Microbial activation of the GLP-2R mitigates gastrointestinal inflammation

Domain: biology
Published: 2026-01-15
Editorial outcome: minor_revision

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 with intact fermentation capacity. 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.

Introduction
Amidst the urgent need for sustainable food systems1 to combat excessive greenhouse gas emmsions, resource depletion, and biodiversity loss2, cultured microbial proteins are emerging as low-emission, resource-efficient alternatives to animal-derived products1. While the ecological benefits of these cultured proteins are well established1, 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 gut barrier integrity3.

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 microbiota4. These interactions underpin immune education, as evidenced by the influence of diet on vaccine efficacy5 and food allergy development6. 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) cells7. pTregs, induced by the gut microbiota8 and their cellular components9, suppress aberrant immune responses to food antigens10 and commensal microbes11. Th17 cells, regulated by both host- and microbially derived signals12, adopt context-dependent roles—ranging from barrier reinforcement to pathogenic inflammation13—dictated by the local microenvironment’s inflammatory milieu14,15. 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 disorders16–20, with contemporary Western diets epidemiologically linked to rising prevalence of GI inflammation21–23. 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 mice24, 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. Moreover, to what extend McB interacts with the native gut microbiota 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, T-cell-independent, pan-GI protection that requires functional GLP-2 receptor (GLP-2R) signaling. This GLP-2R signaling is further dependent on microbial processing of the McB lysate to liberate compound(s) conferring 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 feeding24 was diet-dependent, we compared the simple compositionally defined diet (SD) hitherto used to a complex diet with a humanized nutrient composition (CD), both with and without McB lysate as the protein source (Fig. 1A and Supplementary Table 1). Fecal shotgun sequencing revealed rapid changes in gut microbiota composition (Fig. 1B, C), where McB presence explained 27% variance compared to only 10% from all other nutrients combined (Fig. 1B). The observed microbiota changes were accompanied by major changes to the metabolic potential of the McB-associated microbiota (Fig. 1D–E; >460 differentially regulated pathways), already a few days after McB-feeding and regardless of background diet. Noteworthy metabolic pathways enriched in feces of McB-fed mice were fermentation processes, including metabolic processes leading to production of short-chain fatty acids (SCFAs), supporting our finding of elevated cecal SCFA in monoculture-fed mice24. 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 (Fig. 1F). Still, the McB-induced changes to the microbial community remained stable after their initial shift (Fig. 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 Bacteroidaceae families (Fig. 1C), we used our shotgun analysis to assess species-level resolution (Fig. 1G). This revealed that changes were largely driven by few species (Fig. 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 (Fig. 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 (Fig. S1B–E). Notably, McB-feeding also induced a ~50% increase in Th17 cells exclusively in the small intestine (Fig. S1F, G). Although Th17 cells originally were conceived as proinflammatory culprits, recent literature points towards their pleiotropic nature being instrumental for maintaining GI immune balance13 and metabolic homeostasis upon HFD feeding7. Indeed, the McB-attuned Th17 landscape exhibited immunoregulatory traits, indicated by an increase in IL-10+ Th17 cells paralleled by a reduction of IFN-γ+ Th17 cells, suggesting a shift towards tolerogenic immunity (Fig. 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 (Fig. S1H–I). McB-induced immune imprinting seemed targeted towards RORγt+ T cell subsets, as neither Th1 nor thymic-derived Treg (nTreg) populations were affected by McB-feeding (Fig. S1J–Q).

As GI pTreg11 and Th17 cell25,26 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 (Fig. 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 (Fig. 2A). Despite ABX-induced disruption of gut homeostasis, as evident from the ABX-induced increase in cecal size (Fig. 2B), McB instantly enhanced pTreg abundance in the small intestine of both ABX-treated and non-treated mice (Fig. 2C). This trait was, however, not fully recapitulated in LP of the large intestine, but pTreg induction was notably accelerated in ABX-treated mice (Fig. 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 (Fig. 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 (Fig. 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 (Fig. 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, Fig. 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 treatment27, likely due to disruptions in gut barrier integrity, impaired mucus production, reduced microbial load, and a lack of fiber fermentation28,29. During ABX treatment, we observed that McB-fed mice were protected against ABX-induced increases in cecum weight (Fig. 2B). These findings suggest that McB preserves gut barrier function alongside enhanced tolerogenic immunity (Fig. 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 atrophy30–32, 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) (Fig. 3A). Following 5-FU challenge, mice displayed acute weight loss over three days, then rapidly regained weight and returned to baseline within six days (Fig. 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 (Fig. 3C) and induced villus atrophy (Fig. 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 challenge32–34, we observed deeper crypts in reference-fed mice. In contrast, McB-fed mice consistently exhibited deeper crypts regardless of 5-FU exposure (Fig. 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 (Fig. 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 (Fig. 3H). McB-fed mice were fully protected against DSS-induced weight loss (Fig. 3I) and exhibited a substantially milder disease trajectory (Fig. 3J), with Disease Activity Index (DAI) scores approximately 2-fold lower than controls by study end (Fig. 3K). McB feeding also preserved colon length after DSS exposure (Fig. 3L), and large intestines from McB-fed mice displayed firmer contents and fewer hemorrhagic lesions (Fig. 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 (Fig. 3N). Histopathological analysis of colon Swiss rolls supported these findings, showing a >2-fold reduction in colonic damage scores in McB-fed mice (Fig. 3O–P).

As McB modulated mucosal T-cells towards a more regulatory phenoype, we asked whether the McB-induced T-cell imprinting was required for acute GI protection. To functionally test this, we performed CD4+ T-cell depletion in both disease models (Fig. S3). Despite effective depletion (Fig. S3A), McB-mediated protection remained intact (Fig. S3B–I), indicating that acute epithelial protection is independent of CD4+ T cells.

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, although these effects are seemingly not hierachical.

GLP-2 receptor signaling is essential for McB-induced gastrointestinal protection
Given the robust protection conferred by McB throughout the GI tract and lack of T-cell involvement, we hypothesized that its tissue-protective 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 wild type (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 intestine35,36, and the necessity of intact GLP-2R signaling in McB-mediated gut protection.

In the 5-FU-induced small intestinal injury model (Fig. 4A), GLP-2R WT mice on a reference diet lost significant weight (Fig. 4B), exhibited dramatically reduced small intestinal weights (Fig. 4C), and developed villus atrophy (Figs. 4D, E, N and S4). Still, McB-fed mice were protected against intestinal damage (Fig. 4C–G, N). 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 of reference-fed mice (Fig. 4F–G). Importantly, McB feeding significantly reduced MPO accumulation, further supporting its barrier-protective role in WT mice. However, these protective effects were completely abolished in GLP-2R KO mice (Figs. 4H–N and S4), confirming that McB requires functional GLP-2R signaling for restoring small intestinal homeostasis. Notably, while 5-FU induced colonic damage, GLP-2R KO mice also exhibited complete loss of McB-mediated protection in the colon (Fig. S4F, 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 (Fig. 5A). In GLP-2R WT mice, McB feeding conferred robust protection, mitigating weight loss (Fig. 5B, C), reducing DAI scores (Fig. 5D, E), preserving colon length (Fig. 5F), and lowering inflammation index (Fig. 5G). These protective effects were abolished in GLP-2R KO mice (Fig. 5H–M), as confirmed by histological scoring of crypt integrity and immune infiltration (Fig. 5N–P).

