Positive modulation of a new reconstructed human gut microbiota by Maitake extract helpfully boosts the intestinal environment in vitro.

The human gut is a complex environment where the microbiota and its metabolites play a crucial role in the maintenance of a healthy state. The aim of the present work is the reconstruction of a new in vitro minimal human gut microbiota resembling the microbe-microbe networking comprising the principal phyla (Bacillota, Bacteroidota, Pseudomonadota, and Actinomycetota), to comprehend the intestinal ecosystem complexity. In the reductionist model, we mimicked the administration of Maitake extract as prebiotic and a probiotic formulation (three strains belonging to Lactobacillus and Bifidobacterium genera), evaluating the modulation of strain levels, the release of beneficial metabolites, and their health-promoting effects on human cell lines of the intestinal environment. The administration of Maitake and the selected probiotic strains generated a positive modulation of the in vitro bacterial community by qPCR analyses, evidencing the prominence of beneficial strains (Lactiplantibacillus plantarum and Bifidobacterium animalis subsp. lactis) after 48 hours. The bacterial community growths were associated with the production of metabolites over time through GC-MSD analyses such as lactate, butyrate, and propionate. Their effects on the host were evaluated on cell lines of the intestinal epithelium and the immune system, evidencing positive antioxidant (upregulation of SOD1 and NQO1 genes in HT-29 cell line) and anti-inflammatory effects (production of IL-10 from all the PBMCs). Therefore, the results highlighted a positive modulation induced by the synergic activities of probiotics and Maitake, inducing a tolerogenic microenvironment.

Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Bacillus halotolerans SW207 alleviates enterotoxigenic Escherichia coli-induced inflammatory responses in weaned piglets by modulating the intestinal epithelial barrier, the TLR4/MyD88/NF-κB pathway, and intestinal microbiota.

Enterotoxigenic Escherichia coli (ETEC) is one of the major pathogens contributing to piglet diarrhea, with significant implications for both piglet health and the economic aspects of the livestock industry. SW207 is an isolate of Bacillus halotolerans isolated from the cold- and disease-resistant Leixiang pigs in Northeastern China. We have discovered that SW207 can survive in the pig's gastrointestinal fluid and under conditions of high bile salt concentration, displaying potent antagonistic activity against ETEC. In this study, we established a weaned piglet diarrhea model infected with ETEC to investigate the role of SW207 in preventing diarrhea and improving intestinal health. Results indicate that SW207 upregulates the expression of tight junction proteins, including claudin-1, occludin, and zonula occludens-1, at both the transcriptional and translational levels. Furthermore, SW207 reduces serum endotoxin, D-lactic acid, and various oxidative stress markers while enhancing piglet mechanical barrier function. In terms of immune barrier, SW207 suppressed the activation of the TLR4/MyD88/NF-κB pathway, reducing the expression of various inflammatory factors and upregulating the expression of small intestine mucosal sIgA. Concerning the biological barrier, SW207 significantly reduces the content of E. coli in the intestines and promotes the abundance of beneficial bacteria, thereby mitigating the microbiota imbalance caused by ETEC. In summary, SW207 has the potential to prevent weaned piglet diarrhea caused by ETEC, alleviate intestinal inflammation and epithelial damage, and facilitate potential beneficial changes in the intestinal microbiota. This contributes to elucidating the potential mechanisms of host-microbe interactions in preventing pathogen infections.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC) has consistently been one of the significant pathogens causing mortality in weaned piglets in pig farming. The industry has traditionally relied on antibiotic administration to control ETEC-induced diarrhea. However, the overuse of antibiotics has led to the emergence of drug-resistant zoonotic bacterial pathogens, posing a threat to public health. Therefore, there is an urgent need to identify alternatives to control pathogens and reduce antibiotic usage. In this study, we assessed the protective effect of a novel probiotic in a weaned piglet model infected with ETEC and analyzed its mechanisms both in vivo and in vitro. The study results provide theoretical support and reference for implementing interventions in the gut microbiota to alleviate early weaned piglet diarrhea and improve intestinal health.

Extraction of Mucins from the Mammalian Intestinal Tract.

