Microbial Activation of the GLP-2R Mitigates Gastrointestinal Inflammation

Microbial Activation of the GLP-2R Mitigates Gastrointestinal Inflammation | 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 Microbial Activation of the GLP-2R Mitigates Gastrointestinal Inflammation Benjamin Jensen, Sune Yang-Jensen, Béatrice Choi, Simone Pærregaard, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6973437/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jan, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract There is an urgent need for sustainable protein sources to meet rising global nutritional demands. Here, we show that a commercially scalable microbial lysate from Methylococcus capsulatus Bath (McB), used as a dietary protein, orchestrates host-diet-microbe interactions that protect against gastrointestinal inflammation. McB administration rapidly reshapes the gut microbiota and upregulates microbial fermentation pathways, while robustly increasing peripherally induced regulatory T cells (pTregs) across intestinal regions, independent of the microbiota. In contrast, McB-driven induction of tolerogenic Th17 cells requires a functional microbiota. In models of mucositis and colitis, McB preserves villus architecture, restores mucosal integrity, and reduces disease severity. Mechanistically, these effects depend on microbial fermentation and functional GLP-2 receptor signalling, yet are independent of endogenous GLP-2 secretion, indicating a fermentation-driven molecular mimicry of GLP-2R activation. Collectively, our findings position microbial lysates as a sustainable nutritional strategy that improves gastrointestinal health through defined immune and microbial pathways. Biological sciences/Biological techniques/Biological models/Gastrointestinal models Biological sciences/Immunology/Inflammation Cultured protein microbial lysate nutritional imprinting microbiota T-cells gut inflammation GLP-2 GLP-2R Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Amidst the urgent need for sustainable food systems 1 to combat excessive green house gass emmsions, resource depletion, and biodiversity loss 2 , cultured microbial proteins are emerging as low-emission, resource-efficient alternatives to animal-derived products 1 . While the ecological benefits of these cultured proteins are well established 1 , their influence on host biology – particularly the impact on gastrointestinal (GI) homeostasis – remains largely unexplored. This knowledge gap is critical, as dietary components actively shape immune function, host-microbe interactions, and barrier integrity. Within the gut, dietary components are digested into smaller entities that interact with the mucosal immune system both directly, through antigen presentation to immune cells such as T cells, and indirectly, by modulating the structure and function of the gut microbiota 3 . These interactions underpin immune education, as evidenced by the influence of diet on vaccine efficacy 4 and food allergy development 5 . Host-diet-microbe dynamics are especially important for the differentiation of RORγt-expressing T cell subsets, including peripherally induced regulatory T cells (pTregs) and T-helper 17 (Th17) cells. pTregs, induced by the gut microbiota 6 and their cellular components 7 , suppress aberrant immune responses to food antigens 8 and commensal microbes 9 . Th17 cells, regulated by both host- and microbially derived signals 10 , adopt context-dependent roles – ranging from barrier reinforcement to pathogenic inflammation 11 – dictated by the local microenvironment’s inflammatory milieu 12,13 . Notably, since both subsets respond to microbial and dietary cues, their phenontype and function hinge on the functional capacity of resident gut microbes. Disruptions to these host-diet-microbe interactions underlie a spectrum of diseases, including inflammatory bowel diseases (IBD) and metabolic disorders 14–18 , with contemporary Western diets epidemiologically linked to rising prevalence of GI inflammation 19–21 . Although microbiota-targeted therapies show promise, their inconsistent efficacy highlights the need for interventions that bypass or reprogram microbial dependencies to promote immune tolerance and tissue repair. Previously, we demonstrated that a bacterial lysate derived from the soil microbe Methylococcus capsulatus Bath (McB) modulates mucus production and mucosal immunity in mice 22 , suggesting enhanced gut barrier function and positioning McB as a next-generation cultured protein. However, the clincal potential of non-scalable monoculture approaches is limited, and the impact of McB on host physiology during inflammation – particularly its interaction with epithelial repair pathways such as glucagon-like peptide-2 (GLP-2) signaling – remains unresolved, hampering translational development. Here, we investigate the immunological and regenerative properties of a sustainable, commercially scalable microbial protein lysate in which ~95% of the biomass is derived from McB (FeedKind®, Calysta UK). We show that dietary delivery of this lysate as the main protein source rapidly alters microbial community structure and enriches for pathways involved in microbial fermentation. These shifts coincide with mucosal immune imprinting across both the small and large intestine, characterized by microbiota-independent expansion of pTregs and microbiota-sensitive reprogramming of Th17 cells toward a tolerogenic, immunoregulatory state. Importantly, in two mechanistically distinct models of GI inflammation targeting regionalized areas of the gut, the scalable McB lysate confers robust, pan-GI protection that is dependent on functional GLP-2 receptor (GLP-2R) signaling. This GLP-2R signaling is further dependent on microbial fermenation of the McB lysate to confer its protective actions. Together, our findings reveal a mode of nutritional immunomodulation that integrates microbial fermentation, mucosal T cell plasticity, and molecular mimicry-driven engagement of host receptors to facilitate tissue repair. This work positions McB-derived lysates as a novel class of microbial proteins with dual functionality: offering scalable protein nutrition while engaging endogenous pathways of immune tolerance and intestinal regeneration. Results McB Induces Rapid and Persistent Gut Microbiota Alterations To address if the previously reported phenotype of McB feeding 22 was diet-dependent, we compared the simple compositionally defined diet (SD) previously used, to a complex diet with a humanized nutrient composition (CD), both with and without McB lysate as the protein source (Figure 1A, Supplementary Table 1). Fecal shotgun sequencing revealed rapid changes in gut microbiota composition (Figure 1B-C), where McB presence explained 27% variance compared to only 10% from all other nutrients combined (Figure 1B). The observed microbiota changes were accompanied by major changes to the metabolic potential of the McB-associated microbiota (Figure 1D-E; >460 differentially regulated pathways) already a few days after McB-feeding and regardless of background diet. Noteworthy metabolic pathways significantly enriched in feces of McB-fed mice were fermentation processes, including metabolic processes leading to production of short-chain fatty acids (SCFAs), supporting our previous finding of elevated cecal SCFA in monoculture-fed mice 22 . Indicative of a near-complete utilization of the lysate within the GI tract, McB was barely, and only transiently, detectable (<0.01%) in fecal samples of a few mice consuming lysate-containing diets (Figure 1F). Still, the McB-induced changes to the microbial community remained stable after their initial shift (Figure 1B-D), highlighting robust and reproducible McB-induced microbiota modulation, despite moving from a monoculture at lab scale in the original report to a commercial product with minimal amount of helper bacteria (FeedKind®, Calysta UK). Considering that the major microbiota shifts appeared to be driven by Lachnospiraceae and Bacteroidacea families (Figure 1C), we used our shotgun analysis to assess species-level resolution (Figure 1G). This revealed that changes were largely driven by few species (Figure 1G), likely better equipped to process the microbial lysate escaping digestion in the small intestine, considering the consistent >30% increase in relative abundance of those species after introduction of McB. Nutritional Immune Imprinting Exhibits Subset Specific Microbiota Dependence We next examined the lamina propria (LP) T-cell landscape (Figure S1A), in both small and large intestine, to study regionalized McB-, microbe-, and diet-dependent effects, by flow cytometry. Independent of background diet, McB-feeding increased the small and large intestinal LP pTreg populations (2.5 and 4 times, respectively), including the relative proportion of triple-positive IL-17 + pTregs (Figure S1B-E). Notably, McB-feeding also induced a ~50% increase in Th17 cells exclusively in the small intestine (Figure S1F-G). Although Th17 cells originally were conceived as proinflammatory culprits, recent literature points towards their pleiotropic nature being instrumental for maintaining GI immune balance 11 and metabolic homeostasis upon HFD feeding 23 . Indeed, the McB-attuned Th17 landscape exhibited immunoregulatory traits, indicated by an increase in IL-10 + Th17 cells paralleled by a reduction of IFN-g + Th17 cells, suggesting a shift towards tolerogenic immunity (Figure S1F-I). Although absolute numbers of large intestinal Th17 cells were unaffected by McB feeding, their phenotype recapitulated our findings from the small intestinal LP (Figure S1H-I). McB-induced immune imprinting seemed targeted towards RORgt + T cell subsets, as neither Th1 nor thymic-derived Treg (nTreg) populations were affected by McB-feeding (Figure S1J-Q). As GI pTreg 9 and Th17 cell 24,25 plasticity can be modulated by the gut microbiota, we next investigated if McB-induced immune alterations were driven by the substantial shift in microbial community structure and functions following McB-feeding (Figure 1) or if McB-induced immune imprinting developed independently of resident gut microbes. To test this, we administered either control drinking water or a broad-spectrum antibiotic cocktail (ABX) for 2, 4, or 6 weeks, blunting the gut microbiota (Figure 2A). Despite ABX-induced disruption of gut homeostasis, as evident from the ABX-induced increases in cecal size (Figure 2B), McB instantly enhanced pTreg abundance in the small intestine of both ABX-treated and non-treated mice (Figure 2C). This trait was, however, not fully recapitulated in LP of the large intestine, but pTreg induction was notably accelerated in ABX-treated mice (Figure 2D). We interpreted the delayed pTreg-induction in colons of conventional mice as a direct consequence of their microbial ‘buffer’ (i.e., niche occupation) limiting direct McB-host interactions. Summarized, these data suggest that the McB lysate can directly increase LP pTregs across the intestines. Next, we turned our attention to the Th17 cells, which were also notably affected by McB feeding (Figure 2E-J). In sharp contrast to the above-mentioned pTreg inductions, we found that numeric and phenotypic McB-mediated Th17 alterations were highly dependent on a functional gut microbiota. Thus, in the small intestine of conventional mice, McB feeding induced a rapid >50% increase in Th17 cell proportions (Figure 2E-G). This effect was dampened the first 4 weeks of ABX treatment. The McB-mediated Th17 induction after 6 weeks of ABX treatment mirrored the gradual increase in total microbial load (Figure S1R). This rise in bacterial load suggested a selective bloom of ABX-resistant species, possibly interacting with McB to drive the observed expansion of Th17 cells, even under sustained ABX exposure. Further corroborating the Th17 cell-gut microbiota dependence, we observed that even in the absence of a numeric increase (large intestine, Figure 2H-J), McB-feeding still facilitated a phenotypic shift, exemplified by a >2-fold increase in IL-10 + Th17 cells, exclusively in conventional mice. Together, these data demonstrate that while pTreg induction occurred independently of gut microbiota composition, Th17 cell induction and phenotypic manipulation relied on complex host-diet-microbe interactions, corroborating that the impact of the gut microbiota on McB-induced immune regulation is subset specific. McB Promotes Gastrointestinal Homeostasis and Reduces Inflammation Diarrhea and a swollen, fluid-filled cecum are common side effects of ABX treatment 26 , likely due to disruptions in gut barrier integrity, impaired mucus production, reduced microbial load, and a lack of fiber fermentation 27,28 . During ABX treatment, we observed that McB-fed mice exhibited less diarrhea (data not shown) and were protected against ABX-induced increases in cecum weight (Figure 2B). These findings suggest that McB preserves gut barrier function alongside enhanced tolerogenic immunity (Figure S1) in both the murine small and large intestine. With this in mind, we used a two-pronged experimental approach to interrogate if McB feeding confers protection against region-specific gut inflammation, namely 1) chemotherapy-induced mucositis affecting the entire GI tract but predominantly damages the small intestine via villus atrophy 29,30 , 31 , and 2) chemically-induced colitis, primarily targeting the large intestine. Mucositis was induced using a single intraperitoneal (i.p.) injection of 5-fluorouracil (5-FU; 400 mg/kg) (Figure 3A). Following 5-FU challenge, mice displayed acute weight loss over three days, then rapidly regained weight and returned to baseline within six days (Figure 3B). McB-fed mice showed a modest but noticeable improvement in recovery rate compared to reference diet-fed controls. As expected, 5-FU significantly reduced small intestinal wet weights (Figure 3C) and induced villus atrophy (Figure 3D–G). Notably, McB feeding partially protected the small intestine from 5-FU-induced damage, with preserved villus architecture observed along the entire small intestinal axis. In line with prior reports describing crypt hyperproliferation during mucosal recovery post-5-FU challenge 31–33 , we observed deeper crypts in reference-fed mice. In contrast, McB-fed mice consistently exhibited deeper crypts regardless of 5-FU exposure (Figure S2A-H), indicating a baseline trophic effect of McB on the intestine, rather than a reactive compensatory proliferation. Although the most pronounced damage from 5-FU occurred in the small intestine, colonic shortening was also evident in the acute phase (Figure S2I-J), again indicating mucosal inflammation – a feature that was attenuated in McB-fed mice. Given the observed mitigation of colonic inflammation in the 5-FU model, we next tested McB’s capacity to prevent large intestinal injury in the DSS-induced colitis model (Figure 3H). McB-fed mice were fully protected against DSS-induced weight loss (Figure 3I) and exhibited a substantially milder disease trajectory (Figure 3J), with Disease Activity Index (DAI) scores approximately 2-fold lower than controls by study end (Figure 3K). McB feeding also preserved colon length after DSS exposure (Figure 3L), and large intestines from McB-fed mice displayed firmer contents and fewer hemorrhagic lesions (Figure 3M). When inflammation was examined by colon wet weight-to-length ratio, a standard index of colonic inflammation, McB-fed mice exhibited significantly lower values (Figure 3N). Histopathological analysis of colon Swiss rolls supported these findings, showing a >2-fold reduction in colonic damage scores in McB-fed mice (Figure 3O–P). Together, these results indicate that McB not only modulates GI immunity and microbial composition but also confers broad protection against inflammation throughout the GI tract. GLP-2 Receptor Signaling is Essential for McB-Induced Gastrointestinal Protection Given the robust protection conferred by McB throughout the GI tract, we hypothesized that its effects were mediated by intestinotrophic pathways. As a central regulator of intestinal growth, GLP-2, a gut hormone with potent intestinotrophic properties, and its receptor (GLP-2R) emerged as key candidates. To test this hypothesis, we utilized GLP-2R wildtype (WT) and knockout (KO) mice in the mucositis and colitis models. This approach allowed us to dissect both regional specificity, given GLP-2’s known preference for the small intestine 34,35 , and the necessity of intact GLP-2R signaling in McB-mediated gut protection. In the 5-FU-induced small intestinal injury model (Figure 4A), GLP-2R WT mice on a reference diet lost significant weight (Figure 4B), exhibited dramatically reduced small intestinal weights (Figure 4C), and developed villus atrophy (Figure 4D-E, 4N & Figure S3A). Still, McB-fed mice were protected against intestinal damage (Figure 4C-G, 4N). Myeloperoxidase (MPO), an enzyme released by neutrophils during inflammation, serves as a key indicator of gut barrier damage and local inflammation. We found that 5-FU markedly increased MPO levels, especially in the ileum (Figure 4F–G). Importantly, McB feeding significantly reduced MPO accumulation, further supporting its barrier-protective role. However, these protective effects were completely abolished in GLP-2R KO mice (Figure 4H–N & Figure S3), confirming that McB requires functional GLP-2R signaling for restoring small intestinal homeostasis. Notably, while 5-FU induced colonic damage, GLP-2R KO mice exhibited complete loss of McB-mediated protection in the colon (Figure S3F, L), extending GLP-2R’s role beyond canonical small intestinal functions to encompass GI-wide therapeutic effects. To validate this GLP-2R-dependent host-microbe signaling axis in a large intestine-focused model, we administered DSS to GLP-2R WT and KO mice (Figure 5A). In GLP-2R WT mice, McB feeding conferred robust protection, mitigating weight loss (Figure 5B–C), reducing DAI scores (Figure 5D–E), preserving colon length (Figure 5F), and lowering inflammation index (Figure 5G). These protective effects were abolished in GLP-2R KO mice (Figure 5H–M), as confirmed by histological scoring of crypt integrity and immune infiltration (Figure 5N–P). Together, these data demonstrate that McB’s gut-protective effects are strictly GLP-2R-dependent across intestinal regions. Given the co-secretion of GLP-2 and its structurally realted hormone GLP-1, we investigated potential GLP-1R involvement. However, McB-fed GLP-1R WT and KO littermates were equally protected in both mucositis and colitis models (Figures S4–S5), excluding a role of GLP-1R and underscoring the specificity of GLP-2R signaling in McB’s therapeutic action. Microbial Fermentation of McB is Required for Protection Against Gut Inflammation To elucidate how McB confers GLP-2R-dependent protection, we first investigated whether McB stimulates endogenous GLP-2 secretion. Using the perfused rat intestinal model (Figure S6A) – which enables real-time assessment of hormone secretion and nutrient absorption – we delivered McB directly to the intestinal lumen. Notably, McB