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 (Figs. S5, S6), indicating that McB’s gut-protective effects are strictly GLP-2R-dependent across intestinal regions.

Although our acute GI disease models did not rely on CD4 T-cell activity, tolerogenic T-cell subsets remain of key interest for long-term clinical scenarios, including recurrent IBD3 and lifestyle-associated gut barrier dysfunction observed in cardiometabolic diseases37. We therefore assessed whether McB’s mucosal immunomodulatory abilities would persist in GLP-1R and GLP-2R KO mice, considering that GLP-1R signaling has been reported to govern certain intraepithelial T-cell populations38. Notably, McB feeding increased pTreg frequencies and induced phenotypic reprogramming of Th17 cells despite receptor KO (Fig. S7A–E), suggesting that McB lysate may mitigate chronic, T-cell-driven inflammation alongside acute barrier protection. These data imply the presence of distinct effector mechanisms within McB, as enhancement of gut barrier function required functional GLP-2R signaling, while T-cell subset modulation occurred independently of both GLP-2R and GLP-1R pathways.

Microbial Fermentation of McB is Required for Protection Against Acute Gut Inflammation
To define the mechanisms whereby McB confers GLP-2R-dependent protection against acute epithelial injury, we first investigated whether McB stimulates endogenous GLP-2 secretion. Using the perfused rat intestinal model (Fig. S8A)—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 (Fig. S8B, 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-immunoassay (RIA) (Fig. S8D). While glucose stimulated GLP-2 release, neither McB nor the reference protein increased circulating GLP-2 (Fig. S8E). 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 (Figs. 1D and 3) the near-complete fecal degradation of McB (Fig. 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 (Fig. 6A). In reference-fed mice, FermInh had no significant effect on colitis severity (Fig. 6B–G). Consistent with prior findings, McB-fed mice exhibited reduced DAI scores, attenuated colonic inflammation, conserved colon length, and reduced histopathological scores compared to controls (Fig. 6H–M). Strikingly, FermInh treatment generally abolished McB’s protective effects, exacerbating DAI scores (Fig. 6H, I), elevating colonic inflammation index (Fig. 6J), and leading to colon shortening (Fig. 6K), reflected by the aggravation of tissue pathology (Fig. 6M). Given McB’s intestinotrophic effects in healthy mice, we assessed intestinal morphology. Remarkably, FermInh eliminated McB-induced elongation of both small and large intestines, even in healthy, non-DSS treated mice (Fig. 6L–M), 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) and thus enzymtic processing, we found no significant increase in circulating GLP-2 in either control or McB-fed mice, regardless of fermentation status (Fig. 6N).

To determine whether the fermentation-driven benefits of McB relate to shifts in gut microbiota composition, we evaluated fecal community structures with or without fermentation blockade (Fig. S9A, B). McB substantially altered the overall microbiota structure, explaining approximately 42% of total compositional variance (Fig. S9C). In contrast, inhibition of microbial fermentation with FermInh had minimal impact on overall community composition or diversity (Fig. S9A–C). Consistently, McB levels in stool were not increased by fermentation blockade, suggesting effective gut utilization of the lysate independent of fermentation status (Fig. S9D). Notably, the expansion of Lachnospiraceae and Bacteroidaceae typically associated with McB feeding was similarly unaltered by inhibition of microbial fermentation (Fig. S9E–G).

Finally, given that McB may serve as a substrate for fermentation-driven GLP-2R activation, we examined whether microbial processing is also necessary for its microbiota-dependent immunomodulatory effects, i.e., Th17 cell reprogramming. Blockade of microbial fermentation did not alter the McB-induced elevation of peripheral pTreg populations (Fig. S10A). Similarly, no effect was observed on nTregs (Fig. S10B). However, the tolerogenic reprogramming as well as expansion of total Th17 cells in response to McB, both microbiota-dependent processes (Fig. 2), were abrogated by fermentation inhibition (Fig. S10C–E).

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 a likely underlying mechanism. Our data support two parallel mechanisms of McB rather than a single hierarchical pathway: (i) mucosal T-cell-mediated immune tolerance and (ii) fermentation-dependent GLP-2R-mediated epithelial protection.

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 mice52 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 described53. 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-h 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°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 miutes at 1000×g at 4°C and stored at −80°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 or CD ) 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 ) 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 . 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 or Complex 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 400 mg/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 or Complex 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 or Complex for one week and split into two cohorts: one subjected to colitis induction, and one for interrogation of lamina propria T-cells. Colitis was induced as described above. To inhibit microbial hindgut fermentation mice were given a 20 ppm solution of beta-acid extracts from Humulus lupulus as previously described54, or control drinking water. For the colitis cohort, body weight and disease activity were monitored daily, and at day five mice were euthanized and tissues harvested. To test if lamina propria T-cells were dependent on microbial fermentation of McB, both numerically and phenotypically, T-cell subsets were studied using flow cytometry as described below.

Depletion of T-cells in inflammatory models. To assess the dependence of McB-mediated protection on adaptive immunity, CD4+ T-cell depletion was performed in both the DSS-induced colitis and 5-fluorouracil (5-FU)–induced mucositis models. Mice were pre-fed experimental diets (±McB) seven days before disease induction (similar to above descriptions). Depletion was achieved using anti-mouse CD4 monoclonal antibody (Nordic Biosite, BE0003-1) administered intravenously at 100 µg per mouse on day −2 and 100 µg per mouse i.p. at days 0, 1, and 3 relative to disease induction. Control animals received equivalent doses of isotype control antibody (Nordic Bioscience). Efficacy of depletion was confirmed by flow cytometry prepared from spleen and intestinal lamina propria as described below. Cells were stained with Live/Dead viability dye, CD45, CD4, and CD8, and analyzed using a LSR-Fortessa X-20 (BD Bioscience). Near-complete systemic depletion (>95%) of CD4+ T cells was confirmed in all treated mice. Local depletion was validated in the leased affected intestinal compartment (small intestine in DSS cohorts; large intestine in 5-FU cohorts), as lamina propria isolation from the severely inflamed tissue was not technically feasible. Disease severity scoring was assessed as described for non-depleted cohorts.

