A human microbiome-derived therapeutic for ulcerative colitis promotes mucosal healing and immune homeostasis: a randomized, controlled Phase 1 trial in healthy volunteers

A human microbiome-derived therapeutic for ulcerative colitis promotes mucosal healing and immune homeostasis: a randomized, controlled Phase 1 trial in healthy volunteers | 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 A human microbiome-derived therapeutic for ulcerative colitis promotes mucosal healing and immune homeostasis: a randomized, controlled Phase 1 trial in healthy volunteers Lutz Krause, Páraic Cuív, Johanna Ljungberg, Joyce Zhou, Michael Nissen, and 26 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6771039/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Using a metagenome-guided, large cohort-based approach, we identified Hominenteromicrobium mulieris as prevalent in healthy individuals but depleted in ulcerative colitis. In murine colitis models, a newly isolated strain of this species, MH27-2, improved disease pathology and accelerated gut healing, marked by epithelial restitution and reduced immune cell infiltration. In vitro, MH27-2 promoted mucosal healing, through accelerating epithelial cell migration and proliferation, and by improving gut barrier integrity and supporting immune homeostasis. Scalable manufacturing processes were developed and the safety of MH27-2 drug product, MAP 315, was evaluated in a randomized, double-blind, placebo-controlled, multiple-dose Phase 1 trial (ACTRN12623000291684). MAP 315 or placebo was administered daily for 14 days to 32 healthy female and male adults. MAP 315 was safe and well-tolerated, with no serious adverse events, bacterial translocation, or clinically significant changes in inflammatory markers, supporting its further clinical development as a novel microbiome-derived therapeutic for ulcerative colitis. Biological sciences/Drug discovery Health sciences/Diseases/Gastrointestinal diseases/Inflammatory bowel disease Biological sciences/Microbiology/Bacteria/Bacterial host response Inflammatory bowel disease Ulcerative colitis Gut Microbiome Metagenomic Hominenteromicrobium mulieris Drug Live biotherapeutic Mucosal healing Gut barrier Inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations Yes there is potential Competing Interest. This project was funded by Microba Life Sciences. Johanna K. Ljungberg, Joyce Zhou, Michael Nissen, Annika Krueger, Joel Boyd, Mareike Bongers, Jimenez Loayza Jeimy, Charlotte Vivian, Andrea Rabellino, Rhys Newell, Liang Fang, Samantha MacDonald, Alena Pribyl, Luke Reid, Nicola Angel, David L.A. Wood, Blake Wills, Trent Munro, Páraic Ó Cuív, Lutz Krause are or have been employees of Microba Life Sciences. Ella Reich completed an industry at Microba Life Sciences. Gene W. Tyson and Philip Hugenholtz are the founders of Microba Life Sciences. Shandelle Caban, Huw McCarthy, Joanne Soh, Simon Keely and Hiram Chipperfield have provided paid services to Microba Life Sciences. Ian H. Frazer is non-executive director and both he and Jakob Begun are members of the medical advisory board at Microba Life Sciences. Microba Life Sciences is a microbial genomics company developing microbiome-based diagnostic tests and therapeutics. Supplementary Files Suppfig.pdf Figure S1. A. Microscopy image of Gram stained MH27-2 cells. B. MH27-2 growth in YG/P is affected by increasing concentrations of bile salts. The specific growth rate of MH27-2 is significantly higher in YG/P as compared to YG/P supplemented with 0.25 or 0.5% bile salts. All data are presented as average and SD. Significance was determined using the ordinary one-way ANOVA. ****, p < 0.0001. Figure S2. A. In a therapeutic model of DSS-induced acute murine colitis (Figure 2A), the body weight changes of mice were monitored over the course of the experiment. The body weight over time in mice treated with MH27-2 and DSS was comparable to that of mice receiving DSS alone. B. Endoscopy was performed on Day -1 for a baseline pre-DSS score, Day 3 one day prior to the commencement of LBP treatments, and Day 9 after six days of LBP treatment. For each time point, the different groups were compared to DSS + Vehicle group, using an uncorrected Brown-Forsythe and Welch ANOVA test with multiple comparisons. Data presented as mean with standard deviation. For all data, ns: not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. C. Lipocalin-2/NGAL ELISA was performed on fecal protein isolates collected at post-mortem and is presented as the concentration of Lipocalin-2/NGAL (pg) to weight of stool (mg). All groups were compared directly to the DSS + Vehicle group using the uncorrected Brown-Forsythe and Welch ANOVA test with multiple comparisons. Data presented as mean ± standard deviation. For all data, ns: not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Figure S3. A. Effects on gut microbial community composition in a therapeutic model of DSS-induced acute murine colitis (Figure 2A). Principal Coordinates Analysis (PCoA) of fecal microbial profiles based on Bray-Curtis distances. Fecal pellet samples were collected from mice in three experimental groups: Vehicle (n=10, dark blue), DSS + Vehicle (n=10, green), and DSS + MH27-2 (n=10, pink). B. Microbial Shannon diversity (left), evenness (middle), and richness (right) at days 7 and 10 post-DSS treatment initiation. Statistical differences were assessed using t-tests with multiple test correction, where * indicates p < 0.05. Figure S4. The prophylactic treatment of MH27-2 was evaluated in the DSS mouse model as presented in Figure 2F. In mice challenged with DSS, the co-treatment with live MH-27-2 significantly ameliorated inflammation (A.), as well as erosion and ulceration (B.), compared to mice exposed to DSS only. Although improvements in abnormalities of mucosal architecture (C.), epithelial regeneration (D.) and the percentage of tissue involved (E.) were also observed in mice treated with both DSS and MH27-2, these changes were not statistically significant. For all data, ns: not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; presented is the mean and standard deviation. The Kruskal-Wallis test with uncorrected Dunn’s multiple comparisons was used for statistical testing. Figure S5. Prophylactic effects of MH27-2 were assessed in a mouse model of acute murine colitis that uses the haptenizing agent 2,4,6-Trinitrobenzene sulfonic acid (TNBS) to cause predominately Th1-driven pro-inflammatory responses in the murine colon characteristic of CD in humans (experimental setup presented in Figure 2K). A. MH27-2 ameliorated TNBS induced colitis as evidenced by the total histological score (total colitis index), that is the sum of five assessed sub-scores including inflammation, epithelial regeneration, erosion/ulceration, abnormalities of mucosal architecture, and