In the intestine, mucus covering the mucosa plays a critical role in maintaining gut homeostasis by protecting the mucosa from invasion by commensal bacteria. The gut mucus is composed primarily of MUC2 mucin secreted by goblet cells. MUC2 is highly O-glycosylated, and O-glycans are necessary for the function and polymer structure of MUC2. In addition, recent evidence revealed that several glycan modifications, such as sialylation and sulfation, confer resistance of mucins to proteolysis and affect the viscosity and lubricity of mucus. Therefore, characterizing glycan structures of mucins is required to understand their functions fully. In this chapter, we describe how to purify secreted mucins from the mammalian intestine for analysis of their glycan structures. This description includes the extraction of MUC2 mucin from the mucosal surface of the mouse colon and colon explants.

Pathogens in Crohn's Disease: The Role of Adherent Invasive Escherichia coli.

In Crohn's disease (CD), gut dysbiosis is marked by the prevalence of pathogenic bacterial species. Although several microbes have been reported as risk factors or causative agents of CD, it is not yet clear which is the real trigger of the disease. Thirty years ago, a new pathovar of Escherichia coli strain was isolated in the ileal mucosa of CD patients. This strain, called adherent invasive E. coli (AIEC), for its ability to invade the intestinal mucosa, could represent the causative agent of the disease. Several authors studied the mechanisms by which the AIEC penetrate and replicate within macrophages, and release inflammatory cytokines sustaining inflammation. In this review we will discuss about the role of AIEC in the pathogenesis of CD, the virulence factors mediating adhesion and invasion of AIEC in mucosal tissue, the environmental conditions improving AIEC survival and replication within macrophages. Finally, we will also give an overview of the new strategies developed to limit AIEC overgrowth.

Microbial interventions in yak colibacillosis: Lactobacillus-mediated regulation of intestinal barrier.

Introduction: The etiology of Escherichia coli in yaks, along with its drug resistance, results in economic losses within the yak breeding industry. The utilization of lactic acid bacteria treatment has emerged as a viable alternative to antibiotics in managing colibacillosis. Methods: To elucidate the therapeutic mechanisms of Lactobacillus against Escherichia coli-induced intestinal barrier damage in yaks, we employed yak epithelial cells as the experimental model and established a monolayer epithelial barrier using Transwell. The study encompassed four groups: a control group, a model group (exposed to E. coli O78), a low-dose Lactobacillus group (E. coli O78 + 1 × 105CFU LAB), and a high-dose Lactobacillus group (E. coli O78 + 1 × 107CFU LAB). Various techniques, including transmembrane resistance measurement, CFU counting, RT-qPCR, and Western Blot, were employed to assess indicators related to cell barrier permeability and tight junction integrity. Results: In the Model group, Escherichia coli O78 significantly compromised the permeability and tight junction integrity of the yak epithelial barrier. It resulted in decreased transmembrane resistance, elevated FD4 flux, and bacterial translocation. Furthermore, it downregulated the mRNA and protein expression of MUC2, Occludin, and ZO-1, while upregulating the mRNA expression and protein expression of FABP2 and Zonulin, thereby impairing intestinal barrier function. Contrastingly, Lactobacillus exhibited a remarkable protective effect. It substantially increased transmembrane resistance, mitigated FD4 flux, and reduced bacterial translocation. Moreover, it significantly upregulated the mRNA and protein expression of MUC2, Occludin, and ZO-1, while downregulating the mRNA and protein expression of FABP2 and Zonulin. Notably, high-dose LAB demonstrated superior regulatory effects compared to the low-dose LAB group. Discussion: In conclusion, our findings suggest that Lactobacillus holds promise in treating yak colibacillosis by enhancing mucin and tight junction protein expression. Furthermore, we propose that Lactobacillus achieves these effects through the regulation of Zonulin.

Development of a new antibiotic-induced dysbiosis model of the canine colonic microbiota.

