The TNF∆ARE Model of Crohn's Disease-like Ileitis.

Crohn's disease (CD) is one of the 2 main phenotypes of inflammatory bowel diseases (IBDs); CD ischaracterized by a discontinuous, spontaneously recurring, transmural immunopathology that largely affects the terminal ileum. Crohn's disease exhibits both a relapsing and progressive course, and its prevalence is on the rise globally, mirroring the trends of industrialization. While the precise pathogenesis of CD remains unknown, various factors including immune cell dysregulation, microbial dysbiosis, genetic susceptibility, and environmental factors have been implicated in disease etiology. Animal models, particularly ileitis mouse models, have provided valuable tools for studying the specific mechanisms underlying CD, allowing longitudinal assessment and sampling in interventional preclinical studies. Furthermore, animal models assess to evaluate the distinct role that bacterial and dietary antigens play in causing inflammation, using germ-free animals, involving the introduction of individual bacteria (monoassociation studies), and experimenting with well-defined dietary components. An ideal animal model for studying IBD, specifically CD, should exhibit an inherent intestinal condition that arises spontaneously and closely mimics the distinct transmural inflammation observed in the human disease, particularly in the terminal ileum. We have recently characterized the impact of disease-relevant, noninfectious microbiota and specific bacteria in a mouse model that replicates CD-like ileitis, capturing the intricate nature of human CD, namely the TNF∆ARE mouse model. Using germ-free mice, we studied the impact of different diets on the expansion of disease-relevant pathobionts and on the severity of inflammation. In this review article, we review some of the currently available ileitis mouse models and discuss in detail the TNF∆ARE model of CD-like Ileitis.

Is Autologous Fecal Microbiota Transfer after Exclusive Enteral Nutrition in Pediatric Crohn's Disease Patients Rational and Feasible? Data from a Feasibility Test.

BACKGROUND: Exclusive enteral nutrition (EEN) is a highly effective therapy for remission induction in pediatric Crohn's disease (CD), but relapse rates after return to a regular diet are high. Autologous fecal microbiota transfer (FMT) using stool collected during EEN-induced clinical remission might represent a novel approach to maintaining the benefits of EEN. METHODS: Pediatric CD patients provided fecal material at home, which was shipped at 4 °C to an FMT laboratory for FMT capsule generation and extensive pathogen safety screening. The microbial community composition of samples taken before and after shipment and after encapsulation was characterized using 16S rRNA amplicon sequencing. RESULTS: Seven pediatric patients provided fecal material for nine test runs after at least three weeks of nutritional therapy. FMT capsules were successfully generated in 6/8 deliveries, but stool weight and consistency varied widely. Transport and processing of fecal material into FMT capsules did not fundamentally change microbial composition, but microbial richness was <30 genera in 3/9 samples. Stool safety screening was positive for potential pathogens or drug resistance genes in 8/9 test runs. CONCLUSIONS: A high pathogen burden, low-diversity microbiota, and practical deficiencies of EEN-conditioned fecal material might render autologous capsule-FMT an unsuitable approach as maintenance therapy for pediatric CD patients.

Diet prevents the expansion of segmented filamentous bacteria and ileo-colonic inflammation in a model of Crohn's disease.