lastly, the percentage involvement. One-way ANOVA with uncorrected Fisher’s LSD test for multiple comparison. B. The macroscopic score is composed of the four sub-scores ulcers/inflammation, strictures, wall thickness, and adhesions. Treatment with MH27-2 significantly improved the occurrence of ulcers and inflammation, but had no effect on the other three macroscopic readouts. Kruskal-Wallis test with uncorrected Dunn’s multiple comparisons. C. Compared to the DSS vehicle control, MH27-2 reduced levels of pro-inflammatory cytokine interleukin-6 (IL-6) in colonic tissue, had no effect on IL-17, TNF, MPO, and increased levels of IL-12. Kruskal-Wallis test with uncorrected Dunn’s multiple comparisons was used for IL-6, and for the remaining cytokines a one-way ANOVA with uncorrected Fisher’s LSD test for multiple comparison. For all data in Figure S5, ns: not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; presented is the mean and standard deviation. Figure S6. A. Proliferation of T84 cells was inhibited by the pro-inflammatory stimulus IFNγ and ameliorated by treatment with MH27-2 extract relative to the medium control extract. B. Proliferation of HT29 cells is inhibited by IFNγ and this effect is ameliorated by treatment MH27-2 extract but not medium control. The bacterial controls, Enterocloster (previously Clostridium ) bolteae (strain ATCC BAA-613), a bacterium positively associated with IBD 38 , and Faecalibacterium prausnitzii (strain A2­165), a well-known anti-inflammatory gut bacterium depleted in IBD and currently evaluated as LBP candidate, did not promote proliferation. C. Endpoint analysis (200 h) for the proliferation data presented in B. with statistical analysis; Dunnett’s multiple comparisons test medium control versus bacterial extracts, ****, p < 0.0001. Figure S7. A. The effect of MH27-2 on barrier function of T84 gut epithelial cells was assessed in a trans-epithelial electrical resistance (TEER) model. For this, IL-6 was used as a barrier disruptor which, after 48 hours, led to an approximately 75% reduction in TEER, indicative of an increased barrier permeability, which naturally and gradually increased upon removal of the stimulus. T84 cells were treated with MH27-2 extract (1X) for one hour prior to challenge with IL-6 (100 ng/mL), and MH27-2 was replenished every 24 hours throughout the duration of the experiment. MH27-2 mitigated IL-6 mediated reduction in battier integrity, and promoted recovery compared to the medium control extract. One-way ANOVA at 48 and 240 hours. B. At experimental endpoint of the TEER assays presented in Figure S7A, cell permeability was assessed by paracellular translocation of FITC-labelled dextran. Compared to the medium control, MH27-2 extract significantly reduced the flux of FITC-dextran across the T84 cell monolayer as assessed through One-way ANOVA. C. In T84 cells and mouse intestinal organoids, MH27-2 metabolite extract (1X) mitigates IFNγ (100 ng/mL) mediated transcriptional changes of ZO1, OCLDN, and select pro-inflammatory drivers after 24 hours. Experiments performed in biological triplicates, two technical replicates each; unpaired two-tailed t-test comparing IFNγ + YG/P medium control to IFNγ + MH27-2. D. MH27-2 culture supernatant (raw and <3kDa filtered), and MH27-2 extract (1X) significantly mitigated IL-6 mediated STAT3 activity in a HEK-Blue IL-6 reporter cell line. E. MH27-2 extract (1X) significantly mitigated IL-6/IL6R mediated STAT3 activity in a HEK-Blue IL-6 reporter cell line. F. Raw MH27-2 culture supernatant and MH27-2 extract (1X) significantly mitigated IFNγ-mediated STAT1 activity in a HEK-Blue IFNγ reporter cell line. Figure S8. A. As assessed through confocal microscopy, one hour pre-treatment with select fractions of MH27-2 extract, known to contain PLA and ILA respectively, significantly mitigated IFNγ-mediated reduction in ZO-1 protein expression following 24 hours stimulation. B. As assessed by HPLC, MH27-2 principally produces D-PLA when grown in YG/V. Supplementation with phenylpyruvic acid results in increased L-PLA, but not D-PLA, production. C. As assessed through confocal microscopy, one hour pre-treatment with the aromatic lactic acids ILA, HPLA, D-PLA, and L-PLA followed by 24 hours stimulation with IFNγ resulted in dose-dependent protective effects on ZO-1 protein expression. D. Representative images of effect of aromatic lactic acids on ZO-1 protein expression. E. As assessed through RT-qPCR, one hour pre-treatment with ILA, HPLA, D-PLA, or L-PLA, followed by 24-hour stimulation with IFNγ significantly mitigated transcriptional changes of ZO1 (HPLA and D-PLA) and OCLDN (HPLA), IL6 (ILA and D-PLA), CCL3 (L-PLA), and ISG15 (ILA, D-PLA, and L-PLA). F. The effect of various concentrations of ILA on barrier function of T84 gut epithelial cells was assessed in a trans-epithelial electrical resistance (TEER) model. T84 cells were pre-treated with ILA of Tofacitinib (TFC; 80µM) for one hour prior to challenge with IFNγ (100 ng/mL), and treatments were replenished every 24 hours throughout the duration of the experiment. ILA mitigated IFNγ-mediated loss of barrier integrity in a dose dependent manner. Figure S9. A. Gating strategy for flow cytometry experiment presented in Figure 4A (Immature monocyte-derived dendritic cells were exposed to MH27-2 bacterial cells, followed by co-culture with naïve CD4+ T cells, resulting in differentiation to Foxp3 + regulatory T cells). Figure S10. A. Testing for virulent phage and for inducible prophage presence. First round of enrichment of virulent phage test (left upper panel), second round of enrichment of virulent phage test (right upper panel), and test for inducible prophage (left lower panel) are represented by typical field of view. Positive control is right lower panel. B. A specific growth rate of 0.33 h -1 was measured and maximum culture density was achieved after ten hours when MH27-2 was cultured in YG/P animal component-free medium. In comparison, a growth rate of 0.25 h -1 and maximum culture density after 13 hours was reached in YG/V animal component-free medium. No growth was observed in PYG medium containing casein-derived tryptone. Figure S11. Images of MAP 315 drug substance (A.) and drug product (B.) Figure S12. A. The therapeutic efficacy of MAP 315 drug substance in three different doses was assessed in a prophylactic model of DSS-induced murine colitis, and compared to freshly grown live MH27-2. Both the drug substance and freshly grown MH27-2 significantly improved the total histopathological score (B.), which includes the five sub-scores inflammation (C.), erosion and ulceration (D.), percentage of the colonic epithelium affected (E.), epithelial regeneration (F.) and abnormalities of mucosal architecture (G.). There was no significant difference in the efficacy of the MAP 315 