Modulation of dextran sodium sulfate-induced colitis in germ-free mice by Enterococcus faecalis-monocolonization

Modulation of dextran sodium sulfate-induced colitis in germ-free mice by Enterococcus faecalis-monocolonization | 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 Article Modulation of dextran sodium sulfate-induced colitis in germ-free mice by Enterococcus faecalis-monocolonization Beate Vestad, Petra Hanzely, Indrė Karaliūtė, Oda Ramberg, Jurgita Skiecevičienė, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6871860/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are characterized by chronic gastrointestinal inflammation and involve complex interactions of genetic, environmental, and immune factors. Enterococcus faecalis , a gut commensal bacterium, has been implicated in IBD pathogenesis. This study investigated the effects of E. faecalis -monocolonization in germ-free (GF) mice subjected to dextran sulfate sodium (DSS)-induced colitis. We assessed the impact of E. faecalis on colitis severity, inflammation, and intestinal barrier function. In the context of DSS, E. faecalis -colonized mice exhibited reduced anemia and lower fecal calprotectin levels, though fecal albumin levels were elevated. Despite translocation of E. faecalis to mesenteric lymph nodes, no systemic dissemination was observed. Histological analysis showed similar inflammatory patterns in DSS-treated mice, regardless of E. faecalis- colonization, but more severe mucosal damage was noted in the colonized mice. These findings support that E. faecalis plays a dual role in modulating colitis, influencing inflammation, and exacerbating epithelial injury. The study highlights the utility of GF models in understanding microbial contributions to IBD and emphasized how specific bacterial strains may influence disease progression through strain-dependent interactions with the host immune system and intestinal barrier. Further research is needed to elucidate these complex mechanisms. Biological sciences/Immunology/Mucosal immunology Biological sciences/Microbiology/Pathogens Biological sciences/Microbiology/Applied microbiology Biological sciences/Microbiology/Bacteria/Bacterial host response Inflammatory bowel disease (IBD) bacterial translocation epithelial barrier dysfunction host-microbe interactions gut microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Inflammatory bowel diseases (IBD), encompass Crohn’s disease (CD) and ulcerative colitis (UC), both characterized by chronic gastrointestinal inflammation [ 1 ]. The pathogenesis of IBD involves a complex interplay of genetic susceptibility, environmental factors, and inappropriate immune activation [ 2 ]. This immune dysregulation includes abnormal activation of innate and adaptive immunity, with pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 contributing to mucosal damage and perpetuating the inflammatory cycle [ 2 – 4 ]. Gut microbiota imbalance is also implicated, with certain bacterial species suspected of modulating intestinal inflammation. However, the role of individual bacterial species in IBD remains poorly understood [ 2 , 4 ]. Germ-free (GF) mice are invaluable tools for studying host-microbiota interactions, allowing researchers to isolate the effects of specific microbes or defined communities on disease processes [ 5 , 6 ]. In the dextran sulfate sodium (DSS)-induced colitis model, oral administration of DSS in drinking water induces severe colitis that mimics key features of human UC, including mucosal ulceration, epithelial barrier disruption, and inflammatory cell infiltration [ 7 , 8 ]. Mice undergoing DSS treatment typically exhibit weight loss, bloody diarrhea, and reduced activity, paralleling clinical symptoms of UC. While the precise mechanisms of DSS toxicity remain unclear, one hypothesis is that the sulphated, negatively charged DSS molecule interacts with dietary medium-chain fatty acids, forming complexes absorbed by colonic epithelial cells that contribute to barrier disruption and inflammation [ 9 ]. Other mechanisms, such as direct epithelial toxicity and activation of immune pathways, are also likely to play a role [ 9 – 12 ]. Unlike human UC, the development of DSS-induced colitis does not require adaptive immune cells, making this model particularly suitable for investigating the role of innate immune responses in intestinal inflammation [ 10 ]. The model is also useful for exploring microbial contributions to colitis progression, as the specificity of DSS to the colon is thought to depend on bacterial activity and local physiological factors such as water and electrolyte absorption [ 7 , 10 ]. Studying monocolonized mice provides a unique opportunity to investigate the effects of individual bacterial strains, helping to delineate their specific contributions to intestinal inflammation and immune responses [ 13 ]. Enterococcus faecalis ( E. faecalis ) is a gram-positive bacterium normally residing in the human gastrointestinal tract. While often considered a commensal organism, E. faecalis is associated with various infections and has been implicated in IBD pathogenesis [ 14 , 15 ]. Previous studies have highlighted the capacity of E. faecalis to translocate across the intestinal barrier, migrate to lymph nodes and disseminate to other tissues. Virulence factors such as the aggregation substance (Agg) and the extracellular surface protein (Esp) are hypothesized to enhance its pathogenicity by increasing surface hydrophobicity, promoting colonization, and evading immune responses [ 14 , 16 , 17 ]. Additionally, secreted hydrolytic enzymes like hyaluronidase may contribute to tissue damage and bacterial spread [ 18 , 19 ]. Although E. faecalis has been shown to modulate inflammation in murine colitis models, most studies have utilized conventional or genetically modified mice with chronic colitis [ 20 , 21 ]. The effect of E. faecalis -monocolonization in acute murine colitis is still underexplored and offers to study the specific effects of an individual bacterial species. In the present study, we investigated the effects of E. faecalis -monocolonization in GF mice subjected to DSS-induced colitis. We aimed to assess whether the presence of E. faecalis alters colitis severity and general disease status, focusing on physical, histological, hematological, and intestinal markers of inflammation and barrier function. Methods Animals Germ-free C57BL/6J mice, originating from the University of Bern Clean Mouse Facility, were bred in open cages (Eurostandard type II, 11bbB, Tecniplast, Buguggiate, Italy) maintained in sterile flexible-film isolators. At 5 weeks of age, the mice were earmarked and exported into an SPF facility in autoclaved GM500 individually ventilated cages (IVC) (Tecniplast) with bedding and nesting material. The animals were acclimatized for 7 days before they underwent monocolonization or remained GF as controls. GF status in the isolator was confirmed by monthly aerobic and anaerobic culture of fecal pellets and mold trap samples from the isolator, as well as yearly PCR-based serology testing according to FELASA recommendations [ 22 ]. In the IVC cages, fecal pellets were cultured weekly during the experiment to monitor sterility and monoculture status of the animals. The animals had ad libitum access to autoclaved chow pellets (LabDiet 5021, IPS Products Supplies, Alfreton, Great Britain) and water. Mice were kept in a humidity and temperature-controlled environment on a 12/12-hour day/night cycle at an approved animal facility at the Oslo University Hospital, Rikshospitalet. The mice were segregated by sex and housed 2–3 mice in each cage during colonization, as well as before and during DSS administration. Cage change and experimental procedures were performed using sterile equipment by one sterile and one unsterile operator by strict aseptic handling of the cages inside of a laminar air flow changing station [ 23 ]. At the end of experiment, the mice were euthanized by heart puncture under isoflurane anesthesia followed by cervical dislocation and harvesting of organs. Monocolonization Freshly prepared cultures of a single Enterococcus faecalis isolate retrieved from the feces of a male patient in his 60-ies with ulcerative colitis in remission was used for colonization. The patient was part of a cohort included in the Norwegian PSC (primary sclerosing cholangitis) Research Center biobank at Oslo University Hospital. Genomic data, including putative virulence factors and antimicrobial resistance genes are available in the Supplementary Material. E. faecalis was cultured on blood agar at 37°C, 5% CO 2 for 48–72 hours. The bacterial stock solution was prepared by adding 20–30 middle big colonies into 1 mL of sterile PBS, equal to an optical density of 1.5–1.6 (2 x 10 9 colony-forming units (CFU)/mL). An additional 3 mL PBS was added to make a final colonization solution of approximately 5 x 10 8 CFU/mL. At 6 weeks of age, mice were colonized by slowly injecting 200 µL of the E. faecalis solution (10 8 CFU) in a single dose via rectal administration using a gavage needle (18G) during manual restraint. After 21 days, a subset of the colonized mice was subjected to DSS administered in autoclaved drinking water for 7 days. Noncolonized GF mice with DSS administration served as colitis controls, whereas colonized GF mice without DSS served as colonization controls. DSS colitis model Dextran sulfate sodium (Cat no. DB001, Batch no. DB001-47, 35866 kDa MW, TdB Labs AB, Uppsala, Sweden) was administered to 9-week-old mice at 2.5% concentration (w/v) in autoclaved drinking water for 7 days to induce acute colitis. Start of DSS dosing was defined as day 0. During DSS administration, the mice were monitored daily with measurements of food and water intake (data not shown), body weight, and scoring according to disease activity. Mice were euthanized according to humane endpoint (HEP) scoring in cases of weight loss above 20% from DSS start, severe rectal prolapse, isolated behavior or reaching HEP score of 14 or higher (Supplementary Table 1). DSS-induced colitis development was evaluated by calculating a separate Disease Activity Index (DAI) score based on weight loss, stool consistency, and rectal bleeding (modified from [ 24 , 25 ]). The DAI was calculated as the average of the total scores consisting of: weight loss (0: 0–5%; 1: 6–10%; 2: 11–15%; 3: 16–20%; 4: >20%), stool consistency (0: none; 2: loose stools; 4: gross diarrhea), and rectal bleeding (0: normal; 2: mild bleeding, visible in stool; 3: moderate bleeding, visible from rectum and/or in bedding/cage; 4: gross bleeding, marked staining in bedding/cage). Sample collection and processing Fresh fecal samples were collected before and after DSS administration for analysis of albumin and calprotectin and kept on ice until storing at -20°C. Blood was collected by cardiac puncture under isoflurane anesthesia into a syringe coated with 0.5M EDTA using a 23G needle after sterilizing the insertion site with 70% ethanol. Mesenteric lymph nodes were collected by aseptic technique and cultured to monitor for bacterial translocation. Briefly, 2–3 lymph nodes were homogenized in 100–200 µL PBS, and 100 µL of the homogenate were cultured on blood agar plates overnight at 37°C, 5% CO 2 . Subsequently, liver, spleen and cecum were collected and weighed. Colon length was measured before the entire colon was processed for histology assessment by “swiss roll” preparation on a 27G needle. Fecal pellets and cecal content for DNA extraction were snap frozen on dry ice and kept at -80°C. Blood plasma was prepared by centrifugation of whole blood twice at 2500 x g for 15 minutes. Hematological parameters Determination of hematological parameters were performed in 10 µL fresh EDTA-blood using an ABX Micros 60 automated hematology instrument (Horiba ABX SAS, Montpellier, France). Histology Tissue sections were fixed in 4% PFA for 18 hours and placed in cold PBS, then paraffin embedded, cut, and stained with hematoxylin and eosin. Two different sections were evaluated per sample, taken 15 µm apart. Sections were scanned using Olympus SLIDEVIEW™ VS200 Slide scanner (Olympus Corporation, Tokyo, Japan) and images processed using QuPath 0.4.3 software [ 26 ]. The images were examined and scored with regards to colitis by one of the authors (B.V.) blinded to group allocations. The colon sections were divided into three main scoring regions: proximal colon, middle colon and distal colon. Histology assessment of the following parameters; I: mononuclear cell infiltration, II: polymorphonuclear cell infiltration, III: epithelial hyperplasia, as well as IV: epithelial injury, comprising visible erosion, ulcerations and alterations to the crypt architecture, were scored for each region of the colon as absent (0), mild (1), moderate (2), or severe (3): [ 27 , 28 ]. A total colon score was created by calculating an average of the three colonic region scores. Albumin and calprotectin measurements Fecal albumin was analyzed using Mouse Albumin ELISA Kit (Cat E99-134, Bethyl Laboratories, Montgomery, Texas, USA) and fecal calprotectin was analyzed using the S100A8/S100A9 ELISA Kit (Ref. KR6936, Immundiagnostik AG, Bensheim, Germany), according to the manufacturer’s recommendations. Briefly, fecal pellets were diluted 1:10 with dilution buffer from the albumin kit, placed on ice for 15 minutes and thoroughly homogenized before centrifugation at 13,000 RPM for 5 minutes at 4°C. The supernatant was collected and used for albumin measurements. The remainder of the sample material was further diluted 1:5 with extraction buffer from the calprotectin kit and centrifugated at 2,000 x g for 10 minutes at 4°C. The supernatant was collected and used for calprotectin measurements. Albumin and calprotectin results were obtained using a BioTek Synergy H1 Hybrid plate reader (Agilent, Santa Clara, California, USA). DNA extraction and real-time quantitative PCR of E. faecalis DNA was extracted from one pellet of mouse feces (20–60 mg) or 200 µL of cecal content per mouse using the Genesig® Easy DNA/RNA extraction Kit (Primerdesign™ Ltd, Eastleigh, United Kingdom) and mouse plasma DNA was extracted using the QIAamp MinElute ccf DNA Kit (Cat 55284, Qiagen, Hilden, Germany), following the manufacturer’s recommendations. A template DNA input of 1 ng/µL was used per reaction, diluted to 8µL volume with DNase/RNase free H 2 O. Copy numbers of Enterococcus faecalis DNA was then quantified using the Enterococcus faecalis qPCR Test Kit (YouSeq Ltd, Winchester, United Kingdom) and a Stratagene Mx3000P real-time PCR cycler and MxPro software (Agilent). The PCR cycling conditions used were: 3 minutes hot start at 95°C followed by 45 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Fluorogenic data was collected both through the FAM ( E. faecalis /sample) and HEX (supplied endogenous control) channels. Statistical analysis Differences between two groups were analyzed using two-tailed unpaired t test for parametric data or two-tailed Mann-Whitney U test for nonparametric data. Comparisons between three or more groups were analyzed by one-way ANOVA, reporting adjusted p-value for multiple comparisons using Bonferroni’s correction. Body weight and disease activity index curves were analyzed either using unpaired t tests between timepoints or 2-way ANOVA using column factor for p-value calculation. Differences were considered significant when p < 0.05. All statistical analyses were performed using GraphPad Prism version 10.2.0 (Boston, Massachusetts, USA). Ethics statement Animal experiments were carried out according to the EU Directive on the protection of animals used for scientific purposes (2010/63/EU) and the Norwegian Animal research legislation. The study is reported in accordance with the ARRIVE guidelines, and all animal experiments were approved by The Norwegian National Animal Research Authority (project license no. 25770/29980/29935). The Enterococcus faecalis isolate used in the study was obtained from a patient sample that was part of the Norwegian PSC Research Biobank. Use of the biobanked patient material was covered under written informed consent, and approved by the Regional Committee for Medical and Health Research Ethics in South-Eastern Norway, with reference number 2015/2140. Results E. faecalis- monocolonization and colitis induction A total of 24 GF C57BL/6J mice (16 males and 8 females) were included in the study. To assess the effect of E. faecalis on colitis development, mice were monocolonized with E. faecalis 21 days (3 weeks) prior to administration of autoclaved drinking water with DSS (n = 11, 6 males and 5 females), or autoclaved water only (n = 7, 4 females and 3 males) for 7 days. Sterile water with DSS was provided to a control group of GF mice (n = 6 males) (Fig. 1 a). The DSS dosage applied was based on titration in a separate pilot study to ensure proper colitis development without reaching unacceptable toxicity (Supplementary Material). At the end of the experiment (day 7 after DSS start, day 35 after acclimatization start), the mice were euthanized and organs harvested. To confirm successful monocolonization or GF status, fresh fecal samples were collected one week after colonization. As expected, colonies of E. faecalis were found in fecal pellets from all colonized mice (Fig. 1 b). To further validate colonization status and evaluate potential bacterial translocation, we quantified E. faecalis DNA copy numbers in samples from feces, cecum and plasma after euthanasia of the animals. In 1 ng of total DNA, we detected a median of 40,000 copies in feces of colonized mice without DSS treatment with a clear trend towards lower numbers in the feces of colonized mice with DSS treatment (p = 0.06) (Fig. 1 c). E. faecalis was also consistently detected in all cecum content samples from colonized mice, however in much lower copy numbers (median around 3,000 copies for colonized mice only and 150 copies for colonized mice treated with DSS). We could not detect any E. faecalis copies in the plasma of any of the mouse groups. This suggests that E. faecalis either did not translocate to the bloodstream, or that the DNA copy numbers were below the detection limit of our analysis. We did not detect any E. faecalis DNA in any of the samples from the GF mice receiving DSS (Fig. 1 c). E. faecalis -colonization alters colitis disease activity and physiological parameters in DSS-treated mice The mice in both DSS-treated groups experienced a clear weight loss on day 7 of DSS administration, without significant difference between the E. faecalis -colonized mice and the GF mice at harvest (Fig. 2 a). The colitis-related disease activity index increased for both groups of DSS-treated mice with significantly higher scores in the mice that were pre-colonized with E. faecalis compared to GF mice (p = 0.02; 2-way ANOVA, column factor) (Fig. 2 b). Both DSS groups had shorter colons compared to colonized mice only (p < 0.001), but with no difference between the DSS groups (Fig. 2 c). Mean cecum weight in GF mice receiving DSS was 18.5% of body weight, and it was lower in both colonized groups, although significantly reduced only in colonized mice receiving DSS (Fig. 2 d). Both DSS groups had a higher degree of anemia compared to the mice that were only E. faecalis -colonized, while the GF mice treated with DSS had lower blood hemoglobin than colonized mice treated with DSS (Fig. 2 e). No changes were observed between the groups in levels of circulating white blood cells (Fig. 2 f). DSS treatment of GF and E. faecalis- monocolonized mice induces similar histology alterations In mice colonized with E. faecalis only, we observed mild to moderate hyperplasia and a few immune cell aggregates. Some of the mice had mild edema and erosion in the mucosa while the epithelium was generally intact (Fig. 3 a). GF mice treated with DSS displayed histological features compatible with loss of crypts, erosion, edema and infiltrations of immune cells into muscle layers (Fig. 3 b). Similarly, E. faecalis -colonized mice with DSS-induced colitis showed comparable histological alterations although with somewhat more pronounced ulcerations and damaged crypt architecture (Fig. 3 c). Histological scoring of the different colonic regions revealed that inflammation and epithelial injury were more pronounced in the middle and distal colon than in the proximal colon (Fig. 3 d-f). There were no statistical differences between the two groups of DSS-treated mice for any of the colonic regions. However, comparing the two groups of colonized mice, DSS-treated mice had higher histoscores than non-DSS-treated mice (Fig. 3 d-g). E. faecalis alters fecal calprotectin and albumin levels and translocates to mesenteric lymph nodes Levels of the intestinal inflammation marker calprotectin, measured before and after DSS treatment, did not differ significantly between colonized and non-colonized mice (Fig. 4 a). However, at Day 7, calprotectin levels were notably lower in colonized mice treated with DSS compared to GF mice treated with DSS (Fig. 4 a). Fecal albumin levels increased significantly from day 0 (before DSS treatment) to day 7, with the highest levels observed in colonized mice receiving DSS (Fig. 4 b). As fecal albumin could be a marker of intestinal leakage and E. faecalis is known to translocate to lymph nodes in mice [ 29 ], we wanted to validate this in the present study. We collected 2–3 mesenteric lymph nodes (MLNs) from a subset of colonized mice during harvest, and overnight cultures revealed that E. faecalis had translocated to MLNs in all these mice independent of DSS intake (Fig. 4 c). Discussion Growing evidence suggests that manipulation of the intestinal microbiota can influence the development and progression of chronic inflammatory diseases such as IBD [ 30 – 32 ]. In this study, we used GF mice and a chemically induced colitis model to investigate the effect of monocolonization using an E. faecalis strain isolated from a patient with UC. We found that 3 weeks of monocolonization (using a single rectal dose of 10 8 CFU) followed by 7 days of 2.5% DSS administration resulted in several disease-modifying effects at the molecular level. Specifically, E. faecalis -monocolonization led to reduced anemia, lower fecal calprotectin levels, and increased fecal albumin compared to GF mice receiving DSS alone. However, no significant differences were observed in body weight, colitis-related disease activity, or colon length, suggesting that the presence of E. faecalis alone may have marginal clinical effects on colitis development in this model. We successfully detected E. faecalis DNA in the feces of all colonized mice one week after colonization and confirmed colonization in cecum and translocation to the mesenteric lymph nodes at the end of experiment, suggesting that the bacterium could migrate beyond the gut. Notably, the lower copy numbers of E. faecalis in cecal DNA compared to fecal DNA may reflect differences in host versus bacterial DNA content in the samples. The DSS-induced colitis model in experimental mice is well-documented and mimics key features of human UC, including rectal bleeding, diarrhea, and weight loss [ 8 , 10 ]. In line with these expected disease traits, the mice in present study showed shortened colons and weight loss towards the end of the experiment. Despite these indications of disease, we observed no significant differences between E. faecalis -colonized and GF mice in terms of weight loss or disease activity index, suggesting that E. faecalis had limited effects on these aspects of colitis progression. However, the reduced anemia observed in E. faecalis -colonized mice could suggest a protective role in general disease status, potentially reducing blood loss associated with colonic inflammation. This is consistent with previous studies where DSS treatment in GF mice has been linked to significant blood loss, which is often used as a marker of disease severity [ 33 , 34 ]. Monocolonization studies using probiotic or disease-associated bacteria have been shown to affect the severity of chemically induced colitis in GF mice. For example, colonization with Bacteroides fragilis has been demonstrated to protect against DSS-induced acute colitis by increasing survival and reducing immune cell infiltration and colon shortening [ 35 , 36 ]. In contrast, certain microbes have been suggested to exacerbate colitis development in chemically induced models. For example, mucosa-associated microbes from patients with UC have been shown to increase susceptibility to DSS-induced colitis in GF BALB/c mice, although they did not induce spontaneous colitis on their own [ 37 ]. Additionally, strains of E. faecalis isolated from the inflamed mucosa of UC patients are highly adherent and are likely to carry virulence-related genes, contributing to disease activity [ 38 ]. In GF mouse models of chronic colitis, E. faecalis -monocolonization has been shown to primarily induce colitis in the distal colon [ 15 , 39 ]. Our findings are consistent with this, as we observed a trend of increased histoscores in the middle and distal colon compared to the proximal colon, both in E. faecalis -colonized mice (with and without DSS treatment) and in GF mice. Notably, E. faecalis -colonization has been shown to form a uniformly distributed biofilm in the GF murine gastrointestinal tract, yet it does not appear to induce a severe inflammatory response in the GF host [ 40 ]. Our findings are in line with this and even suggest a potential beneficial effect on the host during colitis induction. Specifically, monocolonization with E. faecalis in DSS-treated mice reduced anemia and lowered fecal calprotectin levels, suggesting alleviated intestinal inflammation. However, histological analysis showed similar patterns of immune cell infiltration, inflamed areas, and immune cell aggregates in both E. faecalis -colonized and GF mice treated with DSS. Interestingly, fecal albumin levels were higher in the colonized mice receiving DSS than in GF mice receiving DSS, suggesting additional epithelial damage induced by E. faecalis . Histological analysis partially supported this, showing similar overall colonic injury with a tendency towards increased erosion and ulceration in the mucosa and submucosa. Colonization of GF mice with monocultures like E. faecalis prior to the chemical induction of acute colitis may have several effects on colitis development [ 15 ]. One possible mechanism is increased susceptibility to colitis by an enhanced inflammatory response. We found that mice colonized with E. faecalis had reduced levels of calprotectin in feces as compared to GF mice treated with DSS. In general, increased levels of fecal calprotectin are indicative of neutrophil infiltration in the gut, which corresponds with active inflammation. While direct studies on fecal calprotectin in E. faecalis -monocolonized GF mice are limited, existing research on gut inflammation markers suggests that fecal calprotectin would likely be elevated in such conditions [ 41 ]. Our findings contradict this, possibly due to strain-specific differences in E. faecalis virulence factors, which can influence the host immune response via various mechanisms. For example, some strains of E. faecalis may modulate the immune system by inducing production of anti-inflammatory cytokines such as IL-10 [ 42 , 43 ], which may lead to reduced neutrophil recruitment and activation. Additionally, a specific strain of E. faecalis has been shown to reduce the expression of pro-inflammatory cytokines like TNF-α and IL-6, which are crucial in driving neutrophil-mediated inflammation in colitis [ 44 ]. Probiotic E. faecalis strains have also been reported to enhance mucosal integrity and reduce epithelial damage in colitis models, potentially lowering fecal calprotectin levels [ 45 – 47 ]. However, most studies have been conducted in conventional hosts, and current observations suggest that Enterococcus species may provoke a more pro-inflammatory response in disease-susceptible hosts, such as gnotobiotic and IL-10 knockout mice [ 48 , 49 ]. Sequence results from the strain used in our study revealed genes for several putative virulence factors, including surface-binding proteins, degradative enzymes, adhesins and aggregation substance proteins, that may be involved in bacterial aggregation, biofilm formation, and tissue damage in the colonized mice. In summary, monocolonization of GF mice with E. faecalis prior to DSS-induced acute colitis alters disease progression and overall status, as reflected in clinical, histological, and molecular assessments, including markers of inflammation and gut barrier function. While our study addresses only a subset of the complex interactions between host immunity, microbial colonization, and chemically induced colitis, it highlights the potential role of specific bacterial species in modulating the severity of inflammatory bowel diseases. A key strength of this study is the use of GF mice and a controlled DSS model, enabling direct investigation of E. faecalis effects. However, the strain-specific nature of our findings suggests that future studies should examine a broader range of E. faecalis strains and their interactions with other microbial species to elucidate their contribution to colitis modulation and therapeutic potential. Declarations Funding The project and BV have received funding from EEA Grants 2014–2021 - Baltic Research Programme (project No LT08-2-LMT-K-01-060) under grant agreement with the Research Council of Lithuania (LMTLT). PH and JRH were funded by a grant from the European Research Council (no. 802544). Author Contribution JS, JK, HR, JRH and EM conceived the study and provided funding. BV, PH, RL, HR, JRH and EM contributed substantially to the study implementation and experiment planning. BV, PH, IK and OR performed the animal experiments. BV and PH performed laboratory analyses on mouse samples. JVB contributed with microbiology analyses. KH contributed with bioinformatics analyses. BV performed the statistical analyses. All authors contributed to the interpretation of data for the manuscript and drafting, revising and critically reviewing the manuscript for important intellectual content. All authors have read and approved the final manuscript. Acknowledgement The authors wish to thank the staff at Department of Comparative medicine, particularly Nina Kjølen, Vibeke Stensrud Krohn and Elisabeth Jensen. We also thank the Core Facility for Advanced Light Microscopy at Oslo University Hospital, Montebello, for use of the Olympus SLIDEVIEW™ VS200 Slide scanner. Data Availability The raw numerical data and statistical analyses from which the data presented in the manuscript and figures have been derived, in addition to the Supplementary material and ARRIVE checklist are available on Zenodo at https://doi.org/10.5281/zenodo.15282425. The raw sequencing reads for Enterococcus faecalis are available in the NCBI Sequence Read Archive (SRA) under accession number SRR32735232, linked to BioProject PRJNA1236225. Gene predictions and typing results from RVFScan, CARD scan, and PubMLST are available on Zenodo at https://doi.org/10.5281/zenodo.15023961. 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Housing Gnotobiotic Mice in Conventional Animal Facilities. Curr. Protoc. Mouse Biol. 9 , e59. https://doi.org/10.1002/cpmo.59 (2019). Cooper, H. S., Murthy, S. N., Shah, R. S. & Sedergran, D. J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab. Invest. 