BACKGROUND: Crohn's disease (CD) is associated with changes in the microbiota, and murine models of CD-like ileo-colonic inflammation depend on the presence of microbial triggers. Increased abundance of unknown Clostridiales and the microscopic detection of filamentous structures close to the epithelium of Tnf ΔARE mice, a mouse model of CD-like ileitis pointed towards segmented filamentous bacteria (SFB), a commensal mucosal adherent bacterium involved in ileal inflammation. RESULTS: We show that the abundance of SFB strongly correlates with the severity of CD-like ileal inflammation in two mouse models of ileal inflammation, including Tnf ΔARE and SAMP/Yit mice. SFB mono-colonization of germ-free Tnf ΔARE mice confirmed the causal link and resulted in severe ileo-colonic inflammation, characterized by elevated tissue levels of Tnf and Il-17A, neutrophil infiltration and loss of Paneth and goblet cell function. Co-colonization of SFB in human-microbiota associated Tnf ΔARE mice confirmed that SFB presence is indispensable for disease development. Screening of 468 ileal and colonic mucosal biopsies from adult and pediatric IBD patients, using previously published and newly designed human SFB-specific primer sets, showed no presence of SFB in human tissue samples, suggesting a species-specific functionality of the pathobiont. Simulating the human relevant therapeutic effect of exclusive enteral nutrition (EEN), EEN-like purified diet antagonized SFB colonization and prevented disease development in Tnf ΔARE mice, providing functional evidence for the protective mechanism of diet in modulating microbiota-dependent inflammation in IBD. CONCLUSIONS: We identified a novel pathogenic role of SFB in driving severe CD-like ileo-colonic inflammation characterized by loss of Paneth and goblet cell functions in Tnf ΔARE mice. A purified diet antagonized SFB colonization and prevented disease development in Tnf ΔARE mice in contrast to a fiber-containing chow diet, clearly demonstrating the important role of diet in modulating a novel IBD-relevant pathobiont and supporting a direct link between diet and microbial communities in mediating protective functions. Video Abstract.

TNF ΔARE Pigs: A Translational Crohn's Disease Model.

BACKGROUND AND AIMS: Crohn's disease [CD] is a major subtype of inflammatory bowel diseases [IBD] with increasing incidence and prevalence. Results of studies using available small and large animal models are often poorly translatable to patients, and few CD models show small intestinal pathology. Due to its similarities to humans, the pig has emerged as a highly suitable translational disease model, particularly for testing novel nutritional and technological interventions. Our goal was to develop a physiologically relevant porcine CD model to facilitate translation of findings and interventions towards the clinic. METHODS: We generated pigs bearing a 93-bp deletion of the adenosine-uracil-rich element [ARE] and a constitutive-decay element within the 3' untranslated region of the TNF gene. Comparative analysis of physiological, molecular, histological and microbial characteristics was performed between wild-type, TNFΔARE/+ and TNFΔARE/ΔARE animals. Alterations in the microbiome were compared to the TNFΔARE mouse model and IBD patients. RESULTS: TNF ΔARE pigs recapitulate major characteristics of human CD, including ulcerative transmural ileocolitis, increased abundance of proinflammatory cytokines, immune cell infiltration and dysbiotic microbial communities. 16S rRNA gene amplicon sequencing revealed enrichment in members belonging to Megasphaera, Campylobacter, Desulfovibrio, Alistipes and Lachnoclostridum in faecal or mucosa-associated bacteria compared to wild-type littermates. Principal components analysis clustering with a subset of TNFΔARE/+ mice and human IBD patients suggests microbial similarity based on disease severity. CONCLUSIONS: We demonstrate that the TNFΔARE pig resembles a CD-like ileocolitis pathophenotype recapitulating human disease. The ability to conduct long-term studies and test novel surgical procedures and dietary interventions in a physiologically relevant model will benefit future translational IBD research studies.

Microbiome risk profiles as biomarkers for inflammatory and metabolic disorders.

The intestine harbours a complex array of microorganisms collectively known as the gut microbiota. The past two decades have witnessed increasing interest in studying the gut microbiota in health and disease, largely driven by rapid innovation in high-throughput multi-omics technologies. As a result, microbial dysbiosis has been linked to many human pathologies, including type 2 diabetes mellitus and inflammatory bowel disease. Integrated analyses of multi-omics data, including metagenomics and metabolomics along with measurements of host response and cataloguing of bacterial isolates, have identified many bacteria and bacterial products that are correlated with disease. Nevertheless, insight into the mechanisms through which microbes affect intestinal health requires going beyond correlation to causation. Current understanding of the contribution of the gut microbiota to disease causality remains limited, largely owing to the heterogeneity of microbial community structures, interindividual differences in disease evolution and incomplete understanding of the mechanisms that integrate microbiota-derived signals into host signalling pathways. In this Review, we provide a broad insight into the microbiome signatures linked to inflammatory and metabolic disorders, discuss outstanding challenges in this field and propose applications of multi-omics technologies that could lead to an improved mechanistic understanding of microorganism-host interactions.

Modeling microbiota-associated human diseases: from minimal models to complex systems.