drug substance and freshly grown MH27-2 cells, indicating that the drug substance manufacturing process did not negatively impact therapeutic efficacy (Total histology score MH27 live vs. MAP 315 (5E7) p=0.56; MAP 315 (5E6) p=0.25; MAP 315 (5E4) p = 0.07; For the subscores, all comparisons p>0.05 except for epithelial regeneration MH27-2 live vs MAP 315 5e4 p=0.02). The statistical test for the total histology score was a one-way ANOVA with uncorrected Fisher’s LSD test for multiple comparisons. For the five subscores, a Kruskal-Wallis test with uncorrected Dunn’s multiple comparisons was used. Data is presented as the mean and SD, and ns: not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Figure S13. Representative images of histopathologic findings from the murine tolerability study. Healthy C57BL/6 mice were treated with 5 x 10 6 , 5 x 10 7 or 1 x 10 8 CFU/mouse/day of MAP 315 for 14 days by oral administration to evaluate safety and tolerability. The gastrointestinal tracts, spleens and mesenteric lymph nodes of the treated mice were evaluated by histopathology and there were no atypical findings. Representative pathology images from the colon, ileum, jejunum, rectum, duodenum, and stomach of the (A.) vehicle group and (B.) high dose groups (1 x 10 8 CFU/mouse/day of MAP 315) are shown. Figure S14. A Phase 1 clinical study was conducted to assess the safety and tolerability of the MAP 315 drug product in humans. This single-center, randomized, double-blind, placebo-controlled study involved multiple doses administered to 32 healthy adults. Participants were randomized in a 3:1 ratio to receive either MAP 315 or a matching placebo for 14 consecutive days and were divided into two cohorts based on dosage: a low-dose group (one MAP 315 or placebo capsule daily) and a high-dose group (eight MAP 315 or placebo capsules daily). The study was completed as planned, with all participants finishing the trial. Cite Share Download PDF Status: Under Review 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-6771039","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":468806877,"identity":"86935807-77b8-4b71-9ed6-2c76829d0fb8","order_by":0,"name":"Lutz Krause","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBACPjB5AEwyPmAwAFISBLSwMTDDtTAbkKyFDaKYoBaJ/MMfGM7ckzc4f/hY1Y0Cu3x+6eYHDD8qtuHRkgx0zY1iww030tJu5xgkW86cc8yAsefMbbxaEhg+JDBuuMFjBtRywMDgRoIBM2Mbfi0HgFrsN5w//60YoiX9AyEtjA0MNxISNxzIYWOGaMkhYAvPY2OGhDMJyTNvpBlLA/1iIDnnTMFBfH7hZ098/OHDsQTbvvOHH37O+WNnwC/dvvHBjwrcWsAgAV3gAH71o2AUjIJRMAoIAQB6HVWRDZlChQAAAABJRU5ErkJggg==","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":true,"prefix":"","firstName":"Lutz","middleName":"","lastName":"Krause","suffix":""},{"id":468806878,"identity":"606f6f78-2d6a-4c5e-b67d-fe84c6d9372a","order_by":1,"name":"Páraic Cuív","email":"","orcid":"","institution":"Microba Life Sciences, Translational Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Páraic","middleName":"","lastName":"Cuív","suffix":""},{"id":468806879,"identity":"ed0e3f64-df04-4df0-bfd4-5eeb0fc144a8","order_by":2,"name":"Johanna Ljungberg","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Johanna","middleName":"","lastName":"Ljungberg","suffix":""},{"id":468806880,"identity":"5fc2d971-2ec5-42e0-b182-1114d3a19555","order_by":3,"name":"Joyce Zhou","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Joyce","middleName":"","lastName":"Zhou","suffix":""},{"id":468806881,"identity":"1252da61-6018-4905-ab0b-863e19926dcc","order_by":4,"name":"Michael Nissen","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Nissen","suffix":""},{"id":468806882,"identity":"5125b1ea-1ea0-4235-8917-d523e59eb05e","order_by":5,"name":"Annika Krueger","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Annika","middleName":"","lastName":"Krueger","suffix":""},{"id":468806883,"identity":"70b25469-61d8-4097-8683-2fa6b4555e76","order_by":6,"name":"Joel Boyd","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Joel","middleName":"","lastName":"Boyd","suffix":""},{"id":468806884,"identity":"da8ed021-50c0-44f4-8576-64ca7c030f58","order_by":7,"name":"Mareike Bongers","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mareike","middleName":"","lastName":"Bongers","suffix":""},{"id":468806885,"identity":"3ea453ca-ac69-498c-8acf-e2ed80f1422b","order_by":8,"name":"Jeimy Jimenez Loayza","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jeimy","middleName":"Jimenez","lastName":"Loayza","suffix":""},{"id":468806886,"identity":"15408ccc-c9a1-4f1d-b9d7-40897c33e1ec","order_by":9,"name":"Charlotte Vivian","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Charlotte","middleName":"","lastName":"Vivian","suffix":""},{"id":468806887,"identity":"c1b6d861-279b-4ee9-bc0c-9614c61962b7","order_by":10,"name":"Ella Reich","email":"","orcid":"https://orcid.org/0009-0007-6240-6013","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ella","middleName":"","lastName":"Reich","suffix":""},{"id":468806888,"identity":"bc1bd752-06b4-47d7-85f7-e6e2c9202193","order_by":11,"name":"Andrea Rabellino","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Rabellino","suffix":""},{"id":468806889,"identity":"7ceab52d-d6e9-4295-b175-ef8a44c99d6b","order_by":12,"name":"Rhys Newel","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Rhys","middleName":"","lastName":"Newel","suffix":""},{"id":468806890,"identity":"8e35b1e1-90a1-493c-9235-656ee6a7ac28","order_by":13,"name":"Liang Fang","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Fang","suffix":""},{"id":468806891,"identity":"ff801357-1538-4ff9-823b-72681498936d","order_by":14,"name":"Samantha MacDonald","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Samantha","middleName":"","lastName":"MacDonald","suffix":""},{"id":468806892,"identity":"2b338c7d-7a22-45c5-929d-2e585afd394e","order_by":15,"name":"Alena Pribyl","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Alena","middleName":"","lastName":"Pribyl","suffix":""},{"id":468806893,"identity":"e15bb678-43f5-4f38-86d9-c8f513ff9c6b","order_by":16,"name":"Shandelle Caban","email":"","orcid":"","institution":"The University of Newcastle","correspondingAuthor":false,"prefix":"","firstName":"Shandelle","middleName":"","lastName":"Caban","suffix":""},{"id":468806894,"identity":"d552c620-fe44-4196-a3cc-a137e268920d","order_by":17,"name":"Huw McCarthy","email":"","orcid":"","institution":"The University of Newcastle","correspondingAuthor":false,"prefix":"","firstName":"Huw","middleName":"","lastName":"McCarthy","suffix":""},{"id":468806895,"identity":"9be259f9-391e-4e08-8ff6-7b0972141dbb","order_by":18,"name":"Joanne Soh","email":"","orcid":"","institution":"The University of Newcastle","correspondingAuthor":false,"prefix":"","firstName":"Joanne","middleName":"","lastName":"Soh","suffix":""},{"id":468806896,"identity":"a9e0e1a3-6cfc-40a9-a1e8-9e1c1c57de0d","order_by":19,"name":"Luke