AIMS:Hygienic monitoring (HM) of germ-free (GF) mouse colonies is exceptionally challenging. The test accuracy of the applied diagnostic methodology has to be outstanding to provide proof of absence of all living microorganisms confirming the GF status. In this context, microscopy of native intestinal content serves as a highly sensitive diagnostic tool for the detection of bacterial contaminants. However, with this method residual microorganisms may be detected. To overcome this risk of false-positive results, we complemented our analyses with a bacterial viability staining of the intestinal content of GF mice. METHODS AND RESULTS:Intestinal contents of GF mice (n = 13) from five isolators were analyzed by bacterial culture and phase-contrast microscopy. Additionally, 16S rRNA gene PCR analysis and metagenomic sequencing were performed. To distinguish between live and dead bacteria, intestinal content was stained by a Bacterial Viability Kit and analyzed by fluorescence microscopy. While culture medium proved sterility of the sample material, increased amounts of scattered bacterial structures were detected during microscopic analysis, indicating potential contamination. Molecular techniques pointed to a presence of environmental bacteria. However, viability staining revealed the presence of only dead (double-stained) bacteria in all samples. Likewise, non-viable bacteria have been identified in samples obtained from irradiated feed, probably being the source of bacterial structures found in GF mice. CONCLUSIONS:Altogether, detected bacterial structures were proven to be non-viable and therefore should not be interpreted as isolator contaminants. Thus, in our hands, with the herein described report of suspected contamination, we prove that bacterial viability staining served as a highly valuable screening tool, enhancing diagnostic quality of the HM of GF colonies.

BACKGROUND:Colorectal cancer (CRC) exhibits increased levels of arachidonic acid-derived pro-inflammatory derivatives indicating an uptake of dietary polyunsaturated fatty acids (PUFAs). OBJECTIVE:We aimed to investigate uptake of extrinsic fatty acids (FAs) in tumours and their relevance for CRC lipid metabolism and progression. DESIGN:Total FAs were quantified using gas chromatography-mass spectrometry in non-diseased mucosa and tumour tissue from patients with CRC of a discovery cohort (n=152), validated in an independent cohort (n=28) and associated with clinical, genomic and microbiome data. The genetic mouse tumour model Apc1638N was used to track the flux of stable isotope-labelled FAs in tumours from the intestinal lumen. The relationship between FA uptake and tumour progression was investigated in 2D and 3D cell models. RESULTS:Extrinsic long chain PUFAs, including arachidonic acid, accumulate in CRC, particularly in right-sided tumours, and in tumours of Apc1638N mice. The CRC-specific FA profiles were independent of sex, molecular subtypes, early-disease or late-disease onset. The absorption of FAs from the intestinal lumen in tumours was confirmed in specific pathogen-free Apc1638N mice. In the absence of the microbiome, in germ-free Apc1638N mice, fewer tumours were developed, and survival was increased. Inhibition of FA import or β-oxidation reduces cancer cell proliferation. CONCLUSION:Extrinsic FAs accumulate in CRC, verifying a central role of arachidonic acid-derived inflammatory mediators, but also suggesting a relevance of dietary FAs for cancer cell proliferation. It will be intriguing to explore to what extent targeting this flux pathway together with the interrelated microbiome opens new therapeutic avenues for CRC in humans.

The gut microbiota influences host metabolism, but the mechanisms of lipid uptake from food remain mysterious. Here we used stable isotope-labelled tracers in gnotobiotic mouse models, which revealed that host uptake of dietary lipids depends on microbial colonization. Systemic lipid metabolism modelling predicted that the gut microbiota restricts intestinal lipid absorption, and labelled lipid administration verified that the gut contents of microbiota-colonized mice contained up to 12-fold more lipids than those of germ-free animals. A combination of lipidomics and proteomics showed that gut microbes trigger Myd88 signalling, leading to a downregulation of hepatic Cyp7b1 activity and increased taurocholate production. Taurocholate stimulates phospholipase A1 activity in bile, causing the degradation of phosphatidylcholine that is essential for luminal micelle formation and lipid uptake. A diverse microbiome was associated with lower phosphatidylcholine content. This previously unrecognized host-gut microbiota interplay via enzymes in bile could provide future targets to modulate dietary lipid absorption.