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 (ThermoFischer), 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, 15 mM 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 2 mM EDTA. After each wash, samples were incubated at 37°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 s., 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°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×g for 7 min at 4°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×g 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×g at 4°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°C and 5% CO2 for 4 h. After 1 h, 10 µg/mL brefeldin A was added to all samples, and continued incubation. After stimulation, plates were centrifuged for 5 min at 500×g at 4°C and washed with PBS. After additional centrifugation for 5 min at 500×g at 4°C, cells were resuspended in PBS containing Live/Dead stain (Zombie UV) and placed at 4°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×g at 4°C. Cells were resuspended in MACS buffer and surface stained with primary antibodies (see Table 1 below) for 30 min at 4°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°C overnight with primary antibodies (see Table 1 below).

Flow cytometry
Flow cytometry was carried out by standard procedures, and data were acquired using an LSRFortessa X-20 (BD Bioscience). The complete gating strategy can be found in Supplementary Fig. 1A. Data were analyzed using the FlowJo Software v10 (TreeStar).

Measurements of circulating gut hormones and perfusion of rat intestine
Eight-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 µL/g, cat. no. 333-B1070, Nordic Biosite, Sweden) and sitagliptin (dipeptidyl peptidase-4 inhibitor, 10 mg/kg, 5 µL/g, Xelevia) 30 min 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-min post gavage. GLP-2 levels were measured by radio-immunoassay (RIA) as described previously55. Rat small intestines were perfused as described previously55 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 (v3.8), 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 systems56,57, 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).

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 3.058.

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 abundance calculated using HUMAnN 3.0 based on the Metacyc database59. 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 min, followed by 50 cycles of (i) denaturation at 95°C for 10 seconds, (ii) annealing at 60°C for 20 s, and (iii) extension at 72°C for 20 s. 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 50 mM K-Phosphate buffer for 6 min 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 (16,000×g, 30 min at RT), supernatant was collected and diluted 10x for MPO measurement. Samples were mixed with substrate buffer (O-dianisidine dihydrochloride in 50 mM K-Phosphate buffer with 0.0005% H2O2) on a 96-well plate, in duplicates, and measurements were taken every 30 s for 5 min at 450 nm. MPO activity was calculated using all readings and corrected for total protein content of each sample.