Reid","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Luke","middleName":"","lastName":"Reid","suffix":""},{"id":468806897,"identity":"b5db0461-77d3-4f06-aa54-de052a5725f1","order_by":20,"name":"Blake Wills","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Blake","middleName":"","lastName":"Wills","suffix":""},{"id":468806898,"identity":"23b92462-903e-4327-99db-059c007c26f2","order_by":21,"name":"Duncan McHale","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Duncan","middleName":"","lastName":"McHale","suffix":""},{"id":468806899,"identity":"bf65fddd-2cf5-4507-891f-b0f7d6a7957e","order_by":22,"name":"David Wood","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Wood","suffix":""},{"id":468806900,"identity":"1c5fa1ce-13c2-4cf0-83df-5c746bf1e3de","order_by":23,"name":"Ian Frazer","email":"","orcid":"https://orcid.org/0000-0002-8002-4680","institution":"University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Ian","middleName":"","lastName":"Frazer","suffix":""},{"id":468806901,"identity":"62484f48-8a4a-43fc-9658-0e8e63657920","order_by":24,"name":"Nicola Angel","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nicola","middleName":"","lastName":"Angel","suffix":""},{"id":468806902,"identity":"3e3a9fee-460c-40be-a4d0-a01e8f4f56c5","order_by":25,"name":"Hiram Chipperfield","email":"","orcid":"","institution":"Beyond Drug Development Pty Ltd","correspondingAuthor":false,"prefix":"","firstName":"Hiram","middleName":"","lastName":"Chipperfield","suffix":""},{"id":468806903,"identity":"ac7d7a28-6f8a-4996-9460-6695c101aca2","order_by":26,"name":"Jakob Begun","email":"","orcid":"https://orcid.org/0000-0001-5256-7672","institution":"Mater Research Institute, University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Jakob","middleName":"","lastName":"Begun","suffix":""},{"id":468806904,"identity":"9d4a16aa-d6cc-4069-83ab-d62310863b5c","order_by":27,"name":"Simon Keely","email":"","orcid":"https://orcid.org/0000-0002-1248-9590","institution":"NHMRC Centre of Research Excellence in Digestive Health, The University of Newcastle","correspondingAuthor":false,"prefix":"","firstName":"Simon","middleName":"","lastName":"Keely","suffix":""},{"id":468806905,"identity":"63716c57-7ceb-4b85-ae99-162606eeada4","order_by":28,"name":"Gene Tyson","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Gene","middleName":"","lastName":"Tyson","suffix":""},{"id":468806906,"identity":"762b956c-a2dc-4823-a500-cdcfde201898","order_by":29,"name":"Philip Hugenholtz","email":"","orcid":"","institution":"Australian Centre for Ecogenomics, University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Philip","middleName":"","lastName":"Hugenholtz","suffix":""},{"id":468806907,"identity":"9978bba6-0d78-4bd4-858c-05bf18eda3ed","order_by":30,"name":"Trent Munro","email":"","orcid":"","institution":"Microba Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Trent","middleName":"","lastName":"Munro","suffix":""}],"badges":[],"createdAt":"2025-05-28 21:01:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6771039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6771039/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85911267,"identity":"39c94504-613f-4bf6-8fd4-d88238964d7a","added_by":"auto","created_at":"2025-07-03 05:34:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":743680,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic trees of \u003cem\u003eHominenteromicrobium mulieris \u003c/em\u003e(s__\u003cem\u003eUBA1417 sp003531055\u003c/em\u003e) and surrounding species with their prevalence in IBD patients and healthy subjects. \u003cstrong\u003eA\u003c/strong\u003e. Genome tree constructed using publicly available genomes from NCBI using GTDBtk. Each leaf is a single representative of a species within the bacterial family \u003cem\u003eAcutalibacteraceae\u003c/em\u003e. Colored nodes represent the bootstrap support values (white ≥50%, grey ≥75%, black 100%). \u003cstrong\u003eB. \u003c/strong\u003ePhylogenetic tree showing the broader phylogenetic context of \u003cem\u003eH. mulieris\u003c/em\u003e. Genera with ≥5 member species are labelled, with \u003cem\u003eH. mulieris \u003c/em\u003eindicated by a red node. Each leaf of the tree connects to a bar in the adjacent bar plot, which represents the difference in prevalence of that species in Healthy (n) vs IBD (n), where green bars represent positive associations (higher in healthy), and red bars show negative associations (higher in IBD).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6771039/v1/1732774f02e16732876f6494.png"},{"id":85911266,"identity":"265be185-9b11-4983-9bf9-8ada9153fb5c","added_by":"auto","created_at":"2025-07-03 05:34:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1403717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eLive cultures of MH27-2 were assessed in a therapeutic dextran sulphate sodium (DSS) mouse model of colitis (n=10 per experimental group, except DSS control group n=20). Treatment with MH27-2 ameliorated DSS induced colitis as illustrated by representative gut histology images (\u003cstrong\u003eB.\u003c/strong\u003e) and as evidenced by a reduction of the histopathological score (\u003cstrong\u003eC.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e$\u003c/strong\u003e\u003c/sup\u003e), including the sub-score measures of inflammation (\u003cstrong\u003eD.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e$\u003c/strong\u003e\u003c/sup\u003e) and epithelial injury (\u003cstrong\u003eE.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003e), compared to the DSS vehicle control. \u003cstrong\u003eF. \u003c/strong\u003eLive cultures of MH27-2 were assessed in a prophylactic DSS mouse model (n=20 per group). Treatment with MH27-2 ameliorated DSS induced colitis as illustrated by representative gut histology images (\u003cstrong\u003eG.\u003c/strong\u003e) and as evidenced by a reduction of the histopathological score (\u003cstrong\u003eH.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e$\u003c/strong\u003e\u003c/sup\u003e), including the sub-score measures of inflammation (\u003cstrong\u003eI.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003e) as well as erosion and ulceration (\u003cstrong\u003eJ.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003e), compared to the DSS vehicle control. \u003cstrong\u003eK. \u003c/strong\u003eProphylactic effects of live MH27-2 were assessed in a mouse model of acute murine colitis that uses the haptenizing agent 2,4,6-Trinitrobenzene sulfonic acid (TNBS) to cause predominately Th1-driven pro-inflammatory responses in the murine colon characteristic of CD in humans. MH27-2 ameliorated TNBS induced colitis as illustrated by representative gut histology images (\u003cstrong\u003eL.\u003c/strong\u003e) and as evidenced by a reduction of the histopathological score (\u003cstrong\u003eM.