Abstract Limited resource availability in the gut promotes competitive interactions between bacteria, which drive adaptive within-host evolution ( 1–3 ). While adaptive evolution of bacterial communities has been increasingly studied in the recent years ( 4–7 ), its functional implications for host physiology remain unknown. Here, we show that within-host evolution of the human commensal Enterococcus faecalis boosts colonization resistance to enteric Salmonella enterica serovar Typhimurium ( S . Typhimurium) infection. During gut colonization, E. faecalis evolves the ability to metabolize fructoselysine, an abundant Amadori rearrangement product generated by thermal food processing. The depletion of this diet-derived nutrient prevents S . Typhimurium colonization by restricting an essential resource. This protective mechanism was conserved across independent mouse colonies and arises via diverse evolutionary trajectories, including nucleotide polymorphisms, gene amplifications, and a horizontal gene transfer event. Additionally, analysis of E. faecalis isolates from human infants revealed that adaptation to fructoselysine availability occurs in a diet-dependent manner. Isolates from infants fed with fructoselysine-rich formula were able to utilize fructoselysine, whereas those from infants fed with fructoselysine-poor breast milk were not. Conclusively, our results identify an inherent microbiome-driven self-healing mechanism, wherein bacterial evolution restores colonization resistance against enteric pathogens through evolved nutrient depletion. Understanding these evolutionary dynamics will inform microbiome-targeted approaches to prevent and treat infectious diseases by harnessing adaptive bacterial metabolism.

IntroductionThe intestinal immune system is organized into regionally specialized lymphatic drainage networks that coordinate adaptive responses according to the anatomical site of microbial encounter. During Citrobacter rodentium (C. rodentium) infection in mice, initial colonization occurs in the cecum and proximal colon, where antigen‑presenting cells drain to the corresponding mesenteric lymph nodes (mLN), before the pathogen progresses to dominant attachment in the distal colon at peak infection. These segment‑specific patterns of colonization are matched by segment‑specific lymphatic drainage, such that distinct lymph node compartments support immune priming in different intestinal regions. Understanding how these proximal draining mLN contribute to the development of colonic immunity, despite the later dominance of distal‑colonic infection, remains an important open question.MethodsTo examine the contribution of the colon‑draining mLN, we surgically removed either the entire mLN chain or only the nodes draining the cecum and colon, followed by C. rodentium infection. Immune responses were analyzed at day 10 and day 18, corresponding to phases in which distal‑colonic colonization dominates.ResultsWe found that Th17 effector responses in the colon were maintained independently of the removed mLN, consistent with the fact that local colonic activation is the primary driver of the observed T cell phenotype at these time points. In contrast, B cell differentiation was markedly impaired in mLN‑resected animals: both plasma cell frequencies in the colon and pathogen‑specific serum IgG1 and IgG2a responses were significantly reduced.DiscussionThese findings indicate that, although distal‑colonic T cell activation proceeds locally, effective B cell activation and class switching depend on the presence of the appropriate draining lymph nodes. Overall, this study highlights the segment‑specific organization of intestinal immunity and demonstrates that B cell responses during C. rodentium infection are critically dependent on the lymph nodes draining the cecum and colon, whereas local T cell activation in the distal colon can occur independently of these structures.

Understanding the complex interactions between microbes and their environment requires robust model systems such as synthetic communities (SynComs). We developed a functionally directed approach to generate SynComs by selecting strains that encode key functions identified in metagenomes. This approach enables the rapid construction of SynComs tailored to any ecosystem. To optimize community design, we implemented genome-scale metabolic models, providing in silico evidence for cooperative strain coexistence prior to experimental validation. Using this strategy, we designed multiple host-specific SynComs, including those for the rumen, mouse, and human microbiomes. By weighting functions differentially enriched in diseased versus healthy individuals, we constructed SynComs that capture complex host-microbe interactions. We designed an inflammatory bowel disease SynCom of 10 members that successfully induced colitis in gnotobiotic IL10-/- mice, demonstrating the potential of this method to model disease-associated microbiomes. Our study establishes a framework for designing functionally representative SynComs of any microbial ecosystem, facilitating mechanistic study.

Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis in humans. The intestinal microbiota provides colonization resistance to enteric pathogens such as S. Typhimurium. Akkermansia muciniphila is an anaerobic bacterium commonly found in the intestinal tract of humans and other mammals and specializes in the degradation of mucin. To study the role of A. muciniphila in affecting the outcome of S. Typhimurium colonization and pathology, we used gnotobiotic mice colonized with a defined simplified human (SIHUMI) or mouse (OMM11) intestinal microbiota and infected them with the attenuated S. Typhimurium ΔaroA strain. By comparing SIHUMI and OMM11 mice to mice additionally colonized with A. muciniphila, we demonstrate that the presence of A. muciniphila leads to a decrease in intestinal Salmonella colonization. In addition, Salmonella-induced colitis is significantly reduced in the presence of A. muciniphila including improved histopathological changes as well as decreased levels of inflammatory cytokines. Furthermore, we demonstrate that viable A. muciniphila inhibit adhesion of Salmonella to the intestinal epithelium in vivo as well as to differentiated, polarized HT29-MTX-E12 epithelial cells. These data indicate that A. muciniphila plays an important role in mediating protection from S. Typhimurium colitis by inhibiting adhesion of Salmonella to the intestinal epithelium.