Statistics and reproducibility
Statistical analyses were performed using GraphPad Prism software (version 10). All data are presented as means ± standard error of the mean (SEM) unless otherwise specified in the figure legends, with individual data points included to illustrate data distribution. For comparisons between two groups, two-tailed unpaired t-tests were conducted to assess statistical significance. When comparing three or more 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 three or more 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. No statistical method was used to pre-determine sample size, and no data were deliberately excluded from the analyses. All animals were randomly allocated to experimental groups upon arrival.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Yang-Jensen et al. investigate the nutritional potential of a microbial lysate derived from Methylococcus capsulatus Bath (McB) in mitigating gastrointestinal (GI) inflammation. This study builds on their previous work published in Nature Communications (2021). Since the same material was used, some of the key mechanisms overlap, particularly the McB-induced immune modulation mediated by pTreg induction and Th17 modulation. The novelty of this work lies in defining the involvement of host GLP-2R signaling, which is dependent on microbial fermentation, and in expanding the disease relevance to GI inflammatory disorders. These findings suggest the translational potential of McB for modulating inflammatory symptoms.\n\nHowever, further studies are required to identify the GLP 2-mimicking components derived from the McB-fermentation, elucidating the underlying mechanism of pTreg generation. In addition, although the current study advances our understanding by identifying GLP-2R signaling as a novel component, it remains unclear whether these findings in animal models can be recapitulated in human samples. Moreover, while the authors propose molecular mimicry as a potential mechanism of GLP-2R activation, they do not provide specific evidence identifying the effector metabolites or molecules responsible for GLP-2R-mediated health benefits.\n\nRegarding microbiota dependency, the authors describe an interplay between microbiota-independent pTreg induction and microbiota-dependent tolerogenic Th17 responses. An in-depth analysis is needed to define the role of metabolites and microbiota-host interactions, ideally using germ-free mouse models.\n\nSpecific Comments:\nFigure 1:\nThe elevated cecal SCFAs upon McB administration appear to be driven by Lachnospiraceae and Bacteroidaceae families. Could some of the bacterial species enriched by McB administration mimic the function of McB? This could be tested by administering defined bacterial species to conventional SPF mice under a standard chow diet or to germ-free mice mono-associated with these bacteria.\n\nMcB feeding increased RORγt⁺ pTreg populations. What is the underlying mechanism of pTreg generation? Is there any enhancement in the effector function of nTreg cells, despite the authors’ statement that there was “no increase in nTreg population”?\n\nFigure 2:\n1) Despite ABX treatment, McB enhanced pTreg abundance. Are there changes in the Lachnospiraceae and Bacteroidaceae families and species under ABX treatment? Do they still show increased abundance?\n2) Based on Figure 2, the authors conclude that “pTreg induction occurred independently of gut microbiota composition, while Th17 cell induction and phenotypic manipulation relied on complex host-diet-microbe interactions.” However, the possibility that pTreg generation is mediated by ABX-resistant bacteria cannot be ruled out. Does McB feeding induce pTreg populations in germ-free mice? This experiment would provide conclusive support for the authors’ claim that pTreg induction by McB is a microbiota-independent mechanism.\n3) Additionally, Figure S6 shows that McB failed to elicit gut hormone release or modulate amino acid absorption, as it did not liberate bioactive components that stimulate GLP-2 secretion, suggesting that gut microbiota are required to release the active components from McB. Does protease-treated McB induce pTregs in germ-free mice? Such experiments would further substantiate the claim that pTreg induction is independent of gut microbiota.\n\nMechanistic Questions\n1) What was the rationale for focusing on GLP-2R signaling in McB-mediated gastrointestinal protection? More in-depth studies, such as RNA-seq analysis from intestinal inflammation models, could help determine whether GLP-2R pathways are altered explicitly in conjunction with other pathways.\n2) The authors suggest microbial fermentation of McB generates metabolites that influence GLP-2 release or activate GLP-2R signaling. They propose “molecular mimicry” as a possible mechanism, but what is the rationale for this claim without identifying the mimicry components?\n3) What are the actual compositions of the McB lysate besides proteins? Are McB-derived peptides or amino acids significantly different from those in conventional chow diets or humanized nutrient compositions? Do heat-stable versus heat-labile components have different effects on immune modulation?\n\nTreg and GLP-2R Crosstalk\n1) The authors propose two mechanisms for McB-induced tolerance: Treg generation and GLP-2R signaling. Which pathway plays the predominant role?\n2) What are the underlying mechanisms for pTreg and tolerogenic Th17 cell generation? Do any types of antigen-presenting cells (APCs) in the gut contribute to this process, or is it APC-independent?\n3) Is there crosstalk between Tregs and GLP-2R signaling in McB-mediated tolerance?\n4) Does McB treatment mitigate already established gut inflammation?\n5) Does monoassociation with live Methylococcus capsulatus induce Tregs and activate GLP-2R signaling pathways similarly to its extract?\n\nMinor Comments\n1) The authors should provide representative FACS plots and gating strategies for each T helper cell population and cytokine levels, in addition to bar graphs.\n2) Typographical errors should be corrected (e.g., “stiumlate” → “stimulate”).", "reviewer_2": "The manuscript describes dietary delivery of a microbial protein lysate mainly derived from Methylococcus capsulatus Bath (McB) rapidly alters microbiota composition and shifts T cell population in both small and large intestine. The McB-induced concurrent increase in IL-10+ Th17 cell and decrease in IFN- + Th17 is dependent on the gut microbiota. In contrast, McB-induced increase in peripherally induced regulatory T cells (pTreg) cells is independent of the gut microbiota. McB also protects against gut injury and inflammation during animal models of mucositis and colitis. The authors further demonstrate that microbial fermentation of McB produces a molecular mimicry that activates GLP-2R, the underlying mechanism behind the beneficial effects of McB in the GI tract. This work is important in the context of new providing nutritional advances in literate food sources, which appeared to be the original goal, but in the end using a commercially scalable microbial lysate found a way to regulate gut barrier function and dampen intestinal inflammation, which is also important.\n\nOverall, I found this manuscript compelling, expanding our knowledge on host-diet-microbe interaction during gastrointestinal inflammation. This manuscript is well written, experiments are generally well designed, data are clearly presented with appropriate analyses. There are many strength in the manuscript, particularly the advance in the concept of microbial mimicry and effects on the host through novel microbial compounds that work on host receptors such as GLP-2R. It is a strength that GLP-2R is a good candidate for engaging microbial products given its location and importance. Another strength is the evidence and advancement of the concept of microbial lysate-induced immune imprinting and reprogramming of the gut immune environment toward more tolerogenic responses. The use of both simple compositionally defined diet and a complex human-like diet, both with and without McB, is a considerable strength. The use of 2 different models of intestinal injury/damage is a strength. The experiments comparing GLP-2KO and GLP-1 showing specificity of the receptor are a strength.\n\nNevertheless, there are still some questions to address:\nMajor:\n1. It is a very interesting finding that inhibiting bacterial fermentation can mitigate the benefits of McB on gut inflammation and barrier function. Ther is a good series of experiments leading up to the concept of the requirement of fermentation and empirical test, where the effect of McB requires fermentation, but it does not alter GLP-2 levels. It is likely beyond the scope of this manuscript to find the exact molecule or molecules in McB that engage GLP-2. However, some simple experiments are missing. The authors should test if it is a protein that is responsible. Experiments using proteases, heat inactivation etc. would be very informative to understand if it is a protein produced by McB or if other microbial metabolites can mimic GLP-2 and have these effects. This should be addressed experimentally.