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e$\u003c/strong\u003e\u003c/sup\u003e), including the sub-score measures of inflammation (\u003cstrong\u003eN.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e$\u003c/strong\u003e\u003c/sup\u003e) as well as epithelial regeneration (\u003cstrong\u003eO.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003e), and reduced levels of pro-inflammatory cytokine interleukin-6 (IL-6) in colonic tissue (\u003cstrong\u003eP.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003e) compared to the DSS vehicle control.\u003c/p\u003e\n\u003cp\u003eFor all data, ns: not significant; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001; presented is the mean and standard deviation. For statistical testing: \u003csup\u003e\u003cstrong\u003e$\u003c/strong\u003e\u003c/sup\u003eOne-way ANOVA with uncorrected Fisher’s LSD test for multiple comparison. \u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003eKruskal-Wallis test with uncorrected Dunn’s multiple comparisons.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6771039/v1/89bcfc96a771f8c5d6279615.png"},{"id":85909964,"identity":"19f4caa6-c4bc-4f97-aadd-d2a46330102e","added_by":"auto","created_at":"2025-07-03 05:10:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1103073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\n\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eSchematic representation of Transwell cell migration. \u003cstrong\u003eB. \u003c/strong\u003eCompared to the medium control, MH27-2 extract induced faster migration of HCT116 gut epithelial cells. Unpaired two-tailed t-test; biological triplicates, four technical replicates each. \u003cstrong\u003eC. \u003c/strong\u003eThe impact of MH27-2 on intestinal epithelial cell migration was also assessed using an \u003cem\u003ein vitro \u003c/em\u003escratch wound assay. The rate of wound closure of T84 gut epithelial cells challenged with IFNγ was monitored for up to 200 hours post-scratch. MH27-2 extract (1X) promoted wound closure as evidenced by representative images of biological triplicates. \u003cstrong\u003eD. \u003c/strong\u003eQuantitation of wound confluency of the experiment presented in Figure 3C. T84 cells showed a significantly accelerated rate of wound closure in the presence of MH27-2 secretome extract 80, 140 and 200 hours after wounding, compared to the control cells treated with bacterial medium extract control. \u003cstrong\u003eE. \u003c/strong\u003eIn mouse intestinal organoids, MH27-2 metabolite extract (1X) significantly mitigates IFNγ (100 ng/mL) mediated transcriptional changes of 13-catenin (\u003cem\u003eCtnnb1\u003c/em\u003e) and multiple WNT ligands after 24 hours. Experiment performed in biological triplicates, two technical replicates each; unpaired two-tailed t-test comparing IFNγ + YGV medium control to IFNγ + MH27-2. \u003cstrong\u003eF. \u003c/strong\u003eAt the protein level, as assessed by ELISA, suppression of 13-catenin by IFNγ is ameliorated by treatment with MH27-2 extract (1X) after 24 hours in mouse intestinal organoids and T84 gut epithelial cells. Biological triplicates, technical duplicates; unpaired two-tailed t-test comparing IFNγ + YGV medium control to IFNγ + MH27-2. \u003cstrong\u003eG\u003c/strong\u003e. 13-catenin protein expression in mouse intestinal organoids as assessed by immunofluorescence straining and confocal microscopy. Representative images of three independent biological replicates. Image quantitation is presented to the right (the average ratio of 13-catenin area of expression relative to the area of expression of DAPI (µm\u003csup\u003e2\u003c/sup\u003e) and normalized to the untreated organoids). \u003cstrong\u003eH. \u003c/strong\u003eThe effect of MH27-2 on barrier function of T84 gut epithelial cells was assessed in two trans-epithelial electrical resistance (TEER) models. In both models, IFNγ was used as a barrier disruptor which, after 48 hours, led to an approximately 80% reduction in TEER, indicative of an increased barrier permeability, which naturally and gradually recovered after removing of the stimulus. The prophylactic model is presented to the left, where T84 cells were treated with MH27-2 extract (1X) for one hour prior to challenge with IFNγ, and MH27-2 was replenished every 24 hours throughout the duration of the experiment. In the prophylactic model, to the right, T84 cells received MH27-2 treatment only after barrier disruption occurred in response to IFNγ. In both models, MH27-2 promoted recovery compared to the medium control extract, and prophylactic treatment was more effective than therapeutic administration. One-way ANOVA at 48 and 168 hours. \u003cstrong\u003eI. \u003c/strong\u003eAt experimental endpoint of the TEER assays presented in Figure 3H, cell permeability was assessed by paracellular translocation of FITC-labelled dextran. Compared to the medium control, MH27-2 extract significantly reduced the flux of FITC-dextran across the T84 cell monolayer. One-way ANOVA. For all bar graphs presented in Figure 3, data is presented as the mean and standard deviation (SD), and ns: not significant; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6771039/v1/ccaa4340e3f634b8dd116561.png"},{"id":85909962,"identity":"ca32bc17-06df-4b93-b7ef-dec26692d4e1","added_by":"auto","created_at":"2025-07-03 05:10:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eImmature monocyte-derived dendritic cells were exposed to MH27-2 bacterial cells, followed by co-culture with naïve CD4+ T cells, resulting in differentiation to Foxp3\u003csup\u003e+\u003c/sup\u003e regulatory T cells. \u003cstrong\u003eB. \u003c/strong\u003eMH27-2 did not induce secretion of the anti-inflammatory cytokine IL-10 by dendritic cells, indicating that Treg differentiation was probably IL-10-independent. \u003cstrong\u003eC. \u003c/strong\u003eIn TL1A reporter cells challenged with recombinant human TL1A (100 ng/mL), MH27-2 15% culture supernatant significantly mitigates TL1A signaling activation, compared to the bacterial medium control; Data presents mean ± SD from 3 biological replicates, 3 technical replicates each; unpaired two-tailed t-test \u003cem\u003ep \u003c/em\u003e= 0.025. \u003cstrong\u003eD. \u003c/strong\u003eRelative gene expression of CCL5, CXCL10, and TNF in DR3-expressing TF-1 cells challenged with TL1A is significantly reduced in cells treated with MH27-2 1x extract, compared to the medium extract. Three biological replicates, two technical replicates; mean ± SD, unpaired two-tailed t-test. \u003cstrong\u003eE. \u003c/strong\u003eCo-incubating MH27-2 bacterial cells with human PBMCs, resulted in PBMCs secreting higher levels of anti-inflammatory IL-10 than pro-inflammatory IL-12.