purified using the SP3 protocol\u0026nbsp;[31,32]\u0026nbsp;to eliminate detergents prior to in solution protein digestion. Purified proteins were digested overnight at 37 ºC with 100 ng of sequencing-grade trypsin (Promega) diluted in 50 mM ammonium bicarbonate and the reaction was stopped by addition of 0.1% trifluoroacetic acid.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS/MS analysis.\u003c/strong\u003e Liquid chromatography (LC) was performed by loading 200 ng of digested peptides (diluted in 20 µL of 0.1% formic acid) onto an Evotip Pure tip (EvoSep) following the manufacturer’s instructions, followed by separation using the Evosep One system on an analytical column Performance 15 cm × 150 μm, 1.5 μm (Evosep). The eluted peptides were ionized in a captive Spray with 1700 V at 200 ºC and tandem mass spectrometry analysis (MS/MS) was performed in a TimsTOF fleX mass spectrometer (Bruker) in data-independent acquisition Parallel Accumulation-Serial Fragmentation (diaPASEF) mode.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData processing and protein quantification.\u003c/strong\u003e The PASER system (Bruker) was used to send the raw data for subsequent analysis and quantification with DIA-NN v1.8 software (https://github.com/vdemichev/DiaNN). First, an \u003cem\u003ein silico\u003c/em\u003e-predicted spectral library was built from the Uniprot\u0026nbsp;Mus musculus\u0026nbsp;database using DIA-NN v1.8, using QuantUMS (high precision) as the quantification strategy. After raw data normalization and quantification, proteins were filtered at 1% false discovery rate (FDR) to ensure confidence in protein identification. The proteomic analysis was carried out at the Proteomics Unit of the Central Support Service for Experimental Research (SCSIE) of the University of Valencia (Spain), a member of the Instituto de Salud Carlos III (ISCIII) ProteoRed Proteomics Platform.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u0026nbsp;\u003c/strong\u003ePeak intensities of all the conditions were transformed to the logarithm with base \u003cem\u003ee using the log1p function in R. Next, a\u003c/em\u003e stringent statistical pipeline employing three distinct methodologies was applied to identify differentially expressed proteins (DEPs) with high robustness, as previously described\u0026nbsp;[33]. \u003cem\u003eFirst, the glmnet package in R was employed to fit an elastic-net regularised regression model to select those variables (proteins) that better explain each experimental condition\u0026nbsp;\u003c/em\u003e[34]\u003cem\u003e\u0026nbsp;(\u003cstrong\u003eFig. S1\u003c/strong\u003e).\u0026nbsp;\u003c/em\u003eTo refine the model, the nearZeroVar function from the caret package in R was applied to identify variables with near-zero variance, indicating very low variability across observations. These proteins were discarded from subsequent analysis. \u003cem\u003eThe values of the regularisation parameters (alpha and lambda) were optimized with the train function of the caret package through cross-validation re-sampling to obtain the regularised model that best fit our data. The log-transformed expression values of the selected proteins were standardized through Z-score normalization to\u0026nbsp;\u003c/em\u003eensure direct comparability of protein expression levels across samples\u003cem\u003e, followed by representation in heatmaps. Second, feature selection by elastic-net was validated through Partial Least Squares Discriminant Analysis (PLS-DA) using the mixOmics package in R\u0026nbsp;\u003c/em\u003e[35]\u003cem\u003e\u0026nbsp;(\u003cstrong\u003eFig. S2\u003c/strong\u003e). This package contains the vip function, which estimates the importance of each variable in the projection. When vip\u0026gt;1.5, the influence of the variable on the response is very high\u0026nbsp;\u003c/em\u003e[33]\u003cem\u003e. Third, differential expression analysis between selected pairs of experimental conditions was performed using the limma package in R to get fold-change (FC) differences in protein expression along with the corresponding p-values\u0026nbsp;\u003c/em\u003e[36]\u003cem\u003e.\u0026nbsp;\u003c/em\u003eOnly proteins that were selected by both elastic-net and PLS-DA (vip\u0026gt;1.5) and exhibited a significant fold-change (p-value\u0026lt;0.05), as calculated by limma, were considered differentially expressed\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThis analysis was performed by the Statistics and Omics Data Analysis Unit of the Central Support Service for Experimental Research (SCSIE) of the University of Valencia, Spain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of interaction effects.\u003c/strong\u003e To identify proteins whose differential expression was specifically driven by the interaction of FhNEJ and PLG, rather than by either factor alone, an interaction effect analysis was conducted on the list of DEPs identified using the abovementioned statistical pipeline in the Untreated vs. FhNEJ+PLG comparison. To ensure that only proteins influenced by the interaction between FhNEJ and PLG were retained, proteins that were also differentially expressed in either the Untreated vs. FhNEJ or Untreated vs. PLG comparisons were excluded unless their expression in the combined treatment deviated significantly from the expected additive effect.\u0026nbsp;This approach is conceptually similar to established methodologies in gene interaction studies aimed at detecting synergistic genetic perturbations, where interaction effects are assessed by comparing the observed combined effects to the expected additive effects of individual perturbations [37,38]. To this end, an expected additive FC was computed as the sum of the individual effects of FhNEJ and PLG, based on FC values obtained from the Untreated vs. FhNEJ and Untreated vs. PLG comparisons. An interaction score was then calculated as the difference between the observed FC in the Untreated vs. FhNEJ+PLG comparison and the expected additive FC. Proteins common to all three comparisons were considered to be differentially expressed as a result of the interaction between FhNEJ and PLG only if their absolute interaction score exceeded a threshold.\u0026nbsp;To avoid arbitrary thresholding, interaction scores were filtered using the 1.5 × IQR rule, where IQR is the interquartile range, a standard method for outlier detection based on Tukey’s definition of extreme values [39]. Therefore,\u0026nbsp;the interaction threshold was defined as: median interaction score + (1.5 × IQR); where the median interaction score is the median of all the interaction scores calculated across proteins in the Untreated vs. FhNEJ+PLG condition. This analysis ensured that only proteins exhibiting substantial interaction effects—whether synergistic or antagonistic—were retained for further investigation. In addition to this analysis, a complementary approach was performed to identify proteins whose differential expression in the FhNEJ+PLG condition, when compared to FhNEJ-alone, could not be explained solely by the effect of PLG. In this case, proteins were filtered based on their FC values in the Untreated vs. PLG comparison. A threshold was defined as the median absolute FC ± (1.5 × IQR) in this comparison, capturing the typical range of PLG-induced expression changes. Only proteins in the FhNEJ vs. FhNEJ+PLG comparison with an absolute FC exceeding this threshold were retained.\u003c/p\u003e\n\u003ch2\u003eAnimal ethics protocol\u003c/h2\u003e\n\u003cp\u003eAll animals received human care in accordance with the guidelines for the protection of animals used for scientific purposes (Directive 2010/63EU, Decision 2012/707/UE, and RD 53/2013). All procedures were approved by the Ethical Animal Experimentation Committee of the University of Córdoba and by the Junta de Andalucía (project nr. 2021P|/22).