failed to elicit gut hormone release or modulate amino acid absorption (Figure S6B–C). Since this model bypasses digestion, we posited that enzymatic processing might be needed to liberate bioactive components stimulating GLP-2 secretion. To test this, we gavaged mice with McB or a control protein mix following administration of dipeptidyl peptidase 4 (DPP-4) and neprilysin inhibitors, and then measured plasma GLP-2 via radio-immuno-assay (RIA) (Figure S6D). While glucose stimulated GLP-2 release, neither McB nor the reference protein increased circulating GLP-2 (Figure S6E). These findings demonstrate that McB does not acutely induce endogenous GLP-2 secretion. However, given 1) microbial fermentation’s known role in L-cell stimulation, 2) our observation of enriched fermentative microbial pathways (Figure 1D), and 3) the near-complete fecal degradation of McB (Figure 1F), we hypothesized that microbial fermentation of McB generates metabolites capable of either stimulating GLP-2 release or directly activating the GLP-2R via molecular mimicry, a prerequisite for its beneficial effects. To test this hypothesis, we employed the DSS colitis model and administered a fermentation inhibitor (FermInh; 20 ppm beta-acid extract from Humulus lupulus ) to block microbial hindgut fermentation (Figure 6A). In reference-fed mice, FermInh had no significant effect on colitis severity (Figure 6B–F). Consistent with prior findings, McB-fed mice exhibited reduced DAI scores, attenuated colonic inflammation, and conserved colon length compared to controls (Figure 6G-K). Strikingly, FermInh treatment abolished McB’s protective effects, exacerbating DAI scores (Figure 6G–H), elevating colonic inflammation index (Figure 6I), leading to colon shortening (Figure 6J). Given McB’s intestinotrophic effects in healthy mice (Figure 3, 4, S2-S4), we assessed intestinal morphology. Remarkably, FermInh eliminated McB-induced elongation of both small and large intestines, even in healthy, non-DSS treated mice (Figure 6J-K), directly linking microbial fermentation to McB-mediated gut homeostasis. Because fermentation yields microbial metabolites known to stimulate gut hormone secretion, we asked whether blocking fermentation would lower systemic GLP-2 levels. Despite extended McB feeding (~2 weeks), we found no significant increase in circulating GLP-2 in either control or McB-fed mice, regardless of fermentation status (Figure 6L). Together, these findings demonstrate that McB protects against gut injury via a microbial fermentation-dependent mechanism that engages GLP-2R signaling independently of endogenous GLP-2 secretion, highlighting molecular mimicry as the underlying mechanism. Discussion Food demands are soaring 1,2 and Western food habits 19 are closely associated with increasing prevalence of GI inflammatory diseases 20,21 . It is thus imperative to develop nutritional strategies that can counter these trajectories and potentially aid alleviating already established inflammation and disturbed gut health. Unfortunately, current means of food production drain our natural resources, with great environmental implications 1,2 . A proposed strategy to overcome these hurdles is the usage of alternative foods, such as cultured, microbe-based proteins. However, the effects of microbial protein sources on host health remains inadequately described. In this study, we utilized a microbial lysate derived from a non-native soil bacterium, McB, as a potent nutritional modulator of GI immunity, microbiota composition, and host resilience against intestinal injury (Figure 6M). Building on previous observations in simplified dietary contexts 22 , we demonstrate that McB retains its immunomodulatory and protective functions even when introduced into complex, humanized diets. The translational relevance of this lies not only in McB’s robust effects across diet types but in its ability to trigger subset-specific immune imprinting, remodel gut microbial ecology, and protect against both small and large intestinal inflammation through GLP-2R-dependent mechanisms. A central breakthrough of this work is the demonstration that McB directly induces pTregs in both the small and large intestine, independent of the gut microbiota. This is in stark contrast to most known dietary or microbial interventions, where previous reports on microbial cell component-induced increases of pTregs exclusively precipitate in the colon 7 where microbial density is highest. Here, even in microbiota-depleted mice, McB rapidly expanded pTreg populations, highlighting a direct host-microbe interaction that bypasses gut microbiota derived intermediates. Such a finding not only decouples microbial dependence from tolerogenic T cell induction but also offers a novel route for immune modulation in various disease indications where microbiota-mediated therapies fail. In contrast to pTreg expansion, McB-driven Th17 cell modulation was strictly gut microbiota-dependent. McB not only increased Th17 cell numbers exclussively in conventional mice but also skewed their phenotype toward a tolerogenic IL-10 high IFN-γ low state, highlighting the microbiota’s essential role in shaping adaptive immune responses. Th17 cells are particularly interesting for gut homeostasis, as these cells are known for their plasticity and ability to adapt their cytokine production depending on the local microenvironment 36 . As such, their functions are highly context-dependent; Th17 cells can be implicated in inflammatory diseases 11 but have also been demonstrated to orchestrate gut barrier integrity 37,38 and metabolic regulation 23 . The importance of Th17 cells is further highlighted by dietary influences. To this end, high-sugar diets deplete Th17 cell-inducing microbes, explaining the detrimentally low abundance of these cells following Western Diet feeding 39 , a contributing factor to the development of metabolic syndrome 23,39 . The microbiota-independent induction of pTregs by McB, coupled with the microbiota-dependent modulation of tolerogenic Th17 cells, is highly intriguing. The dual mechanism suggests broad therapeutic applicability across diverse enterotypes and microbiota states, including antibiotic-perturbed ecosystems. From a microbial ecology perspective, McB triggered rapid, stable shifts in gut microbiota composition and function, dominated by taxa within the Lachnospiraceae and Bacteroidaceae families. Crucially, this effect persisted across two distinct dietary contexts and using a standardized, commercially produced lysate, posistioning McB as a reproducible modulator of microbioal communities. McB was nearly undetectable in feces, indicating near-complete GI utilization and suggesting its role as a precision substrate for microbial fermentation. The concomittant enrichment of microbial fermentation pathways mechanistically connect McB-induced microbiota remodelling to some of its downstream benefits in tolerogenic immunity. Given the tolerogenic T-cell populations and microbial functional changes, we subsequently explored if consumption of the lysate would confer health benefits locally within the GI tract. To this end, we used two distinct models of GI inflammation: 5-FU-induced mucositis and DSS-induced colitis. Functionally, McB conferred significant protection against both chemotherapy-induced mucositis and chemically-induced colitis – two mechanistically distinct models affecting the small and large intestine, respectively. These protective effects were associated with preservation of tissue architecture, reduction of inflammatory markers (e.g., MPO), and mitigation of weight loss and histopathological scores. Consistently, McB-fed healthy control mice exhibited elongated small intestinal villi, deeper crypts, and increased colon length, demonstrating direct intestinotrophic remodelling of intestinal architecture. We mechanistically dissected the McB-mediated protection using our two-pronged approach to modelling gut inflammation, combined with RIA and high-resolution intestinal perfusion models. Through this integrated approach, we systematically investigated the proposed role of GLP-2 and its receptor in McB’s protective effects. The GLP-2 axis is well-described to directly affect mesenteric blood flow 40–42 and epithelial proliferation and repair 32,43,44 . As a testimony to that therapeutic potential, GLP-2 analogues are clinically used to treat short-bowel syndrome 45 . Using GLP-2R WT and KO mice, we demonstrated that McB’s protective effects were entirely abolished in GLP-2R KO mice, underscoring the necessity of intact GLP-2 signaling for McB-mediated intestinal resilience. Importantly, GLP-2R KO alone did not alter baseline disease progression in any model, suggesting the GLP-2R is dispensable for physiological recovery but can be therapeutically engaged to enhance repair. Critically, McB did not stiumlate endegnous GLP-2 secretion, pointing to direct engagement of GLP-2R signaling rather than hormonal induction. Mechanistically this interaction dependents on microbial fermentation of McB, as inhibition of hindgut fermentation abolished GI protection, linking microbial metabolisn to host receptor activation (Figure 6M). Collectively, these findings position McB as a novel class of bioactive cultured dietary proteins that transcend classic nutrition, functioning as precision modulators of intestinal immunity and gut barrier integrity . Its unique ability to orchestrate both microbiota-independent (direct pTreg induction) and microbiota-dependent (Th17 tolerogenesis) immune reprogramming, while simultanously driving GLP-2R-mediated epithelial repair, establishes a new paradigm in dietary-microbiota crosstalk. To our knowledge, our work is the first to demonstrate that a microbial lysate can engage the GLP-2 pathway through dietary means, bypassing exogenous hormone administration. This discovery opens avenues for developing McB or its bioactive derivatives into next-generation therapeutics that concurrently address intestinal inflammation, immune dysregulation, and epithelial damage – three critical unmet needs in conditions like IBD, chemotherapy-induced mucositis, and environmental enteropathy. By linking microbial fermentation to receptor mimicry, our findings advance the broader field of dietary-microbiota therapeutics, where nutritional interventions can be engineered to target host pathways. Materials and Methods Rodents and ethical statements . Animal experiments were approved by the Danish Animal Experiments Inspectorate (#2021-15-0201-01031 and # 2023-15-0201-01409). Six- to seven-week-old male and female C57BL/6 mice and male Sprague Dawley were purchased from vendors as detailed below. Both GLP-1 receptor knockout (GLP-1R -/- ) and GLP-2 receptor knockout (GLP-2R -/- ) mice were used in this study. GLP-2R knockout mice 46 were generated by Taconic using CRISPR/Cas9-mediated gene editing, with the parental strain designated as C57BL/6NTac-Glp2rem5153Tac. The GLP-1R knockout mice were created by deleting exons 4 and 5 of the Glp1r gene in a Cre-dependent manner, and the conditional Glp1r knockout strain (C57BL/6N-Glp1rtm1c(KOMP)MbpH) was obtained from the MRC Harwell Institute as previously described 47 . All knockout mice were maintained through heterozygous breeding, with wild-type (WT) littermates serving as controls. Upon arrival mice and rats were allowed to acclimatize in the animal facility environment for two weeks before to study initiation. All rodents were housed under specific pathogen-free conditions in 12-hour light/dark cycle (6 AM-6 PM). Male mice were housed three mice per cage and female mice were housed up to five per cage. Rats were housed four per cage. All data from animal studies used in this manuscript can be found in the supplementary material. Diets and experimental setups . All special diets were obtained from Sniff Spezialdiäten GmBH, Germany, and stored at -20 o C. Several customized diets were designed depending on the specific research question. Dietary compositions can be found in the supplementary material. In all protocols, mice were fed ad libitum , and at termination mice were anesthetized with 2.5% isoflurane and euthanized by cervical dislocation following cardiac puncture blood collection. Cardiac blood samples were taken using EDTA coated needles and syringes. Plasma was collected by centrifugation of blood samples for 10 minutes at 1000 rcf at 4 o C and stored at -80 o C. Nutrient comparison protocol . Male mice were purchased from Taconic Laboratories, Denmark. After two weeks of acclimatization mice were given one of two diets, a diet with few simple sources of protein, fat, and carbohydrates (referred to as Simple Diet, SD ) or an isocaloric macronutrient-matched diet with a complex composition of humanized sources of protein, fat, and carbohydrates (referred to as Complex Diet, CD ) for eight weeks. At week eight a subgroup of mice was maintained on the respective simple or complex diets or fed an experimental diet of which the protein source was exchanged with solely McB lysates (contain <5% biomass from helper bacteria, sold under the commercial name Feedkind®, Calysta UK Ltd) ( SD MCB or CD MCB ) for an additional 4 weeks. To avoid any secondary effect of caloric density, we modified the complex low-fat-low-fiber reference (referred to as Complex ref ) to also contain McB lysate as the protein source. Microbiota-dependency protocol . Male mice were purchased from Taconic Laboratories (Denmark). After two weeks of acclimatization, mice were split into two groups: one group given control drinking water and one group given a broad-spectrum antibiotics cocktail of 0.5 g/L Neomycin (Sigma #N1876-25G) and 1 g/L Ampicillin (Sigma #A9518-25G) in the drinking water to reduce gut microbial load. After acclimatization, mice underwent dietary intervention and were split into two groups per drinking water condition: one group switched to the CD , and one group switched to the CD MCB . Mice were maintained on control drinking water or antibiotics during the dietary intervention period. Mice were then euthanized after two, four, or six weeks to look at the temporal effects of McB-consumption on blunted gut microbiota and mucosal T-cell populations. Intestinal mucositis protocols . This study protocol used either female WT mice purchased from Janvier (Le Genest-Saint-Isle, France), or female GLP-1R -/- and GLP-2R -/- mice. After at least a week of acclimatization, mice were fed either the Complex ref or Complex McB for up to 13 days. After a week of dietary intervention, at experimental day 0, mice were intraperitoneally injected once with control saline or 5-fluorouracil (5-FU, Hospira Nordic AB, Stockholm, Sweden) at 400mg/kg to induce intestinal mucositis. Body weight development was subsequently monitored daily. Mice were euthanized at post-injection days 3, 4, or 6 (as indicated in figure legends) to investigate the acute and recovery phases of 5-FU-induced mucositis. Occasionally, mice were single-housed due to fighting and otherwise housed between two-five per cage. Colitis protocols . This study protocol used either male mice purchased from Janvier (Le Genest-Saint-Isle, France), or male GLP-1R -/- and GLP-2R -/- mice. After at least a week of acclimatization, mice were fed the reference Complex ref or Complex McB for one week before induction of colitis. Colitis was induced by adding 2.5% DSS (Thermofisher, #J63606.14) to the drinking water for five days. DSS was freshly dissolved in regular drinking water and filter-sterilized before being given to the mice. Body weight and disease activity (can be found in supplementary material) were monitored daily. At day five, mice were euthanized and tissues harvested. Mice were housed between one-three per cage, depending on in-cage fighting. Inhibition of microbial fermentation . Male mice were ordered from Janvier (Le Genest-Saint-Isle, France) and allowed to acclimatize for at least a week. First, mice were fed the reference Complex ref or Complex McB for one week before induction of colitis as described above. A subgroup of mice were given 20 ppm solution of beta-acid extracts from Humulus lupulus to inhibit microbial hindgut fermentation as previously described 48 . Body weight and disease activity were monitored daily. At day five, mice were euthanized and tissues harvested. Mice were housed three per cage. Measurements of circulating gut hormones and perfusion of rat intestine . 8-week-old Sprague Dawley rats and C57BL/6J were purchased from Janvier (Le Genest-Saint-Isle, France) and allowed to acclimatize for 1 week. Mice and rats were gavaged with an inhibitor cocktail consisting of sacubitril (neprilysin inhibitor, 0.3 mg/kg, 5 uL/g, cat. no. 333-B1070, Nordic Biosite, Sweden) and sitagliptin (dipeptidyl peptidase-4 inhibitor, 10 mg/kg, 5 uL/g, Xelevia) 30 minutes before oral delivery of microbial McB lysates of control protein. At time point 0, mice and rats were gavaged with a 20% w/v (200 mg/mL in sterile saline) solution of McB lysate, a control protein solution (Albumin Fraction V, cat. no. 1.12018.0500, Sigma-Aldrich) or glucose. In mice, blood was collected by cardiac puncture and in rats blood was collected sublingual bleeding after 0, 5, 10, and 20 minutes post gavage. GLP-2 levels were measured by radio-immuno-assay (RIA) as described previously 49 . Rat small intestines were perfused as described previously 49 with lysate or control protein solutions. Vascular effluents were collected every minute and gut hormone levels measured by RIA. Histology . Intestinal sections were fixed in 10% paraformaldehyde and embedded in paraffin following standard procedures. Tissue slides were stained with H&E and samples were randomized and blinded before histological analyses. For small intestinal sections, villus height was measured by identifying three villi with visible extrusion zone, to ensure measurement of full-length villi, from each sample, and measuring from the tip until the crypt border. Crypt depth of small and large intestinal samples was assessed by measuring the depth of three full-length crypts per slide, only when the entire crypt epithelium was visible from the lamina muscularis mucosa to the intestinal lumen. Villus height and crypt depth were analyzed using the Zeiss Zen Desk Software, from three separate areas within each section and reported as an average of measurements from all individuals. For assessment of colonic injury following DSS administration, fecal matter was mechanically removed, colons were opened longitudinally and rinsed. Next, colons were rolled into Swiss rolls and fixed in 10% paraformaldehyde and stained with H&E according to standard practice. Histopathological scoring was conducted based on previously described scoring systems 50,51 , where each section was given a score based on 1) degree of inflammation, 2) degree of crypt damage, and 3) extent of inflammation and damage as described in the supplementary material. The sum of all parameters was multiplied by 1-4 depending on the area of the section affected, giving a dynamic scoring range of 0-40. Digital images were acquired using an Axioscan Z.1 (Zeiss, Jena, Germany). Isolation of small and large intestine lamina propria (LP) cells . In protocols involving flow cytometry, after removal of 1 cm gut sections for histology, the small intestine was flushed with cold 1X HBSS (Gibco) containing 15 mM HEPES and 5% FBS (Thermo Fischer), and had feces removed mechanically prior to flushing with cold 1X PBS. Peyer’s Patches were carefully removed from the small intestine. Small and large intestines were opened longitudinally and cut into 1 cm pieces in 1X HBSS, 15mM HEPES, and 5% FBS. Gut pieces were incubated thrice in prewarmed EDTA wash-buffer containing 1X HBSS, 15 mM HEPES, 2.5% heat-inactivated (HIA) FBS, 1 mM sodium pyruvate, 10.000 U/mL Penicillin/Streptomycin, 50 mg/mL Gentamycin, and 2mM EDTA. After each wash, samples were incubated at 37 o C for 10 min during first incubation and 15 min during second and third incubation. During incubation steps, large intestine samples, but not small intestine samples, were shaken on an orbital shaker at 450 rpm. After each incubation step small intestine samples were vigorously shaken by hand for 10 seconds., and media containing cell debris and epithelial cells were discarded by filtration through a 250 µm nylon mesh. The remaining tissue was digested for 20-25 min at 37 o C under magnetic stirring at 450 rpm in R10 medium (RPMI 1640 with L-glutamine, 1 mM sodium pyruvate, 15 mM HEPES, 10.000 U/mL Penicillin/Streptomycin, 50 mg/mL Gentamycin, 10% HIA FBS) containing 1 mg/mL Collagenase P (Roche) and 30 µg/mL DNAse I (Roche). After digestion, samples were mechanically agitated, filtered through a 100 µm cell strainer, and centrifugated at 500 rcf for 7 minutes at 4 o C and supernatant removed. The cell pellets were resuspended in 40% isotonic Percoll (GE Healthcare) and LP cells were purified by density centrifugation using 40/70% Percoll gradient, centrifuged at 800 rcf for 20 min at room temperature, acceleration 5 and brake 0. The lymphocyte interface was collected in fresh R10 medium followed by centrifugation for 7 min at 500 rcf at 4 o C. Subsequently, the supernatant was removed, and the cell pellet was resuspended in R10 medium and transferred to 96 well plates for ex vivo stimulation of LP cells and ensuing flow cytometry. Ex vivo stimulation of LP cells and staining . Small intestine-LP and large intestine-LP cells were restimulated ex vivo in R10 medium as follows. 