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6771039/v1/d28c38151c642a475c691eff.png"},{"id":85911278,"identity":"29fdfd49-34d6-459a-aa71-dcdf98fe80aa","added_by":"auto","created_at":"2025-07-03 05:35:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1101921,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic summarizing the proposed therapeutic effects and mode of action of MH27-2 in the context of IBD. Wound healing in the gut involves the migration of epithelial cells to cover the injury site, followed by their proliferation to restore the integrity and functionality of the intestinal barrier. MH27-2 promotes both the migration and proliferation of injured gut epithelial cells. In IBD, gut barrier function is compromised due to disrupted epithelial integrity, increased permeability, and impaired tight junctions, leading to heightened immune activation and chronic inflammation. MH27-2, secretes both known (ALAs = aromatic lactic acids) and unknown bioactives, that influence several of these processes to promote restoration of gut barrier integrity and immune homeostasis.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6771039/v1/ebcdccbbce2f8845cd7cc7ab.png"},{"id":86879767,"identity":"76e1ae1e-798c-4031-be3c-f79bf92489bf","added_by":"auto","created_at":"2025-07-16 16:09:39","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1955545,"visible":true,"origin":"","legend":"Article File","description":"","filename":"MAP315Manuscript29052025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6771039/v1_covered_a0fa86c6-7e44-4a5c-aaca-1e0ecfe38779.pdf"},{"id":85911269,"identity":"7f603cb3-9213-446f-a33e-33f151ce1912","added_by":"auto","created_at":"2025-07-03 05:35:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1484264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1. A. \u003c/strong\u003eMicroscopy image of Gram stained MH27-2 cells. \u003cstrong\u003eB. \u003c/strong\u003eMH27-2 growth in YG/P is affected by increasing concentrations of bile salts. The specific growth rate of MH27-2 is significantly higher in YG/P as compared to YG/P supplemented with 0.25 or 0.5% bile salts. All data are presented as average and SD. Significance was determined using the ordinary one-way ANOVA. ****, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2. A. \u003c/strong\u003eIn a therapeutic model of DSS-induced acute murine colitis (Figure 2A), the body weight changes of mice were monitored over the course of the experiment. The body weight over time in mice treated with MH27-2 and DSS was comparable to that of mice receiving DSS alone. \u003cstrong\u003eB. \u003c/strong\u003eEndoscopy was performed on Day -1 for a baseline pre-DSS score, Day 3 one day prior to the commencement of LBP treatments, and Day 9 after six days of LBP treatment. For each time point, the different groups were compared to DSS + Vehicle group, using an uncorrected Brown-Forsythe and Welch ANOVA test with multiple comparisons. Data presented as mean with standard deviation. For all data, ns: not significant; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001. \u003cstrong\u003eC. \u003c/strong\u003eLipocalin-2/NGAL ELISA was performed on fecal protein isolates collected at post-mortem and is presented as the concentration of Lipocalin-2/NGAL (pg) to weight of stool (mg). All groups were compared directly to the DSS + Vehicle group using the uncorrected Brown-Forsythe and Welch ANOVA test with multiple comparisons. Data presented as mean ± standard deviation. For all data, ns: not significant; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S3. A. \u003c/strong\u003eEffects on gut microbial community composition in a therapeutic model of DSS-induced acute murine colitis (Figure 2A). Principal Coordinates Analysis (PCoA) of fecal microbial profiles based on Bray-Curtis distances. Fecal pellet samples were collected from mice in three experimental groups: Vehicle (n=10, dark blue), DSS + Vehicle (n=10, green), and DSS + MH27-2 (n=10, pink). \u003cstrong\u003eB. \u003c/strong\u003eMicrobial Shannon diversity (left), evenness (middle), and richness (right) at days 7 and 10 post-DSS treatment initiation. Statistical differences were assessed using t-tests with multiple test correction, where * indicates p \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S4. \u003c/strong\u003eThe prophylactic treatment of MH27-2 was evaluated in the DSS mouse model as presented in Figure 2F. In mice challenged with DSS, the co-treatment with live MH-27-2 significantly ameliorated inflammation (\u003cstrong\u003eA.\u003c/strong\u003e), as well as erosion and ulceration (\u003cstrong\u003eB.\u003c/strong\u003e), compared to mice exposed to DSS only. Although improvements in abnormalities of mucosal architecture (\u003cstrong\u003eC.\u003c/strong\u003e), epithelial regeneration (\u003cstrong\u003eD.\u003c/strong\u003e) and the percentage of tissue involved (\u003cstrong\u003eE.\u003c/strong\u003e) were also observed in mice treated with both DSS and MH27-2, these changes were not statistically significant. For all data, ns: not significant; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001; presented is the mean and standard deviation. The Kruskal-Wallis test with uncorrected Dunn’s multiple comparisons was used for statistical testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S5. \u003c/strong\u003eProphylactic effects of MH27-2 were assessed in a mouse model of acute murine colitis that uses the haptenizing agent 2,4,6-Trinitrobenzene sulfonic acid (TNBS) to cause predominately Th1-driven pro-inflammatory responses in the murine colon characteristic of CD in humans (experimental setup presented in Figure 2K). \u003cstrong\u003eA. \u003c/strong\u003eMH27-2 ameliorated TNBS induced colitis as evidenced by the total histological score (total colitis index), that is the sum of five assessed sub-scores including inflammation, epithelial regeneration, erosion/ulceration, abnormalities of mucosal architecture, and lastly, the percentage involvement. One-way ANOVA with uncorrected Fisher’s LSD test for multiple comparison. \u003cstrong\u003eB. \u003c/strong\u003eThe macroscopic score is composed of the four sub-scores ulcers/inflammation, strictures, wall thickness, and adhesions. Treatment with MH27-2 significantly improved the occurrence of ulcers and inflammation, but had no effect on the other three macroscopic readouts. Kruskal-Wallis test with uncorrected Dunn’s multiple comparisons. \u003cstrong\u003eC. \u003c/strong\u003eCompared to the DSS vehicle control, MH27-2 reduced levels of pro-inflammatory cytokine interleukin-6 (IL-6) in colonic tissue, had no effect on IL-17, TNF, MPO, and increased levels of IL-12. Kruskal-Wallis test with uncorrected Dunn’s multiple comparisons was used for IL-6, and for the remaining cytokines a one-way ANOVA with uncorrected Fisher’s LSD test for multiple comparison. For all data in Figure S5, ns: not