Mohamad El–Zaatari

Interleukin-1β Promotes Gastric Atrophy Through Suppression of Sonic Hedgehog

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. METHODS Shh-LacZ reporter mice were infected with Helicobacter felis for 3 and 8 weeks. Changes in Shh expression were monitored using beta-galactosidase staining and immunohistochemistry. Gastric acidity was measured after infection, and interleukin (IL)-1beta was quantified by quantitative reverse-transcription polymerase chain reaction. Mice were injected with either IL-1beta or omeprazole before measuring Shh mRNA expression and acid secretion. Organ cultures of gastric glands from wild-type or IL-1R1 null mice were treated with IL-1beta then Shh expression was measured. Primary canine parietal or mucous cells were treated with IL-1beta. Shh protein was determined by immunoblot analysis. Changes in intracellular calcium were measured by Fura-2. RESULTS All major cell lineages of the corpus including surface pit, mucous neck, zymogenic, and parietal cells expressed Shh. Helicobacter infection reduced gastric acidity and inhibited Shh expression in parietal cells by 3 weeks. IL-1beta produced during Helicobacter infection inhibited gastric acid, intracellular calcium, and Shh expression through the IL-1 receptor. Suppression of parietal cell Shh expression by IL-1beta and omeprazole was additive. IL-1beta did not suppress Shh expression in primary gastric mucous cells. CONCLUSIONS IL-1beta suppresses Shh gene expression in parietal cells by inhibiting acid secretion and subsequently the release of intracellular calcium.

Stress-Induced Corticotropin-Releasing Hormone-Mediated NLRP6 Inflammasome Inhibition and Transmissible Enteritis in Mice

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.

Dual oxidases control release of hydrogen peroxide by the gastric epithelium to prevent Helicobacter felis infection and inflammation in mice.

BACKGROUND & AIMS Dual oxidases (DUOX) are conserved reduced nicotinamide adenine dinucleotide phosphate oxidases that produce H2O2 at the epithelial cell surface. The DUOX enzyme comprises the DUOX and DUOX maturation factor (DUOXA) subunits. Mammalian genomes encode 2 DUOX isoenzymes (DUOX1/DUOXA1 and DUOX2/DUOXA2). Expression of these genes is up-regulated during bacterial infections and chronic inflammatory diseases of the luminal gastrointestinal tract. The roles of DUOX in cellular interactions with microbes have not been determined in higher vertebrates. METHODS Mice with disruptions of Duoxa1 and Duoxa2 genes (Duoxa(-/-) mice) and control mice were infected with Helicobacter felis to create a model of Helicobacter pylori infection--the most common human chronic infection. RESULTS Infection with H. felis induced expression of Duox2 and Duoxa2 in the stomachs of wild-type mice, and DUOX protein specifically localized to the apical surface of epithelial cells. H. felis colonized the mucus layer in the stomachs of Duoxa(-/-) mice to a greater extent than in control mice. The increased colonization persisted into the chronic phase of infection and correlated with an increased, yet ineffective, inflammatory response. H. felis colonization also was increased in Duoxa(+/-) mice, compared with controls. We observed reduced expression of the H2O2-inducible katA gene in H. felis that colonized Duoxa(-/-) mice, compared with that found in controls (P = .0002), indicating that Duox causes oxidative stress in these bacteria. In vitro, induction of oxidative defense by H. felis failed to prevent a direct bacteriostatic effect at sustained levels of H2O2 as low as 30 μmol/L. CONCLUSIONS Based on studies of Duoxa(-/-) mice, the DUOX enzyme complex prevents gastric colonization by H. felis and the inflammatory response. These findings indicate the nonredundant function of epithelial production of H2O2 in restricting microbial colonization.

Bone Morphogenetic Protein Signaling Regulates Gastric Epithelial Cell Development and Proliferation in Mice

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 Smad 1, 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 (Bandeiraea) simplicifolia lectin 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.