250 ng/mL PMA (Sigma-Aldrich) in combination with 0.5 µg/mL Ionomycin (Sigma-Aldrich) was added to restimulate samples and incubated at 37 o C and 5% CO 2 for four hours. After one hour 10 µg/mL brefeldin A was added to all samples, and continued incubation. After stimulation, plates were centrifuged for 5 min at 500 rcf at 4 o C and washed with PBS. After additional centrifugation for 5 min at 500 rcf at 4 o C, cells were resuspended in PBS containing Live/Dead stain (Zombie UV) and placed at 4 o C in the dark for 20 min. Cells were washed with MACS buffer (containing PBS, HIA FBS, EDTA) and centrifuged for 5 min at 500 rcf at 4 o C. Cells were resuspended in MACS buffer and surface stained with primary antibodies (see table below) for 30 min at 4 o C in the dark followed by washing and centrifugation. Cells were fixed and subsequently permeabilized using the FoxP3/Transcription Factor Staining Buffer Set from eBioscience according to manufacturer’s instructions. Intracellular staining with primary antibodies was performed at 4 o C overnight with primary antibodies (see table below). Flow cytometry . Flow cytometry was carried out by standard procedures and data acquired using an LSRFortessa X-20 (BD Bioscience). The complete gating strategy can be found in Supplementary Figure 1A. Data was analyzed using the FlowJo Software (Tree Star). Antibodies . Antibodies Source Identifier BD Horizon™ BUV395 Rat Anti-Mouse CD4 BD Bioscences Cat # 563790; RRID AB_2738426 BD Horizon™ BV480 Rat Anti-Mouse CD8a BD Bioscences Cat # 566096; RRID AB_2739500 Brilliant Violet 421™ anti-mouse TCR β chain Antibody BioLegend Cat # 109229; RRID AB_10933263 FOXP3 Monoclonal Antibody (FJK-16s), APC, eBioscience™ Invitrogen Cat # 17-5773-82; RRID AB_469457 BD Pharmingen™ APC-Cy™7 Rat Anti-Mouse CD45 BD Bioscences Cat # 557659; RRID AB_396774 ROR gamma (t) Monoclonal Antibody (B2D), PE-eFluor™ 610, eBioscience™ Invitrogen Cat # 50-112-4863; RRID N/A IL-10 Monoclonal Antibody (JES5-16E3), PE, eBioscience™ Invitrogen Cat # 12-7101-82; RRID AB_466176 Gata-3 Monoclonal Antibody (TWAJ), PE-Cyanine5, eBioscience™ Invitrogen Cat # 15-9966-42; RRID AB_2811756 T-bet Monoclonal Antibody (eBio4B10 (4B10)), PE-Cyanine7, eBioscience™ Invitrogen Cat # 25-5825-82; RRID AB_11042699 Brilliant Violet 605™ anti-mouse IFN-γ Antibody BioLegend Cat # 505839; RRID AB_2561438 BD Pharmingen™ Alexa Fluor® 488 Rat anti-Mouse IL-17A BD Bioscences Cat # 560220; RRID AB_1645194 Microbiome analyses and bioinformatics processing . Input sample was weighed before extraction, and DNA was extracted using the NucleoSpin Soil kit (Macherey-Nagel) following the manufacturer's protocol. DNA libraries were prepared using the MGIEasy DNA Library Prep Kit (MGI) following the manufacturer’s protocol. The MGIEasy DNA Adapters-96 (Plate) Kit (MGI) was used for adapter ligation. DNA cleanup was performed with the MGIEasy DNA Clean Beads (MGI). Circularization of libraries was carried out using the MGIEasy Circularization Module V2.0 (MGI). Sequencing was performed on the DNBSEQ-G400RS platform using the G400-PE150 sequencing kit (MGI), generating paired-end reads of 150 bp. Taxonomic profiling was performed with MetaPhlAn 3.0, while functional profiling of microbial pathways was conducted using HUMAnN 3.0 52 . Beta Diversity Analysis . Beta diversity was assessed using the Bray-Curtis dissimilarity index (vegan package), which measures compositional differences based on species abundances. Principal coordinate analysis (PCoA) was used for visualization, and statistical significance of group differences was tested using PERMANOVA (adonis function). Differential Abundance Analysis of Taxa . A linear mixed model (lme4 package) was applied to assess species-level differences, with dietary intervention as a fixed effect. Post-hoc pairwise comparisons were performed using glht (multcomp package) with multiple testing correction. Effect sizes, t-statistics, and p-values were extracted to identify significant taxa. Analysis of Microbial Pathways . Metabolic pathways were annotated and abundanced calculated using HUMAnN 3.0 based on the Metacyc database 53 . Pathway-level differences were analyzed using ALDEx2 , which applies Monte Carlo Dirichlet sampling and log-ratio transformations to account for compositional data constraints. Statistical testing was performed using Welch's t-test. False discovery rate (FDR) correction was applied using the Benjamini-Hochberg method, with p < 0.05 considered significant. Quantification of fecal bacterial load . Bacterial 16S rRNA gene copies were quantified by qPCR on a Light Cycler 480 II (Roche) using V4 region-specific Primers 505F (5’-GTGYCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACNVGGGTWTCTAAT-3’). The thermal cycling conditions started with a DNA-denaturation step at 95° C for 5 minutes, followed by 50 cycles of i) denaturation at 95°C for 10 seconds, ii) annealing at 60°C for 20 seconds, and iii) extension at 72°C for 20 seconds. All samples were run in triplicates. A 2-fold standard curve was produced by serially diluting the DNA pool (starting dilution: 1:20) in sterile MilliQ Water. qPCR threshold cycle (Ct) values were converted to estimated bacterial genomes present in 1 mg of feces and used as a proxy for bacterial abundance. Tissue MPO levels . Approximately 1 cm of gut was homogenized in 50 mM HTAB in 50mM K-Phosphate buffer for 6 minutes with a steel bead. Tissues were snap frozen on dry ice, thawed in water and homogenized again; this cycle was done a total of 3 times. After centrifugation (16 000g, 30 minutes at RT), supernatant was collected and diluted 10x for MPO measurement. Samples were mixed with substrate buffer (O-dianisidine dihydrochloride in 50mM K-Phosphate buffer with 0.0005% H 2 O 2 ) on a 96-well plate, in duplicates, and measurements were taken every 30 seconds for 5 minutes at 450nm. MPO activity was calculated using all readings and corrected for total protein content of each sample. Statistical analyses . Statistical analyses were performed using GraphPad Prism software (version 10). All data are presented as means ± standard error of the mean (SEM), with individual data points included to illustrate data distribution. For comparisons between two groups, unpaired t-tests were conducted to assess statistical significance. When comparing more than three groups with a single independent variable, a one-way analysis of variance (ANOVA) was utilized, followed by Tukey's post hoc test to identify specific group differences. In instances where more than three groups with two independent variables were analyzed, a two-way ANOVA was performed to evaluate the effects of both independent variables and their interactions, testing main effects for multiple testing by Fisher’s LSD post hoc test. A p-value of <0.05 was considered statistically significant. A full overview of all statistical tests sorted by figures can be found in the supplementary material. Declarations Funding : This work was primarily funded by an Excellence Emerging Investigator grant (NNF21OC0066931) issued to BAHJ by Novo Nordisk Foundation, and partly by a grant from the Louis Hansen Fonden (24-2B-16713), issued to SKYJ. Calysta provided the Feedkind® material. BSYC is supported by the BRIDGE – Translational Excellence Program, funded by the Novo Nordisk Foundation (NNF20SA0064340) and by a postdoctoral fellowship from the Fonds de recherche du Québec – Santé (FRQS). The funding bodies had no share in data integration and presentation. Conflicts of Interest : BAHJ, JBH, and KK are co-inventors of an issued patent (US and EU) related to McB and gut dysbiosis. BAHJ, SKYJ, and BSYC are co-inventors of a patent application related to McB’s potential in treating or preventing gastrointestinal barrier dysfunction. The remaining authors declare that there are no conflicts of interest. Contributions : Study design: SKYJ, BSYC, SIP, and BAHJ. SKYJ, BSYC, NN, SBS, NG, IMM, and SIP, performed experiments. JBH and KK were responsible for metagenomic sequencing and subsequent analyses with SKYJ and BAHJ. HK, JJH, and BH were involved in breeding GLP-1R and GLP-2R mice, 5-FU experiments, and gut hormone measurements. SKYJ wrote the first manuscript draft, revised together with BSYC, and finalized it with BAHJ. All authors contributed significantly to the manuscript and approved the final version. Acknowledgements : We extend our gratitude to the Core Facility for Integrated Microscopy at the Faculty of Health and Medical Sciences, University of Copenhagen, as well as the members of UCPH Histolab—Associate Professor Jens Brings Jacobsen, Biomedical Laboratory Technologists Bente Stærgaard and Heidi Paulsen, and Associate Professor Steen Seier Poulsen for their valuable support with histological processing and imaging. We would also like to thank the Core Facility for Flow Cytometry and Single Cell Analysis, Faculty of Health and Medical Sciences, University of Copenhagen, for inputs regarding flow cytometry analysis. We gratefully acknowledge Calysta (UK) Ltd. for providing access to commercial McB lysates (Feedkind ®). We want to express our sincere gratitude to Staff Scientist Si Brask Sonne for invaluable help and instruction in the laboratory, and likewise Laboratory Scientists Lene Brus Albæk and Anette Bjerregaard for instrumental assistance with radioimmunoassays and genotyping the GLP-2R and GLP-1R mice. Finally, a thank to Isabella Paul, Cecilie Anastacia Stokkeby Koch, and Kobe Neven for their help, input, and discussions during their traineeship. Data availability: All data used to generate this manuscript will be available on Mendeley Data. References Malila, Y. et al. Current challenges of alternative proteins as future foods. npj Sci. Food 8 , 53 (2024). Xu, X. et al. Global greenhouse gas emissions from animal-based foods are twice those of plant-based foods. Nat. Food 2 , 724–732 (2021). Fackelmann, G. et al. Gut microbiome signatures of vegan, vegetarian and omnivore diets and associated health outcomes across 21,561 individuals. Nat. Microbiol. (2025) doi:10.1038/s41564-024-01870-z. Nunes-Cabaço, H., Moita, D., Rôla, C., Mendes, A. M. & Prudêncio, M. Impact of Dietary Protein Restriction on the Immunogenicity and Efficacy of Whole-Sporozoite Malaria Vaccination. Front. Immunol. 13 , (2022). Jensen, B. A. H. et al. Small intestine vs. colon ecology and physiology: Why it matters in probiotic administration. Cell Reports Med. 4 , 101190 (2023). Sefik, E. et al. MUCOSAL IMMUNOLOGY. Individual intestinal symbionts induce a distinct population of RORγ + regulatory T cells. Science 349 , 993–997 (2015). Verma, R. et al. Cell surface polysaccharides of Bifidobacterium bifidum induce the generation of Foxp3 + regulatory T cells. Sci. Immunol. 3 , (2018). Abdel-Gadir, A. et al. Microbiota therapy acts via a regulatory T cell MyD88/RORγt pathway to suppress food allergy. Nat. Med. 25 , 1164–1174 (2019). Caspar, O. et al. The microbiota regulates type 2 immunity through RORγt+ T cells. Science (80-. ). 349 , 989–993 (2015). Ivanov, I. I. et al. Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria. Cell 139 , 485–498 (2009). Wen, Y., Wang, H., Tian, D. & Wang, G. TH17 cell: a double-edged sword in the development of inflammatory bowel disease. Therap. Adv. Gastroenterol. 17 , 17562848241230896 (2024). Zielinski, C. E. et al. Pathogen-induced human TH17 cells produce IFN-γ or IL-10 and are regulated by IL-1β. Nature 484 , 514–518 (2012). Cui, H. et al. The dynamic shifts of IL-10-producing Th17 and IL-17-producing Treg in health and disease: a crosstalk between ancient ‘Yin-Yang’ theory and modern immunology. Cell Commun. Signal. 22 , 99 (2024). Liu, T. C. et al. Western diet induces Paneth cell defects through microbiome alterations and farnesoid X receptor and type I interferon activation. Cell Host Microbe 29 , 988-1001.e6 (2021). Koh, A. et al. Microbially Produced Imidazole Propionate Impairs Insulin Signaling through mTORC1. Cell 175 , 947-961.e17 (2018). Pendyala, S., Walker, J. M. & Holt, P. R. A high-fat diet is associated with endotoxemia that originates from the gut. Gastroenterology 142 , 1100-1101.e2 (2012). Thaiss, C. A. et al. Hyperglycemia drives intestinal barrier dysfunction and risk for enteric infection. Science (80-. ). 359 , 1376–1383 (2018). Lee, J. C. et al. Obesogenic diet-induced gut barrier dysfunction and pathobiont expansion aggravate experimental colitis. PLoS One 12 , e0187515 (2017). Srour, B. et al. Ultra-processed foods and human health: from epidemiological evidence to mechanistic insights. Lancet Gastroenterol. Hepatol. 7 , 1128–1140 (2022). Adolph, T. E. & Zhang, J. Diet fuelling inflammatory bowel diseases: preclinical and clinical concepts. Gut 71 , 2574 LP – 2586 (2022). Christensen, C. et al. Diet, Food, and Nutritional Exposures and Inflammatory Bowel Disease or Progression of Disease: an Umbrella Review. Adv. Nutr. 15 , 100219 (2024). Jensen, B. A. H. et al. Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3(+)RORgammat(+)IL-17(+) Tregs and improve metabolism. Nat Commun 12 , 1093 (2021). Hong, C.-P. et al. Gut-Specific Delivery of T-Helper 17 Cells Reduces Obesity and Insulin Resistance in Mice. Gastroenterology 152 , 1998–2010 (2017). Sun, C.-Y., Yang, N., Zheng, Z.-L., Liu, D. & Xu, Q.-L. T helper 17 (Th17) cell responses to the gut microbiota in human diseases. Biomed. Pharmacother. 161 , 114483 (2023). Evans-Marin, H. et al. Microbiota-Dependent Involvement of Th17 Cells in Murine Models of Inflammatory Arthritis. Arthritis Rheumatol. 70 , 1971–1983 (2018). Shaughnessy, M. P., Park, C. J., Salvi, P. S. & Cowles, R. A. Jejunoileal mucosal growth in mice with a limited microbiome. PLoS One 17 , e0266251 (2022). Krigul, K. L. et al. A history of repeated antibiotic usage leads to microbiota-dependent mucus defects. Gut Microbes 16 , 2377570 (2024). Sawaed, J. et al. Antibiotics damage the colonic mucus barrier in a microbiota-independent manner. Sci. Adv. 10 , 4119 (2024). Rosemann, E., Schaefer, H., Esmaty, A., Stintzing, S. & Karthaus, M. Incidence, severity, and onset of oral mucositis in 5-FU based chemotherapy for gastrointestinal cancer. J. Clin. Oncol. 40 , 77 (2024). McQuade, R. M. et al. Gastrointestinal dysfunction and enteric neurotoxicity following treatment with anticancer chemotherapeutic agent 5-fluorouracil. Neurogastroenterol. Motil. 28 , 1861–1875 (2016). Billeschou, A. et al. Intestinal Adaptation upon Chemotherapy-Induced Intestinal Injury in Mice Depends on GLP-2 Receptor Activation. Biomedicines (2021) doi:10.3390/biomedicines9010046. Hytting-Andreasen, R. et al. Endogenous glucagon-like peptide- 1 and 2 are essential for regeneration after acute intestinal injury in mice. PLoS One (2018) doi:10.1371/journal.pone.0198046. Taminiau, J. A., Gall, D. G. & Hamilton, J. R. Response of the rat small-intestine epithelium to methotrexate. Gut 21 , 486 LP – 492 (1980). Drucker, D. J., Deforest, L. & Brubaker, P. L. Intestinal response to growth factors administered alone or in combination with human [Gly2]glucagon-like peptide 2. Am. J. Physiol. Liver Physiol. 273 , G1252–G1262 (1997). Brubaker, P. L. & Drucker, D. J. Minireview: Glucagon-Like Peptides Regulate Cell Proliferation and Apoptosis in the Pancreas, Gut, and Central Nervous System. Endocrinology 145 , 2653–2659 (2004). Pan, Y. et al. The protective and pathogenic role of Th17 cell plasticity and function in the tumor microenvironment. Front. Immunol. 14 , (2023). Ariana, P. & D., M. E. Precarious Balance: Th17 Cells in Host Defense . Infect. Immun. 78 , 32–38 (2010). Liang, S. C. et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides . J. Exp. Med. 203 , 2271–2279 (2006). Kawano, Y. et al. Microbiota imbalance induced by dietary sugar disrupts immune-mediated protection from metabolic syndrome. Cell 185 , 3501-3519.e20 (2022). Bremholm, L., Hornum, M., Henriksen, B. M., Larsen, S. & Holst, J. J. Glucagon-like peptide-2 increases mesenteric blood flow in humans. Scand. J. Gastroenterol. 44 , 314–319 (2009). Guan, X. et al. GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets 1 1This work is a publication of the USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine . Gastroenterology 125 , 136–147 (2003). Bremholm, L. et al. The effect of Glucagon-Like Peptide-2 on mesenteric blood flow and cardiac parameters in end-jejunostomy short bowel patients. Regul. Pept. 168 , 32–38 (2011). Drucker, D. J., Erlich, P., Asa, S. L. & Brubaker, P. L. Induction of intestinal epithelial proliferation by glucagon-like peptide 2. Proc Natl Acad Sci U S A 93 , 7911–7916 (1996). Estall, J. L. & Drucker, D. J. Dual Regulation of Cell Proliferation and Survival via Activation of Glucagon-Like Peptide-2 Receptor Signaling. J. Nutr. 133 , 3708–3711 (2003). Jeppesen, P. B. et al. Glepaglutide, a Long-acting Glucagon-like Peptide-2 Analog, Reduces Parenteral Support in Patients with Short Bowel Syndrome: a Phase 3, Randomized, Controlled Trial. Gastroenterology (2024) doi:https://doi.org/10.1053/j.gastro.2024.11.023. Hunt, J. E. et al. Pharmacological activation of TGR5 promotes intestinal growth via a GLP-2-dependent pathway in mice. Am. J. Physiol. Liver Physiol. 