As in humans, antibiotics are widely used in dogs to treat gastrointestinal infections, contributing to the global burden of antimicrobial resistance on both human and animal health. Close contact between pets and their owners can lead to horizontal transfer of gut microbes, including transmission of antibiotic resistance. Nevertheless, until now, the impact of antibiotics on the canine gut microbiota has been poorly described. The aim of this study was to adapt the canine mucosal artificial colon (CANIM-ARCOL) model, reproducing the main nutritional, physicochemical and microbial parameters found in the large intestine of the dog to simulate an antibiotic-induced perturbation. Following initial investigation of five antibiotic cocktails at in-field doses, a 5-day regimen of metronidazole/enrofloxacin (ME) was selected for further model development. Two CANIM-ARCOL bioreactors were inoculated with a faecal sample (n=2 donors) and run in parallel for 26 days under control or antibiotic conditions. ME reduced microbial diversity and induced major shifts in bacterial populations, leading to a state of dysbiosis characterized by an increase in the relative abundance of Streptococcaceae, Lactobacillaceae and Enterobacteriaceae, and a decrease in the relative abundance of Bacteroidaceae, Fusobacteriota and Clostridiaceae. Overall, mucus-associated microbiota were less impacted by antibiotics than luminal microbes. Microbial alterations were associated with drastic decreases in gas production and short-chain fatty acid concentrations. Finally, the model was well validated through in-vitro-in-vivo comparisons in a study in dogs. The CANIM-ARCOL model provides a relevant platform as an alternative to in-vivo assays for an in-depth understanding of antibiotic-microbiota interactions and further testing of restoration strategies at individual level.

A bacterial sialidase mediates early-life colonization by a pioneering gut commensal.

The early microbial colonization of the gastrointestinal tract can have long-term impacts on development and health. Keystone species, including Bacteroides spp., are prominent in early life and play crucial roles in maintaining the structure of the intestinal ecosystem. However, the process by which a resilient community is curated during early life remains inadequately understood. Here, we show that a single sialidase, NanH, in Bacteroides fragilis mediates stable occupancy of the intestinal mucosa in early life and regulates a commensal colonization program. This program is triggered by sialylated glycans, including those found in human milk oligosaccharides and intestinal mucus. NanH is required for vertical transmission from dams to pups and promotes B. fragilis dominance during early life. Furthermore, NanH facilitates commensal resilience and recovery after antibiotic treatment in a defined microbial community. Collectively, our study reveals a co-evolutionary mechanism between the host and microbiota mediated through host-derived glycans to promote stable colonization.

Gut complement induced by the microbiota combats pathogens and spares commensals.

Canonically, the complement system is known for its rapid response to remove microbes in the bloodstream. However, relatively little is known about a functioning complement system on intestinal mucosal surfaces. Herein, we report the local synthesis of complement component 3 (C3) in the gut, primarily by stromal cells. C3 is expressed upon commensal colonization and is regulated by the composition of the microbiota in healthy humans and mice, leading to an individual host's specific luminal C3 levels. The absence of membrane attack complex (MAC) components in the gut ensures that C3 deposition does not result in the lysis of commensals. Pathogen infection triggers the immune system to recruit neutrophils to the infection site for pathogen clearance. Basal C3 levels directly correlate with protection against enteric infection. Our study reveals the gut complement system as an innate immune mechanism acting as a vigilant sentinel that combats pathogens and spares commensals.

Research Note: Intestinal avian defensin 2 and robustness of chicks.

Poultry production is an important agricultural sector for human food worldwide. Chicks after hatch often face health problems leading to economic losses that are deleterious for breeders. Avian defensin 2 (AvBD2) is a prominent host defense peptide of the intestinal mucosa of cecum and is involved in the resistance of poultry to bacterial pathogens. This peptide could thus represent an innate immunity marker of robustness of birds. To test this hypothesis by comparing fast-growing and slow-growing lines in different conditions of breeding, the chick's cecal AvBD2 content was analyzed according to animal quality and immunity indicators. Chick's cecal tissue sections labeled by immunohistochemistry with newly developed specific antibodies revealed the localization of AvBD2 in the mucosa with high individual variability, without showing differences attributable to quality indicators, but interestingly showing inverse correlation with seric IgM levels in the fast-growing line. The availability of our anti-AvBD2 antibodies to the scientific community opens perspectives to identify the cellular sources of this defensin in the cecal mucosa and to investigate the organization and function of innate immune arsenal of birds.

Crohn's disease-associated Escherichia coli LF82 in the gut damage of germ-free honeybees: A laboratory study.