69 , 238–249 (1993). Bang, B. & Lichtenberger, L. M. Methods of Inducing Inflammatory Bowel Disease in Mice. Curr Protoc. Pharmacol 72 , 5 58 51–55 58 42 (2016). https://doi.org/10.1002/0471141755.ph0558s72 Bankhead, P. et al. QuPath: Open source software for digital pathology image analysis. Sci. Rep. 7 , 16878. https://doi.org/10.1038/s41598-017-17204-5 (2017). Garrett, W. S. et al. Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system. Cell 131 , 33–45. https://doi.org/10.1016/j.cell.2007.08.017 (2007). Neurath, M. F. et al. The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn's disease. J. Exp. Med. 195 , 1129–1143. https://doi.org/10.1084/jem.20011956 (2002). Wells, C. L., Jechorek, R. P. & Erlandsen, S. L. Evidence for the translocation of Enterococcus faecalis across the mouse intestinal tract. J. Infect. Dis. 162 , 82–90. https://doi.org/10.1093/infdis/162.1.82 (1990). Heilpern, D. & Szilagyi, A. Manipulation of intestinal microbial flora for therapeutic benefit in inflammatory bowel diseases: review of clinical trials of probiotics, pre-biotics and synbiotics. Rev. Recent. Clin. Trials . 3 , 167–184. https://doi.org/10.2174/157488708785700302 (2008). Damman, C. J., Miller, S. I., Surawicz, C. M. & Zisman, T. L. The microbiome and inflammatory bowel disease: is there a therapeutic role for fecal microbiota transplantation? Am. J. Gastroenterol. 107 , 1452–1459. https://doi.org/10.1038/ajg.2012.93 (2012). Cohen, L. J., Cho, J. H., Gevers, D. & Chu, H. Genetic Factors and the Intestinal Microbiome Guide Development of Microbe-Based Therapies for Inflammatory Bowel Diseases. Gastroenterology 156 , 2174–2189. https://doi.org/10.1053/j.gastro.2019.03.017 (2019). Kitajima, S., Morimoto, M., Sagara, E., Shimizu, C. & Ikeda, Y. Dextran sodium sulfate-induced colitis in germ-free IQI/Jic mice. Exp. Anim. 50 , 387–395. https://doi.org/10.1538/expanim.50.387 (2001). Maslowski, K. M. et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 461 , 1282–1286. https://doi.org/10.1038/nature08530 (2009). Chiu, C. C. et al. Monocolonization of germ-free mice with Bacteroides fragilis protects against dextran sulfate sodium-induced acute colitis. Biomed Res Int 675786 (2014). (2014). https://doi.org/10.1155/2014/675786 He, Q. et al. Protective effects of a new generation of probiotic Bacteroides fragilis against colitis in vivo and in vitro. Sci. Rep. 13 , 15842. https://doi.org/10.1038/s41598-023-42481-8 (2023). Du, Z. et al. Development of gut inflammation in mice colonized with mucosa-associated bacteria from patients with ulcerative colitis. Gut Pathog . 7 , 32. https://doi.org/10.1186/s13099-015-0080-2 (2015). Golinska, E. et al. Virulence factors of Enterococcus strains isolated from patients with inflammatory bowel disease. World J. Gastroenterol. 19 , 3562–3572. https://doi.org/10.3748/wjg.v19.i23.3562 (2013). Kim, S. C. et al. Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria. Gastroenterology 128 , 891–906. https://doi.org/10.1053/j.gastro.2005.02.009 (2005). Barnes, A. M. T. et al. Enterococcus faecalis readily colonizes the entire gastrointestinal tract and forms biofilms in a germ-free mouse model. Virulence 8 , 282–296. https://doi.org/10.1080/21505594.2016.1208890 (2017). Amara, J. et al. Circadian Rhythm Disruption Aggravates DSS-Induced Colitis in Mice with Fecal Calprotectin as a Marker of Colitis Severity. Dig. Dis. Sci. 64 , 3122–3133. https://doi.org/10.1007/s10620-019-05675-7 (2019). Are, A. et al. Enterococcus faecalis from newborn babies regulate endogenous PPARgamma activity and IL-10 levels in colonic epithelial cells. Proc. Natl. Acad. Sci. U. S. A. 105, 1943–1948 (2008). https://doi.org/10.1073/pnas.0711734105 Takada, Y. et al. Monocyte chemoattractant protein-1 contributes to gut homeostasis and intestinal inflammation by composition of IL-10-producing regulatory macrophage subset. J. Immunol. 184 , 2671–2676. https://doi.org/10.4049/jimmunol.0804012 (2010). Takahashi, K. et al. Effect of Enterococcus faecalis 2001 on colitis and depressive-like behavior in dextran sulfate sodium-treated mice: involvement of the brain-gut axis. J. Neuroinflammation . 16 , 201. https://doi.org/10.1186/s12974-019-1580-7 (2019). Chen, Y. et al. Probiotic mixtures with aerobic constituent promoted the recovery of multi-barriers in DSS-induced chronic colitis. Life Sci. 240 , 117089. https://doi.org/10.1016/j.lfs.2019.117089 (2020). Chen, L. L. et al. Therapeutic effects of four strains of probiotics on experimental colitis in mice. World J. Gastroenterol. 15 , 321–327. https://doi.org/10.3748/wjg.15.321 (2009). Naveed, U. et al. Inhibitory Effect of Lactococcus and Enterococcus faecalis on Citrobacter Colitis in Mice. Microorganisms 12 https://doi.org/10.3390/microorganisms12040730 (2024). Hoffmann, M., Messlik, A., Kim, S. C., Sartor, R. B. & Haller, D. Impact of a probiotic Enterococcus faecalis in a gnotobiotic mouse model of experimental colitis. Mol. Nutr. Food Res. 55 , 703–713. https://doi.org/10.1002/mnfr.201000361 (2011). Seishima, J. et al. Gut-derived Enterococcus faecium from ulcerative colitis patients promotes colitis in a genetically susceptible mouse host. Genome Biol. 20 , 252. https://doi.org/10.1186/s13059-019-1879-9 (2019). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialSciRepFinal.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6871860","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":477061209,"identity":"6a6376a5-8109-4f5a-9367-76cfe0a7f43f","order_by":0,"name":"Beate Vestad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIiWNgGAWjYBACA2SOxAcGBsY2BsYGEBufFrAKBgY2BgbJGSRrkeaB8/AAc/bm5w8+MByWk5/f/PC2bY6NbB//4bYHjG0WcgzSzQewabHsOWbYOIPhsLHBMTZj69xtacZtEontBoxtEsYMMscSsDrsRoJhMw9DWuIGNgYz6dxthxPbJBjBKLFBIscAq5b7zz82/2FIq5/fxv5N2nLb/8Q2/oMEtNzgMWxmYLBJYDjGYybNuO1AYhtDIn4tlj05hTN7DGwMNxzLKbbs3ZYM8kubRMI5CWM2iTSsfjFnP77hw48KCXn55uMbb/zcZic7v//4M4kPZXVy/BLJWEMM6jx0AZDxbLjVj4JRMApGwSggAAC0mVr7Toj4pgAAAABJRU5ErkJggg==","orcid":"","institution":"Oslo University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Beate","middleName":"","lastName":"Vestad","suffix":""},{"id":477061215,"identity":"40663132-45f7-4296-a056-3afe2e04864e","order_by":1,"name":"Petra Hanzely","email":"","orcid":"","institution":"Oslo University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Petra","middleName":"","lastName":"Hanzely","suffix":""},{"id":477061223,"identity":"28de111a-5f32-4846-9c14-46b0ebc5aec6","order_by":2,"name":"Indrė Karaliūtė","email":"","orcid":"","institution":"Lithuanian University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Indrė","middleName":"","lastName":"Karaliūtė","suffix":""},{"id":477061224,"identity":"cba76e51-0ec1-4500-91ba-a7cf060a2c48","order_by":3,"name":"Oda Ramberg","email":"","orcid":"","institution":"Oslo University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Oda","middleName":"","lastName":"Ramberg","suffix":""},{"id":477061227,"identity":"8839c8f0-f7a9-43bb-9764-28422b542eae","order_by":4,"name":"Jurgita Skiecevičienė","email":"","orcid":"","institution":"Lithuanian University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jurgita","middleName":"","lastName":"Skiecevičienė","suffix":""},{"id":477061230,"identity":"d751b9ff-82ea-404c-b73c-882313d5be40","order_by":5,"name":"Rokas Lukoševičius","email":"","orcid":"","institution":"Lithuanian University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Rokas","middleName":"","lastName":"Lukoševičius","suffix":""},{"id":477061231,"identity":"bf6ae55e-1cec-4335-b773-bc141691134b","order_by":6,"name":"Jørgen V. Bjørnholt","email":"","orcid":"","institution":"University of Oslo","correspondingAuthor":false,"prefix":"","firstName":"Jørgen","middleName":"V.","lastName":"Bjørnholt","suffix":""},{"id":477061232,"identity":"99514ba6-0be6-4b75-8e15-a971c2bfcb26","order_by":7,"name":"Kristian Holm","email":"","orcid":"","institution":"Oslo University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kristian","middleName":"","lastName":"Holm","suffix":""},{"id":477061235,"identity":"dc6b0d21-b895-4f33-bef1-c03f8362db46","order_by":8,"name":"Juozas Kupčinskas","email":"","orcid":"","institution":"Lithuanian University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Juozas","middleName":"","lastName":"Kupčinskas","suffix":""},{"id":477061236,"identity":"10bd7bf2-b602-4d4c-b80a-36afe0e88422","order_by":9,"name":"Henrik Rasmussen","email":"","orcid":"","institution":"University of Oslo","correspondingAuthor":false,"prefix":"","firstName":"Henrik","middleName":"","lastName":"Rasmussen","suffix":""},{"id":477061238,"identity":"8dcdbf75-1186-4ab9-b19e-d9a68090f742","order_by":10,"name":"Johannes R. Hov","email":"","orcid":"","institution":"Oslo University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Johannes","middleName":"R.","lastName":"Hov","suffix":""},{"id":477061239,"identity":"0d436fbb-708f-44af-a154-6391dcd96ca0","order_by":11,"name":"Espen Melum","email":"","orcid":"","institution":"Oslo University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Espen","middleName":"","lastName":"Melum","suffix":""}],"badges":[],"createdAt":"2025-06-11 12:23:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6871860/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6871860/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85548989,"identity":"3697ddf4-bb18-45e8-b77b-39fd2d38e54a","added_by":"auto","created_at":"2025-06-27 09:21:29","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3128364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy design and validation of colonization.\u003c/strong\u003e a) Study design and experimental group allocation. b) Culture of mouse fecal samples after one week of colonization with \u003cem\u003eE. faecalis\u003c/em\u003e, individual values are shown with mean (column height) and error bars indicate SD, p-value from two-tailed unpaired \u003cem\u003et\u003c/em\u003e test. c) qPCR copy numbers of \u003cem\u003eE. faecalis\u003c/em\u003eper ng of total DNA from feces, cecum and plasma. Median values are indicated by the line, p-value calculated by Mann-Whitney \u003cem\u003eU\u003c/em\u003e test comparing feces from colonized mice with \u003cem\u003evs\u003c/em\u003e without DSS treatment.\u003c/p\u003e","description":"","filename":"Figure1Studydesign600dpi.