318 , G980–G987 (2020). Balk-Møller, E. et al. Glucagon-Like Peptide 1 and Atrial Natriuretic Peptide in a Female Mouse Model of Obstructive Pulmonary Disease. J. Endocr. Soc. 4 , bvz034 (2020). Singh, V. et al. Dysregulated Microbial Fermentation of Soluble Fiber Induces Cholestatic Liver Cancer. Cell 175 , 679-694.e22 (2018). Modvig, I. M. et al. Amino acids differ in their capacity to stimulate GLP-1 release from the perfused rat small intestine and stimulate secretion by different sensing mechanisms. Am. J. Physiol. Metab. 320 , E874–E885 (2021). Katakura, K. et al. Toll-like receptor 9–induced type I IFN protects mice from experimental colitis. J. Clin. Invest. 115 , 695–702 (2005). Chassaing, B. et al. Fecal Lipocalin 2, a Sensitive and Broadly Dynamic Non-Invasive Biomarker for Intestinal Inflammation. PLoS One 7 , e44328 (2012). Beghini, F. et al. Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3. Elife 10 , e65088 (2021). Caspi, R. et al. The MetaCyc database of metabolic pathways and enzymes - a 2019 update. Nucleic Acids Res. 48 , D445–D453 (2020). Additional Declarations Yes there is potential Competing Interest. BAHJ, JBH, and KK are co-inventors of an issued patent (US and EU) related to McB and gut dysbiosis. BAHJ, SKYJ, and BSYC are co-inventors of a patent application related to McB’s potential in treating or preventing gastrointestinal barrier dysfunction. The remaining authors declare that there are no conflicts of interest. Supplementary Files Supplementarymateriallegends.docx YangJensenetal2025ExtendedData.pdf Extended data figures S1-S6 SupplementaryMaterialFigureS1.xlsx Supplementary Material Figure S1 SupplementaryMaterialFigure2.xlsx Supplementary Material Figure 2 SupplementaryMaterialFigure3AGS2.xlsx Supplementary Material Figure 3A-G + S2 SupplementaryMaterialFigure3HO.xlsx Supplementary Material Figure 3H-O SupplementaryMaterialFigure4S3.xlsx Supplementary Material Figure 4 + S3 SupplementaryMaterialFigureS4.xlsx Supplementary Material Figure S4 SupplementaryMaterialFigureS5.xlsx Supplementary Material Figure S5 SupplementaryMaterialFigure5.xlsx Supplementary Material Figure 5 SupplementaryMaterialFigure6.xlsx Supplementary Material Figure 6 SupplementaryMaterialFigureS6AC.xlsx Supplementary Material Figure S6 A-C SupplementaryMaterialFigureS6DE.xlsx Supplementary Material Figure S6 D-E SupplementaryMaterialTables13.xlsx Supplementary Material Tables 1-3 Cite Share Download PDF Status: Published Journal Publication published 15 Jan, 2026 Read the published version in Nature Communications → 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-6973437","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":479135478,"identity":"040aaf6e-4d09-4e46-9bed-a4da4791add6","order_by":0,"name":"Benjamin Jensen","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-6991-0828","institution":"University of copenhagen","correspondingAuthor":true,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"Jensen","suffix":""},{"id":479135479,"identity":"76e0e684-2e32-470d-9d12-488c1aad5ebf","order_by":1,"name":"Sune Yang-Jensen","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Sune","middleName":"","lastName":"Yang-Jensen","suffix":""},{"id":479135480,"identity":"cfd2321a-e2bd-4cb3-ae82-35598ca24699","order_by":2,"name":"Béatrice Choi","email":"","orcid":"https://orcid.org/0000-0001-8160-9448","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Béatrice","middleName":"","lastName":"Choi","suffix":""},{"id":479135481,"identity":"fc8ae48f-183b-4fd4-96a4-e69d045b22c5","order_by":3,"name":"Simone Pærregaard","email":"","orcid":"https://orcid.org/0000-0001-6879-0529","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Simone","middleName":"","lastName":"Pærregaard","suffix":""},{"id":479135482,"identity":"cadbcbe6-609c-4e5f-8c81-1801afc49567","order_by":4,"name":"Nora Nägele","email":"","orcid":"https://orcid.org/0009-0007-4711-1954","institution":"University of copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Nora","middleName":"","lastName":"Nägele","suffix":""},{"id":479135483,"identity":"2aec711e-0def-48f9-882e-964e5a4661c8","order_by":5,"name":"Nazuk Gupta","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Nazuk","middleName":"","lastName":"Gupta","suffix":""},{"id":479135484,"identity":"3d0e5dd9-1790-4ca2-b9fb-b2cdd457d3ae","order_by":6,"name":"Ida Modvig","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Ida","middleName":"","lastName":"Modvig","suffix":""},{"id":479135485,"identity":"2c9e75d7-adec-49b4-9b32-8e8febe63968","order_by":7,"name":"Jacob Holm","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Jacob","middleName":"","lastName":"Holm","suffix":""},{"id":479135486,"identity":"029a74fa-326a-4e77-bd94-b8ef22498147","order_by":8,"name":"Karsten Kristiansen","email":"","orcid":"https://orcid.org/0000-0002-6024-0917","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Karsten","middleName":"","lastName":"Kristiansen","suffix":""},{"id":479135487,"identity":"7e45f691-7bb7-4f4d-91d9-71b11de43f38","order_by":9,"name":"Hannelouise Kissow","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Hannelouise","middleName":"","lastName":"Kissow","suffix":""},{"id":479135488,"identity":"1e48545d-cad7-4caf-afea-8285db865aca","order_by":10,"name":"Jens Holst","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Jens","middleName":"","lastName":"Holst","suffix":""},{"id":479135489,"identity":"9d29bf28-5d7c-454e-a68f-425a70e67ad7","order_by":11,"name":"Bolette Hartmann","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Bolette","middleName":"","lastName":"Hartmann","suffix":""}],"badges":[],"createdAt":"2025-06-25 10:10:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6973437/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6973437/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-68551-9","type":"published","date":"2026-01-15T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86327415,"identity":"183de577-d37e-4a1a-a659-fd57fbf3e418","added_by":"auto","created_at":"2025-07-09 11:12:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1448632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMcB lysates rapidly imprints the gut microbiota and alters microbial function. \u003c/strong\u003e(A) Study design. (B) Principal component plot of Bray-Curtis dissimilarity of experimental diets over time from two independent experiments. Each circle indicates one mouse, radiating from centroids with indicated experimental day. (C) Plot of longitudinal family-level relative abundances. (D) Bubble plot of top 10 significantly regulated microbial metabolic pathways differentiating reference diets and McB-containing diets. Size of each dot indicated the center log-ration transformed pathway abundance as determined by ALDEx2. (E) Top 20 significantly regulated microbial pathways sorted by level of significance, determined by ALDEx2. (F) Relative abundance levels of \u003cem\u003eMethylococcus capsulatus \u003c/em\u003eBath across diets and time. (G) Temporal relative abundance plots of top 12 species differentially regulated by McB as determined by general mixed linear modelling with subject as a random variable.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/3f431431baeb129b17edc870.png"},{"id":86327413,"identity":"8e2b616b-6104-4dc9-9d88-fba0b6aae6d7","added_by":"auto","created_at":"2025-07-09 11:12:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":770087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMcB-mediated immune imprinting is subset specific. \u003c/strong\u003e(A) Study design. (B) Relative cecum sizes over time in response to antibiotics (ABX) treatment, shown as fold-change (FC) compared to water groups. (C) Levels of small intestinal \u003cem\u003elamina propria \u003c/em\u003eFoxP3\u003csup\u003e+\u003c/sup\u003eROR𝛾t\u003csup\u003e+\u003c/sup\u003e peripherally induced regulatory T-cells (pTregs) over 2, 4, and 6 weeks. (D) Levels of large intestinal \u003cem\u003elamina propria \u003c/em\u003epTregs over time. (E-G) Levels of FoxP3\u003csup\u003e-\u003c/sup\u003eROR𝛾t\u003csup\u003e+\u003c/sup\u003e T-helper 17 (Th17) cells in small intestinal \u003cem\u003elamina propria\u003c/em\u003e after 2 (E), 4 (F), and 6 weeks (G) each with proportions of Th17 cells also expressing IL-10 or IFN𝛾. (H-J) Levels of FoxP3\u003csup\u003e-\u003c/sup\u003eROR𝛾t\u003csup\u003e+\u003c/sup\u003e Th17 cells in large intestinal \u003cem\u003elamina propria\u003c/em\u003e after 2 (H), 4 (I), and 6 weeks (J) with levels of IL-10 and IFN𝛾-expressing cells. Data are presented as mean ± SEM (n = 3-4 per group) with individual data points representing each mouse. Statistical significance was determined by a Two-Way ANOVA with Fisher’s LSD multiple comparisons post hoc test. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/44d9ac3f9dc6068492c95858.png"},{"id":86327414,"identity":"57fb5740-f920-4145-b65b-5283cf25fb86","added_by":"auto","created_at":"2025-07-09 11:12:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4541934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMcB consumption protects against gastrointestinal injury. \u003c/strong\u003e(A) Small intestinal mucositis study design. (B) Body weight development post injection with 5-fluorouracil (5-FU) (400 mg/kg) as relative to baseline weight. Acute phase is considered day 1-3 and recovery phase considered day 4-6. The area-under-the-curve is visible for each group. (C) Small intestinal wet weights in grams at the acute phase (Day 3) and recovery phase (Day 6). (D-F) Small intestinal villus length in µm at the acute phase and recovery phase, spanning the entire small intestine axis from duodenum (D), jejunm (E), and ileum (F). (G) Representative HE images of jejunum and ileum villi at Day 3, representing the median within each group. Scalebars = 50 µm. (H) Large intestinal DSS colitis study design. (I) Relative body weight development during colitis. (J) Progression of disease as assessed by the Disease Activity Index (DAI). (K) DAI at termination. (L) Large intestinal lengths in cm. (M) Representative images of large intestines, chosen as the median representatives within each group. (N) Colonic inflammation index in mg colon wet weight/cm colon length. (O) Colitis histopathological scores, assessed blinded, with median representative images (O). Scale bars = 100 µm. Data are presented as mean ± SEM (n = 9 per group in the mucositis protocol, n = 8-9 per group in the colitis protocol) with individual data points denoting each mouse. Statistical significance was determined by a Two-Way ANOVA followed by Fisher’s LSD multiple comparisons post hoc test. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/f8314b08d35a6e29af7565d9.png"},{"id":86327564,"identity":"800d3bed-a66c-48c8-b92c-2658e5d946e8","added_by":"auto","created_at":"2025-07-09 11:20:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4598481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMcB-mediated small intestinal protection is GLP-2R dependent.\u003c/strong\u003e (A) Study design of beginning of recovery phase after induction of small intestinal mucositis using the 5-fluorouracil model in whole-body GLP-2R wildtype (WT) and knockout (KO) mice. Body weight development following small intestinal injury (B), small intestinal wet weights (C), Jejunum (D) and ileum (E) villus lengths in µm, and jejunum (F) and ileum (G) myeloperoxidase (MPO) levels corrected for total protein content, in GLP-2R WT mice. (H-M) Body weight development following small intestinal injury (H), small intestinal wet weights (I), Jejunum (J) and ileum (K) villus lengths in µm, and jejunum (L) and ileum (M) MPO levels corrected for total protein content, in GLP-2R KO mice. (N) Representative histological HE images of jejunum and ileum sections of GLP-2R WT and KO mice, representing group medians. Scalebars = 50 µm. Data are from four independent experiments and presented as mean ± SEM (n = 6-10 per group) with individual data points. Statistical significance was determined by a Two-Way ANOVA followed by Fisher’s LSD multiple comparisons post hoc test. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/b439cd602aec3f55962e3476.png"},{"id":86327431,"identity":"02db4b48-22fb-4571-98fd-44e946c74b1e","added_by":"auto","created_at":"2025-07-09 11:12:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2861274,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe GLP-2R is necessary for McB-mediated protection against colitis.\u003c/strong\u003e(A) Study design of large intestinal colitis using the dextran sodium sulphate (DSS) model in whole-body GLP-2R wildtype (WT) and knockout (KO) mice. DSS was administered at a 2.5% concentration in drinking water. (B-G) Disease assessment in GLP-2R WT mice. (B) Body weight development over all 5 study days, relative to initial starting weight. (C) Body weight comparisons at termination. (D) Heatmap of disease activity index (DAI) over time and at termination (E). (F) Large intestinal length in cm and weight-to-length inflammation index (G). (H-M) Disease assessment in GLP-2R KO mice. (H) Body weight development over all 5 study days, relative to initial starting weight. (I) Body weight comparisons at termination. (J) Heatmap of DAI over time and at termination (K). (L) Large intestinal length in cm and weight-to-length inflammation index (M). Histopathological scoring of colitis assessed blinded, in GLP-2R WT (N) and KO (O) mice. (P) Histological images of colon swissroll sections of GLP-2R WT and KO mice, representing group medians. Scalebars = 200 µm. Data are from four independent experiments and presented as mean ± SEM (n = 7-8 per group) with individual data points. Statistical significance was determined by a Two-Way ANOVA followed by Fisher’s LSD multiple comparisons post hoc test. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/7025be86efe8030c7206f417.png"},{"id":86327423,"identity":"da060e8e-bf2f-4ee2-b9bf-6e56aa547575","added_by":"auto","created_at":"2025-07-09 11:12:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1140649,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrobial fermentation is essential to confer McB-mediated gut protection\u003c/strong\u003e. (A) Study design of the colitis protocol. Mice were given control drinking water or a 20 ppm solution of an inhibitor of microbial hindgut fermentation (FermInh). (B-F) Disease assessment in reference diet-fed mice. (B) Disease activity index (DAI) over the entire study period. (C) DAI at termination. (D) Colon weight-to-length inflammation index. (E) Colon length in cm. (F) Small intestine lengths in cm. (G-K) Disease assessment in McB-fed mice. (G) DAI over the entire study period. (H) DAI at termination. (I) Colon weight-to-length inflammation index. (J) Colon length in cm. (K) Small intestine lengths in cm. (L) GLP-2 levels in plasma of mice fed the reference diet or McB-diet with and without FermInh. (M) Proposed mode-of-action of McB in vivo. Data are presented as mean ± SEM (n = 12 per group) with individual data points. Statistical significance was determined by a Two-Way ANOVA followed by Fisher’s LSD multiple comparisons post hoc test. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/3feed6555240d56c7eb36b71.png"},{"id":103052883,"identity":"accd5f26-3a45-47cc-98fb-8bb7efc84e52","added_by":"auto","created_at":"2026-02-20 08:07:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15642765,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/24a1dd95-8879-447f-a053-928fdd708864.pdf"},{"id":86327412,"identity":"39fe7e5b-1df0-4600-84cd-c73b510d6e85","added_by":"auto","created_at":"2025-07-09 11:12:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17011,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymateriallegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/bb1b4cd2631aac81aab9d145.docx"},{"id":86327427,"identity":"5de865b7-e670-47b4-a64a-305cfaa4bee6","added_by":"auto","created_at":"2025-07-09 11:12:43","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14095710,"visible":true,"origin":"","legend":"Extended data figures S1-S6","description":"","filename":"YangJensenetal2025ExtendedData.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/f062cd03cc5cff4421c624a5.pdf"},{"id":86327563,"identity":"d552f062-f256-4ca5-b8cb-12af35fec2c1","added_by":"auto","created_at":"2025-07-09 11:20:42","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14718,"visible":true,"origin":"","legend":"Supplementary Material Figure S1","description":"","filename":"SupplementaryMaterialFigureS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/5bd62e619d583fc6b4036945.xlsx"},{"id":86327421,"identity":"3ba7559f-a20b-45ca-ad8e-02ef3425681e","added_by":"auto","created_at":"2025-07-09 11:12:42","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":22909,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material Figure 2\u003c/p\u003e","description":"","filename":"SupplementaryMaterialFigure2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/cf10bd03df135f81000d9c24.xlsx"},{"id":86327425,"identity":"96e8e25b-ddc6-4b21-b88d-5449be70f288","added_by":"auto","created_at":"2025-07-09 11:12:43","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":104944,"visible":true,"origin":"","legend":"Supplementary Material Figure 3A-G + S2","description":"","filename":"SupplementaryMaterialFigure3AGS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/2b009900515ccfb26d81825b.xlsx"},{"id":86327432,"identity":"b5e2b156-8e26-41ea-ad5b-6c64f7bb3832","added_by":"auto","created_at":"2025-07-09 11:12:43","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":108763,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material Figure 3H-O\u003c/p\u003e","description":"","filename":"SupplementaryMaterialFigure3HO.