The Fecal Microbiome in Quiescent Crohn's Disease with Persistent Gastrointestinal Symptoms Show Enrichment of Oral Microbes But Depletion of Butyrate and Indole Producers.

Background and Aims: Even in the absence of inflammation, persistent symptoms in Crohn's disease (CD) are prevalent and negatively impact quality of life. We aimed to determine whether quiescent CD patients with persistent symptoms ( qCD+symptoms ) have changes in microbial structure and functional potential compared to those without symptoms ( qCD-symptoms ). Methods: We performed a prospective multi-center observational study nested within the SPARC IBD study. CD patients were included if they had evidence of quiescent disease as defined by fecal calprotectin level < 150 mcg/g. Persistent symptoms were defined by the CD-PRO2 questionnaire. Active CD ( aCD ), diarrhea-predominant irritable bowel syndrome ( IBS-D ), and healthy controls ( HC ) were included as controls. Stool samples underwent whole genome shotgun metagenomic sequencing. Results: A total of 424 patients were analyzed, including 39 qCD+symptoms, 274 qCD-symptoms, 21 aCD, 40 IBS-D, and 50 HC. Patients with qCD+symptoms had a less diverse microbiome, including significant reductions in Shannon diversity ( P <.001) and significant differences in microbial community structure ( P <.0001), compared with qCD-symptoms, IBS-D, and HC. Further, patients with qCD+symptoms showed significant enrichment of bacterial species that are normal inhabitants of the oral microbiome, including Klebsiella pneumoniae (q=.003) as well as depletion of important butyrate and indole producers, such as Eubacterium rectale (q=.001), Lachnospiraceae spp . (q<.0001), and Faecalibacterium prausnitzii (q<.0001), compared with qCD-symptoms. Finally, qCD+symptoms showed significant reductions in bacterial tnaA genes, which mediate tryptophan metabolism, as well as significant tnaA allelic variation, compared with qCD-symptoms. Conclusion: The microbiome in patients with qCD+symptoms show significant changes in diversity, community profile, and composition compared with qCD-symptoms. Future studies will focus on the functional significance of these changes. What You Need to Know: Background: Persistent symptoms in quiescent Crohn's disease (CD) are prevalent and lead to worse outcomes. While changes in the microbial community have been implicated, the mechanisms by which altered microbiota may lead to qCD+symptoms remain unclear.Findings: Quiescent CD patients with persistent symptoms demonstrated significant differences in microbial diversity and composition compared to those without persistent symptoms. Specifically, quiescent CD patients with persistent symptoms were enriched in bacterial species that are normal inhabitants of the oral microbiome but depleted in important butyrate and indole producers compared to those without persistent symptoms.Implications for Patient Care: Alterations in the gut microbiome may be a potential mediator of persistent symptoms in quiescent CD. Future studies will determine whether targeting these microbial changes may improve symptoms in quiescent CD.

Patients with breath test positive are necessary to be identified from irritable bowel syndrome: a clinical trial based on microbiomics and rifaximin sensitivity.

BACKGROUND: As a non-invasive and effective diagnostic method for small intestinal bacterial overgrowth (SIBO), wild-use of breath test (BT) has demonstrated a high comorbidity rate in patients with diarrhea-predominant irritable bowel syndrome (IBS-D) and SIBO. Patients overlapping with SIBO respond better to rifaximin therapy than those with IBS-D only. Gut microbiota plays a critical role in both of these two diseases. We aimed to determine the microbial difference between IBS-D overlapping with/without SIBO, and to study the underlying mechanism of its sensitivity to rifaximin. METHODS: Patients with IBS-D were categorized as BT-negative (IBSN) and BT-positive (IBSP). Healthy volunteers (BT-negative) were enrolled as healthy control. The patients were clinically evaluated before and after rifaximin treatment (0.4 g bid, 4 weeks). Blood, intestine, and stool samples were collected for cytokine assessment and gut microbial analyses. RESULTS: Clinical complaints and microbial abundance were significantly higher in IBSP than in IBSN. In contrast, severe systemic inflammation and more active bacterial invasion function that were associated with enrichment of opportunistic pathogens were seen in IBSN. The symptoms of IBSP patients were relieved in different degrees after therapy, but the symptoms of IBSN rarely changed. We also found that the presence of IBSN-enriched genera ( Enterobacter and Enterococcus ) are unaffected by rifaximin therapy. CONCLUSIONS: IBS-D patients overlapping with SIBO showed noticeably different fecal microbial composition and function compared with IBS-D only. The better response to rifaximin in those comorbid patients might associate with their different gut microbiota, which suggests that BT is necessary before IBS-D diagnosis and use of rifaximin. REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1800017911.