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eIn vivo\u003c/em\u003e model\u003c/h2\u003e\n\u003cp\u003e20 wild-type, 49–55-day-old female C57BL/6 mice (Charles River Laboratories) were divided in two groups: one group (n=10) was injected intraperitoneally with mouse PLG activator inhibitor 1 (PAI-1) recombinant stable mutant (50\u0026nbsp;µg per mouse; Innovative Research) to inhibit the fibrinolytic route, as previously described\u0026nbsp;[40]; and the control group (n=10) was injected intraperitoneally with sterile phosphate-buffered saline (PBS). All mice were challenged with \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae 30 min after injection, following a protocol recently described by our lab\u0026nbsp;[29]. First, mice were orally infected with 125 \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae as follows: metacercariae were resuspended in a total volume of 50\u0026nbsp;µL of PBS and oral infection was performed using a sterile 20–200\u0026nbsp;µL pipette tip trimmed 0.5 cm and pre-washed with PBS containing 0.01% Triton X-100 to prevent the metacercariae from sticking to the plastic tip. Second, 8 days after challenge with \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae, mice were euthanised after 24 h fasting through CO\u003csub\u003e2\u003c/sub\u003e overdose followed by cervical dislocation. Prior to dissection, 2 mL of sterile PBS were injected into the peritoneal cavity, taking care to avoid puncturing any organs. Peritoneal fluid was then collected by making a small abdominal incision, followed by careful aspiration with a pipette tip. The liquid was transferred to 1.5 mL tubes and immediately frozen at\u0026nbsp;–80 ºC. For the recovery of juvenile worms from the hepatic parenchyma, livers were carefully dissected with tweezers under a stereomicroscope and the number of juvenile parasites was manually counted by independent, trained, and experimental group-blinded researchers that were unfamiliar with the objective of the experiment. The experiment was conducted over two consecutive days, using 5 mice per group per day, with the same reagents and personnel. Animal handling was performed by qualified staff at the Experimental Animal Facility of the University of Córdoba (Spain). Mice were housed in groups of 4 per cage, with aspen wood bedding, and provided food and water \u003cem\u003ead libitum\u003c/em\u003e on a standard dry pellet diet for rodents. The experiment was conducted after 1 week of acclimatization at 22 ± 3 ºC, 50-60% relative humidity, and a 12 h light/dark cycle.\u003c/p\u003e\n\u003ch2\u003ePAI-1 and t-PA activity assays\u003c/h2\u003e\n\u003cp\u003eThe activities of PAI-1 and t-PA in peritoneal fluids of mice were measured using the commercially available Mouse active PAI1 ELISA Kit (Innovative Research) and Mouse Active tPA ELISA Kit (Innovative Research), following the manufacturer’s instructions. The amounts of active PAI-1 and t-PA in the samples were quantified by fitting a regression line to the linear portion of a standard curve with known active PAI-1/t-PA concentrations. All the reactions were performed in technical duplicate.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003ePlots were created, and statistical analyses were performed using Prism 10 software (GraphPad Software). Unless otherwise stated, graph bars represent the mean of three cell culture replicates, each calculated as the average of two or three technical replicates. Error bars represent either the standard error of the mean (SEM) or the standard deviation (SD) to reflect the precision of the sample mean as an estimate of the population mean or the variability among individual technical measurements, respectively. The data from the \u003cem\u003ein vivo\u003c/em\u003e experiment was analysed using a two-way analysis of variance (ANOVA), considering both treatment groups and the day on which the experiment was conducted as factors. Based on the result of this analysis, the data was plotted separately for each day and presented as mean ± SEM. Since normality in data distribution cannot be confidently determined with small sample sizes [41], non-parametric methods were chosen to determine statistically significant differences between experimental conditions across biological replicates. Specifically, the Mann-Whitney U test was used to assess differences between two experimental conditions; and comparisons between more than two experimental conditions were performed via Kruskal-Wallis test followed by Dunn’s post hoc analysis of pairwise comparisons. Unless otherwise stated, the threshold for significance was set at α ≤ 0.1.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFhNEJ stimulate plasmin generation in the pericellular space of mouse intestinal epithelial cells\u003c/p\u003e\n\u003cp\u003eIn order to study whether FhNEJ stimulate plasmin generation from host PLG in an \u003cem\u003ein vitro\u003c/em\u003e system of host-parasite interactions [27,28], mPSIEC were co-incubated for 24 hours with FhNEJ in the presence of host PLG and without the exogenous addition of PLG activators. A condition where cells were co-incubated with FhNEJ, PLG, and the lysine analogue \u0026epsilon;-ACA was included to assess whether FhNEJ-induced plasmin generation is mediated by lysine residues present in FhNEJ-derived PLG-interacting proteins, as previously described [14]. Plasmin generation in cell culture supernatants of mPSIEC was assessed using a plasmin-specific chromogenic substrate, which revealed that cells incubated with PLG alone showed a small degree of plasmin generation that was increased in the presence of FhNEJ. This effect was observed across three independent cell culture replicates and was completely abrogated in the presence of \u0026epsilon;-ACA, indicating that the observed increased plasmin generation is mediated by lysine residues (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eFhNEJ increase u-PA levels in mPSIEC culture supernatants and promote collagen degradation in a PLG-dependent manner\u003c/p\u003e\n\u003cp\u003eWe next addressed how mPSIEC respond to the presence of FhNEJ in terms of u-PA release and collagen degradation, and whether these responses would be modulated by the interaction between FhNEJ and PLG. The levels of u-PA in cell culture supernatants of mPSIEC left unstimulated, stimulated with FhNEJ and PLG, or simulated with either component alone were assessed through ELISA, and total collagen in these samples was quantified by measuring the amount of N-Gly terminal peptides generated upon treatment of the samples with a collagenolytic enzyme. These experiments revealed that, in contrast to cell culture supernatants from mPSIEC single-treated with either FhNEJ or PLG, cell culture supernatants from mPSIEC stimulated with both FhNEJ and PLG exhibited a statistically significant increase in u-PA levels (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) and a decrease in collagen (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, S3A) compared to supernatants from untreated mPSIEC. In line with this, standard zymography performed with increasing amounts of recombinant plasmin revealed that this protease degrades the ECM coating solution used to culture mPSIEC (\u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eB\u003c/strong\u003e). Altogether, these results show that the stimulation of mPSIEC with both FhNEJ and PLG results in increased u-PA release and ECM degradation in this \u003cem\u003ein vitro\u003c/em\u003e cell system.\u003c/p\u003e\n\u003cp\u003eRegulation of MMP levels in mPSIEC culture supernatants by FhNEJ\u003c/p\u003e\n\u003cp\u003eWe went on to evaluate whether FhNEJ influence the secretion of different ECM-degrading MMPs by mPSIEC, and whether this would be influenced by PLG. To that end, the levels of MMP-2, -3, -8, -12, and pro-MMP-9 were analysed through multiplexed assays in cell culture supernatants of mPSIEC left unstimulated, stimulated with FhNEJ and PLG, or stimulated with either component alone (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). This experiment revealed that MMP-2 and pro-MMP-9 are the MMPs showing greater concentrations in the cell culture supernatants of mPSIEC. Specifically, the levels of MMP-2 were similar across the different experimental conditions, whereas those of pro-MMP-9 were only detectable in the conditions incubated with FhNEJ (FhNEJ-only and FhNEJ\u0026thinsp;+\u0026thinsp;PLG). Similar results were obtained for MMP-3, -8, and \u0026minus;\u0026thinsp;12, despite their levels in mPSIEC culture supernatants being much lower than those observed for MMP-2 and pro-MMP-9. Overall, although some experimental conditions could not be quantified due to values falling below the detection limits of the technique, these results indicate that MMP levels in cell culture supernatants substantially increase upon incubation of mPSIEC with FhNEJ, regardless of PLG availability.