Aging manifests a decline of immune function, induces microbiome dysbiosis, drives organ inflammation, and impedes the resolution of inflammation. However, the mechanisms underlying age-related intestinal inflammation remain poorly described. Here, we find that the resolution of T cell-initiated intestinal inflammation is impaired with aging. This impairment is mediated by disrupting the immune-microbiota interplay, controlled by intestinal eicosanoid metabolism. Pharmacologically inhibiting eicosanoid biosynthesis, blocking the prostaglandin E receptor subtype 4 (EP4), or genetically ablating EP4 diminishes age-related impairment of intestinal inflammation resolution. Mechanistically, mononuclear phagocyte-intrinsic eicosanoid-EP4 signaling impedes the resolution of intestinal inflammation through fostering gut microbial dysbiosis and, more importantly, interrupting segmented filamentous bacterial adhesion to the intestinal epithelium. Colonization with EP4-ablated mouse microbiota or segmented filamentous bacteria improves the resolution of intestinal inflammation. These findings reveal that eicosanoid-dependent immune-microbiota interactions impair inflammation resolution in the aged intestine, highlighting potential intervention strategies for improving age-related gut health.

IntroductionRecent evidence supports the hypothesis of an association between gut microbiota and the pathogenesis of retinal and eye diseases, suggesting the existence of a gut-eye axis. However, no data are available on the possible effect of the gut microbiota on the optic nerve fiber maturation and myelin development.MethodsWe investigated the impact of gut microbiota on the optic nerves collected from neonatal and young adult germ-free (GF), gnotobiotic (stably colonized with 12 bacteria strains, OMM12) and control (colonized with a complex gut microbiota, CGM) mice, by performing stereological and morphoquantitative analyses with transmission electron microscopy and gene expression analysis by quantitative real-time PCR.ResultsYoung adult GF and OMM12 optic nerve axons are smaller and hypermyelinated compared to CGM ones, while no such differences were detected in neonatal optic nerves. The transcription factors Olig1, Olig2, and Sox10 (oligodendrocyte myelination positive regulators) are downregulated in CGM and OMM12 young adult mice compared to the respective neonates. Such developmental downregulation was not observed in GF optic nerves, suggesting that the absence of the gut microbiota prolongs the stimulation of optic nerve fiber myelination, possibly through mechanisms that are yet to be identified.DiscussionAltogether, these data underscore the gut microbiota pivotal role in driving optic nerve myelination, contributing to our knowledge about both the gut-eye axis and the gut-brain axis, and opening new horizons for further investigations that will explore the role of the microbiota also in pathologies, injuries and regeneration associated with the optic nerve.

Anti-viral immunity can vary tremendously from individual to individual but mechanistic understanding is still scarce. Here, we show that a defined, low complex bacterial community (OMM12) but not the general absence of microbes in germ-free mice leads to a more potent immune response compared to the microbiome of specific-pathogen-free (SPF) mice after a systemic viral infection with LCMV Clone-13. Consequently, gnotobiotic mice colonized with OMM12 have more severe LCMV-induced disease pathology but also enhance viral clearance in the intestinal tract. Mechanistically, single-cell RNA sequencing analysis of adoptively transferred virus-specific T helper cells and endogenous T helper cells in the intestinal tract reveal a stronger pro-inflammatory Th1 profile and a more vigorous expansion in OMM12 than SPF mice. Altogether, our work highlights the causative function of the intestinal microbiome for shaping adaptive anti-viral immunity with implications for vaccination strategies and anti-cancer treatment regimens.