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6871860/v1/3c07c26a89168cef600c7654.jpeg"},{"id":85549813,"identity":"aee81605-48e0-4bba-ab62-78bc129512bb","added_by":"auto","created_at":"2025-06-27 09:29:29","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2946358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisease activity measures during experiment and harvest measures at Day 7 of DSS administration\u003c/strong\u003e. a) Body weight as percentage of starting weight shown as mean values with SD. P-value was calculated by unpaired \u003cem\u003et\u003c/em\u003e test between the two DSS groups at Day 7. b) Disease activity index encompassing weight loss, stool consistency, and rectal bleeding shown as mean values with SD. P-value calculated by 2-way ANOVA analysis representing column factor as curve difference between the two DSS groups. c) Colon length in cm. d) Cecum weight as percentage of body weight. e) Hemoglobin levels. f) White blood cell count. Data in c), d), e) and f) represent measurements at the end of experiment and are shown as individual values with mean value indicated by the line. Adjusted p-values calculated by one-way ANOVA with correction for multiple comparisons. Only significant p-values are displayed.\u003c/p\u003e","description":"","filename":"Figure2Diseaseactivitymeasures600dpi.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6871860/v1/5855e641cdd2b72abdb4b883.jpeg"},{"id":85548992,"identity":"94bca56f-4042-47ef-b305-dd518dec9a83","added_by":"auto","created_at":"2025-06-27 09:21:29","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10097352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological alterations following DSS treatment in GF and monocolonized mice.\u003c/strong\u003e The images show representative histology images from a) an \u003cem\u003eE. faecalis\u003c/em\u003e-colonized mouse, b) a GF mouse treated with DSS and c) a \u003cem\u003eE. faecalis\u003c/em\u003e-colonized mouse treated with DSS. Histological scores were calculated from d) proximal, e) middle, f) distal and g) total colon (average of the three regions). Individual scores are shown with the lines representing mean scores. P-values were calculated using one-way ANOVA with multiple comparisons and Bonferroni corrections.\u003c/p\u003e","description":"","filename":"Figure3Histologicalalterations600dpi.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6871860/v1/6511979597e3a8bb90c5f75d.jpeg"},{"id":85548995,"identity":"1f3562c5-2c4d-4a47-aeb3-2cb7e2634a4e","added_by":"auto","created_at":"2025-06-27 09:21:30","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3044371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMarkers of gut inflammation and translocation.\u003c/strong\u003e a) Levels of fecal calprotectin in ng per g of feces at day 0 (Col n=6, GF DSS n=5, Col+DSS n=5) and day 7 (Col n=6, GF DSS n=6, Col+DSS n=11). b) Levels of fecal albumin in ng per g of feces at day 0 (Col n=5, GF DSS n=6, Col+DSS n=5) and day 7 (Col n=7, GF DSS n=6, Col+DSS n=11). P-values in a and b were calculated by unpaired \u003cem\u003et\u003c/em\u003e tests. c) Representative overnight culture of mesenteric lymph nodes (MLNs) from an \u003cem\u003eE. faecalis\u003c/em\u003e-colonized mouse collected at the end of experiment. CFU: colony forming unit.\u003c/p\u003e","description":"","filename":"Figure4Markersofgutinflandtransloc600dpi.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6871860/v1/cdd5709732bcf089ae1a7b85.jpeg"},{"id":86304842,"identity":"0f30318c-173f-44bb-ac8a-f53084889411","added_by":"auto","created_at":"2025-07-09 07:02:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19950833,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6871860/v1/7a6e9094-83e4-4bef-b579-f4bb44d45f4b.pdf"},{"id":85548991,"identity":"badb129a-f914-44f3-95ad-ca5239e5db5d","added_by":"auto","created_at":"2025-06-27 09:21:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1484047,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialSciRepFinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6871860/v1/e98a6bfca36730d24b8afe35.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modulation of dextran sodium sulfate-induced colitis in germ-free mice by Enterococcus faecalis-monocolonization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInflammatory bowel diseases (IBD), encompass Crohn’s disease (CD) and ulcerative colitis (UC), both characterized by chronic gastrointestinal inflammation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The pathogenesis of IBD involves a complex interplay of genetic susceptibility, environmental factors, and inappropriate immune activation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This immune dysregulation includes abnormal activation of innate and adaptive immunity, with pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 contributing to mucosal damage and perpetuating the inflammatory cycle [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e–\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Gut microbiota imbalance is also implicated, with certain bacterial species suspected of modulating intestinal inflammation. However, the role of individual bacterial species in IBD remains poorly understood [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGerm-free (GF) mice are invaluable tools for studying host-microbiota interactions, allowing researchers to isolate the effects of specific microbes or defined communities on disease processes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the dextran sulfate sodium (DSS)-induced colitis model, oral administration of DSS in drinking water induces severe colitis that mimics key features of human UC, including mucosal ulceration, epithelial barrier disruption, and inflammatory cell infiltration [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mice undergoing DSS treatment typically exhibit weight loss, bloody diarrhea, and reduced activity, paralleling clinical symptoms of UC. While the precise mechanisms of DSS toxicity remain unclear, one hypothesis is that the sulphated, negatively charged DSS molecule interacts with dietary medium-chain fatty acids, forming complexes absorbed by colonic epithelial cells that contribute to barrier disruption and inflammation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Other mechanisms, such as direct epithelial toxicity and activation of immune pathways, are also likely to play a role [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Unlike human UC, the development of DSS-induced colitis does not require adaptive immune cells, making this model particularly suitable for investigating the role of innate immune responses in intestinal inflammation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The model is also useful for exploring microbial contributions to colitis progression, as the specificity of DSS to the colon is thought to depend on bacterial activity and local physiological factors such as water and electrolyte absorption [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudying monocolonized mice provides a unique opportunity to investigate the effects of individual bacterial strains, helping to delineate their specific contributions to intestinal inflammation and immune responses [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (\u003cem\u003eE. faecalis\u003c/em\u003e) is a gram-positive bacterium normally residing in the human gastrointestinal tract. While often considered a commensal organism, \u003cem\u003eE. faecalis\u003c/em\u003e is associated with various infections and has been implicated in IBD pathogenesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Previous studies have highlighted the capacity of \u003cem\u003eE. faecalis\u003c/em\u003e to translocate across the intestinal barrier, migrate to lymph nodes and disseminate to other tissues. Virulence factors such as the aggregation substance (Agg) and the extracellular surface protein (Esp) are hypothesized to enhance its pathogenicity by increasing surface hydrophobicity, promoting colonization, and evading immune responses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, secreted hydrolytic enzymes like hyaluronidase may contribute to tissue damage and bacterial spread [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although \u003cem\u003eE. faecalis\u003c/em\u003e has been shown to modulate inflammation in murine colitis models, most studies have utilized conventional or genetically modified mice with chronic colitis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The effect of \u003cem\u003eE. faecalis\u003c/em\u003e-monocolonization in acute murine colitis is still underexplored and offers to study the specific effects of an individual bacterial species.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the effects of \u003cem\u003eE. faecalis\u003c/em\u003e-monocolonization in GF mice subjected to DSS-induced colitis. We aimed to assess whether the presence of \u003cem\u003eE. faecalis\u003c/em\u003e alters colitis severity and general disease status, focusing on physical, histological, hematological, and intestinal markers of inflammation and barrier function.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eAnimals\u003c/p\u003e\u003cp\u003eGerm-free C57BL/6J mice, originating from the University of Bern Clean Mouse Facility, were bred in open cages (Eurostandard type II, 11bbB, Tecniplast, Buguggiate, Italy) maintained in sterile flexible-film isolators. At 5 weeks of age, the mice were earmarked and exported into an SPF facility in autoclaved GM500 individually ventilated cages (IVC) (Tecniplast) with bedding and nesting material. The animals were acclimatized for 7 days before they underwent monocolonization or remained GF as controls. GF status in the isolator was confirmed by monthly aerobic and anaerobic culture of fecal pellets and mold trap samples from the isolator, as well as yearly PCR-based serology testing according to FELASA recommendations [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the IVC cages, fecal pellets were cultured weekly during the experiment to monitor sterility and monoculture status of the animals.\u003c/p\u003e\u003cp\u003eThe animals had \u003cem\u003ead libitum\u003c/em\u003e access to autoclaved chow pellets (LabDiet 5021, IPS Products Supplies, Alfreton, Great Britain) and water. Mice were kept in a humidity and temperature-controlled environment on a 12/12-hour day/night cycle at an approved animal facility at the Oslo University Hospital, Rikshospitalet. The mice were segregated by sex and housed 2–3 mice in each cage during colonization, as well as before and during DSS administration. Cage change and experimental procedures were performed using sterile equipment by one sterile and one unsterile operator by strict aseptic handling of the cages inside of a laminar air flow changing station [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. At the end of experiment, the mice were euthanized by heart puncture under isoflurane anesthesia followed by cervical dislocation and harvesting of organs.