\u003c/p\u003e\n\u003cp\u003eProteomic profiling reveals mPSIEC responses driven by the interaction between FhNEJ and host PLG\u003c/p\u003e\n\u003cp\u003eTo characterise in greater detail how mPSIEC respond to FhNEJ and whether these responses are modulated by their interaction with host PLG, a proteomic analysis was conducted in whole-cell lysates from mPSIEC left unstimulated, stimulated with FhNEJ and PLG, or stimulated with either component alone. This analysis identified a total of 8136 proteins with FDR\u0026thinsp;\u0026gt;\u0026thinsp;1% that were included in the differential expression analysis. We compared the protein expression profiles of untreated cells to cells stimulated with both FhNEJ and PLG to identify proteins that are differentially regulated by the interaction between both factors, which results in increased plasmin activity in cell culture supernatants (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In order to keep only proteins regulated by the combination of FhNEJ and PLG, and not by either component alone, DEPs identified in both the untreated vs. FhNEJ and the untreated vs. PLG comparisons that overlapped with DEPs identified in the untreated vs. FhNEJ\u0026thinsp;+\u0026thinsp;PLG comparison were not considered as differentially regulated by FhNEJ\u0026thinsp;+\u0026thinsp;PLG unless their FC values showed an interaction effect. As a result of this interaction analysis, we identified a total of 84 DEPs in mPSIEC treated with FhNEJ and PLG that are specifically attributable to the combined effect of both components (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDEPs in untreated vs. FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated mPSIEC. A negative fold-change indicates that proteins were overexpressed in FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated vs. untreated mPSIEC. The opposite is true when the fold-change is positive.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elogFC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eadjPVal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elogFC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eadjPVal\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCap1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-11.5641\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.16E-08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMrgbp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.315304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.041192\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEif1ad11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-10.8284\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044884\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSgo2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.321909\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044884\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZfp606\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-9.98762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.16E-08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTmem184b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.337123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.042419\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerpinb5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-9.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.29E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCdca2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.337796\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044267\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA130006I12Rik\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.8194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.9E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAurka\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.337847\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.037707\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS1pr2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.79117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.89E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGlul\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.353169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.029492\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePptc7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-7.5837\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.26E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.365228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.032615\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTmem14c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.70435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044884\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eActn4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.36718\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRab3b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.67455\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031743\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRab38\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.367389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.026216\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMmp11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.67003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAurkb\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.370126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.047626\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDennd3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.4902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004343\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSash1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.377697\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044267\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDesi2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.44875\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044884\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRpl35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.398069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.012174\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDhx38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.44049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.035042\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcsl4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.398548\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007476\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSrsf2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.43185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.041835\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCox6a1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.405347\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.028322\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGorasp1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.38835\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.011505\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEsco2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.419492\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.018064\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCcn2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.37626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0610010K14Rik\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.476796\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044884\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAtrx\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.32326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04661\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eArhgap20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.48969\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044267\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSlc25a30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.31699\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCmip\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.490543\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP4ha2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.26881\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAven\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.490844\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.041192\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSlc12a7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.2512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.026216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGmnn\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.495586\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.047626\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