significant; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001; presented is the mean and standard deviation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S6. A. \u003c/strong\u003eProliferation of T84 cells was inhibited by the pro-inflammatory stimulus IFNγ and ameliorated by treatment with MH27-2 extract relative to the medium control extract. \u003cstrong\u003eB. \u003c/strong\u003eProliferation of HT29 cells is inhibited by IFNγ and this effect is ameliorated by treatment MH27-2 extract but not medium control. The bacterial controls, \u003cem\u003eEnterocloster \u003c/em\u003e(previously \u003cem\u003eClostridium\u003c/em\u003e) \u003cem\u003ebolteae \u003c/em\u003e(strain ATCC\u003c/p\u003e\n\u003cp\u003eBAA-613), a bacterium positively associated with IBD\u003csup\u003e38\u003c/sup\u003e, and \u003cem\u003eFaecalibacterium prausnitzii \u003c/em\u003e(strain A2­165), a well-known anti-inflammatory gut bacterium depleted in IBD and currently evaluated as LBP candidate, did not promote proliferation. \u003cstrong\u003eC. \u003c/strong\u003eEndpoint analysis (200 h) for the proliferation data presented in \u003cstrong\u003eB. \u003c/strong\u003ewith statistical analysis; Dunnett’s multiple comparisons test medium control versus bacterial extracts, ****, p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S7. A. \u003c/strong\u003eThe effect of MH27-2 on barrier function of T84 gut epithelial cells was assessed in a trans-epithelial electrical resistance (TEER) model. For this, IL-6 was used as a barrier disruptor which, after 48 hours, led to an approximately 75% reduction in TEER, indicative of an increased barrier permeability, which naturally and gradually increased upon removal of the stimulus. T84 cells were treated with MH27-2 extract (1X) for one hour prior to challenge with IL-6 (100 ng/mL), and MH27-2 was replenished every 24 hours throughout the duration of the experiment. MH27-2 mitigated IL-6 mediated reduction in battier integrity, and promoted recovery compared to the medium control extract. One-way ANOVA at 48 and 240 hours. \u003cstrong\u003eB. \u003c/strong\u003eAt experimental endpoint of the TEER assays presented in Figure S7A, cell permeability was assessed by paracellular translocation of FITC-labelled dextran. Compared to the medium control, MH27-2 extract significantly reduced the flux of FITC-dextran across the T84 cell monolayer as assessed through One-way ANOVA. \u003cstrong\u003eC. \u003c/strong\u003eIn T84 cells and mouse intestinal organoids, MH27-2 metabolite extract (1X) mitigates IFNγ (100 ng/mL) mediated transcriptional changes of ZO1, OCLDN, and select pro-inflammatory drivers after 24 hours. Experiments performed in biological triplicates, two technical replicates each; unpaired two-tailed t-test comparing IFNγ + YG/P medium control to IFNγ + MH27-2. \u003cstrong\u003eD. \u003c/strong\u003eMH27-2 culture supernatant (raw and \u0026lt;3kDa filtered), and MH27-2 extract (1X) significantly mitigated IL-6 mediated STAT3 activity in a HEK-Blue IL-6 reporter cell line. \u003cstrong\u003eE. \u003c/strong\u003eMH27-2 extract (1X) significantly mitigated IL-6/IL6R mediated STAT3 activity in a HEK-Blue IL-6 reporter cell line. \u003cstrong\u003eF. \u003c/strong\u003eRaw MH27-2 culture supernatant and MH27-2 extract (1X) significantly mitigated IFNγ-mediated STAT1 activity in a HEK-Blue IFNγ reporter cell line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S8. A. \u003c/strong\u003eAs assessed through confocal microscopy, one hour pre-treatment with select fractions of MH27-2 extract, known to contain PLA and ILA respectively, significantly mitigated IFNγ-mediated reduction in ZO-1 protein expression following 24 hours stimulation. \u003cstrong\u003eB. \u003c/strong\u003eAs assessed by HPLC, MH27-2 principally produces D-PLA when grown in YG/V. Supplementation with phenylpyruvic acid results in increased L-PLA, but not D-PLA, production. \u003cstrong\u003eC. \u003c/strong\u003eAs assessed through confocal microscopy, one hour pre-treatment with the aromatic lactic acids ILA, HPLA, D-PLA, and L-PLA followed by 24 hours stimulation with IFNγ resulted in dose-dependent protective effects on ZO-1 protein expression. \u003cstrong\u003eD. \u003c/strong\u003eRepresentative images of effect of aromatic lactic acids on ZO-1 protein expression. \u003cstrong\u003eE. \u003c/strong\u003eAs assessed through RT-qPCR, one hour pre-treatment with ILA, HPLA, D-PLA, or L-PLA, followed by 24-hour stimulation with IFNγ significantly mitigated transcriptional changes of \u003cem\u003eZO1 \u003c/em\u003e(HPLA and D-PLA) and \u003cem\u003eOCLDN \u003c/em\u003e(HPLA), \u003cem\u003eIL6 \u003c/em\u003e(ILA and D-PLA), \u003cem\u003eCCL3 \u003c/em\u003e(L-PLA), and \u003cem\u003eISG15 \u003c/em\u003e(ILA, D-PLA, and L-PLA). \u003cstrong\u003eF. \u003c/strong\u003eThe effect of various concentrations of ILA on barrier function of T84 gut epithelial cells was assessed in a trans-epithelial electrical resistance (TEER) model. T84 cells were pre-treated with ILA of Tofacitinib (TFC; 80µM) for one hour prior to challenge with IFNγ (100 ng/mL), and treatments were replenished every 24 hours throughout the duration of the experiment. ILA mitigated IFNγ-mediated loss of barrier integrity in a dose dependent manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S9. A. \u003c/strong\u003eGating strategy for flow cytometry experiment presented in Figure 4A (Immature monocyte-derived dendritic cells were exposed to MH27-2 bacterial cells, followed by co-culture with naïve CD4+ T cells, resulting in differentiation to Foxp3\u003csup\u003e+\u003c/sup\u003e regulatory T cells).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S10. A. \u003c/strong\u003eTesting for virulent phage and for inducible prophage presence. First round of enrichment of virulent phage test (left upper panel), second round of enrichment of virulent phage test (right upper panel), and test for inducible prophage (left lower panel) are represented by typical field of view. Positive control is right lower panel. \u003cstrong\u003eB. \u003c/strong\u003eA specific growth rate of 0.33 h\u003csup\u003e-1\u003c/sup\u003e was measured and maximum culture density was achieved after ten hours when MH27-2 was cultured in YG/P animal component-free medium. In comparison, a growth rate of 0.25 h\u003csup\u003e-1\u003c/sup\u003e and maximum culture density after 13 hours was reached in YG/V animal component-free medium. No growth was observed in PYG medium containing casein-derived tryptone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S11. \u003c/strong\u003eImages of MAP 315 drug substance (\u003cstrong\u003eA.\u003c/strong\u003e) and drug product (\u003cstrong\u003eB.