\u003c/p\u003e\u003cp\u003eMonocolonization\u003c/p\u003e\u003cp\u003eFreshly prepared cultures of a single \u003cem\u003eEnterococcus faecalis\u003c/em\u003e isolate retrieved from the feces of a male patient in his 60-ies with ulcerative colitis in remission was used for colonization. The patient was part of a cohort included in the Norwegian PSC (primary sclerosing cholangitis) Research Center biobank at Oslo University Hospital. Genomic data, including putative virulence factors and antimicrobial resistance genes are available in the Supplementary Material. \u003cem\u003eE. faecalis\u003c/em\u003e was cultured on blood agar at 37°C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 48–72 hours. The bacterial stock solution was prepared by adding 20–30 middle big colonies into 1 mL of sterile PBS, equal to an optical density of 1.5–1.6 (2 x 10\u003csup\u003e9\u003c/sup\u003e colony-forming units (CFU)/mL). An additional 3 mL PBS was added to make a final colonization solution of approximately 5 x 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. At 6 weeks of age, mice were colonized by slowly injecting 200 µL of the \u003cem\u003eE. faecalis\u003c/em\u003e solution (10\u003csup\u003e8\u003c/sup\u003e CFU) in a single dose via rectal administration using a gavage needle (18G) during manual restraint. After 21 days, a subset of the colonized mice was subjected to DSS administered in autoclaved drinking water for 7 days. Noncolonized GF mice with DSS administration served as colitis controls, whereas colonized GF mice without DSS served as colonization controls.\u003c/p\u003e\u003cp\u003eDSS colitis model\u003c/p\u003e\u003cp\u003eDextran sulfate sodium (Cat no. DB001, Batch no. DB001-47, 35866 kDa MW, TdB Labs AB, Uppsala, Sweden) was administered to 9-week-old mice at 2.5% concentration (w/v) in autoclaved drinking water for 7 days to induce acute colitis. Start of DSS dosing was defined as day 0. During DSS administration, the mice were monitored daily with measurements of food and water intake (data not shown), body weight, and scoring according to disease activity. Mice were euthanized according to humane endpoint (HEP) scoring in cases of weight loss above 20% from DSS start, severe rectal prolapse, isolated behavior or reaching HEP score of 14 or higher (Supplementary Table\u0026nbsp;1). DSS-induced colitis development was evaluated by calculating a separate Disease Activity Index (DAI) score based on weight loss, stool consistency, and rectal bleeding (modified from [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]). The DAI was calculated as the average of the total scores consisting of: weight loss (0: 0–5%; 1: 6–10%; 2: 11–15%; 3: 16–20%; 4: \u0026gt;20%), stool consistency (0: none; 2: loose stools; 4: gross diarrhea), and rectal bleeding (0: normal; 2: mild bleeding, visible in stool; 3: moderate bleeding, visible from rectum and/or in bedding/cage; 4: gross bleeding, marked staining in bedding/cage).\u003c/p\u003e\u003cp\u003eSample collection and processing\u003c/p\u003e\u003cp\u003eFresh fecal samples were collected before and after DSS administration for analysis of albumin and calprotectin and kept on ice until storing at -20°C. Blood was collected by cardiac puncture under isoflurane anesthesia into a syringe coated with 0.5M EDTA using a 23G needle after sterilizing the insertion site with 70% ethanol. Mesenteric lymph nodes were collected by aseptic technique and cultured to monitor for bacterial translocation. Briefly, 2–3 lymph nodes were homogenized in 100–200 µL PBS, and 100 µL of the homogenate were cultured on blood agar plates overnight at 37°C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequently, liver, spleen and cecum were collected and weighed. Colon length was measured before the entire colon was processed for histology assessment by “swiss roll” preparation on a 27G needle. Fecal pellets and cecal content for DNA extraction were snap frozen on dry ice and kept at -80°C. Blood plasma was prepared by centrifugation of whole blood twice at 2500 x \u003cem\u003eg\u003c/em\u003e for 15 minutes.\u003c/p\u003e\u003cp\u003eHematological parameters\u003c/p\u003e\u003cp\u003eDetermination of hematological parameters were performed in 10 µL fresh EDTA-blood using an ABX Micros 60 automated hematology instrument (Horiba ABX SAS, Montpellier, France).\u003c/p\u003e\u003cp\u003eHistology\u003c/p\u003e\u003cp\u003eTissue sections were fixed in 4% PFA for 18 hours and placed in cold PBS, then paraffin embedded, cut, and stained with hematoxylin and eosin. Two different sections were evaluated per sample, taken 15 µm apart. Sections were scanned using Olympus SLIDEVIEW™ VS200 Slide scanner (Olympus Corporation, Tokyo, Japan) and images processed using QuPath 0.4.3 software [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The images were examined and scored with regards to colitis by one of the authors (B.V.) blinded to group allocations. The colon sections were divided into three main scoring regions: proximal colon, middle colon and distal colon. Histology assessment of the following parameters; I: mononuclear cell infiltration, II: polymorphonuclear cell infiltration, III: epithelial hyperplasia, as well as IV: epithelial injury, comprising visible erosion, ulcerations and alterations to the crypt architecture, were scored for each region of the colon as absent (0), mild (1), moderate (2), or severe (3): [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A total colon score was created by calculating an average of the three colonic region scores.\u003c/p\u003e\u003cp\u003eAlbumin and calprotectin measurements\u003c/p\u003e\u003cp\u003eFecal albumin was analyzed using Mouse Albumin ELISA Kit (Cat E99-134, Bethyl Laboratories, Montgomery, Texas, USA) and fecal calprotectin was analyzed using the S100A8/S100A9 ELISA Kit (Ref. KR6936, Immundiagnostik AG, Bensheim, Germany), according to the manufacturer’s recommendations. Briefly, fecal pellets were diluted 1:10 with dilution buffer from the albumin kit, placed on ice for 15 minutes and thoroughly homogenized before centrifugation at 13,000 RPM for 5 minutes at 4°C. The supernatant was collected and used for albumin measurements. The remainder of the sample material was further diluted 1:5 with extraction buffer from the calprotectin kit and centrifugated at 2,000 x \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4°C. The supernatant was collected and used for calprotectin measurements. Albumin and calprotectin results were obtained using a BioTek Synergy H1 Hybrid plate reader (Agilent, Santa Clara, California, USA).\u003c/p\u003e\u003cp\u003eDNA extraction and real-time quantitative PCR of \u003cem\u003eE. faecalis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eDNA was extracted from one pellet of mouse feces (20–60 mg) or 200 µL of cecal content per mouse using the Genesig® Easy DNA/RNA extraction Kit (Primerdesign™ Ltd, Eastleigh, United Kingdom) and mouse plasma DNA was extracted using the QIAamp MinElute ccf DNA Kit (Cat 55284, Qiagen, Hilden, Germany), following the manufacturer’s recommendations. A template DNA input of 1 ng/µL was used per reaction, diluted to 8µL volume with DNase/RNase free H\u003csub\u003e2\u003c/sub\u003eO. Copy numbers of \u003cem\u003eEnterococcus faecalis\u003c/em\u003e DNA was then quantified using the \u003cem\u003eEnterococcus faecalis\u003c/em\u003e qPCR Test Kit (YouSeq Ltd, Winchester, United Kingdom) and a Stratagene Mx3000P real-time PCR cycler and MxPro software (Agilent). The PCR cycling conditions used were: 3 minutes hot start at 95°C followed by 45 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Fluorogenic data was collected both through the FAM (\u003cem\u003eE. faecalis\u003c/em\u003e/sample) and HEX (supplied endogenous control) channels.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eDifferences between two groups were analyzed using two-tailed unpaired \u003cem\u003et\u003c/em\u003e test for parametric data or two-tailed Mann-Whitney \u003cem\u003eU\u003c/em\u003e test for nonparametric data. Comparisons between three or more groups were analyzed by one-way ANOVA, reporting adjusted p-value for multiple comparisons using Bonferroni’s correction. Body weight and disease activity index curves were analyzed either using unpaired \u003cem\u003et\u003c/em\u003e tests between timepoints or 2-way ANOVA using column factor for p-value calculation. Differences were considered significant when p \u0026lt; 0.05. All statistical analyses were performed using GraphPad Prism version 10.2.0 (Boston, Massachusetts, USA).\u003c/p\u003e\u003cp\u003eEthics statement\u003c/p\u003e\u003cp\u003e Animal experiments were carried out according to the EU Directive on the protection of animals used for scientific purposes (2010/63/EU) and the Norwegian Animal research legislation. The study is reported in accordance with the ARRIVE guidelines, and all animal experiments were approved by The Norwegian National Animal Research Authority (project license no. 25770/29980/29935). The \u003cem\u003eEnterococcus faecalis\u003c/em\u003e isolate used in the study was obtained from a patient sample that was part of the Norwegian PSC Research Biobank. Use of the biobanked patient material was covered under written informed consent, and approved by the Regional Committee for Medical and Health Research Ethics in South-Eastern Norway, with reference number 2015/2140.