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/46a0f1617ca5e481688ac312.xlsx"},{"id":86327424,"identity":"3ed5101b-84bc-42e2-9ab8-5589e912ebc5","added_by":"auto","created_at":"2025-07-09 11:12:43","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":139335,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material Figure 4 + S3\u003c/p\u003e","description":"","filename":"SupplementaryMaterialFigure4S3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/921dffbb5e2dd77785075e1a.xlsx"},{"id":86327428,"identity":"215be8e1-1462-4dd6-abd5-9f2bddaf2097","added_by":"auto","created_at":"2025-07-09 11:12:43","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":68788,"visible":true,"origin":"","legend":"Supplementary Material Figure S4","description":"","filename":"SupplementaryMaterialFigureS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/c5e751945133d81238450a89.xlsx"},{"id":86328500,"identity":"d4388877-b515-4397-9e16-3ae891f64734","added_by":"auto","created_at":"2025-07-09 11:28:42","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":139216,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material Figure S5\u003c/p\u003e","description":"","filename":"SupplementaryMaterialFigureS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/6727c2b092243323dedea65c.xlsx"},{"id":86327566,"identity":"aacd1017-8c07-4f7a-ace4-b942c7be9265","added_by":"auto","created_at":"2025-07-09 11:20:43","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":140835,"visible":true,"origin":"","legend":"Supplementary Material Figure 5","description":"","filename":"SupplementaryMaterialFigure5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/840259e773eb2cedeabbcc02.xlsx"},{"id":86327420,"identity":"a1687866-1da6-4803-a71d-201151312061","added_by":"auto","created_at":"2025-07-09 11:12:42","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":147433,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material Figure 6\u003c/p\u003e","description":"","filename":"SupplementaryMaterialFigure6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/fd2f17e9a43c337a6173f9ae.xlsx"},{"id":86327429,"identity":"d6b20f29-9171-4858-be5b-04c2749b6dd0","added_by":"auto","created_at":"2025-07-09 11:12:43","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":15846,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material Figure S6 A-C\u003c/p\u003e","description":"","filename":"SupplementaryMaterialFigureS6AC.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/fa1d5772b0a3d07ebd032c1f.xlsx"},{"id":86327422,"identity":"e18706e2-f30c-4500-b2ef-66621b90471d","added_by":"auto","created_at":"2025-07-09 11:12:42","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":12899,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material Figure S6 D-E\u003c/p\u003e","description":"","filename":"SupplementaryMaterialFigureS6DE.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/2cdfcd64698d26631bca74cd.xlsx"},{"id":86327567,"identity":"669a6d85-acb7-405a-ba8d-ecfbd0b2ebbe","added_by":"auto","created_at":"2025-07-09 11:20:43","extension":"xlsx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":15822,"visible":true,"origin":"","legend":"Supplementary Material Tables 1-3","description":"","filename":"SupplementaryMaterialTables13.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6973437/v1/b767bab05a09ef2c97568467.xlsx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nBAHJ, JBH, and KK are co-inventors of an issued patent (US and EU) related to McB and gut dysbiosis. BAHJ, SKYJ, and BSYC are co-inventors of a patent application related to McB’s potential in treating or preventing gastrointestinal barrier dysfunction. The remaining authors declare that there are no conflicts of interest.","formattedTitle":"Microbial Activation of the GLP-2R Mitigates Gastrointestinal Inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmidst the urgent need for sustainable food systems\u003csup\u003e1\u003c/sup\u003e to combat excessive green house gass emmsions, resource depletion, and biodiversity loss\u003csup\u003e2\u003c/sup\u003e, cultured microbial proteins are emerging as low-emission, resource-efficient alternatives to animal-derived products\u003csup\u003e1\u003c/sup\u003e. While the ecological benefits of these cultured proteins are well established\u003csup\u003e1\u003c/sup\u003e, their influence on host biology \u0026ndash; particularly the impact on gastrointestinal (GI) homeostasis \u0026ndash; remains largely unexplored. This knowledge gap is critical, as dietary components actively shape immune function, host-microbe interactions, and barrier integrity.\u003c/p\u003e\n\n\u003cp\u003eWithin the gut, dietary components are digested into smaller entities that interact with the mucosal immune system both directly, through antigen presentation to immune cells such as T cells, and indirectly, by modulating the structure and function of the gut microbiota\u003csup\u003e3\u003c/sup\u003e. These interactions underpin immune education, as evidenced by the influence of diet on vaccine efficacy\u003csup\u003e4\u003c/sup\u003e and food allergy development\u003csup\u003e5\u003c/sup\u003e. Host-diet-microbe dynamics are especially important for the differentiation of ROR\u0026gamma;t-expressing T cell subsets, including peripherally induced regulatory T cells (pTregs) and T-helper 17 (Th17) cells. pTregs, induced by the gut microbiota\u003csup\u003e6\u003c/sup\u003e and their cellular components\u003csup\u003e7\u003c/sup\u003e, suppress aberrant immune responses to food antigens\u003csup\u003e8\u003c/sup\u003e and commensal microbes\u003csup\u003e9\u003c/sup\u003e. Th17 cells, regulated by both host- and microbially derived signals\u003csup\u003e10\u003c/sup\u003e, adopt context-dependent roles \u0026ndash; ranging from barrier reinforcement to pathogenic inflammation\u003csup\u003e11\u003c/sup\u003e \u0026ndash; dictated by the local microenvironment\u0026rsquo;s inflammatory milieu\u003csup\u003e12,13\u003c/sup\u003e. Notably, since both subsets respond to microbial and dietary cues, their phenontype and function hinge on the functional capacity of resident gut microbes.\u003c/p\u003e\n\n\u003cp\u003eDisruptions to these host-diet-microbe interactions underlie a spectrum of diseases, including inflammatory bowel diseases (IBD) and metabolic disorders\u003csup\u003e14\u0026ndash;18\u003c/sup\u003e, with contemporary Western diets epidemiologically linked to rising prevalence of GI inflammation\u003csup\u003e19\u0026ndash;21\u003c/sup\u003e. Although microbiota-targeted therapies show promise, their inconsistent efficacy highlights the need for interventions that bypass or reprogram microbial dependencies to promote immune tolerance and tissue repair. Previously, we demonstrated that a bacterial lysate derived from the soil microbe \u003cem\u003eMethylococcus capsulatus\u003c/em\u003e Bath (McB) modulates mucus production and mucosal immunity in mice\u003csup\u003e22\u003c/sup\u003e, suggesting enhanced gut barrier function and positioning McB as a next-generation cultured protein. However, the clincal potential of non-scalable monoculture approaches is limited, and the impact of McB on host physiology during inflammation \u0026ndash; particularly its interaction with epithelial repair pathways such as glucagon-like peptide-2 (GLP-2) signaling \u0026ndash; remains unresolved, hampering translational development.\u003c/p\u003e\n\n\u003cp\u003eHere, we investigate the immunological and regenerative properties of a sustainable, commercially scalable microbial protein lysate in which ~95% of the biomass is derived from McB (FeedKind\u0026reg;, Calysta UK). We show that dietary delivery of this lysate as the main protein source rapidly alters microbial community structure and enriches for pathways involved in microbial fermentation. These shifts coincide with mucosal immune imprinting across both the small and large intestine, characterized by microbiota-independent expansion of pTregs and microbiota-sensitive reprogramming of Th17 cells toward a tolerogenic, immunoregulatory state. Importantly, in two mechanistically distinct models of GI inflammation targeting regionalized areas of the gut, the scalable McB lysate confers robust, pan-GI protection that is dependent on functional GLP-2 receptor (GLP-2R) signaling. This GLP-2R signaling is further dependent on microbial fermenation of the McB lysate to confer its protective actions. Together, our findings reveal a mode of nutritional immunomodulation that integrates microbial fermentation, mucosal T cell plasticity, and molecular mimicry-driven engagement of host receptors to facilitate tissue repair. This work positions McB-derived lysates as a novel class of microbial proteins with dual functionality: offering scalable protein nutrition while engaging endogenous pathways of immune tolerance and intestinal regeneration.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMcB Induces Rapid and Persistent Gut Microbiota Alterations \u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo address if the previously reported phenotype of McB feeding\u003csup\u003e22\u003c/sup\u003e was diet-dependent, we compared the simple compositionally defined diet (SD) previously used, to a complex diet with a humanized nutrient composition (CD), both with and without McB lysate as the protein source (Figure 1A, Supplementary Table 1). Fecal shotgun sequencing revealed rapid changes in gut microbiota composition (Figure 1B-C), where McB presence explained 27% variance compared to only 10% from all other nutrients combined (Figure 1B). The observed microbiota changes were accompanied by major changes to the metabolic potential of the McB-associated microbiota (Figure 1D-E; \u0026gt;460 differentially regulated pathways) already a few days after McB-feeding and regardless of background diet. Noteworthy metabolic pathways significantly enriched in feces of McB-fed mice were fermentation processes, including metabolic processes leading to production of short-chain fatty acids (SCFAs), supporting our previous finding of elevated cecal SCFA in monoculture-fed mice\u003csup\u003e22\u003c/sup\u003e. Indicative of a near-complete utilization of the lysate within the GI tract, McB was barely, and only transiently, detectable (\u0026lt;0.01%) in fecal samples of a few mice consuming lysate-containing diets (Figure 1F). Still, the McB-induced changes to the microbial community remained stable after their initial shift (Figure 1B-D), highlighting robust and reproducible McB-induced microbiota modulation, despite moving from a monoculture at lab scale in the original report to a commercial product with minimal amount of helper bacteria (FeedKind®, Calysta UK). Considering that the major microbiota shifts appeared to be driven by \u003cem\u003eLachnospiraceae\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacteroidacea\u0026nbsp;\u003c/em\u003efamilies (Figure 1C), we used our shotgun analysis to assess species-level resolution (Figure 1G). This revealed that changes were largely driven by few species (Figure 1G), likely better equipped to process the microbial lysate escaping digestion in the small intestine, considering the consistent \u0026gt;30% increase in relative abundance of those species after introduction of McB.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNutritional Immune Imprinting Exhibits Subset Specific Microbiota Dependence\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next examined the \u003cem\u003elamina propria\u003c/em\u003e (LP) T-cell landscape (Figure S1A), in both small and large intestine, to study regionalized McB-, microbe-, and diet-dependent effects, by flow cytometry. Independent of background diet, McB-feeding increased the small and large intestinal LP pTreg populations (2.5 and 4 times, respectively), including the relative proportion of triple-positive IL-17\u003csup\u003e+\u003c/sup\u003e pTregs (Figure S1B-E). Notably, McB-feeding also induced a ~50% increase in Th17 cells exclusively in the small intestine (Figure S1F-G). Although Th17 cells originally were conceived as proinflammatory culprits, recent literature points towards their pleiotropic nature being instrumental for maintaining GI immune balance\u003csup\u003e11\u003c/sup\u003e and metabolic homeostasis upon HFD feeding\u003csup\u003e23\u003c/sup\u003e. Indeed, the McB-attuned Th17 landscape exhibited immunoregulatory traits, indicated by an increase in IL-10\u003csup\u003e+\u003c/sup\u003e Th17 cells paralleled by a reduction of IFN-g\u003csup\u003e+\u003c/sup\u003e Th17 cells, suggesting a shift towards tolerogenic immunity (Figure S1F-I). Although absolute numbers of large intestinal Th17 cells were unaffected by McB feeding, their phenotype recapitulated our findings from the small intestinal LP (Figure S1H-I). McB-induced immune imprinting seemed targeted towards RORgt\u003csup\u003e+\u003c/sup\u003e T cell subsets, as neither Th1 nor thymic-derived Treg (nTreg) populations were affected by McB-feeding (Figure S1J-Q).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs GI pTreg\u003csup\u003e9\u003c/sup\u003e and Th17 cell\u003csup\u003e24,25\u003c/sup\u003e plasticity can be modulated by the gut microbiota, we next investigated if McB-induced immune alterations were driven by the substantial shift in microbial community structure and functions following McB-feeding (Figure 1) or if McB-induced immune imprinting developed independently of resident gut microbes. To test this, we administered either control drinking water or a broad-spectrum antibiotic cocktail (ABX) for 2, 4, or 6 weeks, blunting the gut microbiota (Figure 2A). Despite ABX-induced disruption of gut homeostasis, as evident from the ABX-induced increases in cecal size (Figure 2B), McB instantly enhanced pTreg abundance in the small intestine of both ABX-treated and non-treated mice (Figure 2C). This trait was, however, not fully recapitulated in LP of the large intestine, but pTreg induction was notably accelerated in ABX-treated mice (Figure 2D). We interpreted the delayed pTreg-induction in colons of conventional mice as a direct consequence of their microbial ‘buffer’ (i.e., niche occupation) limiting direct McB-host interactions. Summarized, these data suggest that the McB lysate can directly increase LP pTregs across the intestines.\u003c/p\u003e\n\u003cp\u003eNext, we turned our attention to the Th17 cells, which were also notably affected by McB feeding (Figure 2E-J). In sharp contrast to the above-mentioned pTreg inductions, we found that numeric \u003cem\u003eand\u003c/em\u003e phenotypic McB-mediated Th17 alterations were highly dependent on a functional gut microbiota. Thus, in the small intestine of conventional mice, McB feeding induced a rapid \u0026gt;50% increase in Th17 cell proportions (Figure 2E-G). This effect was dampened the first 4 weeks of ABX treatment. The McB-mediated Th17 induction after 6 weeks of ABX treatment mirrored the gradual increase in total microbial load (Figure S1R). This rise in bacterial load suggested a selective bloom of ABX-resistant species, possibly interacting with McB to drive the observed expansion of Th17 cells, even under sustained ABX exposure. Further corroborating the Th17 cell-gut microbiota dependence, we observed that even in the absence of a numeric increase (large intestine, Figure 2H-J), McB-feeding still facilitated a phenotypic shift, exemplified by a \u0026gt;2-fold increase in IL-10\u003csup\u003e+\u003c/sup\u003e Th17 cells, exclusively in conventional mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, these data demonstrate that while pTreg induction occurred independently of gut microbiota composition, Th17 cell induction and phenotypic manipulation relied on complex host-diet-microbe interactions, corroborating that the impact of the gut microbiota on McB-induced immune regulation is subset specific.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMcB Promotes Gastrointestinal Homeostasis and Reduces Inflammation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiarrhea and a swollen, fluid-filled cecum are common side effects of ABX treatment\u003csup\u003e26\u003c/sup\u003e,\u0026nbsp;likely due to disruptions in gut barrier integrity, impaired mucus production, reduced microbial load, and a lack of fiber fermentation\u003csup\u003e27,28\u003c/sup\u003e.\u0026nbsp;During ABX treatment, we observed that McB-fed mice exhibited less diarrhea (data not shown) and were protected against ABX-induced increases in cecum weight (Figure 2B). These findings suggest that McB preserves gut barrier function alongside enhanced tolerogenic immunity (Figure S1) in both the murine small and large intestine.\u003c/p\u003e\n\u003cp\u003eWith this in mind, we used a two-pronged experimental approach to interrogate if McB feeding confers protection against region-specific gut inflammation, namely 1) chemotherapy-induced mucositis\u0026nbsp;affecting the entire GI tract but predominantly damages the small intestine via villus atrophy\u003csup\u003e29,30\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e31\u003c/sup\u003e, and 2) chemically-induced colitis, primarily targeting the large intestine.\u003c/p\u003e\n\u003cp\u003eMucositis was induced using a single intraperitoneal (i.p.) injection of 5-fluorouracil (5-FU; 400 mg/kg) (Figure 3A). Following 5-FU challenge, mice displayed acute weight loss over three days, then rapidly regained weight and returned to baseline within six days (Figure 3B). McB-fed mice showed a modest but noticeable improvement in recovery rate compared to reference diet-fed controls. As expected, 5-FU significantly reduced small intestinal wet weights (Figure 3C) and induced villus atrophy (Figure 3D–G). Notably, McB feeding partially protected the small intestine from 5-FU-induced damage, with preserved villus architecture observed along the entire small intestinal axis. In line with prior reports describing crypt hyperproliferation during mucosal recovery post-5-FU challenge\u003csup\u003e31–33\u003c/sup\u003e, we observed deeper crypts in reference-fed mice. In contrast, McB-fed mice consistently exhibited deeper crypts regardless of 5-FU exposure (Figure S2A-H), indicating a baseline trophic effect of McB on the intestine, rather than a reactive compensatory proliferation. Although the most pronounced damage from 5-FU occurred in the small intestine, colonic shortening was also evident in the acute phase (Figure S2I-J), again indicating mucosal inflammation – a feature that was attenuated in McB-fed mice.\u003c/p\u003e\n\u003cp\u003eGiven the observed mitigation of colonic inflammation in the 5-FU model, we next tested McB’s capacity to prevent large intestinal injury in the DSS-induced colitis model (Figure 3H). McB-fed mice were fully protected against DSS-induced weight loss (Figure 3I) and exhibited a substantially milder disease trajectory (Figure 3J), with Disease Activity Index (DAI) scores approximately 2-fold lower than controls by study end (Figure 3K). McB feeding also preserved colon length after DSS exposure (Figure 3L), and large intestines from McB-fed mice displayed firmer contents and fewer hemorrhagic lesions (Figure 3M). When inflammation was examined by colon wet weight-to-length ratio, a standard index of colonic inflammation, McB-fed mice exhibited significantly lower values (Figure 3N). Histopathological analysis of colon Swiss rolls supported these findings, showing a \u0026gt;2-fold reduction in colonic damage scores in McB-fed mice (Figure 3O–P).\u003c/p\u003e\n\u003cp\u003eTogether, these results indicate that McB not only modulates GI immunity and microbial composition but also confers broad protection against inflammation throughout the GI tract.