Secondary bile acids mediate high-fat diet-induced upregulation of R-spondin 3 and intestinal epithelial proliferation.

A high-fat diet (HFD) contributes to the increased incidence of colorectal cancer, but the mechanisms are unclear. We found that R-spondin 3 (Rspo3), a ligand for leucine-rich, repeat-containing GPCR 4 and 5 (LGR4 and LGR5), was the main subtype of R-spondins and was produced by myofibroblasts beneath the crypts in the intestine. HFD upregulated colonic Rspo3, LGR4, LGR5, and ß-catenin gene expression in specific pathogen-free rodents, but not in germ-free mice, and the upregulations were prevented by the bile acid (BA) binder cholestyramine or antibiotic treatment, indicating mediation by both BA and gut microbiota. Cholestyramine or antibiotic treatments prevented HFD-induced enrichment of members of the Lachnospiraceae and Rumincoccaceae, which can transform primary BA into secondary BA. Oral administration of deoxycholic acid (DCA), or inoculation of a combination of the BA deconjugator Lactobacillus plantarum and 7α-dehydroxylase-containing Clostridium scindens with an HFD to germ-free mice increased serum DCA and colonic Rspo3 mRNA levels, indicating that formation of secondary BA by gut microbiota is responsible for HFD-induced upregulation of Rspo3. In primary myofibroblasts, DCA increased Rspo3 mRNA via TGR5. Finally, we showed that cholestyramine or conditional deletion of Rspo3 prevented HFD- or DCA-induced intestinal proliferation. We conclude that secondary BA is responsible for HFD-induced upregulation of Rspo3, which, in turn, mediates HFD-induced intestinal epithelial proliferation.

WITHDRAWN: Gut Microbiota Alter Visceral Pain Sensation and Inflammation via Modulation of Synthesis of Resolvin D1 in Colonic Tuft Cells

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/policies/article-withdrawal

Mast cell mediation of visceral sensation and permeability in irritable bowel syndrome.

Abnormalities of mast cell structure or function may play prominent roles in irritable bowel syndrome (IBS) symptom genesis. Mast cells show close apposition to sensory nerves and release bioactive substances in response to varied stimuli including infection, stress, and other neuroendocrine factors. Most studies focus on patients who develop IBS after enteric infection or who report diarrhea-predominant symptoms. Three topics underlying IBS pathogenesis have been emphasized in recent investigations. Visceral hypersensitivity to luminal stimulation is found in most IBS patients and may contribute to abdominal pain. Mast cell dysfunction also may disrupt epithelial barrier function which alters mucosal permeability potentially leading to altered bowel function and pain. Mast cell products including histamine, proteases, prostaglandins, and cytokines may participate in hypersensitivity and permeability defects, especially with diarrhea-predominant IBS. Recent experimental evidence indicates that the pronociceptive effects of histamine and proteases are mediated by the generation of prostaglandins in the mast cell. Enteric microbiome interactions including increased mucosal bacterial translocation may activate mast cells to elicit inflammatory responses underlying some of these pathogenic effects. Therapies to alter mast cell activity (mast cell stabilizers) or function (histamine antagonists) have shown modest benefits in IBS. Future investigations will seek to define patient subsets with greater potential to respond to therapies that address visceral hypersensitivity, epithelial permeability defects, and microbiome alterations secondary to mast cell dysfunction in IBS.

Mucosal Serotonin Reuptake Transporter Expression in Irritable Bowel Syndrome Is Modulated by Gut Microbiota Via Mast Cell-Prostaglandin E2.