RNA-sensing TLRs are strategically positioned in the endolysosome to detect incoming nonself RNA. RNase T2 plays a critical role in processing long, structured RNA into short oligoribonucleotides that engage TLR7 or TLR8. In addition to its positive regulatory role, RNase T2 also restricts RNA recognition through unknown mechanisms, as patients deficient in RNase T2 suffer from neuroinflammation. Consistent with this, mice lacking RNase T2 exhibit interferon-dependent neuroinflammation, impaired hematopoiesis, and splenomegaly. However, the mechanism by which RNase T2 deficiency unleashes inflammation in vivo remains unknown. Here, we report that the inflammatory phenotype found in Rnaset2-/- mice is completely reversed in the absence of TLR13, suggesting aberrant accumulation of an RNA ligand for this receptor. Interestingly, this TLR13-driven inflammatory phenotype is also fully present in germ-free mice, suggesting a role for RNase T2 in limiting erroneous TLR13 activation by an as yet unidentified endogenous ligand. These results establish TLR13 as a potential self-sensor that is kept in check by RNase T2.

Neutrophils are readily activated immune cells after ischemic stroke in mice and patients. Still, the impact of gut microbiota on neutrophil activation and its influence on inflammatory brain injury remain undefined. We report that natural microbiota colonization of germ-free (GF) mice induces substantial neutrophil activation and deteriorates stroke pathology. The colonized Ex-GF stroke mice had considerably larger infarct sizes and higher sensorimotor deficits than GF littermates. Furthermore, employing an antibiotic-based mouse model of microbiota deficiency, we demonstrate that gut microbiota depletion induces a juvenile neutrophil phenotype characterized by the upregulation of resting state surface receptors, reduced inflammatory proteins, and levels of circulating NETs. This disarming of neutrophil responses was associated with decreased expression of brain inflammatory genes, vascular thrombus formation, reduced infarct size, and alleviated behavioral deficits. We conclude that gut microbes strongly influence neutrophil activation after stroke and thus directly contribute to stroke severity.

Aim: The gut microbiota is implicated in the development of intestinal tumors. Furthermore, Western diet is a risk factor for colorectal cancer and induces alterations in both the microbiota and bile acid metabolism. Therefore, we aimed to investigate the causal role of Western diet-induced changes in the microbiota and secondary bile acid production, which were linked to disease exacerbation in APC 1311/+ pigs. Methods: We performed fecal microbiota transfer experiments by inoculating germfree Apc 1368N/+ mice with stool from genetically engineered APC 1311/+ pigs. A control group of Apc 1368N/+ mice stayed germfree. All mice were fed either a control diet, or the same diet supplemented with the primary bile acid cholic acid (CA) to stimulate secondary bile acid production. Results: Unexpectedly, the germfree mice fed CA had a high number of lesions in the upper small intestine, which was reduced by the colonization with microbes. The same mice (germfree, CA diet) were characterized by a remarkable lengthening of the small intestine (approximately +10 cm on average). Colonic lesions were rare and only observed in the mice that received stool from control pigs and fed the CA diet. Diversity and composition analyses showed that the microbiota transfer was incomplete. Nevertheless, mice receiving the Western diet-associated microbiota clustered separately from control animals. The effects of the CA diet on the microbiota were less pronounced and were observed primarily in mice that received stool from control pigs. Bile acid analysis in the recipient mice revealed associations between the phenotype and specific bile acid species in bile and cecum. Conclusion: This descriptive study highlights the importance of diet-microbiota-bile acid interactions in intestinal morphogenesis and tumorigenesis.

Inflammation has a pronounced impact on the intestinal ecosystem by driving an expansion of facultative anaerobic bacteria at the cost of obligate anaerobic microbiota. This pathogen “blooming” is also a hallmark of enteric Salmonella enterica serovar Typhimurium ( S . Tm) infection. Here, we analyzed the contribution of bacterial and host factors to S . Tm “blooming” in a gnotobiotic mouse model for S. Tm-induced enterocolitis. Mice colonized with the Oligo-Mouse-Microbiota (OMM 12 ), a minimal bacterial community, develop fulminant colitis by day 4 after oral infection with wild type S . Tm but not with an avirulent mutant. Inflammation leads to pronounced reduction in overall intestinal bacterial loads, distinct microbial community shifts and pathogen blooming (relative abundance >50%). S. Tm mutants attenuated in inducing gut inflammation generally elicit less pronounced microbiota shifts and reduction in total bacterial loads. In contrast, S. Tm mutants in nitrate respiration, salmochelin production and ethanolamine utilization induced strong inflammation and S . Tm “blooming”. Therefore, individual Salmonella -specific inflammation-fitness factors seem to be of minor importance for competition against this minimal microbiota in the inflamed gut. Finally, we show that antibody-mediated neutrophil depletion normalized gut microbiota loads but not intestinal inflammation or microbiota shifts. This suggests that neutrophils equally reduce pathogen and commensal bacterial loads in the inflamed gut.