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGLP-2 Receptor Signaling is Essential for\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003e\u003cem\u003eMcB-Induced Gastrointestinal Protection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the robust protection conferred by McB throughout the GI tract, we hypothesized that its effects were mediated by intestinotrophic pathways. As a central regulator of intestinal growth, GLP-2, a gut hormone with potent intestinotrophic properties, and its receptor (GLP-2R) emerged as key candidates. To test this hypothesis, we utilized GLP-2R wildtype (WT) and knockout (KO) mice in the mucositis and colitis models. This approach allowed us to dissect both regional specificity, given GLP-2’s known preference for the small intestine\u003csup\u003e34,35\u003c/sup\u003e, and the necessity of intact GLP-2R signaling in McB-mediated gut protection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the 5-FU-induced small intestinal injury model (Figure 4A), GLP-2R WT mice on a reference diet lost significant weight (Figure 4B), exhibited dramatically reduced small intestinal weights (Figure 4C), and developed villus atrophy (Figure 4D-E, 4N \u0026amp; Figure S3A). Still, McB-fed mice were protected against intestinal damage (Figure 4C-G, 4N). Myeloperoxidase (MPO), an enzyme released by neutrophils during inflammation, serves as a key indicator of gut barrier damage and local inflammation.\u0026nbsp;We found that 5-FU markedly increased MPO levels, especially in the ileum (Figure 4F–G). Importantly, McB feeding significantly reduced MPO accumulation, further supporting its barrier-protective role. However, these protective effects were completely abolished in GLP-2R KO mice (Figure 4H–N \u0026amp; Figure S3), confirming that McB requires functional GLP-2R signaling for restoring small intestinal homeostasis.\u0026nbsp;Notably, while\u0026nbsp;5-FU induced colonic damage, GLP-2R KO mice exhibited complete loss of McB-mediated protection in the colon (Figure S3F, L), extending GLP-2R’s role beyond canonical small intestinal functions to encompass GI-wide therapeutic effects.\u003c/p\u003e\n\u003cp\u003eTo validate this GLP-2R-dependent host-microbe signaling axis in a large intestine-focused model, we administered DSS to GLP-2R WT and KO mice (Figure 5A). In GLP-2R WT mice, McB feeding conferred robust protection, mitigating weight loss (Figure 5B–C), reducing DAI scores (Figure 5D–E), preserving colon length (Figure 5F), and lowering inflammation index (Figure 5G). These protective effects were abolished in GLP-2R KO mice (Figure 5H–M), as confirmed by histological scoring of crypt integrity and immune infiltration (Figure 5N–P). Together, these data demonstrate that McB’s gut-protective effects are strictly GLP-2R-dependent across intestinal regions.\u003c/p\u003e\n\u003cp\u003eGiven the co-secretion of GLP-2 and its structurally realted hormone GLP-1, we investigated potential GLP-1R involvement. However, McB-fed GLP-1R WT and KO littermates were equally protected in both mucositis and colitis models (Figures S4–S5), excluding a role of GLP-1R and underscoring the specificity of GLP-2R signaling in McB’s therapeutic action.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMicrobial Fermentation of McB is Required for Protection Against Gut Inflammation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate how McB confers GLP-2R-dependent protection, we first investigated whether McB stimulates endogenous GLP-2 secretion. Using the perfused rat intestinal model (Figure S6A) – which enables real-time assessment of hormone secretion and nutrient absorption – we delivered McB directly to the intestinal lumen. Notably, McB failed to elicit gut hormone release or modulate amino acid absorption (Figure S6B–C). Since this model bypasses digestion, we posited that enzymatic processing might be needed to liberate bioactive components stimulating GLP-2 secretion.\u003c/p\u003e\n\u003cp\u003eTo test this, we gavaged mice with McB or a control protein mix following administration of dipeptidyl peptidase 4 (DPP-4) and neprilysin inhibitors, and then measured plasma GLP-2 via radio-immuno-assay (RIA) (Figure S6D). While glucose stimulated GLP-2 release, neither McB nor the reference protein increased circulating GLP-2 (Figure S6E).\u0026nbsp;These findings demonstrate that McB does not acutely induce endogenous GLP-2 secretion. However, given 1) microbial fermentation’s known role in L-cell stimulation, 2) our observation of enriched fermentative microbial pathways (Figure 1D), and 3) the near-complete fecal degradation of McB (Figure 1F), we hypothesized that \u003cstrong\u003emicrobial fermentation of McB\u003c/strong\u003e generates metabolites capable of either stimulating GLP-2 release or directly activating the GLP-2R via molecular mimicry, a prerequisite for its beneficial effects.\u003c/p\u003e\n\u003cp\u003eTo test this hypothesis, we employed the DSS colitis model and administered a \u003cstrong\u003efermentation inhibitor\u003c/strong\u003e (FermInh; 20 ppm beta-acid extract from \u003cem\u003eHumulus lupulus\u003c/em\u003e) to block microbial hindgut fermentation (Figure 6A).\u0026nbsp;In reference-fed mice, FermInh had no significant effect on colitis severity (Figure 6B–F). Consistent with prior findings, McB-fed mice exhibited reduced DAI scores, attenuated colonic inflammation, and conserved colon length compared to controls (Figure 6G-K). Strikingly,\u0026nbsp;FermInh treatment abolished McB’s protective effects, exacerbating DAI scores (Figure 6G–H), elevating colonic inflammation index (Figure 6I), leading to colon shortening (Figure 6J). Given McB’s intestinotrophic effects in healthy mice (Figure 3, 4, S2-S4), we assessed intestinal morphology. Remarkably, FermInh eliminated McB-induced elongation of both small and large intestines, even in healthy, non-DSS treated mice (Figure 6J-K), directly linking microbial fermentation to McB-mediated gut homeostasis.\u003c/p\u003e\n\u003cp\u003eBecause fermentation yields microbial metabolites known to stimulate gut hormone secretion, we asked whether blocking fermentation would lower systemic GLP-2 levels. Despite extended McB feeding (~2 weeks), we found no significant increase in circulating GLP-2 in either control or McB-fed mice, regardless of fermentation status (Figure 6L).\u003c/p\u003e\n\u003cp\u003eTogether, these findings demonstrate that McB protects against gut injury via a microbial fermentation-dependent mechanism that engages GLP-2R signaling independently of endogenous GLP-2 secretion, highlighting molecular mimicry as the underlying mechanism.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFood demands are soaring\u003csup\u003e1,2\u003c/sup\u003e and Western food habits\u003csup\u003e19\u003c/sup\u003e are closely associated with increasing prevalence of GI inflammatory diseases\u003csup\u003e20,21\u003c/sup\u003e. It is thus imperative to develop nutritional strategies that can counter these trajectories and potentially aid alleviating already established inflammation and disturbed gut health. Unfortunately, current means of food production drain our natural resources, with great environmental implications\u003csup\u003e1,2\u003c/sup\u003e. A proposed strategy to overcome these hurdles is the usage of alternative foods, such as cultured, microbe-based proteins. However, the effects of microbial protein sources on host health remains inadequately described. \u003c/p\u003e\n\u003cp\u003eIn this study, we utilized a microbial lysate derived from a non-native soil bacterium, McB, as a potent nutritional modulator of GI immunity, microbiota composition, and host resilience against intestinal injury (Figure 6M). Building on previous observations in simplified dietary contexts\u003csup\u003e22\u003c/sup\u003e, we demonstrate that McB retains its immunomodulatory and protective functions even when introduced into complex, humanized diets. The translational relevance of this lies not only in McB’s robust effects across diet types but in its ability to trigger subset-specific immune imprinting, remodel gut microbial ecology, and protect against both small and large intestinal inflammation through GLP-2R-dependent mechanisms.\u003c/p\u003e\n\u003cp\u003eA central breakthrough of this work is the demonstration that McB directly induces pTregs in both the small and large intestine, independent of the gut microbiota. This is in stark contrast to most known dietary or microbial interventions, where previous reports on microbial cell component-induced increases of pTregs exclusively precipitate in the colon\u003csup\u003e7\u003c/sup\u003e where microbial density is highest. Here, even in microbiota-depleted mice, McB rapidly expanded pTreg populations, highlighting a direct host-microbe interaction that bypasses gut microbiota derived intermediates. Such a finding not only decouples microbial dependence from tolerogenic T cell induction but also offers a novel route for immune modulation in various disease indications where microbiota-mediated therapies fail. In contrast to pTreg expansion, McB-driven Th17 cell modulation was strictly gut microbiota-dependent. McB not only increased Th17 cell numbers exclussively in conventional mice but also skewed their phenotype toward a tolerogenic IL-10\u003csup\u003ehigh\u003c/sup\u003e IFN-γ\u003csup\u003elow\u003c/sup\u003e state, highlighting the microbiota’s essential role in shaping adaptive immune responses. Th17 cells are particularly interesting for gut homeostasis, as these cells are known for their plasticity and ability to adapt their cytokine production depending on the local microenvironment\u003csup\u003e36\u003c/sup\u003e. As such, their functions are highly context-dependent; Th17 cells can be implicated in inflammatory diseases\u003csup\u003e11\u003c/sup\u003e but have also been demonstrated to orchestrate gut barrier integrity\u003csup\u003e37,38\u003c/sup\u003e and metabolic regulation\u003csup\u003e23\u003c/sup\u003e. The importance of Th17 cells is further highlighted by dietary influences. To this end, high-sugar diets deplete Th17 cell-inducing microbes, explaining the detrimentally low abundance of these cells following Western Diet feeding\u003csup\u003e39\u003c/sup\u003e, a contributing factor to the development of metabolic syndrome\u003csup\u003e23,39\u003c/sup\u003e. The microbiota-independent induction of pTregs by McB, coupled with the microbiota-dependent modulation of tolerogenic Th17 cells, is highly intriguing. The dual mechanism suggests broad therapeutic applicability across diverse enterotypes and microbiota states, including antibiotic-perturbed ecosystems.\u003c/p\u003e\n\u003cp\u003eFrom a microbial ecology perspective, McB triggered rapid, stable shifts in gut microbiota composition and function, dominated by taxa within the \u003cem\u003eLachnospiraceae\u003c/em\u003e and \u003cem\u003eBacteroidaceae\u003c/em\u003e families. Crucially, this effect persisted across two distinct dietary contexts and using a standardized, commercially produced lysate, posistioning McB as a reproducible modulator of microbioal communities. McB was nearly undetectable in feces, indicating near-complete GI utilization and suggesting its role as a precision substrate for microbial fermentation. The concomittant enrichment of microbial fermentation pathways mechanistically connect McB-induced microbiota remodelling to some of its downstream benefits in tolerogenic immunity.\u003c/p\u003e\n\u003cp\u003eGiven the tolerogenic T-cell populations and microbial functional changes, we subsequently explored if consumption of the lysate would confer health benefits locally within the GI tract. To this end, we used two distinct models of GI inflammation: 5-FU-induced mucositis and DSS-induced colitis. Functionally, McB conferred significant protection against both chemotherapy-induced mucositis and chemically-induced colitis – two mechanistically distinct models affecting the small and large intestine, respectively. These protective effects were associated with preservation of tissue architecture, reduction of inflammatory markers (e.g., MPO), and mitigation of weight loss and histopathological scores. Consistently, McB-fed healthy control mice exhibited elongated small intestinal villi, deeper crypts, and increased colon length, demonstrating direct intestinotrophic remodelling of intestinal architecture.\u003c/p\u003e\n\u003cp\u003eWe mechanistically dissected the McB-mediated protection using our two-pronged approach to modelling gut inflammation, combined with RIA and high-resolution intestinal perfusion models. Through this integrated approach, we systematically investigated the proposed role of GLP-2 and its receptor in McB’s protective effects. The GLP-2 axis is well-described to directly affect mesenteric blood flow\u003csup\u003e40–42\u003c/sup\u003e and epithelial proliferation and repair\u003csup\u003e32,43,44\u003c/sup\u003e. As a testimony to that therapeutic potential, GLP-2 analogues are clinically used to treat short-bowel syndrome\u003csup\u003e45\u003c/sup\u003e. Using GLP-2R WT and KO mice, we demonstrated that McB’s protective effects were entirely abolished in GLP-2R KO mice, underscoring the necessity of intact GLP-2 signaling for McB-mediated intestinal resilience. Importantly, GLP-2R KO alone did not alter baseline disease progression in any model, suggesting the GLP-2R is dispensable for physiological recovery but can be therapeutically engaged to enhance repair. Critically, McB did not stiumlate endegnous GLP-2 secretion, pointing to direct engagement of GLP-2R signaling rather than hormonal induction. Mechanistically this interaction dependents on microbial fermentation of McB, as inhibition of hindgut fermentation abolished GI protection, linking microbial metabolisn to host receptor activation (Figure 6M).\u003c/p\u003e\n\u003cp\u003eCollectively, these findings position McB as a novel class of bioactive cultured dietary proteins that transcend classic nutrition, functioning as precision modulators of\u003cem\u003e intestinal immunity and gut barrier integrity\u003c/em\u003e. Its unique ability to orchestrate both microbiota-independent (direct pTreg induction) and microbiota-dependent (Th17 tolerogenesis) immune reprogramming, while simultanously driving GLP-2R-mediated epithelial repair, establishes a new paradigm in dietary-microbiota crosstalk. To our knowledge, our work is the first to demonstrate that a microbial lysate can engage the GLP-2 pathway through dietary means, bypassing exogenous hormone administration. This discovery opens avenues for developing McB or its bioactive derivatives into next-generation therapeutics that concurrently address intestinal inflammation, immune dysregulation, and epithelial damage – three critical unmet needs in conditions like IBD, chemotherapy-induced mucositis, and environmental enteropathy. By linking microbial fermentation to receptor mimicry, our findings advance the broader field of dietary-microbiota therapeutics, where nutritional interventions can be engineered to target host pathways. \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eRodents and ethical statements\u003c/strong\u003e. Animal experiments were approved by the Danish Animal Experiments Inspectorate (#2021-15-0201-01031 and # 2023-15-0201-01409). Six- to seven-week-old male and female C57BL/6 mice and male Sprague Dawley were purchased from vendors as detailed below. Both GLP-1 receptor knockout (GLP-1R\u003csup\u003e-/-\u003c/sup\u003e) and GLP-2 receptor knockout (GLP-2R\u003csup\u003e-/-\u003c/sup\u003e) mice were used in this study. GLP-2R knockout mice\u003csup\u003e46\u003c/sup\u003e were generated by Taconic using CRISPR/Cas9-mediated gene editing, with the parental strain designated as C57BL/6NTac-Glp2rem5153Tac. The GLP-1R knockout mice were created by deleting exons 4 and 5 of the \u003cem\u003eGlp1r\u003c/em\u003e gene in a Cre-dependent manner, and the conditional \u003cem\u003eGlp1r\u003c/em\u003e knockout strain (C57BL/6N-Glp1rtm1c(KOMP)MbpH) was obtained from the MRC Harwell Institute as previously described\u003csup\u003e47\u003c/sup\u003e. All knockout mice were maintained through heterozygous breeding, with wild-type (WT) littermates serving as controls. Upon arrival mice and rats were allowed to acclimatize in the animal facility environment for two weeks before to study initiation. All rodents were housed under specific pathogen-free conditions in 12-hour light/dark cycle (6 AM-6 PM). Male mice were housed three mice per cage and female mice were housed up to five per cage. Rats were housed four per cage. All data from animal studies used in this manuscript can be found in the supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiets and experimental setups\u003c/strong\u003e. All special diets were obtained from Sniff Spezialdiäten GmBH, Germany, and stored at -20\u003csup\u003eo\u003c/sup\u003eC. Several customized diets were designed depending on the specific research question. Dietary compositions can be found in the supplementary material. In all protocols, mice were fed \u003cem\u003ead libitum\u003c/em\u003e, and at termination mice were anesthetized with 2.5% isoflurane and euthanized by cervical dislocation following cardiac puncture blood collection. Cardiac blood samples were taken using EDTA coated needles and syringes. Plasma was collected by centrifugation of blood samples for 10 minutes at 1000 \u003cem\u003ercf\u003c/em\u003e at 4\u003csup\u003eo\u003c/sup\u003eC and stored at -80\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNutrient comparison protocol\u003c/em\u003e. Male mice were purchased from Taconic Laboratories, Denmark. After two weeks of acclimatization mice were given one of two diets, a diet with few simple sources of protein, fat, and carbohydrates (referred to as Simple Diet, \u003cem\u003eSD\u003c/em\u003e) or an isocaloric macronutrient-matched diet with a complex composition of humanized sources of protein, fat, and carbohydrates (referred to as Complex Diet, \u003cem\u003eCD\u003c/em\u003e) for eight weeks. At week eight a subgroup of mice was maintained on the respective simple or complex diets or fed an experimental diet of which the protein source was exchanged with solely McB lysates (contain \u0026lt;5% biomass from helper bacteria, sold under the commercial name Feedkind®, Calysta UK Ltd) (\u003cem\u003eSD\u003csub\u003eMCB\u003c/sub\u003e\u003c/em\u003e or \u003cem\u003eCD\u003csub\u003eMCB\u003c/sub\u003e\u003c/em\u003e) for an additional 4 weeks. To avoid any secondary effect of caloric density, we modified the complex low-fat-low-fiber reference (referred to as \u003cem\u003eComplex\u003csub\u003eref\u003c/sub\u003e\u003c/em\u003e) to also contain McB lysate as the protein source.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicrobiota-dependency protocol\u003c/em\u003e. Male mice were purchased from Taconic Laboratories (Denmark). After two weeks of acclimatization, mice were split into two groups: one group given control drinking water and one group given a broad-spectrum antibiotics cocktail of 0.5 g/L Neomycin (Sigma #N1876-25G) and 1 g/L Ampicillin (Sigma #A9518-25G) in the drinking water to reduce gut microbial load. After acclimatization, mice underwent dietary intervention and were split into two groups per drinking water condition: one group switched to the CD\u003csub\u003e,\u003c/sub\u003e and one group switched to the CD\u003csub\u003eMCB\u003c/sub\u003e. Mice were maintained on control drinking water or antibiotics during the dietary intervention period. Mice were then euthanized after two, four, or six weeks to look at the temporal effects of McB-consumption on blunted gut microbiota and mucosal T-cell populations.