BACKGROUND & AIMS: Increased colonic serotonin (5-HT) level and decreased serotonin reuptake transporter (SERT) expression in irritable bowel syndrome (IBS) may contribute to diarrhea and visceral hypersensitivity. We investigated whether mucosal SERT is modulated by gut microbiota via a mast cell-prostaglandin E2 (PGE2) pathway. METHODS: C57Bl/6 mice received intracolonic infusion of fecal supernatant (FS) from healthy controls or patients with diarrhea-predominant irritable bowel syndrome (IBS-D). The role of mast cells was studied in mast cell-deficient mice. Colonic organoids and/or mast cells were used for in vitro experiments. SERT expression was measured by quantitative polymerase chain reaction and Western blot. Visceromotor responses to colorectal distension and colonic transit were assessed. RESULTS: Intracolonic infusion of IBS-D FS in mice caused an increase in mucosal 5-HT compared with healthy control FS, accompanied by ∼50% reduction in SERT expression. Mast cell stabilizers, cyclooxygenase-2 inhibitors, and PGE2 receptor antagonist prevented SERT downregulation. Intracolonic infusion of IBS-D FS failed to reduce SERT expression in mast cell-deficient (W/Wv) mice. This response was restored by mast cell reconstitution. The downregulation of SERT expression evoked by IBS FS was prevented by lipopolysaccharide (LPS) antagonist LPS from Rhodobacter sphaeroides and a bacterial trypsin inhibitor. In vitro LPS treatment caused increased cyclooxygenase-2 expression and PGE2 release from cultured mouse mast cells. Intracolonic infusion of IBS-D FS in mice reduced colonic transit, increased fecal water content, and increased visceromotor responses to colorectal distension. Ondansetron prevented these changes. CONCLUSIONS: Fecal LPS acting in concert with trypsin in patients with IBS-D stimulates mucosal mast cells to release PGE2, which downregulates mucosal SERT, resulting in increased mucosal 5-HT. This may contribute to diarrhea and abdominal pain common in IBS.

Upregulation of acid sensing ion channels is associated with esophageal hypersensitivity in GERD.

Proton pump inhibitors (PPIs) are the mainstay of therapy for gastroesophageal reflux disease (GERD) but up to 60% of patients have inadequate response to therapy. Acid sensing ion channels (ASICs) play important roles in nociception. This study aimed to investigate whether the increased expression of ASICs results in neuronal hyperexcitability in GERD. Esophageal biopsies were taken from GERD patients and healthy subjects to compare expression of ASIC1 and 3. Next, gene and protein expression of ASIC1 and 3 from esophageal mucosa and dorsal root ganglia (DRG) neurons were measured by qPCR, Western-blot and immunofluorescence in rodent models of reflux esophagitis (RE), non-erosive reflux disease (NERD), and sham operated groups. Excitability of DRG neurons in the GERD and sham groups were also tested by whole-cell patch-clamp recordings. We demonstrated that ASIC1 and 3 expression were significantly increased in patients with RE compared with healthy controls. This correlated positively with symptom severity of heartburn and regurgitation (p < .001). Next, ASIC1 and 3 gene and protein expression in rodent models of RE and NERD were similarly increased in esophageal mucosa as well as T3-T5 DRG neurons compared with sham operation. DRG neurons from RE animals showed hyperexcitability compared with sham group. However, intrathecal injection of ASIC specific inhibitors, PcTx1 and APTEx-2, as well as silencing ASIC1 and 3 genes with specific siRNAs prevented visceral hypersensitivity. Overall, upregulation of ASIC1 and 3 may lead to visceral hypersensitivity in RE and NERD and may be a potential therapeutic target for PPI non-responsive patients.

High FODMAP diet causes barrier loss via lipopolysaccharide-mediated mast cell activation.

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) are carbohydrates thought to contribute to the symptoms of IBS. A diet in high in FODMAPs (HFM) induces gastrointestinal symptoms in patients with irritable bowel syndrome (IBS), and a diet low in FODMAPs (LFM) improves symptoms in up to 60% of patients with IBS. However, the mechanism by which FODMAPs affect IBS symptoms is unclear. We showed that mice fed on a HFM diet have mast cell activation and colonic barrier loss. Using mast cell-deficient mice with and without mast cell reconstitution, we showed that HFM-mediated colonic barrier loss is dependent on TLR4-dependent mast cell activation. In in vitro studies, we demonstrated that IBS fecal supernatant stimulates mast cells significantly more compared with fecal supernatant from healthy controls. This effect of IBS fecal supernatant on mast cell stimulation is ameliorated in the absence of the TLR4 receptor and after a LFM diet. We found that a LFM diet improves colonic barrier function and reduces mast cell activation while decreasing fecal LPS levels. Our findings indicate that a HFM diet causes mast cell activation via LPS, which in turn leads to colonic barrier loss, and a LFM diet reverses these pathophysiologic mucosal changes.

Low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols diet compared with traditional dietary advice for diarrhea-predominant irritable bowel syndrome: a parallel-group, randomized controlled trial with analysis of clinical and microbiological factors associated with patient outcomes.