\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S12. A. \u003c/strong\u003eThe therapeutic efficacy of MAP 315 drug substance in three different doses was assessed in a prophylactic model of DSS-induced murine colitis, and compared to freshly grown live MH27-2. Both the drug substance and freshly grown MH27-2 significantly improved the total histopathological score (\u003cstrong\u003eB.\u003c/strong\u003e), which includes the five sub-scores inflammation (\u003cstrong\u003eC.\u003c/strong\u003e), erosion and ulceration (\u003cstrong\u003eD.\u003c/strong\u003e), percentage of the colonic epithelium affected (\u003cstrong\u003eE.\u003c/strong\u003e), epithelial regeneration (\u003cstrong\u003eF.\u003c/strong\u003e) and abnormalities of mucosal architecture (\u003cstrong\u003eG.\u003c/strong\u003e). There was no significant difference in the efficacy of the MAP 315 drug substance and freshly grown MH27-2 cells, indicating that the drug substance manufacturing process did not negatively impact therapeutic efficacy (Total histology score MH27 live vs. MAP 315 (5E7) p=0.56; MAP 315 (5E6) p=0.25; MAP 315 (5E4) p = 0.07; For the subscores, all comparisons p\u0026gt;0.05 except for epithelial regeneration MH27-2 live vs MAP 315 5e4 p=0.02). The statistical test for the total histology score was a one-way ANOVA with uncorrected Fisher’s LSD test for multiple comparisons. For the five subscores, a Kruskal-Wallis test with uncorrected Dunn’s multiple comparisons was used. Data is presented as the mean and SD, and ns: not significant; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S13. \u003c/strong\u003eRepresentative images of histopathologic findings from the murine tolerability study. Healthy C57BL/6 mice were treated with 5 x 10\u003csup\u003e6\u003c/sup\u003e, 5 x 10\u003csup\u003e7\u003c/sup\u003e or 1 x 10\u003csup\u003e8\u003c/sup\u003e CFU/mouse/day of MAP 315 for\u003c/p\u003e\n\u003cp\u003e14 days by oral administration to evaluate safety and tolerability. The gastrointestinal tracts, spleens and mesenteric lymph nodes of the treated mice were evaluated by histopathology and there were no atypical findings. Representative pathology images from the colon, ileum, jejunum, rectum, duodenum, and stomach of the (\u003cstrong\u003eA.\u003c/strong\u003e) vehicle group and (\u003cstrong\u003eB.\u003c/strong\u003e) high dose groups (1 x 10\u003csup\u003e8\u003c/sup\u003e CFU/mouse/day of MAP 315) are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S14. \u003c/strong\u003eA Phase 1 clinical study was conducted to assess the safety and tolerability of the MAP 315 drug product in humans. This single-center, randomized, double-blind, placebo-controlled study involved multiple doses administered to 32 healthy adults. Participants were randomized in a 3:1 ratio to receive either MAP 315 or a matching placebo for 14 consecutive days and were divided into two cohorts based on dosage: a low-dose group (one MAP 315 or placebo capsule daily) and a high-dose group (eight MAP 315 or placebo capsules daily). The study was completed as planned, with all participants finishing the trial.\u003c/p\u003e","description":"","filename":"Suppfig.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6771039/v1/aacc4c72835978138dd889f0.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nThis project was funded by Microba Life Sciences. Johanna K. Ljungberg, Joyce Zhou, Michael Nissen, Annika Krueger, Joel Boyd, Mareike Bongers, Jimenez Loayza Jeimy, Charlotte Vivian, Andrea Rabellino, Rhys Newell, Liang Fang, Samantha MacDonald, Alena Pribyl, Luke Reid, Nicola Angel, David L.A. Wood, Blake Wills, Trent Munro, Páraic Ó Cuív, Lutz Krause are or have been employees of Microba Life Sciences. Ella Reich completed an industry at Microba Life Sciences. Gene W. Tyson and Philip Hugenholtz are the founders of Microba Life Sciences. Shandelle Caban, Huw McCarthy, Joanne Soh, Simon Keely and Hiram Chipperfield have provided paid services to Microba Life Sciences. Ian H. Frazer is non-executive director and both he and Jakob Begun are members of the medical advisory board at Microba Life Sciences. Microba Life Sciences is a microbial genomics company developing microbiome-based diagnostic tests and therapeutics.","formattedTitle":"A human microbiome-derived therapeutic for ulcerative colitis promotes mucosal healing and immune homeostasis: a randomized, controlled Phase 1 trial in healthy volunteers","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Inflammatory bowel disease, Ulcerative colitis, Gut, Microbiome, Metagenomic, Hominenteromicrobium mulieris, Drug, Live biotherapeutic, Mucosal healing, Gut barrier,Inflammation","lastPublishedDoi":"10.21203/rs.3.rs-6771039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6771039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Using a metagenome-guided, large cohort-based approach, we identified Hominenteromicrobium mulieris as prevalent in healthy individuals but depleted in ulcerative colitis. In murine colitis models, a newly isolated strain of this species, MH27-2, improved disease pathology and accelerated gut healing, marked by epithelial restitution and reduced immune cell infiltration. In vitro, MH27-2 promoted mucosal healing, through accelerating epithelial cell migration and proliferation, and by improving gut barrier integrity and supporting immune homeostasis. Scalable manufacturing processes were developed and the safety of MH27-2 drug product, MAP 315, was evaluated in a randomized, double-blind, placebo-controlled, multiple-dose Phase 1 trial (ACTRN12623000291684). MAP 315 or placebo was administered daily for 14 days to 32 healthy female and male adults. MAP 315 was safe and well-tolerated, with no serious adverse events, bacterial translocation, or clinically significant changes in inflammatory markers, supporting its further clinical development as a novel microbiome-derived therapeutic for ulcerative colitis.","manuscriptTitle":"A human microbiome-derived therapeutic for ulcerative colitis promotes mucosal healing and immune homeostasis: a randomized, controlled Phase 1 trial in healthy volunteers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-03 05:10:38","doi":"10.21203/rs.3.rs-6771039/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"66a52be5-3a66-4d8f-9ad9-a060e4caa767","owner":[],"postedDate":"July 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49774024,"name":"Biological sciences/Drug discovery"},{"id":49774025,"name":"Health sciences/Diseases/Gastrointestinal diseases/Inflammatory bowel disease"},{"id":49774026,"name":"Biological sciences/Microbiology/Bacteria/Bacterial host response"}],"tags":[],"updatedAt":"2026-01-23T13:05:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-03 05:10:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6771039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6771039","identity":"rs-6771039","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}