Background & AimsGut dysbiosis is closely involved in the pathogenesis of inflammatory bowel disease (IBD). However, it remains unclear whether IBD-associated gut dysbiosis contributes to disease pathogenesis or is merely secondary to intestinal inflammation. We established a humanized gnotobiotic (hGB) mouse system to assess the functional role of gut dysbiosis associated with 2 types of IBD: Crohn's disease (CD) and ulcerative colitis (UC).MethodsGerm-free mice were colonized by the gut microbiota isolated from patients with CD and UC, and healthy controls. Microbiome analysis, bacterial functional gene analysis, luminal metabolome analysis, and host gene expression analysis were performed in hGB mice. Moreover, the colitogenic capacity of IBD-associated microbiota was evaluated by colonizing germ-free colitis-prone interleukin 10–deficient mice with dysbiotic patients' microbiota.ResultsAlthough the microbial composition seen in donor patients' microbiota was not completely reproduced in hGB mice, some dysbiotic features of the CD and UC microbiota (eg, decreased diversity, alteration of bacterial metabolic functions) were recapitulated in hGB mice, suggesting that microbial community alterations, characteristic for IBD, can be reproduced in hGB mice. In addition, colonization by the IBD-associated microbiota induced a proinflammatory gene expression profile in the gut that resembles the immunologic signatures found in CD patients. Furthermore, CD microbiota triggered more severe colitis than healthy control microbiota when colonized in germ-free interleukin 10–deficient mice.ConclusionsDysbiosis potentially contributes to the pathogenesis of IBD by augmenting host proinflammatory immune responses. Transcript profiling: GSE73882.

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BACKGROUND & AIMS Dual oxidase 2 (DUOX2), a hydrogen-peroxide generator at the apical membrane of gastrointestinal epithelia, is upregulated in patients with inflammatory bowel disease (IBD) before the onset of inflammation, but little is known about its effects. We investigated the role of DUOX2 in maintaining mucosal immune homeostasis in mice. METHODS We analyzed the regulation of DUOX2 in intestinal tissues of germ-free vs conventional mice, mice given antibiotics or colonized with only segmented filamentous bacteria, mice associated with human microbiota, and mice with deficiencies in interleukin (IL)23 and 22 signaling. We performed 16S rRNA gene quantitative PCR of intestinal mucosa and mesenteric lymph nodes of Duoxa−/− mice that lack functional DUOX enzymes. Genes differentially expressed in Duoxa−/− mice compared to co-housed wild type littermates were correlated with gene expression changes in early-stage IBD using gene set enrichment analysis. RESULTS Colonization of mice with segmented filamentous bacteria upregulated intestinal expression of DUOX2. DUOX2 regulated redox-signaling within mucosa-associated microbes and restricted bacterial access to lymphatic tissues of the mice, thereby reducing microbiota-induced immune responses. Induction of Duox2 transcription by microbial colonization did not require the mucosal cytokines IL17 or IL22, although IL22 increased expression of Duox2. Dysbiotic, but not healthy human microbiota, activated a DUOX2 response in recipient germ-free mice that corresponded to abnormal colonization of the mucosa with distinct populations of microbes. In Duoxa−/− mice, abnormalities in ileal mucosal gene expression at homeostasis recapitulated those in patients with mucosal dysbiosis. CONCLUSIONS DUOX2 regulates interactions between the intestinal microbiota and the mucosa to maintain immune homeostasis in mice. Mucosal dysbiosis leads to increased expression of DUOX2, which might be a marker of perturbed mucosal homeostasis in patients with early-stage IBD.

Epithelial tuft cells are named for their characteristic microtubule bundle located at the cell apex where it is exposed to the luminal environment. As such, tuft cells are found in multiple organs, including the gastrointestinal (GI) tract where the apical “tuft” is hypothesized to detect and transmit environmental signals. Thus, the goal of our study was to characterize gastric tuft cells during GI tract development, then subsequently in the normal and metaplastic adult stomach. GI tracts from mouse embryos, newborn and post-natal mice were analyzed. Tuft cells were identified by immunohistochemistry using acetylated-α-tubulin (acTub) antibody to detect the microtubule bundle. Additional tuft cell markers, e.g., doublecortin-like kinase 1 (DCLK1), were used to co-localize with acTub. Tuft cells were quantified in human gastric tissue arrays and in mouse stomachs with or without inflammation. In the developing intestine, tuft cells in both the crypts and villi expressed all markers by E18.5. In the stomach, acTub co-localized with DCLK1 and other established tuft cell markers by E18.5 in the antrum but not until postnatal day 7 in the corpus, with the highest density of tuft cells clustered at the forestomach ridge. Tuft cell numbers increased in hyperplastic human and mouse stomachs. In the adult GI tract, the tuft cell marker acTub co-expressed with DCKL1 and chemosensory markers, e.g., TRPM5. In summary, tuft cells appear in the gastric antrum and intestine at E18.5, but their maximal numbers in the corpus are not achieved until after weaning. Tuft cell numbers increase with inflammation, hyperplasia and metaplasia.