Escherichia coli LF82 (LF82) is associated with Crohn's disease. The simplicity and genetic maneuverability of honeybees' gut microbiota make them suitable for studying host-microbe interactions. To understand the interaction between LF82 and host gut, LF82 was used to infect germ-free honeybees (Apis mellifera) orally. We found that LF82 successfully colonized the gut and shortened the lifespan of germ-free bees. LF82 altered the gut structure and significantly increased gut permeability. RT-qPCR showed that LF82 infection activated anti-infective immune pathways and upregulated the mRNAs levels of antimicrobial peptides in the gut of germ-free bees. The gut transcriptome showed that LF82 significantly upregulated genes involved in Notch signaling, adhesion junctions, and Toll and Imd signaling pathways and downregulated genes involved in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, protein digestion and absorption, and tyrosine metabolism. In conclusion, the human-derived enteropathogenic bacterium LF82 can successfully colonize the gut of germ-free honeybees and cause enteritis-like changes, which provides an ideal model organism for revealing the pathogenesis of bacterial-associated diseases.

Butyrate reduces adherent-invasive E. coli-evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3.

Gut bacteria provide benefits to the host and have been implicated in inflammatory bowel disease (IBD), where adherent-invasive E. coli (AIEC) pathobionts (e.g., strain LF82) are associated with Crohn's disease. E. coli-LF82 causes fragmentation of the epithelial mitochondrial network, leading to increased epithelial permeability. We hypothesized that butyrate would limit the epithelial mitochondrial disruption caused by E. coli-LF82. Human colonic organoids and the T84 epithelial cell line infected with E. coli-LF82 (MOI = 100, 4 h) showed a significant increase in mitochondrial network fission that was reduced by butyrate (10 mM) co-treatment. Butyrate reduced the loss of mitochondrial membrane potential caused by E. coli-LF82 and increased expression of PGC-1α mRNA, the master regulator of mitochondrial biogenesis. Metabolomics revealed that butyrate significantly altered E. coli-LF82 central carbon metabolism leading to diminished glucose uptake and increased succinate secretion. Correlating with preservation of mitochondrial network form/function, butyrate reduced E. coli-LF82 transcytosis across T84-cell monolayers. The use of the G-protein inhibitor, pertussis toxin, implicated GPCR signaling as critical to the effect of butyrate, and the free fatty acid receptor three (FFAR3, GPR41) agonist, AR420626, reproduced butyrate's effect in terms of ameliorating the loss of barrier function and reducing the mitochondrial fragmentation observed in E. coli-LF82 infected T84-cells and organoids. These data indicate that butyrate helps maintain epithelial mitochondrial form/function when challenged by E. coli-LF82 and that this occurs, at least in part, via FFAR3. Thus, loss of butyrate-producing bacteria in IBD in the context of pathobionts would contribute to loss of epithelial mitochondrial and barrier functions that could evoke disease and/or exaggerate a low-grade inflammation.

A consortia of clinical E. coli strains with distinct in vitro adherent/invasive properties establish their own co-colonization niche and shape the intestinal microbiota in inflammation-susceptible mice.

BACKGROUND: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in vitro definition fully predicts mucosal colonization in vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. RESULTS: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortium of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. CONCLUSIONS: Our findings establish the in vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in vivo colonization dynamics of patient-derived bacteria in murine models. Video Abstract.

Estradiol mediates colonic epithelial protection in aged mice after stroke and is associated with shifts in the gut microbiome.

The gut is a major source of bacteria and antigens that contribute to neuroinflammation after brain injury. Colonic epithelial cells (ECs) are responsible for secreting major cellular components of the innate defense system, including antimicrobial proteins (AMP) and mucins. These cells serve as a critical regulator of gut barrier function and maintain host-microbe homeostasis. In this study, we determined post-stroke host defense responses at the colonic epithelial surface in mice. We then tested if the enhancement of these epithelial protective mechanisms is beneficial in young and aged mice after stroke. AMPs were significantly increased in the colonic ECs of young males, but not in young females after experimental stroke. In contrast, mucin-related genes were enhanced in young females and contributed to mucus formation that maintains the distance between the host and gut bacteria. Bacterial community profiling was done using universal amplification of 16S rRNA gene sequences. The sex-specific colonic epithelial defense responses after stroke in young females were reversed with ovariectomy and led to a shift from a predominately mucin response to the enhanced AMP expression seen in males after stroke. Estradiol (E2) replacement prior to stroke in aged females increased mucin gene expression in the colonic ECs. Interestingly, we found that E2 treatment reduced stroke-associated neuronal hyperactivity in the insular cortex, a brain region that interacts with visceral organs such as the gut, in parallel to an increase in the composition of Lactobacillus and Bifidobacterium in the gut microbiota. This is the first study demonstrating sex differences in host defense mechanisms in the gut after brain injury.