\n2. Related to the point above, the bacterial lysate isolated from McB was added to rodent diet as the main protein source. Casein (simple diet) or a mix of protein sources (complex diet) was used in the control diets. This comparison in manuscript gives rise the potential that the different protein source leads to the different findings in control and McB-fed mice. However, it isnot clear if protein in MCb is the active ingredient. Microbial and casein protein are both composed with essential and nonessential amino acids, and the digestion of both protein leads to the production of their constituent amino acids. Unless there is evidence support different amino acid (or combination) could affect gut health and function, it is possible that beneficial effects of McB are more likely due to an unknown microbial product (i.e. postbiotic) in the McB lysate. This concept should be tested experimentally (as in point #1) and thoroughly discussed.\n3. The authors showed that McB can regulate immune cell phenotype and protect mice from chemical-induced intestinal injury and inflammation. It there a connection between these two effects? Does the immune imprinting also present in the mucositis and colitis model? Is the Th17 regulation also dependent on McB microbial fermentation? Does the changes in Th17 cell correlate with tissue or serum IL-17, IL-10, and IFN-  levels? It experimental evidence is not possible, comments on the model and rationale should suffice.\n\nMinor:\nFigure 4 above D and E should be “Villus length”. Same as Figure S4\nSome of the superlatives ad descriptive words in the writing could be toned down and be made more scientifically accurate – such as the use of “instantly” on line 172; the use of italics for and on line 181; “gradual on line 185 etc.\nThe nomenclature for GLP-2R WT versus GLP-2R KO mice should be improved to be easier to understand in the text and reflect that actual genotype of the mice. Each result from experiment using “WT” or “KO mice” needs to clearly state which mice were used. For example, which mice were used in line 255 (I assume WT, but it is not clear)."}, "author_response": "Comments from author: We thank the reviewer for this elegant summary and valuable input. Although we generally agree, we would like to clarify that the material used in the present study is – as described in the manuscript – similar but not identical to that used in our previous Nature Communications (2021) publication. In the earlier work, we utilized a laboratory-grown monoculture of Methylococcus capsulatus Bath (McB), which was not scalable thus minimally relevant for translational and industrial purposes.\n\nIn contrast, the current study employs a commercially scalable microbial lysate (FeedKind®, Calysta UK) in which approximately 95% of the biomass is derived from McB, with the remaining fraction consisting of non-McB “helper” bacteria required for stable industrial fermentation. This distinction is explicitly described in the Introduction, where we emphasize that a central motivation of the present work was to evaluate the biological and translational relevance of this industrially produced formulation that could essentially aid limiting greenhouse emissions from food production.\n\nImportantly, despite minor compositional differences between the two materials, we find that the core immunomodulatory effects of McB, including pTreg induction and Th17 reprogramming, are robustly reproduced in both a simplified rodent diet and under complex, humanized dietary conditions as well as across multiple models of gastrointestinal injury. This reproducibility underscores that McB is the bioactive driver of the observed responses and validates its functional conservation in a scalable, clinically relevant context.\n\nSpecific Comments:\nFigure 1:\nThe elevated cecal SCFAs upon McB administration appear to be driven by Lachnospiraceae and Bacteroidaceae families. Could some of the bacterial species enriched by McB administration mimic the function of McB? This could be tested by administering defined bacterial species to conventional SPF mice under a standard chow diet or to germ-free mice mono-associated with these bacteria.\n\nResponse: We thank the reviewer for this insightpul comment. We agree that pathways leading to SCFA production is enriched upon McB administration, and that this coincides with expansion of taxa within the Lachnospiraceae and Bacteroidaceae families. However, our data demonstrate co-occurrence rather than direct causality. Although certain enriched species may contribute functionally, we cannot attribute the beneficial effects as a whole solely to these taxa. The robust induction of pTregs in microbiota depleted mice (Fig 2), substantiate this claim.\n\nNotably, our findings in the revised manuscript demonstrate that pTreg induction is strictly microbiota-independent, persisting even when microbial fermentation is pharmacologically inhibited – (new Fig. S10).\n\nIntriguingly, the tolerogenic Th17 phenotype is partially dependent on microbial fermentation (new Fig. S10), pointing towards a contribution from fermentation-derived processes akin to the GLP-2R dependency reported in the original submission.\n\nWhile some enriched species - such as the commercially available Bacteroides uniformis – have previously been reported to mitigate colitis; PMID: 39566817; they are unlikely to replicate the full immunological and regenerative profile elicited by McB. We have acknowledged this in the revised result section (lines 506-516) and discussion (lines 659-692). Considering the magnitude of data included in this report (encompassing >850 animals across multiple experimental models, including, but not limited to, receptor-deficient, antibiotic-treated, and fermentation-inhibited cohorts) we respectfully consider such detailed investigations to be beyond the scope of the current manuscript.\n\nFinally, our new analysis of shotgun sequencing of feces from mice treated with fermentation inhibitors revealed no significant differences in Lachnospiraceae or Bacteroidaceae abundance, but still higher levels than in reference-diet fed mice (new Fig. S9), indicating that these taxa are not dependent on microbial fermentation of McB, but rather McB itself. We have now clarified these points in the revised Discussion (lines 659-692).\n\nMcB feeding increased RORγt⁺ pTreg populations. What is the underlying mechanism of pTreg generation? Is there any enhancement in the effector function of nTreg cells, despite the authors’ statement that there was “no increase in nTreg population”?\n\nResponse: We thank the reviewer for this important question regarding. Our comprehensive data indicate that McB lysate itself harbors an inherent capacity to induce pTregs, independent of the gut microbiota or microbial fermentation. Both antibiotic treatment and pharmacological inhibition of fermentation failed to alter McB-mediated pTreg expansion, demonstrating that this response arises from direct host–lysate interactions rather than secondary microbial effects.\n\nThe precise molecular component(s) responsible remain to be identified. Based on our prior work and emerging evidence from other microbial systems, we hypothesize that specific McB-derived cell wall or membrane-associated components interact with antigen-presenting cells (APCs) to promote a tolerogenic phenotype, which in turn drives pTreg differentiation. This concept aligns with previous in-vitro findings showing that McB interacts with human dendritic cells (PMID: 28293233). We have now added a discussion of this potential mechanism to the revised manuscript (lines 591-629).\n\nImportantly, while pTregs were reproducibly, robustly increased, we found no consistent enhancement in the number or activation status of thymus derived nTreg cells, consistent with a selective effect on the peripherally induced subset. We are currently pursuing bioactivity-guided fractionation of McB lysate to identify the molecular fraction(s) responsible for this direct immunomodulatory effect, which will be the focus of future dedicated studies.\n\nFigure 2:\n1) Despite ABX treatment, McB enhanced pTreg abundance. Are there changes in the Lachnospiraceae and Bacteroidaceae families and species under ABX treatment? Do they still show increased abundance?\n\nResponse: We thank the reviewer for this pertinent question. Unfortunately, this point cannot be experimentally addressed in a reliable manner. As shown in Figure 2 and Supplementary Figure S2R, antibiotic (ABX) treatment effectively abolishes detectable bacterial load, with 16S rRNA copy numbers approaching background levels. Under these conditions, metagenomic or 16S-based sequencing yields unreliable compositional data, as previously demonstrated and discussed in our recent work (Yang-Jensen et al., Gut Microbes, 2025, PMID: 40581887). Therefore, taxonomic analyses of Lachnospiraceae or Bacteroidaceae abundance in ABX-treated mice are not technically meaningful.\n\nImportantly, in cohorts treated with fermentation inhibitors, where only species participating in hindgut fermentation are affected, we observed no significant changes in these bacterial families compared with control-fed mice. This suggests that the pTreg induction observed during McB feeding is not dependent on either residual microbial taxa or fermentation-driven microbial shiw s. We have now clarified this limitation and its rationale in the revised text (e.g., lines 506-557 and 659-692).