BACKGROUND & AIMS We investigated the role of bone morphogenetic protein (BMP) signaling in the regulation of gastric epithelial cell growth and differentiation by generating transgenic mice that express the BMP inhibitor noggin in the stomach. METHODS The promoter of the mouse H+/K+-ATPase β-subunit gene, which is specifically expressed in parietal cells, was used to regulate expression of noggin in the gastric epithelium of mice. The transgenic mice were analyzed for noggin expression, tissue morphology, cellular composition of the gastric mucosa, gastric acid content, and plasma levels of gastrin. Tissues were analyzed by immunohistochemical, quantitative real-time polymerase chain reaction, immunoblot, microtitration, and radioimmunoassay analyses. RESULTS In the stomachs of the transgenic mice, phosphorylation of Smad1, 5, and 8 decreased, indicating inhibition of BMP signaling. Mucosa were of increased height, with dilated glands, cystic structures, reduced numbers of parietal cells, and increased numbers of cells that coexpressed intrinsic factor, trefoil factor 2, and griffonia simplicifolialectin II, compared with wild-type mice. In the transgenic mice, levels of the H+/K+-ATPase α-subunit protein and messenger RNA were reduced, whereas those of intrinsic factor increased. The transgenic mice were hypochloridric and had an increased number of Ki67- and proliferating cell nuclear antigen-positive cells; increased levels of plasma gastrin; increased expression of transforming growth factor-α, amphiregulin, and gastrin; and activation of extracellular signal-regulated kinase 2. CONCLUSIONS Inhibiting BMP signaling in the stomachs of mice by expression of noggin causes loss of parietal cells, development of transitional cells that express markers of mucus neck and zymogenic lineages, and activation of proliferation. BMPs are therefore important regulators of gastric epithelial cell homeostasis.

Gastrin is a pro-proliferative, anti-apoptotic hormone with a central role in acid secretion in the gastric mucosa and a long-standing association with malignant progression in transgenic mouse models. However, its exact role in human gastric malignancy requires further validation. Gastrin expression is tightly regulated by two closely associated hormones, somatostatin and gastrin-releasing peptide, and aspects of their interaction may be deregulated during progression to gastric adenocarcinoma. Furthermore, agonists and antagonists of the receptors for all three hormones have shown modest clinical efficacy against gastric adenocarcinoma, which might provide useful information on the future combined use of these agents.

BACKGROUND & AIMS Stress alters brain–gut interactions and could exacerbate intestinal disorders, including irritable bowel syndrome. Alterations in the intestinal microbiota have been associated with irritable bowel syndrome. Maintenance of healthy microbiota requires nucleotide-binding oligomerization domain protein-like receptors, pyrin- domain containing (NLRP)-6 inflammasomes. We investigated the involvement of NLRP6 in water-avoidance stress (WAS)-induced intestinal disorders in mice. METHODS B57BL6 mice were subjected to WAS for 1 hour each day for 10 days; body weights and intestinal inflammation and permeability were analyzed. We investigated signaling via the NLRP3 and NLRP6 inflammasomes, and the role of corticotropin-releasing hormone (CRH) in WAS-associated inflammation and NLRP6 inhibition. Mice that were not exposed to stress were co-housed with mice subjected to WAS to determine the effects of WAS-induced dysbiosis, measured by sequencing bacterial 16S ribosomal RNA. We also assessed the effects of a peroxisome proliferator-activated receptor-γ agonist and probiotics. RESULTS WAS-induced small-bowel inflammation (enteritis) was associated with inhibition of NLRP6, but not NLRP3, and was prevented by a peroxisome proliferator-activated receptor-γ agonist, which induced epithelial expression of NLRP6. CRH was released during WAS and inhibited NLRP6 expression. WAS induced alterations in the gut microbiota of mice; co-housed nonstressed mice developed enteritis associated with increased CRH and decreased levels of NLRP6. Probiotic therapy reduced intestinal inflammation in mice with WAS-induced enteritis. CONCLUSIONS Exposure of mice to stress inhibits NLRP6 and alters the composition of the gut microbiota, leading to intestinal inflammation. These findings might explain the benefits of probiotics for patients with stress-associated gastrointestinal disorders.

Background & Aims-In both human subjects and rodent models, Helicobacter infection leads to a decrease in Shh expression in the stomach. Sonic Hedgehog (Shh) is highly expressed in the gastric corpus and its loss correlates with gastric atrophy. Therefore, we tested the hypothesis that proinflammatory cytokines induce gastric atrophy by inhibiting Shh expression.