Detecting host responses to microbial stimulation using primary epithelial organoids.

The intestinal epithelium is constantly exposed to microbes residing in the lumen. Traditionally, the response to microbial interactions has been studied in cell lines derived from cancerous tissues, e.g. Caco-2. It is, however, unclear how the responses in these cancer cell lines reflect the responses of a normal epithelium and whether there might be microbial strain-specific effects. To address these questions, we derived organoids from the small intestine from a cohort of healthy individuals. Culturing intestinal epithelium on a flat laminin matrix induced their differentiation, facilitating analysis of microbial responses via the apical membrane normally exposed to the luminal content. Here, it was evident that the healthy epithelium across multiple individuals (n = 9) demonstrates robust acute both common and strain-specific responses to a range of probiotic bacterial strains (BB-12Ⓡ, LGGⓇ, DSM33361, and Bif195). Importantly, parallel experiments using the Caco-2 cell line provide no acute response. Collectively, we demonstrate that primary epithelial cells maintained as organoids represent a valuable resource for assessing interactions between the epithelium and luminal microbes across individuals, and that these models are likely to contribute to a better understanding of host microbe interactions.

Lcn2 deficiency accelerates the infection of Escherichia coli O157:H7 by disrupting the intestinal barrier function.

Bacterial infections result in intestinal inflammation and injury, which affects gut health and nutrient absorption. Lipocalin 2 (Lcn2) is a protein that reacts to microbial invasion, inflammatory responses, and tissue damage. However, it remains unclear whether Lcn2 has a protective effect against bacterial induced intestinal inflammation. Therefore, this study endeavors to investigate the involvement of Lcn2 in the intestinal inflammation of mice infected with Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157:H7). Lcn2 knockout (Lcn2-/-) mice were used to evaluate the changes of inflammatory responses. Lcn2 deficiency significantly exacerbated clinical symptoms of E. coli O157:H7 infection by reducing body weight and encouraging bacterial colonization of. Compared to infected wild type mice, infected Lcn2-/- mice had significantly elevated levels of pro-inflammatory cytokines in serum and ileum, including interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α), as well as severe villi destruction in the jejunum. Furthermore, Lcn2 deficiency aggravated intestinal barrier degradation by significantly reducing the expression of tight junction proteins occludin and claudin 1, the content of myeloperoxidase (MPO) in the ileum, and the number of goblet cells in the colon. Our findings indicated that Lcn2 could alleviate inflammatory damage caused by E. coli O157:H7 infection in mice by enhancing intestinal barrier function.

HIV-1 treatment timing shapes the human intestinal memory B-cell repertoire to commensal bacteria.

HIV-1 infection causes severe alterations of gut mucosa, microbiota and immune system, which can be curbed by early antiretroviral therapy. Here, we investigate how treatment timing affects intestinal memory B-cell and plasmablast repertoires of HIV-1-infected humans. We show that only class-switched memory B cells markedly differ between subjects treated during the acute and chronic phases of infection. Intestinal memory B-cell monoclonal antibodies show more prevalent polyreactive and commensal bacteria-reactive clones in late- compared to early-treated individuals. Mirroring this, serum IgA polyreactivity and commensal-reactivity are strongly increased in late-treated individuals and correlate with intestinal permeability and systemic inflammatory markers. Polyreactive blood IgA memory B cells, many of which egressed from the gut, are also substantially enriched in late-treated individuals. Our data establish gut and systemic B-cell polyreactivity to commensal bacteria as hallmarks of chronic HIV-1 infection and suggest that initiating treatment early may limit intestinal B-cell abnormalities compromising HIV-1 humoral response.

Regional changes in intestinal permeability in cirrhosis are associated with mucosal bacteria.