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIntestinal mucositis protocols\u003c/em\u003e. This study protocol used either female WT mice purchased from Janvier (Le Genest-Saint-Isle, France), or female GLP-1R\u003csup\u003e-/-\u003c/sup\u003e and GLP-2R\u003csup\u003e-/-\u003c/sup\u003e mice. After at least a week of acclimatization, mice were fed either the \u003cem\u003eComplex\u003csub\u003eref\u003c/sub\u003e\u003c/em\u003e or \u003cem\u003eComplex\u003c/em\u003e\u003csub\u003eMcB\u003c/sub\u003e for up to 13 days. After a week of dietary intervention, at experimental day 0, mice were intraperitoneally injected once with control saline or 5-fluorouracil (5-FU, Hospira Nordic AB, Stockholm, Sweden) at 400mg/kg to induce intestinal mucositis. Body weight development was subsequently monitored daily. Mice were euthanized at post-injection days 3, 4, or 6 (as indicated in figure legends) to investigate the acute and recovery phases of 5-FU-induced mucositis. Occasionally, mice were single-housed due to fighting and otherwise housed between two-five per cage.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eColitis protocols\u003c/em\u003e. This study protocol used either male mice purchased from Janvier (Le Genest-Saint-Isle, France), or male GLP-1R\u003csup\u003e-/-\u003c/sup\u003e and GLP-2R\u003csup\u003e-/-\u003c/sup\u003e mice. After at least a week of acclimatization, mice were fed the reference \u003cem\u003eComplex\u003csub\u003eref\u003c/sub\u003e\u003c/em\u003e or \u003cem\u003eComplex\u003c/em\u003e\u003csub\u003eMcB\u003c/sub\u003e for one week before induction of colitis. Colitis was induced by adding 2.5% DSS (Thermofisher, #J63606.14) to the drinking water for five days. DSS was freshly dissolved in regular drinking water and filter-sterilized before being given to the mice. Body weight and disease activity (can be found in supplementary material) were monitored daily. At day five, mice were euthanized and tissues harvested. Mice were housed between one-three per cage, depending on in-cage fighting.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInhibition of microbial fermentation\u003c/em\u003e. Male mice were ordered from Janvier (Le Genest-Saint-Isle, France) and allowed to acclimatize for at least a week. First, mice were fed the reference \u003cem\u003eComplex\u003csub\u003eref\u003c/sub\u003e\u003c/em\u003e or \u003cem\u003eComplex\u003c/em\u003e\u003csub\u003eMcB\u003c/sub\u003e for one week before induction of colitis as described above. A subgroup of mice were given 20 ppm solution of beta-acid extracts from \u003cem\u003eHumulus lupulus to inhibit microbial hindgut fermentation as previously described\u003c/em\u003e\u003cem\u003e\u003csup\u003e48\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eBody weight and disease activity were monitored daily. At day five, mice were euthanized and tissues harvested. Mice were housed three per cage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurements of circulating gut hormones and perfusion of rat intestine\u003c/strong\u003e. 8-week-old Sprague Dawley rats and C57BL/6J were purchased from Janvier (Le Genest-Saint-Isle, France) and allowed to acclimatize for 1 week. Mice and rats were gavaged with an inhibitor cocktail consisting of sacubitril (neprilysin inhibitor, 0.3 mg/kg, 5 uL/g, cat. no. 333-B1070, Nordic Biosite, Sweden) and sitagliptin (dipeptidyl peptidase-4 inhibitor, 10 mg/kg, 5 uL/g, Xelevia) 30 minutes before oral delivery of microbial McB lysates of control protein. At time point 0, mice and rats were gavaged with a 20% w/v (200 mg/mL in sterile saline) solution of McB lysate, a control protein solution (Albumin Fraction V, cat. no. 1.12018.0500, Sigma-Aldrich) or glucose. In mice, blood was collected by cardiac puncture and in rats blood was collected sublingual bleeding after 0, 5, 10, and 20 minutes post gavage. GLP-2 levels were measured by radio-immuno-assay (RIA) as described previously\u003csup\u003e49\u003c/sup\u003e. Rat small intestines were perfused as described previously\u003csup\u003e49\u003c/sup\u003e with lysate or control protein solutions. Vascular effluents were collected every minute and gut hormone levels measured by RIA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e. Intestinal sections were fixed in 10% paraformaldehyde and embedded in paraffin following standard procedures. Tissue slides were stained with H\u0026amp;E and samples were randomized and blinded before histological analyses. For small intestinal sections, villus height was measured by identifying three villi with visible extrusion zone, to ensure measurement of full-length villi, from each sample, and measuring from the tip until the crypt border. Crypt depth of small and large intestinal samples was assessed by measuring the depth of three full-length crypts per slide, only when the entire crypt epithelium was visible from the \u003cem\u003elamina muscularis mucosa\u003c/em\u003e to the intestinal lumen. Villus height and crypt depth were analyzed using the Zeiss Zen Desk Software, from three separate areas within each section and reported as an average of measurements from all individuals.\u003c/p\u003e\n\u003cp\u003eFor assessment of colonic injury following DSS administration, fecal matter was mechanically removed, colons were opened longitudinally and rinsed. Next, colons were rolled into Swiss rolls and fixed in 10% paraformaldehyde and stained with H\u0026amp;E according to standard practice. Histopathological scoring was conducted based on previously described scoring systems \u003csup\u003e50,51\u003c/sup\u003e, where each section was given a score based on 1) degree of inflammation, 2) degree of crypt damage, and 3) extent of inflammation and damage as described in the supplementary material. The sum of all parameters was multiplied by 1-4 depending on the area of the section affected, giving a dynamic scoring range of 0-40. Digital images were acquired using an Axioscan Z.1 (Zeiss, Jena, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of small and large intestine lamina propria (LP) cells\u003c/strong\u003e. In protocols involving flow cytometry, after removal of 1 cm gut sections for histology, the small intestine was flushed with cold 1X HBSS (Gibco) containing 15 mM HEPES and 5% FBS (Thermo Fischer), and had feces removed mechanically prior to flushing with cold 1X PBS. Peyer’s Patches were carefully removed from the small intestine. Small and large intestines were opened longitudinally and cut into 1 cm pieces in 1X HBSS, 15mM HEPES, and 5% FBS. Gut pieces were incubated thrice in prewarmed EDTA wash-buffer containing 1X HBSS, 15 mM HEPES, 2.5% heat-inactivated (HIA) FBS, 1 mM sodium pyruvate, 10.000 U/mL Penicillin/Streptomycin, 50 mg/mL Gentamycin, and 2mM EDTA. After each wash, samples were incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 10 min during first incubation and 15 min during second and third incubation. During incubation steps, large intestine samples, but not small intestine samples, were shaken on an orbital shaker at 450 rpm. After each incubation step small intestine samples were vigorously shaken by hand for 10 seconds., and media containing cell debris and epithelial cells were discarded by filtration through a 250 µm nylon mesh. The remaining tissue was digested for 20-25 min at 37\u003csup\u003eo\u003c/sup\u003eC under magnetic stirring at 450 rpm in R10 medium (RPMI 1640 with L-glutamine, 1 mM sodium pyruvate, 15 mM HEPES, 10.000 U/mL Penicillin/Streptomycin, 50 mg/mL Gentamycin, 10% HIA FBS) containing 1 mg/mL Collagenase P (Roche) and 30 µg/mL DNAse I (Roche). After digestion, samples were mechanically agitated, filtered through a 100 µm cell strainer, and centrifugated at 500 \u003cem\u003ercf\u003c/em\u003e for 7 minutes at 4\u003csup\u003eo\u003c/sup\u003eC and supernatant removed. The cell pellets were resuspended in 40% isotonic Percoll (GE Healthcare) and LP cells were purified by density centrifugation using 40/70% Percoll gradient, centrifuged at 800 \u003cem\u003ercf\u003c/em\u003e for 20 min at room temperature, acceleration 5 and brake 0. The lymphocyte interface was collected in fresh R10 medium followed by centrifugation for 7 min at 500 \u003cem\u003ercf\u003c/em\u003e at 4\u003csup\u003eo\u003c/sup\u003eC. Subsequently, the supernatant was removed, and the cell pellet was resuspended in R10 medium and transferred to 96 well plates for ex vivo stimulation of LP cells and ensuing flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEx vivo stimulation of LP cells and staining\u003c/strong\u003e. Small intestine-LP and large intestine-LP cells were restimulated \u003cem\u003eex vivo\u003c/em\u003e in R10 medium as follows. 250 ng/mL PMA (Sigma-Aldrich) in combination with 0.5 µg/mL Ionomycin (Sigma-Aldrich) was added to restimulate samples and incubated at 37\u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e for four hours. After one hour 10 µg/mL brefeldin A was added to all samples, and continued incubation. After stimulation, plates were centrifuged for 5 min at 500 \u003cem\u003ercf\u003c/em\u003e at 4\u003csup\u003eo\u003c/sup\u003eC and washed with PBS. After additional centrifugation for 5 min at 500 \u003cem\u003ercf\u003c/em\u003e at 4\u003csup\u003eo\u003c/sup\u003eC, cells were resuspended in PBS containing Live/Dead stain (Zombie UV) and placed at 4\u003csup\u003eo\u003c/sup\u003eC in the dark for 20 min. Cells were washed with MACS buffer (containing PBS, HIA FBS, EDTA) and centrifuged for 5 min at 500 \u003cem\u003ercf\u003c/em\u003e at 4\u003csup\u003eo\u003c/sup\u003eC. Cells were resuspended in MACS buffer and surface stained with primary antibodies (see table below) for 30 min at 4\u003csup\u003eo\u003c/sup\u003eC in the dark followed by washing and centrifugation. Cells were fixed and subsequently permeabilized using the FoxP3/Transcription Factor Staining Buffer Set from eBioscience according to manufacturer’s instructions. Intracellular staining with primary antibodies was performed at 4\u003csup\u003eo\u0026nbsp;\u003c/sup\u003eC overnight with primary antibodies (see table below).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e. Flow cytometry was carried out by standard procedures and data acquired using an LSRFortessa X-20 (BD Bioscience). The complete gating strategy can be found in Supplementary Figure 1A. Data was analyzed using the FlowJo Software (Tree Star).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibodies\u003c/strong\u003e.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibodies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdentifier\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBD Horizon™ BUV395 Rat Anti-Mouse CD4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBD Bioscences\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 563790; RRID AB_2738426\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBD Horizon™ BV480 Rat Anti-Mouse CD8a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBD Bioscences\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 566096; RRID AB_2739500\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBrilliant Violet 421™ anti-mouse TCR β chain Antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBioLegend\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 109229; RRID AB_10933263\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFOXP3 Monoclonal Antibody (FJK-16s), APC, eBioscience™\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 17-5773-82; RRID AB_469457\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBD Pharmingen™ APC-Cy™7 Rat Anti-Mouse CD45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBD Bioscences\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 557659; RRID AB_396774\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eROR gamma (t) Monoclonal Antibody (B2D), PE-eFluor™ 610, eBioscience™\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 50-112-4863; RRID N/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eIL-10 Monoclonal Antibody (JES5-16E3), PE, eBioscience™\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 12-7101-82; RRID AB_466176\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eGata-3 Monoclonal Antibody (TWAJ), PE-Cyanine5, eBioscience™\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 15-9966-42; RRID AB_2811756\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eT-bet Monoclonal Antibody (eBio4B10 (4B10)), PE-Cyanine7, eBioscience™\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 25-5825-82; RRID AB_11042699\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBrilliant Violet 605™ anti-mouse IFN-γ Antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBioLegend\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 505839; RRID AB_2561438\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBD Pharmingen™ Alexa Fluor® 488 Rat anti-Mouse IL-17A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBD Bioscences\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCat # 560220; RRID AB_1645194\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobiome analyses and bioinformatics processing\u003c/strong\u003e. Input sample was weighed before extraction, and DNA was extracted using the NucleoSpin Soil kit (Macherey-Nagel) following the manufacturer's protocol. DNA libraries were prepared using the MGIEasy DNA Library Prep Kit (MGI) following the manufacturer’s protocol. The MGIEasy DNA Adapters-96 (Plate) Kit (MGI) was used for adapter ligation. DNA cleanup was performed with the MGIEasy DNA Clean Beads (MGI). Circularization of libraries was carried out using the MGIEasy Circularization Module V2.0 (MGI). Sequencing was performed on the DNBSEQ-G400RS platform using the G400-PE150 sequencing kit (MGI), generating paired-end reads of 150 bp. Taxonomic profiling was performed with MetaPhlAn 3.0, while functional profiling of microbial pathways was conducted using HUMAnN 3.0\u003csup\u003e52\u003c/sup\u003e. \u003cem\u003eBeta Diversity Analysis\u003c/em\u003e. Beta diversity was assessed using the \u003cstrong\u003eBray-Curtis dissimilarity index\u003c/strong\u003e (vegan package), which measures compositional differences based on species abundances. Principal coordinate analysis (PCoA) was used for visualization, and statistical significance of group differences was tested using \u003cstrong\u003ePERMANOVA\u003c/strong\u003e (adonis function). \u003cem\u003eDifferential Abundance Analysis of Taxa\u003c/em\u003e. A linear mixed model (lme4 package) was applied to assess species-level differences, with dietary intervention as a fixed effect. Post-hoc pairwise comparisons were performed using glht (multcomp package) with multiple testing correction. Effect sizes, t-statistics, and p-values were extracted to identify significant taxa.\u003cem\u003e\u0026nbsp;Analysis of Microbial Pathways\u003c/em\u003e. Metabolic pathways were annotated and abundanced calculated using HUMAnN 3.0 based on the Metacyc database\u003csup\u003e53\u003c/sup\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003ePathway-level differences were analyzed using \u003cstrong\u003eALDEx2\u003c/strong\u003e, which applies Monte Carlo Dirichlet sampling and log-ratio transformations to account for compositional data constraints. Statistical testing was performed using Welch's t-test. False discovery rate (FDR) correction was applied using the Benjamini-Hochberg method, with p \u0026lt; 0.05 considered significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of fecal bacterial load\u003c/strong\u003e. Bacterial 16S rRNA gene copies were quantified by qPCR on a Light Cycler 480 II (Roche) using V4 region-specific Primers 505F (5’-GTGYCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACNVGGGTWTCTAAT-3’). The thermal cycling conditions started with a DNA-denaturation step at 95° C for 5 minutes, followed by 50 cycles of i) denaturation at 95°C for 10 seconds, ii) annealing at 60°C for 20 seconds, and iii) extension at 72°C for 20 seconds. All samples were run in triplicates. A 2-fold standard curve was produced by serially diluting the DNA pool (starting dilution: 1:20) in sterile MilliQ Water. qPCR threshold cycle (Ct) values were converted to estimated bacterial genomes present in 1 mg of feces and used as a proxy for bacterial abundance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue MPO levels\u003c/strong\u003e. Approximately 1 cm of gut was homogenized in 50 mM HTAB in 50mM K-Phosphate buffer for 6 minutes with a steel bead. Tissues were snap frozen on dry ice, thawed in water and homogenized again; this cycle was done a total of 3 times. After centrifugation (16 000g, 30 minutes at RT), supernatant was collected and diluted 10x for MPO measurement. Samples were mixed with substrate buffer (O-dianisidine dihydrochloride in 50mM K-Phosphate buffer with 0.0005% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) on a 96-well plate, in duplicates, and measurements were taken every 30 seconds for 5 minutes at 450nm. MPO activity was calculated using all readings and corrected for total protein content of each sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e. Statistical analyses were performed using GraphPad Prism software (version 10). All data are presented as means ± standard error of the mean (SEM), with individual data points included to illustrate data distribution. For comparisons between two groups, unpaired t-tests were conducted to assess statistical significance. When comparing more than three groups with a single independent variable, a one-way analysis of variance (ANOVA) was utilized, followed by Tukey's post hoc test to identify specific group differences. In instances where more than three groups with two independent variables were analyzed, a two-way ANOVA was performed to evaluate the effects of both independent variables and their interactions, testing main effects for multiple testing by Fisher’s LSD post hoc test. A p-value of \u0026lt;0.05 was considered statistically significant. A full overview of all statistical tests sorted by figures can be found in the supplementary material.