eScel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.24909\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCdc34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.522914\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.040503\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGpam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.23204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSuco\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.539981\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.018465\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSppl2a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.191843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.042419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCetn2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.553036\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.040283\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSka3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.199186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNifk\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.619508\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLama5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.223652\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.040283\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFnip1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.677284\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006195\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSlc9a3r2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.224492\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046404\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLrig1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.745655\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00283\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDvl2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.239475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSema3a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.831001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031743\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCdv3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.244815\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.035681\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerpind1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.858629\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.032615\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNotch2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.247477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.022438\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFn1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.888985\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031603\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLamb1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.250854\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.018889\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eZfhx4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.130508\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.010738\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePrkd2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.254831\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.048562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eThbs1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.239887\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.035681\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDiaph3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.255139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.018889\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerpinb7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.278409\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003469\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRnf4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.25624\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eA2m\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.423682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.016865\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePrr11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.265523\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eItih4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.425836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.026216\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpdl1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.271143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031743\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTmc5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.469909\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002405\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMelk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.27314\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.048562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.471351\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003517\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCalu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.28254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.022262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eDnah14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.608509\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.026216\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFam50a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.286497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.011574\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMettl25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.646163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.28E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMphosph10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.292922\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.028712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePigr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.709757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.38E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMsmo1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.302965\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031743\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSema3d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.34484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.39E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRor1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.311029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.021179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLamtor3-ps\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.05803\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00283\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTns2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.313294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.017259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlb\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.61229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.51E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn addition to proteins shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, we also considered as differentially regulated as a result of the interaction between FhNEJ and PLG those DEPs identified by comparing mPSIEC stimulated with FhNEJ\u0026thinsp;+\u0026thinsp;PLG to cells stimulated with only FhNEJ. Following a similar rationale as described above, we excluded proteins regulated by PLG alone by discarding those DEPs in the FhNEJ vs. FhNEJ\u0026thinsp;+\u0026thinsp;PLG comparison that were common to those identified in the Untreated vs. PLG comparison unless their fold change under the FhNEJ\u0026thinsp;+\u0026thinsp;PLG condition exceeded the typical range of PLG-induced expression changes. By doing so, we identified a total of 15 proteins that were differentially regulated in mPSIEC as a result of the stimulation with both FhNEJ and PLG (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, this analysis identified PLG (Plg) as significantly upregulated in FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated cells compared to cells treated with FhNEJ alone, highlighting the accuracy of the approach. By combining both analyses, we identified 94 unique DEPs in double-treated mPSIEC, which are involved in cell adhesion and migration, ECM remodelling, mucosal immunity, and fibrinolysis. These included cyclase-associated protein 1 (Cap1), serpin b5, the Ras-related protein Rab3b, actinin-4 (Actn4), MMP-11 (Mmp11), the tissue inhibitor of metalloproteinase 3 (Timp3), fibronectin 1 (Fn1), quiescin sulfhydryl oxidase 1 (Qsox1), semaphorin 3D (Sema3d), the polymeric immunoglobulin receptor (Pigr), carbohydrate sulfotransferase 2 (Chst2), the complement molecule C7, the sphingosine 1-phosphate receptor 2 (S1pr2), alpha-2 macroglobulin (A2m), and carboxipeptidase 2 (Cpb2), also known as the thrombin activatable fibrinolysis inhibitor (TAFI).