\n\n2) Based on Figure 2, the authors conclude that “pTreg induction occurred independently of gut microbiota composition, while Th17 cell induction and phenotypic manipulation relied on complex host-diet-microbe interactions.” However, the possibility that pTreg generation is mediated by ABX-resistant bacteria cannot be ruled out. Does McB feeding induce pTreg populations in germ-free mice? This experiment would provide conclusive support for the authors’ claim that pTreg induction by McB is a microbiota-independent mechanism.\n\nResponse: We appreciate the reviewer’s thoughpul question. Although nominal amounts of ABX-resistant microbes could theoretically persist during ABX treatment, we strongly believe that the likelihood of such process – in this situation – is negligible. As demonstrated in Figure 2 and Supplementary Figure S2, broad-spectrum ABX treatment led to near-complete elimination of bacterial 16S rRNA copies. Under these conditions, McB feeding robustly enhanced pTreg expansion in both the small and large intestine, and even accelerated pTreg induction in the large intestine compared to that of convention mice (Figure 2D).\n\nIn contrast, Th17 cell responses displayed a clear dependency on microbial presence: ABX exposure attenuated Th17 cell expansion and altered their cytokine profile. These findings were mirrored in mice treated with fermentation inhibitors, which lew pTreg frequencies unaffected while reducing the tolerogenic Th17 phenotype. Together, these results demonstrate that pTreg induction by McB is genuinely microbiota-independent, whereas Th17 modulation depends on microbial metabolism of McB-derived components.\n\nThese considerations are emphasized in the revised discussion (lines 659-692).\n\nWhile germ-free mice could potentially address this question, it is worth noting that their perturbed immune repertoire and pTreg profile (pTregs are generally believed to be microbially induced) could significantly compromise experiemtnal readouts of diet-induced gut immunity. Taken together, such additional investigations – with the potential caveats presented above - seem beyond the scope of this report.\n\n3) Additionally, Figure S6 shows that McB failed to elicit gut hormone release or modulate amino acid absorption, as it did not liberate bioactive components that stimulate GLP-2 secretion, suggesting that gut microbiota are required to release the active components from McB. Does protease-treated McB induce pTregs in germ-free mice? Such experiments would further substantiate the claim that pTreg induction is independent of gut microbiota.\n\nResponse: We thank the reviewer for this thoughpul question. Approximately 16,5% of the McB diet (~85% of the McB biomass) is composed of protein material, which is readily digested by endogenous gastrointestinal proteases when incorporated into the diet, as also supported by our shotgun metagenomic data demonstrating complete degradation and/or fermentation during transit.\n\nTherefore, while protease treatment for in vitro experiments is highly useful, it appears less consequential for in vivo dietary experiments, where the combined biomass is subjected to the wealth of host-secreted digestive enzymes, optimized for nutritional degradation.\nIndeed, McB is a multifactorial microbial lysate, not a purified protein source, and its bioactivity likely derives from the concerted actions of several molecular fractions.\nFuture studies will dissect these bioactive fractions systematically, but such work lies beyond the scope of the current manuscript, already integrating multiple dietary, genetic, and pharmacological models.\n\nMechanistic Questions\n1) What was the rationale for focusing on GLP-2R signaling in McB-mediated gastrointestinal protection? More in-depth studies, such as RNA-seq analysis from intestinal inflammation models, could help determine whether GLP-2R pathways are altered explicitly in conjunction with other pathways.\n\nResponse: We thank the reviewer for this thoughpul question. As alluded to in the original version of the manuscript, our focus on GLP-2R signaling was based on clear histological and morphometric observations in McB-fed mice. Specifically, we observed a prolonged intestinal length, deeper crypts, increased villus height, and greater intestinal mass, all hallmark features of GLP-2–mediated intestinotrophic activity. These phenotypic similarities provided a strong biological rationale to investigate whether McB exerts its protective effects through GLP-2–related mechanisms.\n\nBecause the source of this intestinotrophic signal was unknown, we systematically evaluated GLP-2R and GLP-1R KO mice, as well as McB’s ability to stimulate endogenous GLP-2 release. This comprehensive receptor-level analysis allowed us to directly test causality, demonstrating that McB-mediated intestinal protection is GLP-2R-dependent but independent of endogenous GLP-2 release and GLP-1R signaling.\n\nWhile RNA-seq approaches can be valuable for hypothesis generation, they are not suitable for confirming receptor dependency once it has been validated genetically. For this reason, we respectfully consider that the targeted KO-based approach used here provides stronger mechanistic evidence than transcriptomic correlation.\n\n2) The authors suggest microbial fermentation of McB generates metabolites that influence GLP-2 release or activate GLP-2R signaling. They propose “molecular mimicry” as a possible mechanism, but what is the rationale for this claim without identifying the mimicry components?\n\nResponse: We thank the reviewer for raising this important question. Our conclusion that McB-derived fermentation products act through molecular mimicry of GLP-2R activation is based on a combination of negative and dependency evidence. Specifically, McB exposure, whether administered chronically in the diet, acutely by oral gavage, or directly in isolated intestinal perfusion, failed to stimulate endogenous GLP-2 release. Despite this, McB treatment provided robust GLP-2R-dependent protection.\n\nMoreover, when microbial fermentation was inhibited, we observed a marked loss of McB’s protective effects, indicating that microbial processing of the lysate is required to liberate bioactive component(s) capable of engaging GLP-2R. Taken together, these findings demonstrate that the observed protection is not mediated by host-derived GLP-2, but rather by microbially generated molecule(s) that activate GLP-2R in a ligand-mimicking fashion.\n\nWhile the precise chemical identity of these effector(s), whether derived from McB structural components or secondary microbial metabolites, remains to be determined, identifying them will require a dedicated, several-year, fraction-based approach. Such in-depth molecular work it indeed intriguing and should be pursued but clearly lies beyond the scope of the current study, which already encompasses a large array of mechanistic experiments. In respect of this reviewer’s valid point, we have balanced our revised result section and discussion to reflect these thoughts more carefully (e.g., lines 717-724 and elsewhere).\n\n3) What are the actual compositions of the McB lysate besides proteins? Are McB-derived peptides or amino acids significantly different from those in conventional chow diets or humanized nutrient compositions? Do heat-stable versus heat-labile components have different effects on immune modulation?\n\nResponse: We thank the reviewer for this thoughpul question. Comprehensive compositional analysis was published in our previous NCOMMS publication (2021). To accommodate the request of this reviewer, we have also performed our own amino acid profiling (attached Reviewer Table 1+2) confirming that the McB-containing diet does not differ substantially in amino acid composition or overall nutrient profile between the used diets (Supplementary Table 1). Thus, the observed biological effects cannot be attributed to differences in basic nutritional composition.\n\nThe McB material used here is a whole-cell lysate, which in addition to proteins and peptides may contain membrane-associated fatty acids, phospholipids, nucleotides, and glycoproteins. We currently do not know which of these molecular compartments confer the observed health benefits, and it is plausible that multiple fractions contribute synergistically to the broad immunological and regenerative effects observed. This will be meticulously explored in ongoing and future endeavors.\n\nRegarding thermal stability, we have not specifically examined the effects of heat treatment in this study. While previous reports have shown that heat-killed microbes can elicit similar immunomodulatory effects as their live or lysed counterparts, we note that heat treatment of a complex lysate such as McB, representing 20% of the diet, could induce Maillard reactions and other chemical modifications, thereby altering its biological properties. For this reason, such experiments would be difficult to interpret and are not representative of in vivo gastrointestinal digestion. Future studies will systematically dissect and fractionate McB components to identify the specific bioactive molecules responsible for immune modulation and/or tissue regeneration.\n\nTreg and GLP-2R Crosstalk\n1) The authors propose two mechanisms for McB-induced tolerance: Treg generation and GLP-2R signaling. Which pathway plays the predominant role?\n\nResponse: We thank the reviewer for this insightpul question. Our data indicate that pTreg induction and GLP-2R signaling represent independent but complementary mechanisms contributing to McB-mediated gastrointestinal protection. To mechanistically address this question in our models of acute GI perturbation, we set up new experiments in CD4+ T-cells depleted mice and isotype injected controls (new Figure S3).