BACKGROUND: The efficacy and factors associated with patient outcomes for a diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (LFD) compared with traditional dietary advice (TDA) based on modified National Institute for Clinical Excellence guidelines for irritable bowel syndrome with diarrhea (IBS-D) in regions consuming a non-Western diet are unclear. OBJECTIVES: We aimed to determine the efficacy of an LFD compared with TDA for the treatment of IBS-D in Chinese patients and to investigate the factors associated with favorable outcomes. METHODS: One hundred and eight Chinese IBS-D patients (Rome III criteria) were randomly assigned to an LFD or TDA. The primary endpoint was a ≥50-point reduction in the IBS Severity Scoring System at 3 wk. Fecal samples collected before and after the dietary intervention were assessed for changes in SCFAs and microbiota profiles. A logistic regression model was used to identify predictors of outcomes. RESULTS: Among the 100 patients who completed the study, the primary endpoint was met in a similar number of LFD (30 of 51, 59%) and TDA (26 of 49, 53%) patients (∆6%; 95% CI: -13%, 24%). Patients in the LFD group achieved earlier symptomatic improvement in stool frequency and excessive wind than those following TDA. LFD reduced carbohydrate-fermenting bacteria such as Bifidobacterium and Bacteroides, and decreased saccharolytic fermentation activity. This was associated with symptomatic improvement in the responders. High saccharolytic fermentation activity at baseline was associated with a higher symptom burden (P = 0.01) and a favorable therapeutic response to the LFD (log OR: 4.9; 95% CI: -0.1, 9.9; P = 0.05). CONCLUSIONS: An LFD and TDA each reduced symptoms in Chinese IBS-D patients; however, the LFD achieved earlier symptomatic improvements in stool frequency and excessive wind. The therapeutic effect of the LFD was associated with changes in the fecal microbiota and the fecal fermentation index. At baseline, the presence of severe symptoms and microbial metabolic dysbiosis characterized by high saccharolytic capability predicted favorable outcomes to LFD intervention.This trial was registered at clinicaltrials.gov as NCT03304041.

Berberine alleviates visceral hypersensitivity in rats by altering gut microbiome and suppressing spinal microglial activation.

Accumulating evidence shows that agents targeting gut dysbiosis are effective for improving symptoms of irritable bowel syndrome (IBS). However, the potential mechanisms remain unclear. In this study we investigated the effects of berberine on the microbiota-gut-brain axis in two rat models of visceral hypersensitivity, i.e., specific pathogen-free SD rats subjected to chronic water avoidance stress (WAS) and treated with berberine (200 mg· kg-1 ·d-1, ig, for 10 days) as well as germ-free (GF) rats subjected to fecal microbiota transplantation (FMT) from a patient with IBS (designated IBS-FMT) and treated with berberine (200 mg· kg-1 ·d-1, ig, for 2 weeks). Before the rats were sacrificed, visceral sensation and depressive behaviors were evaluated. Then colonic tryptase was measured and microglial activation in the dorsal lumbar spinal cord was assessed. The fecal microbiota was profiled using 16S rRNA sequencing, and short chain fatty acids (SCFAs) were measured. We showed that berberine treatment significantly alleviated chronic WAS-induced visceral hypersensitivity and activation of colonic mast cells and microglia in the dorsal lumbar spinal cord. Transfer of fecal samples from berberine-treated stressed donors to GF rats protected against acute WAS. FMT from a patient with IBS induced visceral hypersensitivity and pro-inflammatory phenotype in microglia, while berberine treatment reversed the microglial activation and altered microbial composition and function and SCFA profiles in stools of IBS-FMT rats. We demonstrated that berberine did not directly influence LPS-induced microglial activation in vitro. In both models, several SCFA-producing genera were enriched by berberine treatment, and positively correlated to the morphological parameters of microglia. In conclusion, activation of microglia in the dorsal lumbar spinal cord was involved in the pathogenesis of IBS caused by dysregulation of the microbiota-gut-brain axis, and the berberine-altered gut microbiome mediated the modulatory effects of the agent on microglial activation and visceral hypersensitivity, providing a potential option for the treatment of IBS.

Evidence of Duodenal Epithelial Barrier Impairment and Increased Pyroptosis in Patients With Functional Dyspepsia on Confocal Laser Endomicroscopy and "Ex Vivo" Mucosa Analysis.