Alterations in the intestinal microbiota are associated with various human diseases of the digestive system, including obesity and its associated metabolic diseases, inflammatory bowel diseases (IBD), and colorectal cancer (CRC). All three diseases are characterized by modifications of the richness, composition, and metabolic functions of the human intestinal microbiota. Despite being multi-factorial diseases, studies in germ-free animal models have unarguably identified the intestinal microbiota as a causal driver of disease pathogenesis. However, for an increased mechanistic understanding of microbial signatures in human diseases, models require detailed refinement to closely mimic the human microbiota and reflect the complexity and range of dysbiosis observed in patients. The transplantation of human fecal microbiota into animal models represents a powerful tool for studying the causal and functional role of the dysbiotic human microbiome in a pathological context. While human microbiota-associated models were initially employed to study obesity, an increasing number of studies have applied this approach in the context of IBD and CRC over the past decade. In this review, we discuss different approaches that allow the functional validation of the bacterial contribution to human diseases, with emphasis on obesity and its associated metabolic diseases, IBD, and CRC. We discuss the utility of simple models, such as in vitro fermentation systems of the human microbiota and ex vivo intestinal organoids, as well as more complex whole organism models. Our focus here lies on human microbiota-associated mouse models in the context of all three diseases, as well as highlighting the advantages and limitations of this approach.

High-Fructose Diet Alters Intestinal Microbial Profile and Correlates with Early Tumorigenesis in a Mouse Model of Barrett's Esophagus.

Esophageal adenocarcinoma (EAC) is mostly prevalent in industrialized countries and has been associated with obesity, commonly linked with a diet rich in fat and refined sugars containing high fructose concentrations. In meta-organisms, dietary components are digested and metabolized by the host and its gut microbiota. Fructose has been shown to induce proliferation and cell growth in pancreas and colon cancer cell lines and also alter the gut microbiota. In a previous study with the L2-IL-1B mouse model, we showed that a high-fat diet (HFD) accelerated EAC progression from its precursor lesion Barrett's esophagus (BE) through changes in the gut microbiota. Aiming to investigate whether a high-fructose diet (HFrD) also alters the gut microbiota and favors EAC carcinogenesis, we assessed the effects of HFrD on the phenotype and intestinal microbial communities of L2-IL1B mice. Results showed a moderate acceleration in histologic disease progression, a mild effect on the systemic inflammatory response, metabolic changes in the host, and a shift in the composition, metabolism, and functionality of intestinal microbial communities. We conclude that HFrD alters the overall balance of the gut microbiota and induces an acceleration in EAC progression in a less pronounced manner than HFD.

Development of a Highly Sensitive Ultra-High-Performance Liquid Chromatography Coupled to Electrospray Ionization Tandem Mass Spectrometry Quantitation Method for Fecal Bile Acids and Application on Crohn's Disease Studies.

In addition to their important role in fat digestion, bile acids are increasingly being used as markers for various diseases. The large diversity of bile acids results from the conversion of primary and conjugated bile acids into secondary bile acids by deconjugation and dehydroxylation reactions mediated by the intestinal microbiota. Here, we describe a fast and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for absolute quantitation of 45 bile acids in human or mouse feces in combination with a simple workup and extraction procedure. Method validation outlined excellent limits of detection and quantitation, linearity, selectivity, recovery, extraction loss, and precision. To investigate the connection between microbiome alterations and bile acid metabolism, the method was applied on a Crohn's disease study including patients with histologically documented active disease or remission as well as on a model using humanized mice. As the complex mechanism including genetic and environmental factors leading to the development of Crohn's disease is so far not completely understood, the study investigates the microbial metabolism of bile acids and the potential use of bile acid profiles to predict disease state.

Modeling microbe-host interaction in the pathogenesis of Crohn's disease.

Alterations in the gut microbiota structure and function are thought to play an important role in the pathogenesis of Crohn's disease (CD). The rapid advancement of high-throughput sequencing technologies led to the identification of microbiome risk signatures associated with distinct disease phenotypes and progressing disease entities. Functional validation of the identified microbiome signatures is essential to understand the underlying mechanisms of microbe-host interactions. Germfree mouse models are available to study the functional role of disease-conditioning complex gut microbial ecosystems (dysbiosis) or pathobionts (single bacteria) in the pathogenesis of CD-like inflammation. Here, we discuss the clinical and mechanistic relevance and limitations of gnotobiotic mouse models in the context of CD. In addition, we will address the role of diet as an essential external factor modulating microbiome changes, potentially underlying disease initiation and development.