\n\nConsistent with the acute nature of these disease models, and a prolonged induction of adaptive immunity, McB fed mice remained protected against both DSS-induced colitis and 5-FU-induced mucositis despite CD4⁺ T-cell depletion. Conversely, GLP-2R knockout completely abrogated McB-mediated intestinal protection but did not prevent pTreg expansion, which remained comparable to that observed in wild-type mice (new Figure S7). Likewise, GLP-1R deficiency did not affect pTreg induction (new Figure S7). These findings demonstrate that pTreg generation occurs independently of functional GLP-2R or GLP-1R signaling, whereas GLP-2R activation is essential for the intestinotrophic and barrier-protective effects of McB in these acute models. We have included this imperative information in the revised manuscript.\n\nTogether, these results suggest that McB promotes tolerance and mucosal protection through dual, mechanistically distinct pathways: (1) direct induction of pTregs, and (2) GLP-2R–dependent epithelial repair and barrier maintenance. While the GLP-2R dependency is predominant for protecting against chemically induced colitis and mucositis, tolerogenic T-cell subsets remain of key interest for long-term clinical scenarios, including recurrent IBD and lifestyle-associated gut barrier dysfunction observed in cardiometabolic diseases. These reflections are included in the revised manuscript (lines 333-343).\n\n2) What are the underlying mechanisms for pTreg and tolerogenic Th17 cell generation? Do any types of antigen-presenting cells (APCs) in the gut contribute to this process, or is it APC-independent?\n\nResponse: We thank the reviewer for this insightpul question. While we did not perform APC-T cell interaction experiments in the current study, these mechanisms have been previously characterized in detail (Indrelid et al., 2017, PMID: 28293233).\n\nIn the mentioned study, the authors demonstrated that McB directly interacts with human dendritic cells (DCs) to modulate T cell responses in co-culture systems. When compared with lysates from E. coli Nissle 1917 (EcN) and LGG, McB uniquely induced a selective IL-2–driven DC response, without broadly affecting other cytokines. This IL-2 secretion promoted T-cell activation and proliferation, evidenced by a ~50% increase in CD25 expression (IL-2 receptor α) and enhanced ³H-thymidine incorporation. As regulatory T-cells constitutively express CD25, contrasting no-Tregs that only upregulare CD25 upon activation, IL-2 is a canonical driver of regulatory T-cell expansion. This support the notion that McB may act through APC conditioning rather than APC-independent mechanisms.\n\nAlthough RORγt expression was not measured in PMID: 28293233, the reported findings suggest that McB can directly prime DCs toward a tolerogenic phenotype, which in turn promotes peripheral Treg induction. Together with our current in vivo data showing that pTreg expansion is independent of the microbiota and microbial fermentation, these results strongly support an APC-mediated mechanism as the primary driver of McB-induced immune tolerance. Still, as tolerogenic Th17 cell generation, in contrast to pTreg induction, relied on microbial fermentation, it remains elusive if these too are instructed by McB-conditioned DCs or if other pathways are involved. We have added this clarification and relevant citation to the Discussion.\n\n3) Is there crosstalk between Tregs and GLP-2R signaling in McB-mediated tolerance?\n\nResponse: Our data does not support functional crosstalk between Treg activity and GLP-2R signaling in the protective effects of McB. In both DSS-induced colitis and 5-FU–induced mucositis models, CD4⁺ T-cell depletion – which effectively eliminates Tregs – did not abolish McB-mediated protection, indicating that epithelial repair and barrier maintenance occur independently of the adaptive immune compartment (new Figure S3).\n\nConversely, GLP-2R knockout completely negated McB’s protective effects (Figure 4-5) despite preserved pTreg induction (new Figure S7), suggesting that these two mechanisms operate in parallel but not hierarchically.\n\n4) Does McB treatment mitigate already established gut inflammation?\n\nResponse: Our previously published work (NCOMMS, 2021) demonstrated therapeutic effects of McB treatment in settings of established low-grade intestinal inflammation. In diet-induced obesity, mice display a mild inflammatory phenotype characterized by approximately two-fold lower pTreg levels compared with low-fat diet (LFD) fed reference mice (Jensen et al, Nat Comms, 2021). When these obese mice were therapeutically treated with McB, we observed a fivefold increase in intestinal pTreg abundance, accompanied by resolution of mucosal inflammation and improved barrier integrity (Jensen et al., Nat Comms., 2021). These findings indicate that McB is capable not only of preventing inflammation but also of reversing – at least mild - pre-existing immune dysregulation in the gut.\n\n5) Does monoassociation with live Methylococcus capsulatus induce Tregs and activate GLP-2R signaling pathways similarly to its extract?\n\nResponse: We thank the reviewer for this question. However, monoassociation with McB is not experimentally feasible in rodent models. McB is a strict methanotrophic bacterium, requiring methane as its sole carbon and energy source. It cannot survive or replicate in the gastrointestinal tract of monogastric animals. Any attempt to administer “live” McB would result in rapid inactivation and lysis in the acidic gastric environment, effectively generating the same cellular lysate that we investigate in the current study.\n\nTherefore, the proposed monoassociation experiment would not yield additional mechanistic insight beyond what is already shown here. Theoretically, the physiological relevance of McB lies precisely in its non-colonizing, lysate-based activity, which mimics what would naturally occur upon ingestion of non-viable microbial biomass.\n\nMinor Comments\n1) The authors should provide representative FACS plots and gating strategies for each T helper cell population and cytokine levels, in addition to bar graphs.\n\nResponse: We thank the reviewer for this valuable suggestion and fully agree that representative FACS plots and gating strategies are essential for clarity. These data were already included in the original version of the manuscript: Supplementary Figure S1 presents the full gating strategy and representative plots for all analyzed T helper cell populations and cytokine-producing subsets. To improve accessibility, we have now added explicit cross-references to this figure in the main text, ensuring the information is easily located by readers.\n\n2) Typographical errors should be corrected (e.g., “stiumlate” → “stimulate”).\n\nResponse: We thank the reviewer for noting this. We have carefully proofread the entire manuscript and corrected all typographical and minor language errors throughout.\n\nReviewer #2 (Remarks to the Author):\n[...]\nWe sincerely thank the reviewer for their generous and thoughpul evaluation of our work. We are grateful for the recognition of the study’s conceptual and experimental strengths, including the integration of dietary models, receptor-specific analyses, and immune imprinting mechanisms. The reviewer’s constructive comments greatly improved the clarity and depth of our revised manuscript.\n\nNevertheless, there are still some questions to address:\nMajor:\n1. It is a very interesting finding that inhibiting bacterial fermentation can mitigate the benefits of McB on gut inflammation and barrier function. There is a good series of experiments leading up to the concept of the requirement of fermentation and empirical test, where the effect of McB requires fermentation, but it does not alter GLP-2 levels. It is likely beyond the scope of this manuscript to find the exact molecule or molecules in McB that engage GLP-2. However, some simple experiments are missing. The authors should test if it is a protein that is responsible. Experiments using proteases, heat inactivation etc. would be very informative to understand if it is a protein produced by McB or if other microbial metabolites can mimic GLP-2 and have these effects. This should be addressed experimentally.\n\nResponse: We thank the reviewer for their positive and insightpul assessment. We fully agree that identifying the bioactive fraction(s) and precise GLP-2R–activating molecule(s) in McB represents an important next step for future research; an endeavor that is currently ongoing in our lab.\n\nMcB is a whole-cell lysate produced under tightly controlled fermentation conditions (methane, ammonia, mineral salts). Different cell disruption techniques (including French press and mechanical shear) all yielding comparable protective effects in vivo (see current manuscript – using commercial products – and Jensen et al 2021, Nat Comms. using french pressed monocultures). Together, these observations indicate that McB’s biological activity does not depend on a single labile protein but likely arises from multiple, chemically diverse molecules, including proteins, lipids, and glycoproteins.