BACKGROUND: Several complications of cirrhosis are theorized to result from the translocation of bacteria or their products across the intestinal epithelium. We aimed to assess epithelial permeability and associations with mucosal bacteria in patients with cirrhosis. APPROACH AND RESULTS: We collected 247 duodenum, ileum, and colon biopsies from 58 consecutive patients with cirrhosis and 33 controls during clinically indicated endoscopies. Patients with cirrhosis were similarly aged to controls (60 vs. 58 y) and had a median Model for End-stage Liver Disease of 8 (interquartile range 7, 10). Biopsies underwent 16S rRNA-encoding gene amplicon sequencing to determine mucosal bacteria composition and transepithelial electrical resistance (TEER) to determine epithelial permeability. In the entire cohort, there were regional differences in TEER with the lowest TEER (ie, more permeable) in the ileum; duodenum TEER was 43% higher and colon TEER 20% higher than ileum TEER (ANOVA p = 0.0004). When comparing patients with cirrhosis and controls, both TEER (26% lower in cirrhosis, p = 0.006) and alpha diversity differed in the duodenum (27% lower in cirrhosis, p = 0.01) but not ileum or colon. A beta-binomial model found that 26 bacteria were significantly associated with TEER. Bifidobacteriaceae Bifidobacterium in duodenal mucosa was protective of epithelial permeability and future hospitalization for hepatic decompensation. CONCLUSIONS: Duodenal epithelial permeability was higher, and mucosal bacteria alpha diversity was lower in cirrhosis compared to controls, while no such differences were seen in the ileum or colon. Specific bacteria were associated with epithelial permeability and future hepatic decompensation.

Identification of differences in gene expression implicated in the Adherent-Invasive Escherichia coli phenotype during in vitro infection of intestinal epithelial cells.

Introduction: Adherent-invasive Escherichia coli (AIEC) is strongly associated with the pathogenesis of Crohn's disease (CD). However, no molecular markers currently exist for AIEC identification. This study aimed to identify differentially expressed genes (DEGs) between AIEC and non-AIEC strains that may contribute to AIEC pathogenicity and to evaluate their utility as molecular markers. Methods: Comparative transcriptomics was performed on two closely related AIEC/non-AIEC strain pairs during Intestine-407 cell infection. DEGs were quantified by RT-qPCR in the same RNA extracts, as well as in 14 AIEC and 23 non-AIEC strains to validate the results across a diverse strain collection. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarkers. Results: Comparative transcriptomics revealed 67 differences in expression between the two phenotypes in the strain pairs, 50 of which (81.97%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were differentially expressed between AIEC and non-AIEC phenotypes (p-value < 0.042), and 42 genes between the supernatant fraction of infected cell cultures and the cellular fraction containing adhered and intracellular bacteria (p-value < 0.049). Notably, six DEGs detected in the strain collection were implicated in arginine biosynthesis and five in colanic acid synthesis. Furthermore, two biomarkers based on wzb and cueR gene expression were proposed with an accuracy of ≥ 85% in our strain collection. Discussion: This is the first transcriptomic study conducted using AIEC-infected cell cultures. We have identified several genes that may be involved in AIEC pathogenicity, two of which are putative biomarkers for identification.

Low dietary fiber intake impairs small intestinal Th17 and intraepithelial T cell development over generations.

Dietary fiber strongly impacts the microbiota. Here, we show that a low-fiber diet changes the small intestinal (SI) microbiota and impairs SI Th17, TCRαß+CD8αß+ and TCRαß+CD8αα+ intraepithelial T cell development. We restore T cell development with dietary fiber supplementation, but this defect becomes persistent over generations with constant low-fiber diets. Offspring of low-fiber diet-fed mice have reduced SI T cells even after receiving a fiber-rich diet due to loss of bacteria important for T cell development. In these mice, only a microbiota transplant from a fiber-rich diet-fed mouse and a fiber-rich diet can restore T cell development. Low-fiber diets reduce segmented filamentous bacteria (SFB) abundance, impairing its vertical transmission. SFB colonization and a fiber-rich diet partially restore T cell development. Finally, we observe that low-fiber diet-induced T cell defects render mice more susceptible to Citrobacter rodentium infection. Together, these results demonstrate the importance of fiber to microbiota vertical transmission and host immune system development.