Gut microbiota is responsible for essential functions in human health. Several communication axes between gut microbiota and other organs via neural, endocrine, and immune pathways have been described, and perturbation of gut microbiota composition has been implicated in the onset and progression of an emerging number of diseases. Here, we analyzed peripheral nerves, dorsal root ganglia (DRG), and skeletal muscles of neonatal and young adult mice with the following gut microbiota status: a) germ-free (GF), b) gnotobiotic, selectively colonized with 12 specific gut bacterial strains (Oligo-Mouse-Microbiota, OMM12), or c) natural complex gut microbiota (CGM). Stereological and morphometric analyses revealed that the absence of gut microbiota impairs the development of somatic median nerves, resulting in smaller diameter and hypermyelinated axons, as well as in smaller unmyelinated fibers. Accordingly, DRG and sciatic nerve transcriptomic analyses highlighted a panel of differentially expressed developmental and myelination genes. Interestingly, the type III isoform of Neuregulin1 (NRG1), known to be a neuronal signal essential for Schwann cell myelination, was overexpressed in young adult GF mice, with consequent overexpression of the transcription factor Early Growth Response 2 (Egr2), a fundamental gene expressed by Schwann cells at the onset of myelination. Finally, GF status resulted in histologically atrophic skeletal muscles, impaired formation of neuromuscular junctions, and deregulated expression of related genes. In conclusion, we demonstrate for the first time a gut microbiota regulatory impact on proper development of the somatic peripheral nervous system and its functional connection to skeletal muscles, thus suggesting the existence of a novel ‘Gut Microbiota-Peripheral Nervous System-axis.’

Germ-free (GF) rodents have become a valuable tool for studying the role of intestinal microbes on the host physiology. The major characteristic of GF rodents is an enlarged cecum. The accumulation of mucopolysaccharides, digestion enzymes and water in the intestinal lumen drives this phenotype. Microbial colonization normalizes the cecum size in ex-GF animals. However, whether strain genetics influences the cecal enlargement is unknown. Here we investigated the impact of mouse genetic background on the cecal size in five GF strains frequently used in biomedical research. The cecal weight of GF mice on B6 background (B6J and B6N) represented up to 20% of total body weight. GF NMRI and BALBc mice showed an intermediate phenotype of 5–10%, and those on the C3H background of up to 5%. Reduced cecal size in GF C3H mice correlated with decreased water content, increased expression of water transporters, and reduced production of acidic mucins, but was independent of the level of digestive enzymes in the lumen. In contrast, GF B6J mice with greatly enlarged cecum showed increased water content and a distinct metabolic profile characterized by altered amino acid and bile acid metabolism, and increased acidic mucin production. Together, our results show that genetic background influences the cecal enlargement by regulating the water transport, production of acidic mucins, and metabolic profiles.

Enteric glial cells (EGCs) were shown to maintain the barrier integrity and immune homeostasis of the bowel. Postnatally, EGCs develop from progenitor cells located in the myenteric plexus and are continuously replenished through adulthood. Both, murine EGC development and replenishment were shown to depend on the microbiome. The underlying mechanisms are still unknown, and we hypothesized that the myeloid differentiation primary response protein 88 (Myd88) or toll-like receptor signaling pathways may be involved. Adult and neonatal C57BL/6 wild-type (wt) and Myd88 −/− mice were housed under specific pathogen-free (SPF) or germ-free (GF) conditions. GF mice were further conventionalized by gavaging stools from, and cohousing with, SPF mice having intact microbiomes. The small bowels were harvested at various time points, and immunohistochemistry and qPCR analysis of EGC markers in the muscularis externa and mucosa were performed. In wt mice, after conventionalization, the glial cell-specific markers, glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein β (S100β), were upregulated in the mucosa and muscularis externa. In Myd88 −/− mice, this upregulation did not occur. Importantly, GFAP (only in the mucosa) and S100β (in both the mucosa and muscularis externa) were significantly reduced in conventionalized Myd88 −/− mice compared with the conventionalized wt mice. In neonatal mice, the gene expressions of GFAP and S100β increased between the day of birth (P0) and postnatal day 15 (P15) in the mucosa and muscularis externa of both wt and Myd88 −/− mice. Notably, in the mucosa but not the muscularis externa, at P15, the gene expressions of GFAP and S100β were significantly reduced in Myd88 −/− . Our data demonstrated that postnatal development and replenishment of EGCs require intestinal microbiota and depend on Myd88. The specific upstream mechanisms may involve toll-like-receptor recognition of the microbiota and will be the subject of further research.