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eE. faecalis-\u003c/em\u003emonocolonization and colitis induction\u003c/p\u003e \u003cp\u003eA total of 24 GF C57BL/6J mice (16 males and 8 females) were included in the study. To assess the effect of \u003cem\u003eE. faecalis\u003c/em\u003e on colitis development, mice were monocolonized with \u003cem\u003eE. faecalis\u003c/em\u003e 21 days (3 weeks) prior to administration of autoclaved drinking water with DSS (n\u0026thinsp;=\u0026thinsp;11, 6 males and 5 females), or autoclaved water only (n\u0026thinsp;=\u0026thinsp;7, 4 females and 3 males) for 7 days. Sterile water with DSS was provided to a control group of GF mice (n\u0026thinsp;=\u0026thinsp;6 males) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The DSS dosage applied was based on titration in a separate pilot study to ensure proper colitis development without reaching unacceptable toxicity (Supplementary Material). At the end of the experiment (day 7 after DSS start, day 35 after acclimatization start), the mice were euthanized and organs harvested. To confirm successful monocolonization or GF status, fresh fecal samples were collected one week after colonization. As expected, colonies of \u003cem\u003eE. faecalis\u003c/em\u003e were found in fecal pellets from all colonized mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). To further validate colonization status and evaluate potential bacterial translocation, we quantified \u003cem\u003eE. faecalis\u003c/em\u003e DNA copy numbers in samples from feces, cecum and plasma after euthanasia of the animals. In 1 ng of total DNA, we detected a median of 40,000 copies in feces of colonized mice without DSS treatment with a clear trend towards lower numbers in the feces of colonized mice with DSS treatment (p\u0026thinsp;=\u0026thinsp;0.06) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). \u003cem\u003eE. faecalis\u003c/em\u003e was also consistently detected in all cecum content samples from colonized mice, however in much lower copy numbers (median around 3,000 copies for colonized mice only and 150 copies for colonized mice treated with DSS). We could not detect any \u003cem\u003eE. faecalis\u003c/em\u003e copies in the plasma of any of the mouse groups. This suggests that \u003cem\u003eE. faecalis\u003c/em\u003e either did not translocate to the bloodstream, or that the DNA copy numbers were below the detection limit of our analysis. We did not detect any \u003cem\u003eE. faecalis\u003c/em\u003e DNA in any of the samples from the GF mice receiving DSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eE. faecalis\u003c/em\u003e-colonization alters colitis disease activity and physiological parameters in DSS-treated mice\u003c/p\u003e \u003cp\u003eThe mice in both DSS-treated groups experienced a clear weight loss on day 7 of DSS administration, without significant difference between the \u003cem\u003eE. faecalis\u003c/em\u003e-colonized mice and the GF mice at harvest (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The colitis-related disease activity index increased for both groups of DSS-treated mice with significantly higher scores in the mice that were pre-colonized with \u003cem\u003eE. faecalis\u003c/em\u003e compared to GF mice (p\u0026thinsp;=\u0026thinsp;0.02; 2-way ANOVA, column factor) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Both DSS groups had shorter colons compared to colonized mice only (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but with no difference between the DSS groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Mean cecum weight in GF mice receiving DSS was 18.5% of body weight, and it was lower in both colonized groups, although significantly reduced only in colonized mice receiving DSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Both DSS groups had a higher degree of anemia compared to the mice that were only \u003cem\u003eE. faecalis\u003c/em\u003e-colonized, while the GF mice treated with DSS had lower blood hemoglobin than colonized mice treated with DSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). No changes were observed between the groups in levels of circulating white blood cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDSS treatment of GF and \u003cem\u003eE. faecalis-\u003c/em\u003emonocolonized mice induces similar histology alterations\u003c/p\u003e \u003cp\u003eIn mice colonized with \u003cem\u003eE. faecalis\u003c/em\u003e only, we observed mild to moderate hyperplasia and a few immune cell aggregates. Some of the mice had mild edema and erosion in the mucosa while the epithelium was generally intact (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). GF mice treated with DSS displayed histological features compatible with loss of crypts, erosion, edema and infiltrations of immune cells into muscle layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Similarly, \u003cem\u003eE. faecalis\u003c/em\u003e-colonized mice with DSS-induced colitis showed comparable histological alterations although with somewhat more pronounced ulcerations and damaged crypt architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistological scoring of the different colonic regions revealed that inflammation and epithelial injury were more pronounced in the middle and distal colon than in the proximal colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f). There were no statistical differences between the two groups of DSS-treated mice for any of the colonic regions. However, comparing the two groups of colonized mice, DSS-treated mice had higher histoscores than non-DSS-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-g).\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. faecalis\u003c/em\u003e alters fecal calprotectin and albumin levels and translocates to mesenteric lymph nodes\u003c/p\u003e \u003cp\u003eLevels of the intestinal inflammation marker calprotectin, measured before and after DSS treatment, did not differ significantly between colonized and non-colonized mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). However, at Day 7, calprotectin levels were notably lower in colonized mice treated with DSS compared to GF mice treated with DSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Fecal albumin levels increased significantly from day 0 (before DSS treatment) to day 7, with the highest levels observed in colonized mice receiving DSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs fecal albumin could be a marker of intestinal leakage and \u003cem\u003eE. faecalis\u003c/em\u003e is known to translocate to lymph nodes in mice [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], we wanted to validate this in the present study. We collected 2\u0026ndash;3 mesenteric lymph nodes (MLNs) from a subset of colonized mice during harvest, and overnight cultures revealed that \u003cem\u003eE. faecalis\u003c/em\u003e had translocated to MLNs in all these mice independent of DSS intake (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGrowing evidence suggests that manipulation of the intestinal microbiota can influence the development and progression of chronic inflammatory diseases such as IBD [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study, we used GF mice and a chemically induced colitis model to investigate the effect of monocolonization using an \u003cem\u003eE. faecalis\u003c/em\u003e strain isolated from a patient with UC. We found that 3 weeks of monocolonization (using a single rectal dose of 10\u003csup\u003e8\u003c/sup\u003e CFU) followed by 7 days of 2.5% DSS administration resulted in several disease-modifying effects at the molecular level. Specifically, \u003cem\u003eE. faecalis\u003c/em\u003e-monocolonization led to reduced anemia, lower fecal calprotectin levels, and increased fecal albumin compared to GF mice receiving DSS alone. However, no significant differences were observed in body weight, colitis-related disease activity, or colon length, suggesting that the presence of \u003cem\u003eE. faecalis\u003c/em\u003e alone may have marginal clinical effects on colitis development in this model.\u003c/p\u003e \u003cp\u003eWe successfully detected \u003cem\u003eE. faecalis\u003c/em\u003e DNA in the feces of all colonized mice one week after colonization and confirmed colonization in cecum and translocation to the mesenteric lymph nodes at the end of experiment, suggesting that the bacterium could migrate beyond the gut. Notably, the lower copy numbers of \u003cem\u003eE. faecalis\u003c/em\u003e in cecal DNA compared to fecal DNA may reflect differences in host versus bacterial DNA content in the samples.\u003c/p\u003e \u003cp\u003eThe DSS-induced colitis model in experimental mice is well-documented and mimics key features of human UC, including rectal bleeding, diarrhea, and weight loss [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In line with these expected disease traits, the mice in present study showed shortened colons and weight loss towards the end of the experiment. Despite these indications of disease, we observed no significant differences between \u003cem\u003eE. faecalis\u003c/em\u003e-colonized and GF mice in terms of weight loss or disease activity index, suggesting that \u003cem\u003eE. faecalis\u003c/em\u003e had limited effects on these aspects of colitis progression. However, the reduced anemia observed in \u003cem\u003eE. faecalis\u003c/em\u003e-colonized mice could suggest a protective role in general disease status, potentially reducing blood loss associated with colonic inflammation. This is consistent with previous studies where DSS treatment in GF mice has been linked to significant blood loss, which is often used as a marker of disease severity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMonocolonization studies using probiotic or disease-associated bacteria have been shown to affect the severity of chemically induced colitis in GF mice. For example, colonization with \u003cem\u003eBacteroides fragilis\u003c/em\u003e has been demonstrated to protect against DSS-induced acute colitis by increasing survival and reducing immune cell infiltration and colon shortening [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In contrast, certain microbes have been suggested to exacerbate colitis development in chemically induced models. For example, mucosa-associated microbes from patients with UC have been shown to increase susceptibility to DSS-induced colitis in GF BALB/c mice, although they did not induce spontaneous colitis on their own [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Additionally, strains of \u003cem\u003eE. faecalis\u003c/em\u003e isolated from the inflamed mucosa of UC patients are highly adherent and are likely to carry virulence-related genes, contributing to disease activity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In GF mouse models of chronic colitis, \u003cem\u003eE. faecalis\u003c/em\u003e-monocolonization has been shown to primarily induce colitis in the distal colon [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Our findings are consistent with this, as we observed a trend of increased histoscores in the middle and distal colon compared to the proximal colon, both in \u003cem\u003eE. faecalis\u003c/em\u003e-colonized mice (with and without DSS treatment) and in GF mice. Notably, \u003cem\u003eE. faecalis\u003c/em\u003e-colonization has been shown to form a uniformly distributed biofilm in the GF murine gastrointestinal tract, yet it does not appear to induce a severe inflammatory response in the GF host [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our findings are in line with this and even suggest a potential beneficial effect on the host during colitis induction. Specifically, monocolonization with \u003cem\u003eE. faecalis\u003c/em\u003e in DSS-treated mice reduced anemia and lowered fecal calprotectin levels, suggesting alleviated intestinal inflammation. However, histological analysis showed similar patterns of immune cell infiltration, inflamed areas, and immune cell aggregates in both \u003cem\u003eE. faecalis\u003c/em\u003e-colonized and GF mice treated with DSS. Interestingly, fecal albumin levels were higher in the colonized mice receiving DSS than in GF mice receiving DSS, suggesting additional epithelial damage induced by \u003cem\u003eE. faecalis\u003c/em\u003e. Histological analysis partially supported this, showing similar overall colonic injury with a tendency towards increased erosion and ulceration in the mucosa and submucosa.