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDEPs in FhNEJ-treated vs. FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated mPSIEC. A negative fold-change indicates that proteins were overexpressed in FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated vs. FhNEJ-treated mPSIEC. The opposite is true when the fold-change is positive.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elogFC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eadjPVal\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCap1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-11.5928\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.45E-08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCpb2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.97008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.76E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUbe2l6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.30553\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.76E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQsox1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-9.00044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.76E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSema3d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.18779\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.76E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKlhl30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.2297\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.84E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChst2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.69985\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.86E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTimp3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.59491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004089\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRab3b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.50349\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.010792\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePlg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.34697\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.014915\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDennd3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.42075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.022473\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhip\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.55656\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.025486\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCcn1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.45733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.043756\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eScel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.36049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.043756\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSlco2a1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.33878\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044832\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe contribution of the fibrinolytic system to FhNEJ migration in a mouse model of fasciolosis\u003c/p\u003e\n\u003cp\u003eFinally, in an effort to understand the contribution of the host fibrinolytic system in the migration of \u003cem\u003eF. hepatica\u003c/em\u003e juveniles during the earliest stages of infection in a physiologically-relevant experimental system, we employed a mouse model of acute fasciolosis [29] to quantify the amount of juvenile flukes that successfully migrate to the livers of control mice and mice whose fibrinolytic route had been pharmacologically inhibited prior to challenge with \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Due to technical constraints, the experiment was performed across two consecutive days, employing five mice per group per day. Statistical analysis using two-way ANOVA revealed that the day of the experiment gave statistically significant differences in FhNEJ counts (\u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/strong\u003e), as PAI-1-treated mice from day 1\u0026mdash;but not control mice\u0026mdash;exhibited significantly different amounts of liver-migrating flukes than their counterparts from day 2 (\u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/strong\u003e). Therefore, PAI-1-treated mice could not be treated as absolute replicates and data was subsequently plotted and analysed separately for each day. This analysis revealed that PAI-1 treatment prior to \u003cem\u003eF. hepatica\u003c/em\u003e infection resulted in a significant decrease in the number of juvenile flukes recovered from the livers of infected mice eight days after challenge with \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae, but only in the experiment conducted on day 1 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). To assess potential differences in PAI-1 treatment efficacy across different experimental days, we measured the levels of fibrinolysis in the peritoneal fluids of all mice, which were collected immediately after euthanasia. However, no statistically significant differences were observed in either PAI-1 or t-PA activities between control and PAI-1-treated mice on any experimental day (\u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAiming to understand the contribution of the fibrinolytic system in biological processes that might facilitate FhNEJ migration, commercially-available primary intestinal epithelial cells derived from the mouse small intestine (mPSIEC) were stimulated with FhNEJ [27,28] and PLG for 24 hours, after which cell culture supernatants and mPSIEC whole-cell lysates were harvested for downstream analyses. Additionally, mPSIEC left unstimulated or stimulated with either FhNEJ or PLG alone served as negative controls, and an additional subset of mPSIEC stimulated with FhNEJ, PLG, and the lysine analogue ε-ACA was used to assess whether plasmin generation depends on the presence of lysine residues in FhNEJ-derived PLG-interacting proteins, as previously described [14]. Although FhNEJ take only a few hours to cross the intestinal wall [19], we extended the stimulation period to up to 24 hours, as previously described [28], to account for potential delays in cellular responses due to the artificial and reductionist nature of the co-culture system.\u003c/p\u003e \u003cp\u003eFirst, our enzymatic assays revealed that cell culture supernatants from mPSIEC stimulated with both FhNEJ and PLG showed increased plasmin activity compared to those from control or single-treated cells, indicating that FhNEJ stimulate plasmin generation in the pericellular space of the mouse small intestine in the presence of host PLG. Since FhNEJ cannot directly cleave and activate PLG [14], and mPSIEC were stimulated in the absence of exogenous addition of either t-PA or u-PA, these results suggest that mPSIEC express the PLG activator(s) required to convert PLG into plasmin, which might have important physiological and pathological implications. Notably, FhNEJ-induced plasmin generation was completely abrogated in the presence of ε-ACA, indicating that this phenomenon depends on the interaction between lysine residues present in PLG-interacting proteins expressed by FhNEJ and the PLG kringle domains, replicating the mechanism of PLG activation utilised by physiologic PLG-Rs.