\n\nWe have conducted additional experiments – to align with the next comments – and we demonstrate that while McB exposure fails to induce endogenous GLP-2 release, it loses efficacy when microbial fermentation is inhibited, implying that fermentation liberates one or more small, molecules that act as GLP-2R mimetics. This could involve surface-associated or lipid-linked microbial structures, similar to those described by Verma et al. (Sci Immunol, 2018, PMID: 30341145), which interact directly with host receptors to elicit immunomodulatory effects.\n\nWe therefore agree that while protease or heat-treatment experiments might theoretically inform on stability, such approaches are not straighporward for complex lysates and could introduce confounding chemical modifications (e.g., Maillard reactions, and not fully recapitulate the in vivo digestive processes). We have clarified in the revised Discussion that future studies will systematically fractionate McB lysates to identify and characterize the molecular effector classes responsible for GLP-2R activation and immune modulation.\n\n2. The bacterial lysate isolated from McB was added to the rodent diet as the main protein source. Casein (simple diet) or a mix of protein sources (complex diet) was used in the control diets. This comparison gives rise to the possibility that different protein sources may account for the observed differences between control and McB-fed mice. It is not clear whether protein in McB is the active ingredient. Microbial and casein proteins are both composed of essential and non-essential amino acids, and digestion of both would yield their constituent amino acids. Unless there is evidence that different amino acid compositions could affect gut health and function, it is possible that the beneficial effects of McB are due to an unknown microbial product (i.e. postbiotic) in the McB lysate. This concept should be tested and discussed.\n\nResponse: We thank the reviewer for this important note. The use of two control diets i) a simple casein-based protein diet and ii) a complex multi-protein diet, was intentional and designed to demonstrate that McB’s effects are independent of the background diet. Across both dietary backgrounds, McB consistently improved barrier function, resolved inflammation, and expanded pTreg populations, indicating that the observed effects depend on the presence of McB itself, not on differences in dietary protein composition.\n\nWe agree with the reviewer that McB protein is unlikely to represent the sole bioactive component. Amino acid profiling shows no meaningful differences in essential or non-essential amino acid composition between McB-containing and control diets (Reviewer Table 1+2). Both microbial and casein proteins are digested to yield similar amino acids.\n\nTo accommodate all reviewer’s questions, we designed a new study with fermentation inhibitors and investigated whether resident gut microbes ferment the McB lysate to products mediating immunological and/or gut-health promoting effects. While pTreg expansion was not dependent on microbial fermentation – similar to our findings with ABX treatment –Th17 cell reprogramming was indeed dependent on microbial fermentation. We therefore concur that – apart from the robust microbe-independent pTreg induction – the beneficial effects of McB (GI tissue homeostasis and Th17 cell tolerogenicity) are most plausibly mediated by microbial components or postbiotic metabolites generated during microbial hindgut fermentation. Whether these are of proteinaceous or fatty acid origins (or something third) has yet to be elucidated in dedicated studies. This interpretation aligns with our hypothesis and with the reviewer’s comment above. We have added the amino acid comparison in Reviewer Table 1+2, and an explicit statement of this rationale in the revised Discussion.\n\n3. The authors showed that McB can regulate immune cell phenotype and protect mice from chemical-induced intestinal injury and inflammation. It there a connection between these two effects? Does the immune imprinting also present in the mucositis and colitis model? Is the Th17 regulation also dependent on McB microbial fermentation? Does the changes in Th17 cell correlate with tissue or serum IL-17, IL-10, and IFN-g levels? It experimental evidence is not possible, comments on the model and rationale should suffice.\n\nResponse:\nWe thank the reviewer for these constructive questions. Below we summarize the experimental evidence and clarify limitations of what can be measured in each model.\n\nConnection between immune imprinting and protection.\nTo answer these consistently raised questions between reviewer #1 and reviewer #2, we directly tested the dependence of McB protection on adaptive CD4⁺ T cells by performing CD4⁺ T-cell depletion in both DSS colitis and 5-FU mucositis models (new Figure S3).\nConsistent with the acute nature of these disease models, and a prolonged induction of adaptive immunity, McB fed mice remained protected against both DSS-induced colitis and 5-FU-induced mucositis despite CD4⁺ T-cell depletion, demonstrating that the protective phenotype against acute epithelial damage occur independently of the adaptive T-cell compartment.\n\nBecause GLP-2R knockout mice lose McB protection despite preserved pTreg induction (new Figure S7), the data support two parallel mechanisms (pTreg-mediated immune tolerance and GLP-2R-mediated epithelial protection) rather than a single hierarchical pathway.\n\nIs immune imprinting present in the disease models?\nAccurate lamina propria T-cell profiling from the disease-affected tissue is practically impossible, as severe inflammation and tissue frailty prevent reliable LP cell isolation from the affected site (e.g., large intestine LP in acute DSS and small intestine LP in acute 5-FU mucositis). For this reason, we assessed T-cell dependence by systemic CD4 depletion, and we validated depletion systemically (spleen) and locally in the less affected gut region (small intestine for DSS and colon for mucositis) by flow cytometry (Live/Dead, CD45, CD4, CD8, new Figure S3).\n\nIs Th17 regulation fermentation-dependent?\nThis is an excellent point, that we initially did not consider. We thus thanks the reviewer for encouraging us to test this experimentally, as it has added significant nuances to the manuscript. Indeed – Th17 modulation shows at least partial dependence on microbial fermentation. While McB increases total Th17 proportions – as shown numerous times – this numeric increase is lost when fermentation is inhibited, and McB’s reduction of IFN-γ⁺ Th17 cells is similarly attenuated by fermentation blockade (new Figure S10). Effects on IL-10⁺ Th17 were more variable with fermentation inhibitors (likely due to responder/non-responder variability induced by the inhibitor combined with experimental variables), which may explain lack of consistent statistical significance in this exact subpopulation – although there could be trends of reduced levels of IL-10⁺ Th17 cells across all groups when microbial fermentation is inhibited. These observations are consistent with our broader conclusion that pTregs are microbiota-independent whereas Th17 reprogramming requires microbial processing of McB components.\n\nCorrelation of Th17 changes with cytokine measures (tissue/serum IL-17, IL-10, IFNγ).\nWe have previously reported related cytokine readouts in plasma (see Jensen et al., Nat Comms, 2021, PMID: 33597537) and the present study quantifies cytokine-producing T cells by intracellular staining, providing cell-specific information on IL-10 and IFN-γ. Given the extensive dataset already presented, we believe the current intracellular flow cytometry data (which are cell-specific and directly link cytokine production to T-cell subsets) are sufficient to support our conclusions. Of course, if the reviewers and/or editors insists, we can add a targeted tissue cytokine panel for key cytokines in a predetermined or random cohort, but we respectfully feel the incremental value relative to the scope of the present revision is limited.\n\nWe have revised both the Results and Discussion sections according to all of the above.\n\nMinor:\nFigure 4 above D and E should be “Villus length”. Same as Figure S4\nSome of the superlatives ad descriptive words in the writing could be toned down and be made more scientifically accurate – such as the use of “instantly” on line 172; the use of italics for and on line 181; “gradual on line 185 etc.\n\nResponse: We thank the reviewer for their attention to these details. We have amended the text accordingly.\n\nThe nomenclature for GLP-2R WT versus GLP-2R KO mice should be improved to be easier to understand in the text and reflect that actual genotype of the mice. Each result from experiment using “WT” or “KO mice” needs to clearly state which mice were used. For example, which mice were used in line 255 (I assume WT, but it is not clear).\n\nResponse: We apologize for any confusion we may have conveyed. We have attempted to improve clarity in this regard."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most of the reviewer’s concerns in the revised manuscript; however, the underlying mechanism of action remains to be fully clarified, as now rationally discussed.", "reviewer_2": "The authors should be commended for thorough, thoughtful, and meaningful responses to all reviewer comments, including the appropriate inclusion of important additional experiments. This is a great paper."}, "author_response": ""}]

Decision letter