Multi-omic modelling of inflammatory bowel disease with regularized canonical correlation analysis.

BACKGROUND: Personalized medicine requires finding relationships between variables that influence a patient's phenotype and predicting an outcome. Sparse generalized canonical correlation analysis identifies relationships between different groups of variables. This method requires establishing a model of the expected interaction between those variables. Describing these interactions is challenging when the relationship is unknown or when there is no pre-established hypothesis. Thus, our aim was to develop a method to find the relationships between microbiome and host transcriptome data and the relevant clinical variables in a complex disease, such as Crohn's disease. RESULTS: We present here a method to identify interactions based on canonical correlation analysis. We show that the model is the most important factor to identify relationships between blocks using a dataset of Crohn's disease patients with longitudinal sampling. First the analysis was tested in two previously published datasets: a glioma and a Crohn's disease and ulcerative colitis dataset where we describe how to select the optimum parameters. Using such parameters, we analyzed our Crohn's disease data set. We selected the model with the highest inner average variance explained to identify relationships between transcriptome, gut microbiome and clinically relevant variables. Adding the clinically relevant variables improved the average variance explained by the model compared to multiple co-inertia analysis. CONCLUSIONS: The methodology described herein provides a general framework for identifying interactions between sets of omic data and clinically relevant variables. Following this method, we found genes and microorganisms that were related to each other independently of the model, while others were specific to the model used. Thus, model selection proved crucial to finding the existing relationships in multi-omics datasets.

Infusion of donor feces affects the gut-brain axis in humans with metabolic syndrome.

OBJECTIVE: Increasing evidence indicates that intestinal microbiota play a role in diverse metabolic processes via intestinal butyrate production. Human bariatric surgery data suggest that the gut-brain axis is also involved in this process, but the underlying mechanisms remain unknown. METHODS: We compared the effect of fecal microbiota transfer (FMT) from post-Roux-en-Y gastric bypass (RYGB) donors vs oral butyrate supplementation on (123I-FP-CIT-determined) brain dopamine transporter (DAT) and serotonin transporter (SERT) binding as well as stable isotope-determined insulin sensitivity at baseline and after 4 weeks in 24 male and female treatment-naïve metabolic syndrome subjects. Plasma metabolites and fecal microbiota were also determined at these time points. RESULTS: We observed an increase in brain DAT after donor FMT compared to oral butyrate that reduced this binding. However, no effect on body weight and insulin sensitivity was demonstrated after post-RYGB donor feces transfer in humans with metabolic syndrome. Increases in fecal levels of Bacteroides uniformis were significantly associated with an increase in DAT, whereas increases in Prevotella spp. showed an inverse association. Changes in the plasma metabolites glycine, betaine, methionine, and lysine (associated with the S-adenosylmethionine cycle) were also associated with altered striatal DAT expression. CONCLUSIONS: Although more and larger studies are needed, our data suggest a potential gut microbiota-driven modulation of brain dopamine and serotonin transporters in human subjects with obese metabolic syndrome. These data also suggest the presence of a gut-brain axis in humans that can be modulated. NTR REGISTRATION: 4488.

Integrated microbiota and metabolite profiles link Crohn's disease to sulfur metabolism.

Gut microbial and metabolite alterations have been linked to the pathogenesis of inflammatory bowel diseases. Here we perform a multi-omics microbiome and metabolite analysis of a longitudinal cohort of Crohn's disease patients undergoing autologous hematopoietic stem cell transplantation, and investigational therapy that induces drug free remission in a subset of patients. Via comparison of patients who responded and maintained remission, responded but experienced disease relapse and patients who did not respond to therapy, we identify shared functional signatures that correlate with disease activity despite the variability of gut microbiota profiles at taxonomic level. These signatures reflect the disease state when transferred to gnotobiotic mice. Taken together, the integration of microbiome and metabolite profiles from human cohort and mice improves the predictive modelling of disease outcome, and allows the identification of a network of bacteria-metabolite interactions involving sulfur metabolism as a key mechanism linked to disease activity in Crohn's disease.