INTRODUCTION: Duodenal epithelial barrier impairment and immune activation may play a role in the pathogenesis of functional dyspepsia (FD). This study was aimed to evaluate the duodenal epithelium of patients with FD and healthy individuals for detectable microscopic structural abnormalities. METHODS: This is a prospective study using esophagogastroduodenoscopy enhanced with duodenal confocal laser endomicroscopy (CLE) and mucosal biopsies in patients with FD (n = 16) and healthy controls (n = 18). Blinded CLE images analysis evaluated the density of epithelial gaps (cell extrusion zones), a validated endoscopic measure of the intestinal barrier status. Analyses of the biopsied duodenal mucosa included standard histology, quantification of mucosal immune cells/cytokines, and immunohistochemistry for inflammatory epithelial cell death called pyroptosis. Transepithelial electrical resistance (TEER) was measured using Ussing chambers. Epithelial cell-to-cell adhesion proteins expression was assessed by real-time polymerase chain reaction. RESULTS: Patients with FD had significantly higher epithelial gap density on CLE in the distal duodenum than that of controls (P = 0.002). These mucosal abnormalities corresponded to significant changes in the duodenal biopsy samples of patients with FD, compared with controls, including impaired mucosal integrity by TEER (P = 0.009) and increased number of epithelial cells undergoing pyroptosis (P = 0.04). Reduced TEER inversely correlated with the severity of certain dyspeptic symptoms. Furthermore, patients with FD demonstrated altered duodenal expression of claudin-1 and interleukin-6. No differences in standard histology were found between the groups. DISCUSSION: This is the first report of duodenal CLE abnormalities in patients with FD, corroborated by biopsy findings of epithelial barrier impairment and increased cell death, implicating that duodenal barrier disruption is a pathogenesis factor in FD and introducing CLE a potential diagnostic biomarker in FD.

Author Correction: Leptin receptor-expressing neuron Sh2b1 supports sympathetic nervous system and protects against obesity and metabolic disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bile acid toxicity in Paneth cells contributes to gut dysbiosis induced by high-fat feeding.

High-fat feeding (HFF) leads to gut dysbiosis through unclear mechanisms. We hypothesize that bile acids secreted in response to high-fat diets (HFDs) may act on intestinal Paneth cells, leading to gut dysbiosis. We found that HFF resulted in widespread taxonomic shifts in the bacteria of the ileal mucosa, characterized by depletion of Lactobacillus and enrichment of Akkermansia muciniphila, Clostridium XIVa, Ruminococcaceae, and Lachnospiraceae, which were prevented by the bile acid binder cholestyramine. Immunohistochemistry and in situ hybridization studies showed that G protein-coupled bile acid receptor (TGR5) expressed in Paneth cells was upregulated in the rats fed HFD or normal chow supplemented with cholic acid. This was accompanied by decreased lysozyme+ Paneth cells and α-defensin 5 and 6 and increased expression of XBP-1. Pretreatment with ER stress inhibitor 4PBA or with cholestyramine prevented these changes. Ileal explants incubated with deoxycholic acid or cholic acid caused a decrease in α-defensin 5 and 6 and an increase in XBP-1, which was prevented by TGR5 antibody or 4PBA. In conclusion, this is the first demonstration to our knowledge that TGR5 is expressed in Paneth cells. HFF resulted in increased bile acid secretion and upregulation of TGR5 expression in Paneth cells. Bile acid toxicity in Paneth cells contributes to gut dysbiosis induced by HFF.

Satiety induced by bile acids is mediated via vagal afferent pathways.

The aim of this study was to elucidate the role and the pathways used by bile acid receptor TGR5 in transmitting satiety signals. We showed TGR5 colocalized with cholecystokinin type A (CCK-A) receptors in a subpopulation of rat nodose ganglia (NG) neurons. Intra-arterial injection of deoxycholic acid (DCA) dose-dependently increased firing rate in NG while a subthreshold dose of DCA and CCK-8 increased firing rates synergistically. TGR5-specific agonist oleanolic acid induced NG neuronal firing in a dose-dependent manner. However, the same units did not respond to GW4064, a nuclear receptor-specific agonist. Quantity of DCA-activated neurons in the hypothalamus was determined by c-Fos expression. Combining DCA and CCK-8 caused a 4-fold increase in c-Fos activation. In the arcuate nucleus, c-Fos-positive neurons coexpressed cocaine and amphetamine regulated transcript and proopiomelanocortin. DCA-induced c-Fos expression was eliminated following truncal vagotomy or silencing of TGR5 in the NG. Feeding studies showed intravenous injection of 1 µg/kg of DCA reduced food intake by 12% ± 3%, 24% ± 5%, and 32% ± 6% in the first 3 hours, respectively. Silencing of TGR5 or CCK-A receptor in the NG enhanced spontaneous feeding by 18% ± 2% and 13.5% ± 2.4%, respectively. When both TGR5 and CCK-A receptor were silenced, spontaneous feeding was enhanced by 37% ± 4% in the first 3 hours, suggesting that bile acid may have a physiological role in regulating satiety. Working in concert with CCK, bile acid synergistically enhanced satiety signals to reduce spontaneous feeding.