\u003c/p\u003e \u003cp\u003eColonization of GF mice with monocultures like \u003cem\u003eE. faecalis\u003c/em\u003e prior to the chemical induction of acute colitis may have several effects on colitis development [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. One possible mechanism is increased susceptibility to colitis by an enhanced inflammatory response. We found that mice colonized with \u003cem\u003eE. faecalis\u003c/em\u003e had reduced levels of calprotectin in feces as compared to GF mice treated with DSS. In general, increased levels of fecal calprotectin are indicative of neutrophil infiltration in the gut, which corresponds with active inflammation. While direct studies on fecal calprotectin in \u003cem\u003eE. faecalis\u003c/em\u003e-monocolonized GF mice are limited, existing research on gut inflammation markers suggests that fecal calprotectin would likely be elevated in such conditions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Our findings contradict this, possibly due to strain-specific differences in \u003cem\u003eE. faecalis\u003c/em\u003e virulence factors, which can influence the host immune response via various mechanisms. For example, some strains of \u003cem\u003eE. faecalis\u003c/em\u003e may modulate the immune system by inducing production of anti-inflammatory cytokines such as IL-10 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], which may lead to reduced neutrophil recruitment and activation. Additionally, a specific strain of \u003cem\u003eE. faecalis\u003c/em\u003e has been shown to reduce the expression of pro-inflammatory cytokines like TNF-α and IL-6, which are crucial in driving neutrophil-mediated inflammation in colitis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Probiotic \u003cem\u003eE. faecalis\u003c/em\u003e strains have also been reported to enhance mucosal integrity and reduce epithelial damage in colitis models, potentially lowering fecal calprotectin levels [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, most studies have been conducted in conventional hosts, and current observations suggest that \u003cem\u003eEnterococcus\u003c/em\u003e species may provoke a more pro-inflammatory response in disease-susceptible hosts, such as gnotobiotic and IL-10 knockout mice [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Sequence results from the strain used in our study revealed genes for several putative virulence factors, including surface-binding proteins, degradative enzymes, adhesins and aggregation substance proteins, that may be involved in bacterial aggregation, biofilm formation, and tissue damage in the colonized mice.\u003c/p\u003e \u003cp\u003eIn summary, monocolonization of GF mice with \u003cem\u003eE. faecalis\u003c/em\u003e prior to DSS-induced acute colitis alters disease progression and overall status, as reflected in clinical, histological, and molecular assessments, including markers of inflammation and gut barrier function. While our study addresses only a subset of the complex interactions between host immunity, microbial colonization, and chemically induced colitis, it highlights the potential role of specific bacterial species in modulating the severity of inflammatory bowel diseases. A key strength of this study is the use of GF mice and a controlled DSS model, enabling direct investigation of \u003cem\u003eE. faecalis\u003c/em\u003e effects. However, the strain-specific nature of our findings suggests that future studies should examine a broader range of \u003cem\u003eE. faecalis\u003c/em\u003e strains and their interactions with other microbial species to elucidate their contribution to colitis modulation and therapeutic potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe project and BV have received funding from EEA Grants 2014\u0026ndash;2021 - Baltic Research Programme (project No LT08-2-LMT-K-01-060) under grant agreement with the Research Council of Lithuania (LMTLT). PH and JRH were funded by a grant from the European Research Council (no. 802544).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJS, JK, HR, JRH and EM conceived the study and provided funding. BV, PH, RL, HR, JRH and EM contributed substantially to the study implementation and experiment planning. BV, PH, IK and OR performed the animal experiments. BV and PH performed laboratory analyses on mouse samples. JVB contributed with microbiology analyses. KH contributed with bioinformatics analyses. BV performed the statistical analyses. All authors contributed to the interpretation of data for the manuscript and drafting, revising and critically reviewing the manuscript for important intellectual content. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors wish to thank the staff at Department of Comparative medicine, particularly Nina Kj\u0026oslash;len, Vibeke Stensrud Krohn and Elisabeth Jensen. We also thank the Core Facility for Advanced Light Microscopy at Oslo University Hospital, Montebello, for use of the Olympus SLIDEVIEW\u0026trade; VS200 Slide scanner.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw numerical data and statistical analyses from which the data presented in the manuscript and figures have been derived, in addition to the Supplementary material and ARRIVE checklist are available on Zenodo at https://doi.org/10.5281/zenodo.15282425. The raw sequencing reads for Enterococcus faecalis are available in the NCBI Sequence Read Archive (SRA) under accession number SRR32735232, linked to BioProject PRJNA1236225. Gene predictions and typing results from RVFScan, CARD scan, and PubMLST are available on Zenodo at https://doi.org/10.5281/zenodo.15023961. Other data generated during the study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSands, B. E. From symptom to diagnosis: clinical distinctions among various forms of intestinal inflammation. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cb\u003e126\u003c/b\u003e, 1518\u0026ndash;1532. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1053/j.gastro.2004.02.072\u003c/span\u003e\u003cspan address=\"10.1053/j.gastro.2004.02.072\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSartor, R. B. 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Gut-derived Enterococcus faecium from ulcerative colitis patients promotes colitis in a genetically susceptible mouse host. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 252. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13059-019-1879-9\u003c/span\u003e\u003cspan address=\"10.1186/s13059-019-1879-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Inflammatory bowel disease (IBD), bacterial translocation, epithelial barrier dysfunction, host-microbe interactions, gut microbiota","lastPublishedDoi":"10.21203/rs.3.rs-6871860/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6871860/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are characterized by chronic gastrointestinal inflammation and involve complex interactions of genetic, environmental, and immune factors. \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, a gut commensal bacterium, has been implicated in IBD pathogenesis. This study investigated the effects of \u003cem\u003eE. faecalis\u003c/em\u003e-monocolonization in germ-free (GF) mice subjected to dextran sulfate sodium (DSS)-induced colitis. We assessed the impact of \u003cem\u003eE. faecalis\u003c/em\u003e on colitis severity, inflammation, and intestinal barrier function. In the context of DSS, \u003cem\u003eE. faecalis\u003c/em\u003e-colonized mice exhibited reduced anemia and lower fecal calprotectin levels, though fecal albumin levels were elevated. Despite translocation of \u003cem\u003eE. faecalis\u003c/em\u003e to mesenteric lymph nodes, no systemic dissemination was observed. Histological analysis showed similar inflammatory patterns in DSS-treated mice, regardless of \u003cem\u003eE. faecalis-\u003c/em\u003ecolonization, but more severe mucosal damage was noted in the colonized mice. These findings support that \u003cem\u003eE. faecalis\u003c/em\u003e plays a dual role in modulating colitis, influencing inflammation, and exacerbating epithelial injury. The study highlights the utility of GF models in understanding microbial contributions to IBD and emphasized how specific bacterial strains may influence disease progression through strain-dependent interactions with the host immune system and intestinal barrier. Further research is needed to elucidate these complex mechanisms.\u003c/p\u003e","manuscriptTitle":"Modulation of dextran sodium sulfate-induced colitis in germ-free mice by Enterococcus faecalis-monocolonization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-27 09:21:25","doi":"10.21203/rs.3.rs-6871860/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"514d7b0f-b823-4575-adef-87fd9afd743b","owner":[],"postedDate":"June 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50660211,"name":"Biological sciences/Immunology/Mucosal immunology"},{"id":50660212,"name":"Biological sciences/Microbiology/Pathogens"},{"id":50660213,"name":"Biological sciences/Microbiology/Applied microbiology"},{"id":50660214,"name":"Biological sciences/Microbiology/Bacteria/Bacterial host response"}],"tags":[],"updatedAt":"2025-07-09T06:54:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-27 09:21:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6871860","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6871860","identity":"rs-6871860","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}