Partial enteral nutrition has no benefit on bone health but improves growth in paediatric patients with quiescent or mild Crohn's disease.

BACKGROUND AND AIMS: Exclusive enteral nutrition induces remission, improves bone health and growth in paediatric Crohn's disease (CD) patients, but is highly demanding for patients. We investigated efficacy of partial enteral nutrition (PEN) on bone health, growth and course in CD patients and assessed microbial and metabolic changes induced by PEN. METHODS: We performed a two centre, non-randomized controlled intervention study in quiescent CD patients aged <19 years. Patients in intervention group received a liquid formula providing ~25% of daily energy for one year. At baseline, after 3, 6, 9 and 12 months, we collected data on bone, muscle (peripheral quantitative computertomography), anthropometry, disease activity (weighted paediatric CD activity index), metabolomic profile (liquid chromatography mass spectrometry), and faecal microbiome (16S rRNA gene sequencing). RESULTS: Of 41 CD patients, 22 received the intervention (PEN) (mean age 15.0 ± 1.9 years, 50% male), 19 served as controls (non-PEN) (12.8 ± 3.1 years, 58% male). At baseline, mean bone quality was comparable to reference population with no improvement during the intervention. Relapse rate was low (8/41, PEN 4/22 and non-PEN 4/19, ns). PEN was not associated with microbiota community changes (beta diversity) but significantly reduced species diversity. Metabolome changes with upregulation of phosphatidylcholines in PEN patients are likely related to lipid and fatty acid composition of the formula. PEN significantly improved growth in a subgroup with Tanner stage 1-3. CONCLUSION: In our cohort of paediatric CD patients, PEN did not affect bone health but improved growth in patients with a potential to grow.

Mitochondrial impairment drives intestinal stem cell transition into dysfunctional Paneth cells predicting Crohn's disease recurrence.

OBJECTIVE: Reduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn's disease (CD). PCs reside in proximity to Lgr5+ intestinal stem cells (ISC) and mitochondria are critical for ISC-renewal and differentiation. Here, we characterise ISC and PC appearance under inflammatory conditions and describe the role of mitochondrial function for ISC niche-maintenance. DESIGN: Ileal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60Δ/ΔISC) and CD-like ileitis (TNFΔARE), and intestinal organoids were used to characterise PCs and ISCs in relation to mitochondrial function. RESULTS: In patients with CD and TNFΔARE mice, inflammation correlated with reduced numbers of Lysozyme-positive granules in PCs and decreased Lgr5 expression in crypt regions. Disease-associated changes in PC and ISC appearance persisted in non-inflamed tissue regions of patients with CD and predicted the risk of disease recurrence after surgical resection. ISC-specific deletion of Hsp60 and inhibition of mitochondrial respiration linked mitochondrial function to the aberrant PC phenotype. Consistent with reduced stemness in vivo, crypts from inflamed TNFΔARE mice fail to grow into organoids ex vivo. Dichloroacetate-mediated inhibition of glycolysis, forcing cells to shift to mitochondrial respiration, improved ISC niche function and rescued the ability of TNFΔARE mice-derived crypts to form organoids. CONCLUSION: We provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.

Strain-Level Diversity in the Gut: The P. copri Case.

In this issue of Cell Host & Microbe, De Filippis et al. (2019) report that Prevotella copri strain-level diversity in the gut microbiome can be shaped by host diet. Individual signatures were analyzed by marker gene profiling in assembled pangenomes, providing a strong rationale for the functional adaptation of individual microbial ecosystems in response to diet.

Microbial Signatures as a Predictive Tool in IBD-Pearls and Pitfalls.

Studies of microbial signatures have improved our understanding of the role of dysbiosis in gut microbiota for the pathogenesis of inflammatory bowel disease (IBD). New technological advances such as next-generation sequencing facilitate investigations on large patient cohorts, but require methodological considerations regarding study design, sample processing, data analysis, and integration. Here, we summarize recent study approaches in microbial ecology with respect to IBD research and discuss crucial process steps for the production and integration of adequate data sets.