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was primarily funded by an Excellence Emerging Investigator grant (NNF21OC0066931) issued to BAHJ by Novo Nordisk Foundation, and partly by a grant from the Louis Hansen Fonden (24-2B-16713), issued to SKYJ. Calysta provided the Feedkind® material. BSYC is supported by the BRIDGE – Translational Excellence Program, funded by the Novo Nordisk Foundation (NNF20SA0064340) and by a postdoctoral fellowship from the Fonds de recherche du Québec – Santé (FRQS). The funding bodies had no share in data integration and presentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e: BAHJ, JBH, and KK are co-inventors of an issued patent (US and EU) related to McB and gut dysbiosis. BAHJ, SKYJ, and BSYC are co-inventors of a patent application related to McB’s potential in treating or preventing gastrointestinal barrier dysfunction. The remaining authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e: Study design: SKYJ, BSYC, SIP, and BAHJ. SKYJ, BSYC, NN, SBS, NG, IMM, and SIP, performed experiments. JBH and KK were responsible for metagenomic sequencing and subsequent analyses with SKYJ and BAHJ. HK, JJH, and BH were involved in breeding GLP-1R and GLP-2R mice, 5-FU experiments, and gut hormone measurements. SKYJ wrote the first manuscript draft, revised together with BSYC, and finalized it with BAHJ. All authors contributed significantly to the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: We extend our gratitude to the Core Facility for Integrated Microscopy at the Faculty of Health and Medical Sciences, University of Copenhagen, as well as the members of UCPH Histolab—Associate Professor Jens Brings Jacobsen, Biomedical Laboratory Technologists Bente Stærgaard and Heidi Paulsen, and Associate Professor Steen Seier Poulsen for their valuable support with histological processing and imaging. We would also like to thank the Core Facility for Flow Cytometry and Single Cell Analysis, Faculty of Health and Medical Sciences, University of Copenhagen, for inputs regarding flow cytometry analysis. We gratefully acknowledge Calysta (UK) Ltd. for providing access to commercial McB lysates (Feedkind ®). We want to express our sincere gratitude to Staff Scientist Si Brask Sonne for invaluable help and instruction in the laboratory, and likewise Laboratory Scientists Lene Brus Albæk and Anette Bjerregaard for instrumental assistance with radioimmunoassays and genotyping the GLP-2R and GLP-1R mice. Finally, a thank to Isabella Paul, Cecilie Anastacia Stokkeby Koch, and Kobe Neven for their help, input, and discussions during their traineeship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e All data used to generate this manuscript will be available on Mendeley Data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMalila, Y. \u003cem\u003eet al.\u003c/em\u003e Current challenges of alternative proteins as future foods. \u003cem\u003enpj Sci. Food\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 53 (2024).\u003c/li\u003e\n\u003cli\u003eXu, X. \u003cem\u003eet al.\u003c/em\u003e Global greenhouse gas emissions from animal-based foods are twice those of plant-based foods. \u003cem\u003eNat. Food\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 724\u0026ndash;732 (2021).\u003c/li\u003e\n\u003cli\u003eFackelmann, G. \u003cem\u003eet al.\u003c/em\u003e Gut microbiome signatures of vegan, vegetarian and omnivore diets and associated health outcomes across 21,561 individuals. \u003cem\u003eNat. Microbiol.\u003c/em\u003e (2025) doi:10.1038/s41564-024-01870-z.\u003c/li\u003e\n\u003cli\u003eNunes-Caba\u0026ccedil;o, H., Moita, D., R\u0026ocirc;la, C., Mendes, A. M. \u0026amp; Prud\u0026ecirc;ncio, M. Impact of Dietary Protein Restriction on the Immunogenicity and Efficacy of Whole-Sporozoite Malaria Vaccination. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eJensen, B. A. H. \u003cem\u003eet al.\u003c/em\u003e Small intestine vs. colon ecology and physiology: Why it matters in probiotic administration. \u003cem\u003eCell Reports Med.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 101190 (2023).\u003c/li\u003e\n\u003cli\u003eSefik, E. \u003cem\u003eet al.\u003c/em\u003e MUCOSAL IMMUNOLOGY. Individual intestinal symbionts induce a distinct population of ROR\u0026gamma;\u003csup\u003e+\u003c/sup\u003e regulatory T cells. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e349\u003c/strong\u003e, 993\u0026ndash;997 (2015).\u003c/li\u003e\n\u003cli\u003eVerma, R. \u003cem\u003eet al.\u003c/em\u003e Cell surface polysaccharides of Bifidobacterium bifidum induce the generation of Foxp3 + regulatory T cells. \u003cem\u003eSci. Immunol.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003eAbdel-Gadir, A. \u003cem\u003eet al.\u003c/em\u003e Microbiota therapy acts via a regulatory T cell MyD88/ROR\u0026gamma;t pathway to suppress food allergy. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1164\u0026ndash;1174 (2019).\u003c/li\u003e\n\u003cli\u003eCaspar, O. \u003cem\u003eet al.\u003c/em\u003e The microbiota regulates type 2 immunity through ROR\u0026gamma;t+ T cells. \u003cem\u003eScience (80-. ).\u003c/em\u003e \u003cstrong\u003e349\u003c/strong\u003e, 989\u0026ndash;993 (2015).\u003c/li\u003e\n\u003cli\u003eIvanov, I. I. \u003cem\u003eet al.\u003c/em\u003e Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e139\u003c/strong\u003e, 485\u0026ndash;498 (2009).\u003c/li\u003e\n\u003cli\u003eWen, Y., Wang, H., Tian, D. \u0026amp; Wang, G. TH17 cell: a double-edged sword in the development of inflammatory bowel disease. \u003cem\u003eTherap. Adv. Gastroenterol.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 17562848241230896 (2024).\u003c/li\u003e\n\u003cli\u003eZielinski, C. E. \u003cem\u003eet al.\u003c/em\u003e Pathogen-induced human TH17 cells produce IFN-\u0026gamma; or IL-10 and are regulated by IL-1\u0026beta;. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e484\u003c/strong\u003e, 514\u0026ndash;518 (2012).\u003c/li\u003e\n\u003cli\u003eCui, H. \u003cem\u003eet al.\u003c/em\u003e The dynamic shifts of IL-10-producing Th17 and IL-17-producing Treg in health and disease: a crosstalk between ancient \u0026lsquo;Yin-Yang\u0026rsquo; theory and modern immunology. \u003cem\u003eCell Commun. Signal.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 99 (2024).\u003c/li\u003e\n\u003cli\u003eLiu, T. C. \u003cem\u003eet al.\u003c/em\u003e Western diet induces Paneth cell defects through microbiome alterations and farnesoid X receptor and type I interferon activation. \u003cem\u003eCell Host Microbe\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 988-1001.e6 (2021).\u003c/li\u003e\n\u003cli\u003eKoh, A. \u003cem\u003eet al.\u003c/em\u003e Microbially Produced Imidazole Propionate Impairs Insulin Signaling through mTORC1. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e175\u003c/strong\u003e, 947-961.e17 (2018).\u003c/li\u003e\n\u003cli\u003ePendyala, S., Walker, J. M. \u0026amp; Holt, P. R. A high-fat diet is associated with endotoxemia that originates from the gut. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cstrong\u003e142\u003c/strong\u003e, 1100-1101.e2 (2012).\u003c/li\u003e\n\u003cli\u003eThaiss, C. A. \u003cem\u003eet al.\u003c/em\u003e Hyperglycemia drives intestinal barrier dysfunction and risk for enteric infection. \u003cem\u003eScience (80-. ).\u003c/em\u003e \u003cstrong\u003e359\u003c/strong\u003e, 1376\u0026ndash;1383 (2018).\u003c/li\u003e\n\u003cli\u003eLee, J. C. \u003cem\u003eet al.\u003c/em\u003e Obesogenic diet-induced gut barrier dysfunction and pathobiont expansion aggravate experimental colitis. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e0187515 (2017).\u003c/li\u003e\n\u003cli\u003eSrour, B. \u003cem\u003eet al.\u003c/em\u003e Ultra-processed foods and human health: from epidemiological evidence to mechanistic insights. \u003cem\u003eLancet Gastroenterol. Hepatol.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 1128\u0026ndash;1140 (2022).\u003c/li\u003e\n\u003cli\u003eAdolph, T. E. \u0026amp; Zhang, J. Diet fuelling inflammatory bowel diseases: preclinical and clinical concepts. \u003cem\u003eGut\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 2574 LP \u0026ndash; 2586 (2022).\u003c/li\u003e\n\u003cli\u003eChristensen, C. \u003cem\u003eet al.\u003c/em\u003e Diet, Food, and Nutritional Exposures and Inflammatory Bowel Disease or Progression of Disease: an Umbrella Review. \u003cem\u003eAdv. Nutr.\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 100219 (2024).\u003c/li\u003e\n\u003cli\u003eJensen, B. A. H. \u003cem\u003eet al.\u003c/em\u003e Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3(+)RORgammat(+)IL-17(+) Tregs and improve metabolism. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1093 (2021).\u003c/li\u003e\n\u003cli\u003eHong, C.-P. \u003cem\u003eet al.\u003c/em\u003e Gut-Specific Delivery of T-Helper 17 Cells Reduces Obesity and Insulin Resistance in Mice. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cstrong\u003e152\u003c/strong\u003e, 1998\u0026ndash;2010 (2017).\u003c/li\u003e\n\u003cli\u003eSun, C.-Y., Yang, N., Zheng, Z.-L., Liu, D. \u0026amp; Xu, Q.-L. T helper 17 (Th17) cell responses to the gut microbiota in human diseases. \u003cem\u003eBiomed. Pharmacother.\u003c/em\u003e \u003cstrong\u003e161\u003c/strong\u003e, 114483 (2023).\u003c/li\u003e\n\u003cli\u003eEvans-Marin, H. \u003cem\u003eet al.\u003c/em\u003e Microbiota-Dependent Involvement of Th17 Cells in Murine Models of Inflammatory Arthritis. \u003cem\u003eArthritis Rheumatol.\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 1971\u0026ndash;1983 (2018).\u003c/li\u003e\n\u003cli\u003eShaughnessy, M. P., Park, C. J., Salvi, P. S. \u0026amp; Cowles, R. A. Jejunoileal mucosal growth in mice with a limited microbiome. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e0266251 (2022).\u003c/li\u003e\n\u003cli\u003eKrigul, K. L. \u003cem\u003eet al.\u003c/em\u003e A history of repeated antibiotic usage leads to microbiota-dependent mucus defects. \u003cem\u003eGut Microbes\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 2377570 (2024).\u003c/li\u003e\n\u003cli\u003eSawaed, J. \u003cem\u003eet al.\u003c/em\u003e Antibiotics damage the colonic mucus barrier in a microbiota-independent manner. \u003cem\u003eSci. Adv.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 4119 (2024).\u003c/li\u003e\n\u003cli\u003eRosemann, E., Schaefer, H., Esmaty, A., Stintzing, S. \u0026amp; Karthaus, M. Incidence, severity, and onset of oral mucositis in 5-FU based chemotherapy for gastrointestinal cancer. \u003cem\u003eJ. Clin. Oncol.\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 77 (2024).\u003c/li\u003e\n\u003cli\u003eMcQuade, R. M. \u003cem\u003eet al.\u003c/em\u003e Gastrointestinal dysfunction and enteric neurotoxicity following treatment with anticancer chemotherapeutic agent 5-fluorouracil. \u003cem\u003eNeurogastroenterol. Motil.\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 1861\u0026ndash;1875 (2016).\u003c/li\u003e\n\u003cli\u003eBilleschou, A. \u003cem\u003eet al.\u003c/em\u003e Intestinal Adaptation upon Chemotherapy-Induced Intestinal Injury in Mice Depends on GLP-2 Receptor Activation. \u003cem\u003eBiomedicines\u003c/em\u003e (2021) doi:10.3390/biomedicines9010046.\u003c/li\u003e\n\u003cli\u003eHytting-Andreasen, R. \u003cem\u003eet al.\u003c/em\u003e Endogenous glucagon-like peptide- 1 and 2 are essential for regeneration after acute intestinal injury in mice. \u003cem\u003ePLoS One\u003c/em\u003e (2018) doi:10.1371/journal.pone.0198046.\u003c/li\u003e\n\u003cli\u003eTaminiau, J. A., Gall, D. G. \u0026amp; Hamilton, J. R. Response of the rat small-intestine epithelium to methotrexate. \u003cem\u003eGut\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 486 LP \u0026ndash; 492 (1980).\u003c/li\u003e\n\u003cli\u003eDrucker, D. J., Deforest, L. \u0026amp; Brubaker, P. L. Intestinal response to growth factors administered alone or in combination with human [Gly2]glucagon-like peptide 2. \u003cem\u003eAm. J. Physiol. Liver Physiol.\u003c/em\u003e \u003cstrong\u003e273\u003c/strong\u003e, G1252\u0026ndash;G1262 (1997).\u003c/li\u003e\n\u003cli\u003eBrubaker, P. L. \u0026amp; Drucker, D. J. Minireview: Glucagon-Like Peptides Regulate Cell Proliferation and Apoptosis in the Pancreas, Gut, and Central Nervous System. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cstrong\u003e145\u003c/strong\u003e, 2653\u0026ndash;2659 (2004).\u003c/li\u003e\n\u003cli\u003ePan, Y. \u003cem\u003eet al.\u003c/em\u003e The protective and pathogenic role of Th17 cell plasticity and function in the tumor microenvironment. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eAriana, P. \u0026amp; D., M. E. Precarious Balance: Th17 Cells in Host Defense . \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, 32\u0026ndash;38 (2010).\u003c/li\u003e\n\u003cli\u003eLiang, S. C. \u003cem\u003eet al.\u003c/em\u003e Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides . \u003cem\u003eJ. Exp. Med.\u003c/em\u003e \u003cstrong\u003e203\u003c/strong\u003e, 2271\u0026ndash;2279 (2006).\u003c/li\u003e\n\u003cli\u003eKawano, Y. \u003cem\u003eet al.\u003c/em\u003e Microbiota imbalance induced by dietary sugar disrupts immune-mediated protection from metabolic syndrome. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e185\u003c/strong\u003e, 3501-3519.e20 (2022).\u003c/li\u003e\n\u003cli\u003eBremholm, L., Hornum, M., Henriksen, B. M., Larsen, S. \u0026amp; Holst, J. J. Glucagon-like peptide-2 increases mesenteric blood flow in humans. \u003cem\u003eScand. J. Gastroenterol.\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 314\u0026ndash;319 (2009).\u003c/li\u003e\n\u003cli\u003eGuan, X. \u003cem\u003eet al.\u003c/em\u003e GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets 1 1This work is a publication of the USDA/ARS Children\u0026rsquo;s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine . \u003cem\u003eGastroenterology\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 136\u0026ndash;147 (2003).\u003c/li\u003e\n\u003cli\u003eBremholm, L. \u003cem\u003eet al.\u003c/em\u003e The effect of Glucagon-Like Peptide-2 on mesenteric blood flow and cardiac parameters in end-jejunostomy short bowel patients. \u003cem\u003eRegul. Pept.\u003c/em\u003e \u003cstrong\u003e168\u003c/strong\u003e, 32\u0026ndash;38 (2011).\u003c/li\u003e\n\u003cli\u003eDrucker, D. J., Erlich, P., Asa, S. L. \u0026amp; Brubaker, P. L. Induction of intestinal epithelial proliferation by glucagon-like peptide 2. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e93\u003c/strong\u003e, 7911\u0026ndash;7916 (1996).\u003c/li\u003e\n\u003cli\u003eEstall, J. L. \u0026amp; Drucker, D. J. Dual Regulation of Cell Proliferation and Survival via Activation of Glucagon-Like Peptide-2 Receptor Signaling. \u003cem\u003eJ. Nutr.\u003c/em\u003e \u003cstrong\u003e133\u003c/strong\u003e, 3708\u0026ndash;3711 (2003).\u003c/li\u003e\n\u003cli\u003eJeppesen, P. B. \u003cem\u003eet al.\u003c/em\u003e Glepaglutide, a Long-acting Glucagon-like Peptide-2 Analog, Reduces Parenteral Support in Patients with Short Bowel Syndrome: a Phase 3, Randomized, Controlled Trial. \u003cem\u003eGastroenterology\u003c/em\u003e (2024) doi:https://doi.org/10.1053/j.gastro.2024.11.023.\u003c/li\u003e\n\u003cli\u003eHunt, J. E. \u003cem\u003eet al.\u003c/em\u003e Pharmacological activation of TGR5 promotes intestinal growth via a GLP-2-dependent pathway in mice. \u003cem\u003eAm. J. Physiol. Liver Physiol.\u003c/em\u003e \u003cstrong\u003e318\u003c/strong\u003e, G980\u0026ndash;G987 (2020).\u003c/li\u003e\n\u003cli\u003eBalk-M\u0026oslash;ller, E. \u003cem\u003eet al.\u003c/em\u003e Glucagon-Like Peptide 1 and Atrial Natriuretic Peptide in a Female Mouse Model of Obstructive Pulmonary Disease. \u003cem\u003eJ. Endocr. Soc.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, bvz034 (2020).\u003c/li\u003e\n\u003cli\u003eSingh, V. \u003cem\u003eet al.\u003c/em\u003e Dysregulated Microbial Fermentation of Soluble Fiber Induces Cholestatic Liver Cancer. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e175\u003c/strong\u003e, 679-694.e22 (2018).\u003c/li\u003e\n\u003cli\u003eModvig, I. M. \u003cem\u003eet al.\u003c/em\u003e Amino acids differ in their capacity to stimulate GLP-1 release from the perfused rat small intestine and stimulate secretion by different sensing mechanisms. \u003cem\u003eAm. J. Physiol. Metab.\u003c/em\u003e \u003cstrong\u003e320\u003c/strong\u003e, E874\u0026ndash;E885 (2021).\u003c/li\u003e\n\u003cli\u003eKatakura, K. \u003cem\u003eet al.\u003c/em\u003e Toll-like receptor 9\u0026ndash;induced type I IFN protects mice from experimental colitis. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, 695\u0026ndash;702 (2005).\u003c/li\u003e\n\u003cli\u003eChassaing, B. \u003cem\u003eet al.\u003c/em\u003e Fecal Lipocalin 2, a Sensitive and Broadly Dynamic Non-Invasive Biomarker for Intestinal Inflammation. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e44328 (2012).\u003c/li\u003e\n\u003cli\u003eBeghini, F. \u003cem\u003eet al.\u003c/em\u003e Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e65088 (2021).\u003c/li\u003e\n\u003cli\u003eCaspi, R. \u003cem\u003eet al.\u003c/em\u003e The MetaCyc database of metabolic pathways and enzymes - a 2019 update. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, D445\u0026ndash;D453 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"Cultured protein, microbial lysate, nutritional imprinting, microbiota, T-cells, gut inflammation, GLP-2, GLP-2R","lastPublishedDoi":"10.21203/rs.3.rs-6973437/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6973437/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"There is an urgent need for sustainable protein sources to meet rising global nutritional demands. Here, we show that a commercially scalable microbial lysate from Methylococcus capsulatus Bath (McB), used as a dietary protein, orchestrates host-diet-microbe interactions that protect against gastrointestinal inflammation. McB administration rapidly reshapes the gut microbiota and upregulates microbial fermentation pathways, while robustly increasing peripherally induced regulatory T cells (pTregs) across intestinal regions, independent of the microbiota. In contrast, McB-driven induction of tolerogenic Th17 cells requires a functional microbiota. In models of mucositis and colitis, McB preserves villus architecture, restores mucosal integrity, and reduces disease severity. Mechanistically, these effects depend on microbial fermentation and functional GLP-2 receptor signalling, yet are independent of endogenous GLP-2 secretion, indicating a fermentation-driven molecular mimicry of GLP-2R activation. Collectively, our findings position microbial lysates as a sustainable nutritional strategy that improves gastrointestinal health through defined immune and microbial pathways.","manuscriptTitle":"Microbial Activation of the GLP-2R Mitigates Gastrointestinal Inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-09 11:12:37","doi":"10.21203/rs.3.rs-6973437/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":"94f5bb77-3289-4403-a9cd-516fdb16dda3","owner":[],"postedDate":"July 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50876132,"name":"Biological sciences/Biological techniques/Biological models/Gastrointestinal models"},{"id":50876133,"name":"Biological sciences/Immunology/Inflammation"}],"tags":[],"updatedAt":"2026-02-20T08:07:43+00:00","versionOfRecord":{"articleIdentity":"rs-6973437","link":"https://doi.org/10.1038/s41467-026-68551-9","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-01-15 05:00:00","publishedOnDateReadable":"January 15th, 2026"},"versionCreatedAt":"2025-07-09 11:12:37","video":"","vorDoi":"10.1038/s41467-026-68551-9","vorDoiUrl":"https://doi.org/10.1038/s41467-026-68551-9","workflowStages":[]},"version":"v1","identity":"rs-6973437","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6973437","identity":"rs-6973437","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}