\u003c/p\u003e \u003cp\u003eSince u-PA is expressed by epithelial cells of the small intestine, presumably to loosen intercellular junctions and facilitate epithelial cell turnover at the top of the villi [42], we studied whether the combination of FhNEJ and PLG would increase the availability of this PLG activator in the mPSIEC extracellular space. Results revealed that cell culture supernatants of mPSIEC stimulated with both FhNEJ and PLG contained significantly more u-PA than untreated or single-treated cells, suggesting that plasmin generation in the pericellular space may initiate a positive feedback loop that enhances u-PA release by double-treated cells. These findings indicate that plasmin may enhance fibrinolysis within the small intestinal micro-environment through two complementary mechanisms: the well-established ability of this protease to cleave and activate the zymogen form of u-PA [43,44] and the stimulation of u-PA secretion by intestinal cells, as demonstrated in the present study.\u003c/p\u003e \u003cp\u003ePlasmin is considered a master regulator of cell migration through the degradation of different ECM components [5,6], including collagen type IV and laminin [11,45], two major structural proteins of the basement membrane underlying the intestinal epithelium [25]. To determine whether FhNEJ-mediated plasmin generation enhances the degradation of intestinal ECM components, we quantified the levels of total collagen in cell culture supernatants of mPSIEC incubated with FhNEJ, PLG, or both. Decreased collagen levels in cell culture supernatants was interpreted as increased degradation of this ECM protein, as previously described [46]. Our results revealed that collagen levels slightly decrease in mPSIEC supernatants treated with either FhNEJ or PLG, but the reduction becomes significant only when both are combined, indicating that FhNEJ-induced plasmin generation promotes collagen degradation. This notion is supported by the observation that plasmin is capable of degrading the ECM coating solution employed for mPSIEC culture. Although the interaction between helminth parasites and the host fibrinolytic system is well-established and has long been proposed to facilitate tissue migration through enhanced ECM degradation [1\u0026ndash;3], to our knowledge, this is the first experimental demonstration of increased ECM degradation resulting from parasite-induced plasmin generation in a trematode, as this phenomenon had only been shown in the nematode \u003cem\u003eDirofilaria immitis\u003c/em\u003e [46]. Considering the potent collagenolytic properties of FhCL3 [47,48], which is highly expressed and secreted by FhNEJ [49], the residual, non-statistically significant collagen degradation that we detected in cell culture supernatants from mPSIEC only treated with FhNEJ is somewhat unexpected. However, this could be explained by the fact that the commercial kit that we used in our experiments fails to detect the specific collagen isoforms that are degraded by FhCL3, that such isoforms are not present in the ECM coating employed here to culture mPSIEC, or that the extent of collagen degradation by FhNEJ under physiologic conditions is less pronounced than that observed in \u003cem\u003ein vitro\u003c/em\u003e experiments performed in the absence of host cells [47,48].\u003c/p\u003e \u003cp\u003eIn addition to directly degrading ECM components, plasmin activates the precursor zymogens of MMP-1, -2, -3, -9, -10, -12, and \u0026minus;\u0026thinsp;13, either via direct cleavage or by activating other MMPs that, in turn, cleave and activate the MMP precursor [50\u0026ndash;57]. MMPs are highly proficient at degrading ECM components [12], and given their close relationship with the fibrinolytic system [5], we addressed whether FhNEJ-mediated plasmin generation would simulate MMP release by mPSIEC [58], which could facilitate FhNEJ trans-intestinal migration by expanding the arsenal of enzymes that are available in the extracellular space for ECM degradation. Our results showed that FhNEJ substantially stimulate the secretion of MMP-3, -8, -12, and pro-MMP-9 by mPSIEC, a response that was not further stimulated in the presence of PLG, indicating that FhNEJ-induced plasmin generation does not affect the secretion of these enzymes by host cells. Since MMP-like enzymes in the FhNEJ secretome constitute only 0.04% of the total protein content [59], we believe that their contribution to the measured MMP levels in cell culture supernatants is likely negligible. Regardless of MMP secretion, it remains to be addressed whether FhNEJ-induced plasmin generation in the intestinal epithelium facilitates the activation of MMP precursors that could contribute to the observed collagen degradation and ultimately facilitate the migration of these parasites across the host intestine.\u003c/p\u003e \u003cp\u003eSince plasmin influences biological processes not only by regulating the activity of proteolytic enzymes but also by modulating intracellular signalling pathways that culminate in changes in gene expression [60\u0026ndash;63], we conducted a proteomic analysis on whole-cell lysates of mPSIEC treated with both FhNEJ and PLG to investigate how these cells respond to elevated plasmin levels in the pericellular space. To this end, we applied a stringent statistical pipeline [33] combined with an analysis of interaction effects to identify proteins with statistically different abundances in FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated mPSIEC compared to untreated cells that are specific to the combination of FhNEJ and PLG. This analysis included elastic-net penalised regression, which has emerged as a powerful and validated approach for robust feature selection, enabling the reliable identification of condition-specific gene and protein expression signatures in diverse biomedical fields [64\u0026ndash;67]. Additionally, differential expression was performed with the limma package, which applies a combination of statistical principles that outperform ordinary \u003cem\u003et\u003c/em\u003e-tests in proteomics data analysis when sample sizes are small [36,68]. In parallel to this, we also compared the proteomes of FhNEJ-treated and FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated mPSIEC to reduce the overall variability and enhance the detection of subtle but biologically meaningful proteomic changes specifically induced by the combined action of FhNEJ and PLG that may be overlooked in the broader untreated vs. FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated contrast. Supporting the accuracy of this approach, only the comparison between FhNEJ-treated and FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated cells captured PLG as a significantly abundant protein in whole-cell lysates of double-treated mPSIEC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Given that mPSIEC were thoroughly washed before harvesting, we assume that the amount of residual PLG in these samples was negligible, reinforcing that comparing the protein expression profiles of FhNEJ-treated and FhNEJ\u0026thinsp;+\u0026thinsp;PLG-treated cells effectively fine-tunes the analysis to detect subtle proteomic shifts that are undetectable in the untreated vs. FhNEJ\u0026thinsp;+\u0026thinsp;PLG differential expression analysis.\u003c/p\u003e \u003cp\u003eSome of the DEPs resulting from the interaction between FhNEJ and PLG are involved in biological processes that could be beneficial to FhNEJ trans-intestinal migration and survival within the mammalian organism, including cell adhesion and migration, cytoskeleton dynamics, ECM remodelling, mucosal immunity, and fibrinolysis. For instance, we identified Cap1, serpin b5, Rab3b, and Actn4 as differentially regulated upon mPSIEC stimulation with both FhNEJ and PLG. Cap1 is involved in cell adhesion and motility through the regulation of actin cytoskeleton dynamics [69], serpin b5 is a non-inhibitory serpin that regulates u-PA/u-PAR-mediated cell adhesion [70], and Rab3b is required for the distribution of the major tight junction (TJ) adaptor protein zonula occludens 1 to the apical membrane of epithelial cells [71,72]. Similarly, depletion of Actn4 inhibits the recruitment of occludin to TJs, thereby disrupting the formation of functional intercellular junctions [73]. Differential expression of these proteins in mPSIEC stimulated with both FhNEJ and PLG indicates that plasmin might regulate parasite adhesion to the intestinal epithelium and modulate epithelial junction dynamics, potentially compromising epithelial barrier integrity and facilitating FhNEJ migration across the host intestine.\u003c/p\u003e \u003cp\u003eAdditionally, some of the DEPs identified in mPSIEC incubated with both FhNEJ and PLG play important roles in ECM remodelling. Among others, we found differential expression of Mmp11 and Timp3 in double-treated cells, suggesting that FhNEJ-induced plasmin generation alters the protease/anti-protease balance of the MMP system to ultimately regulate ECM degradation. Overexpression of Mmp-11 is particularly compelling as this metalloproteinase degrades collagen type IV [74], a major structural component of the intestinal basement membrane [25]. In the context of ECM remodelling, it is noteworthy that we also detected downregulation of the ECM protein fibronectin in whole-cell lysates of mPSIEC incubated with both FhNEJ and PLG. Decreased Fn1 expression in double-treated mPSIEC aligns with Mmp-11 overexpression in these cells, as MMP-11 was shown to supress fibronectin gene expression in an \u003cem\u003ein vivo\u003c/em\u003e zebrafish model of ECM remodelling [75]. Moreover, the overexpression of Qsox1, which is required for the incorporation of laminin into the ECM [76], provides further evidence that FhNEJ regulate tissue integrity through the modulation of ECM dynamics/deposition in a plasmin(ogen)-dependent manner.\u003c/p\u003e \u003cp\u003eAnother notable functional aspect of the interaction between FhNEJ and PLG, highlighted by our proteomic analysis, is the identification of proteins linked to host immune responses and, potentially, to FhNEJ immune evasion mechanisms. Among others, we found downregulation of Sema3d, Pigr, Chst2, and the complement molecule C7 in mPSIEC treated with both FhNEJ and PLG. Sema3d has been recently shown to promote pancreatic cancer progression by stimulating macrophage polarization into a protumourigenic M2 phenotype [77]. Since M2 polarization is a hallmark of anti-\u003cem\u003eFasciola\u003c/em\u003e immune responses [78], this finding offers novel insights into how these responses might be regulated at the intestinal level during infection with FhNEJ. In the context of FhNEJ trans-intestinal migration, the downregulation of Pigr in mPSIEC upon stimulation with FhNEJ and PLG is particularly noteworthy, as this protein is a key regulator of mucosal immunity by facilitating the translocation of immunoglobulin A (IgA) to the apical compartment of the intestinal epithelium [79]. In fact, mice lacking Pigr expression show increased bacterial infiltration into the liver due to impaired IgA-mediated antimicrobial defence in the gut [80]. In line with this, the downregulation of Chst2 upon stimulation with FhNEJ and PLG may also represent a mechanism to evade mucosal immunity, as this protein is involved in lymphocyte homing to peripheral lymphoid organs and presumably to inflamed sites by modulating the synthesis of carbohydrate ligands that interact with L-selectin molecules expressed on the lymphocyte surface [81,82].\u003c/p\u003e \u003cp\u003eThe complement molecule C7 is essential for the assembly of the lethal, pore-forming membrane attack complex, a key effector in antibacterial defence. Previous studies had shown that \u003cem\u003eF. hepatica\u003c/em\u003e expresses molecules that inhibit complement proteases, thereby evading complement-mediated responses [83]. To the best of our knowledge, the present study provides the first evidence that FhNEJ modulate the expression of C7 in mouse intestinal cells, an effect observed only upon co-stimulation with both FhNEJ and PLG. Intriguingly, we also observed a downregulation of the complement protein C3 in FhNEJ-treated mPSIEC compared to untreated controls (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), suggesting that FhNEJ modulate the expression of complement molecules through plasmin(ogen)-dependent and independent mechanisms. The S1pr2 is another immune-related protein that we identified as differentially regulated in double-treated mPSIEC. This protein regulates the onset of type 2 immune responses in the lung by inhibiting the secretion of interleukin 33 (IL-33) by lung macrophages [84]. IL-33 is also secreted by intestinal epithelial cells and has recently been shown to represent an indispensable trigger of intestinal type 2 immune responses that lead to successful gastrointestinal nematode expulsion [85]. Based on this, the observed S1pr2 overexpression in mPSIEC upon stimulation with both FhNEJ and PLG may serve to reduce IL-33 release by intestinal epithelial cells and suppress the onset of anti-helminth immunity in the intestine, providing an intriguing direction for future investigation.\u003c/p\u003e \u003cp\u003eMoreover, our proteomic analysis indicated that plasmin itself might also be regulating fibrinolysis in the small intestine tissue microenvironment, as double-treated mPSIEC differentially regulate the expression of two major fibrinolysis inhibitors: A2m and TAFI, which were downregulated and overexpressed, respectively, in mPSIEC upon stimulation with FhNEJ and PLG. This finding suggests that fine-tuning fibrinolysis may benefit the parasite by balancing proteolytic activity such that it facilitates tissue penetration while preventing excessive ECM degradation, which could inadvertently lead to severe tissue damage and exacerbated inflammatory responses in the intestine.\u003c/p\u003e \u003cp\u003eFinally, we employed a mouse model of acute fasciolosis [29] to conclusively determine whether the fibrinolytic system is involved in the intra-mammalian migration of \u003cem\u003eF. hepatica\u003c/em\u003e juveniles during the early infection. To that end, mice were injected with either PBS or PAI-1 to inhibit the fibrinolytic route prior to infection with \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae. We utilised a recombinant and stable-mutant version of PAI-1, as previously described [40], because the wild-type inhibitor has a very short half-life of approximately two hours [86]. Eight days post-infection was selected as the time point for quantifying infection success based on previous studies indicating that juvenile recovery from the livers of infected mice is highest at this time [29]. Results showed that the number of liver-migrating flukes in PAI-1-treated mice was significantly lower than that observed in control mice, but only in the experiment conducted on day 1.\u003c/p\u003e \u003cp\u003eSince differences in juvenile counts across experimental days were observed only in PAI-1-treated mice, but not in their control counterparts, we suspected that variations in PAI-1 treatment efficacy between days might explain these results. To investigate this possibility, fibrinolysis was evaluated by measuring the levels of active PAI-1 and t-PA in peritoneal fluids collected immediately after euthanasia. Although the differences in active PAI-1 and t-PA levels between PAI-1-treated and control mice on day 1 were more consistent with the expected inhibition of fibrinolysis than those observed in mice from day 2, these differences were small and did not reach statistical significance. This is likely due to the fact that peritoneal fluids were collected eight days after PAI-1 treatment, exceeding the maximum half-life of six days achieved for this factor through random mutagenesis [86]. Altogether, these results highlight the complexity of working with genetically diverse organisms such as \u003cem\u003eF. hepatica\u003c/em\u003e, which likely constitutes one of several factors contributing to the inconsistent and variable outcomes reported in vaccine trials over the past decades [87,88]. This variability not only hinders data interpretation but also raises ethical concerns regarding the use of large animal cohorts to overcome inter-individual heterogeneity and meet significance thresholds that are inherently arbitrary. Notwithstanding these points, these findings provide unprecedented insights into the role of the fibrinolytic system in FhNEJ migration \u003cem\u003ein vivo\u003c/em\u003e, supporting the long-standing hypothesis that FhNEJ-stimulated plasmin generation from host PLG contributes to the dissemination of \u003cem\u003eF. hepatica\u003c/em\u003e juveniles through mammalian tissues.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study demonstrates that plasmin is generated in the pericellular space of mouse intestinal epithelial cells in the presence of FhNEJ and host-derived PLG. This process enhances collagen degradation and u-PA release into the extracellular environment, potentially facilitating FhNEJ migration across the intestinal wall. Additionally, FhNEJ-induced plasmin generation triggers changes in the expression levels or abundance of proteins involved in biological processes that could be critical during the earliest stages of infection, including cell adhesion and migration, ECM remodelling, immune evasion, and fibrinolysis. Finally, despite the inherent challenges associated to \u003cem\u003ein vivo\u003c/em\u003e models of \u003cem\u003eF. hepatica\u003c/em\u003e infection, our findings provide the first functional evidence supporting the long-proposed role of the fibrinolytic system in facilitating the migration of \u003cem\u003eF. hepatica\u003c/em\u003e juveniles through mammalian tissues. Collectively, these results underscore the contribution of the fibrinolytic system in parasite dissemination during early-stage fasciolosis, providing interesting insights into host-parasite relationships established in the initial stages of infection and highlighting potential avenues for future research in this area.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eActn4: actinin-4; ANOVA: analysis of variance; A2m: alpha-2 macroglobulin; Cbp2: carboxipeptidase; Chst2: carbohydrate sulfotransferase 2; C7: complement molecule 7; DEP: differentially expressed protein; ECM: extracellular matrix; ELISA: enzyme-linked immunosorbent assay; FC: fold change; FDR: false discovery rate; FhNEJ: \u003cem\u003eF. hepatica\u003c/em\u003e newly excysted juvenile; Fn1: fibronectin 1; IgA: immunoglobulin A; IL-33: interleukin 33; IQR: interquartile range; MMPs: matrix metalloproteinases; MMP11: matrix metalloproteinase 11; mPSIEC: mouse primary small intestinal epithelial cells; MS/MS: tandem mass spectrometry; PBS: phosphate-buffered saline; Pigr: polymeric immunoglobulin receptor; PLG: plasminogen; PLG-R: plasminogen receptor; PLS-DA: partial least squares discriminant analysis; pro-u-PA: precursor of the urokinase-type plasminogen activator; Qsox1: quiescin sulfhydryl oxidase 1; Sema3d: semaphorin 3D; SDS: sodium dodecyl sulphate; SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SD: standard deviation; SEM: standard error of the mean; S1pr2: sphingosine 1-phosphate receptor 2; u-PA: urokinase-type plasminogen activator; TAFI: thrombin activatable fibrinolysis inhibitor; Timp3: tissue inhibitor of metalloproteinase 3; TJ: tight junction; \u0026epsilon;-ACA: 6-aminocaproic acid.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJS planned and carried out all the experiments, analysed the data, and wrote the main manuscript text. MT provided technical assistance with the experiments. MLG, VMH, and MTRC contributed to the conception and implementation of the in vivo mouse model. MSL interpreted the results and was involved in their planning. JGM conceived the study, secured funding, planned the experiments, and contributed to their analysis and interpretation. All authors participated in revising the manuscript and have approved the submitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eJGM acknowledges funding received from projects ULYSSES (RTI2018-093463-J-100) and PERSEUS (PID2023-152150OB-C21) funded by MICIU/AEI/10.13039/501100011033 and FEDER, EU, and URANUS (CNS2022-135561) funded by MICIU/AEI/10.13039/501100011033 and NextGenerationEU/PRTR. JS acknowledges the support of the Junta de Castilla y Le\u0026oacute;n for her Predoctoral contract. MLG acknowledges the support of the Spanish Ministry of Science and Innovation for her FPU Predoctoral contract. We thank Miguel \u0026Aacute;ngel S\u0026aacute;nchez Luengo and Teresa Osteso Ib\u0026aacute;\u0026ntilde;ez (CNB-CSIC, Madrid, Spain) for their support with the multiplexed experiment, Raquel Gavidia (SCSIE, Valencia, Spain) for the statistical analysis of proteomics data, and Laura Barrios (Department of Applied Statistics, CSIC, Madrid, Spain) for her valuable advice on how to analyse the in vivo data.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDiosdado A, Sim\u0026oacute;n F, Serrat J, Gonz\u0026aacute;lez-Miguel J. Interaction of helminth parasites with the haemostatic system of their vertebrate hosts: a scoping review. Parasite. 2022;29:35.\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez-Miguel J, Siles-Lucas M, Kartashev V, Morch\u0026oacute;n R, Sim\u0026oacute;n F. 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Vaccines. 2022 Jan 20;10(2).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fasciola hepatica, fibrinolysis, plasminogen, plasmin, migration","lastPublishedDoi":"10.21203/rs.3.rs-6822677/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6822677/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eFasciola hepatica\u003c/em\u003e is the most common etiologic agent of fasciolosis, a parasitic disease that affects millions of ruminants worldwide and a zoonotic human infection of public health concern. Upon ingestion of infective metacercariae, \u003cem\u003eF. hepatica\u003c/em\u003e newly excysted juveniles (FhNEJ) emerge in the duodenum and cross the intestinal wall to initiate a migration route that culminates with their establishment within the hepatic bile ducts. The ability of FhNEJ to exploit the broad-spectrum activities of host plasmin, the central protease of the fibrinolytic system, has been proposed as a strategy employed by these parasites to migrate across the intestinal wall while minimising energy expenditure.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMouse intestinal epithelial cells (mPSIEC) were stimulated with FhNEJ and plasminogen (PLG), the zymogen of plasmin, to understand whether FhNEJ-stimulated plasmin generation modulates processes relevant to parasite migration through the intestinal wall, including extracellular matrix (ECM) degradation and the secretion of ECM-degrading enzymes. Plasmin-mediated cellular responses were further examined by proteomic analysis of mPSIEC whole-cell lysates. In parallel, the contribution of the fibrinolytic system in FhNEJ migration was studied \u003cem\u003ein vivo\u003c/em\u003e by infecting mice with \u003cem\u003eF. hepatica\u003c/em\u003e metacercariae following pharmacological inhibition of fibrinolysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCo-stimulation of mPSIEC with FhNEJ and PLG led to increased plasmin generation in the intestinal pericellular space, which was associated with enhanced collagen degradation and secretion of the urokinase-type plasminogen activator (u-PA). In addition, using independent cell culture replicates and a stringent statistical pipeline, we identified a robust set of differentially expressed proteins in mPSIEC following stimulation with FhNEJ and PLG. These proteins were involved in cell adhesion, migration, ECM remodelling, immune evasion, and fibrinolysis. Despite inter-experimental variability, FhNEJ migration in mice was reduced upon pharmacological inhibition of fibrinolysis, supporting the contribution of host fibrinolysis to parasite invasion \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAltogether, this work provides unprecedented insights into the role of the host fibrinolytic system to FhNEJ migration across mammalian host tissues, thereby advancing our understanding of host-parasite relationships during early-stage fasciolosis and highlighting interesting directions for future research in this area.\u003c/p\u003e","manuscriptTitle":"Early host-parasite interaction models reveal a key role for fibrinolysis in Fasciola hepatica intestinal migration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-05 08:41:30","doi":"10.21203/rs.3.rs-6822677/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-17T17:07:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-17T16:48:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T17:33:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332056222194191886665142898880510249625","date":"2025-06-26T13:44:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156253626973716272041560351169459967697","date":"2025-06-25T11:26:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-24T21:04:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-07T06:23:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-07T05:54:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-06-04T16:58:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6770efa4-0ffb-4222-b14c-70329c592d54","owner":[],"postedDate":"June 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:02:04+00:00","versionOfRecord":{"articleIdentity":"rs-6822677","link":"https://doi.org/10.1186/s13071-025-06992-9","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-09-24 15:58:09","publishedOnDateReadable":"September 24th, 2025"},"versionCreatedAt":"2025-06-05 08:41:30","video":"","vorDoi":"10.1186/s13071-025-06992-9","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06992-9","workflowStages":[]},"version":"v1","identity":"rs-6822677","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6822677","identity":"rs-6822677","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}