Anti-Inflammatory Activity of Bone Morphogenetic Protein Signaling Pathways in Stomachs of Mice

BACKGROUND & AIMS Bone morphogenetic protein (BMP)4 is a mesenchymal peptide that regulates cells of the gastric epithelium. We investigated whether BMP signaling pathways affect gastric inflammation after bacterial infection of mice. METHODS We studied transgenic mice that express either the BMP inhibitor noggin or the β- galactosidase gene under the control of a BMP-responsive element and BMP4(βgal/+) mice. Gastric inflammation was induced by infection of mice with either Helicobacter pylori or Helicobacter felis. Eight to 12 weeks after inoculation, gastric tissue samples were collected and immunohistochemical, quantitative, reverse-transcription polymerase chain reaction and immunoblot analyses were performed. We used enzyme-linked immunosorbent assays to measure cytokine levels in supernatants from cultures of mouse splenocytes and dendritic cells, as well as from human gastric epithelial cells (AGS cell line). We also measured the effects of BMP-2, BMP-4, BMP-7, and the BMP inhibitor LDN-193189 on the expression of interleukin (IL)8 messenger RNA by AGS cells and primary cultures of canine parietal and mucus cells. The effect of BMP-4 on NFkB activation in parietal and AGS cells was examined by immunoblot and luciferase assays. RESULTS Transgenic expression of noggin in mice increased H pylori- or H felis-induced inflammation and epithelial cell proliferation, accelerated the development of dysplasia, and increased expression of the signal transducer and activator of transcription 3 and activation-induced cytidine deaminase. BMP-4 was expressed in mesenchymal cells that expressed α-smooth muscle actin and activated BMP signaling pathways in the gastric epithelium. Neither BMP-4 expression nor BMP signaling were detected in immune cells of C57BL/6, BRE-β-galactosidase, or BMP-4(βgal/+) mice. Incubation of dendritic cells or splenocytes with BMP-4 did not affect lipopolysaccharide-stimulated production of cytokines. BMP-4, BMP-2, and BMP-7 inhibited basal and tumor necrosis factor α-stimulated expression of IL8 in canine gastric epithelial cells. LDN-193189 prevented BMP4-mediated inhibition of basal and tumor necrosis factor α-stimulated expression of IL8 in AGS cells. BMP-4 had no effect on TNFα-stimulated phosphorylation and degradation of IκBα, or on TNFα induction of a NFκβ reporter gene. CONCLUSIONS BMP signaling reduces inflammation and inhibits dysplastic changes in the gastric mucosa after infection of mice with H pylori or H felis.

ZBP-89 regulates expression of tryptophan hydroxylase i and mucosal defense against salmonella typhimurium in mice

BACKGROUND & AIMS ZBP-89 (also ZNF148 or Zfp148) is a butyrate-inducible zinc finger transcription factor that binds to GC-rich DNA elements. Deletion of the N-terminal domain is sufficient to increase mucosal susceptibility to chemical injury and inflammation. We investigated whether conditional deletion of ZBP-89 from the intestinal and colonic epithelium of mice increases their susceptibility to pathogens such as Salmonella typhimurium. METHODS We generated mice with a conditional null allele of Zfp148 (ZBP-89(FL/FL)) using homologous recombination to flank Zfp148 with LoxP sites (ZBP-89(FL/FL)), and then bred the resulting mice with those that express VillinCre. We used microarray analysis to compare gene expression patterns in colonic mucosa between ZBP-89(ΔInt) and C57BL/6 wild-type mice (controls). Mice were gavaged with 2 isogenic strains of S. typhimurium after administration of streptomycin. RESULTS Microarray analysis revealed that the colonic mucosa of ZBP-89(ΔInt) mice had reduced levels of tryptophan hydroxylase 1 (Tph1) messenger RNA, encoding the rate-limiting enzyme in enterochromaffin cell serotonin (5-hydroxytryptamine [5HT]) biosynthesis. DNA affinity precipitation demonstrated direct binding of ZBP-89 to the mouse Tph1 promoter, which was required for its basal and butyrate-inducible expression. ZBP-89(ΔInt) mice did not increase mucosal levels of 5HT in response to S. typhimurium infection, and succumbed to the infection 2 days before control mice. The ΔhilA isogenic mutant of S. typhimurium lacks this butyrate-regulated locus and stimulated, rather than suppressed, expression of Tph1 approximately 50-fold in control, but not ZBP-89(ΔInt), mice, correlating with fecal levels of butyrate. CONCLUSIONS ZBP-89 is required for butyrate-induced expression of the Tph1 gene and subsequent production of 5HT in response to bacterial infection in mice. Reductions in epithelial ZBP-89 increase susceptibility to colitis and sepsis after infection with S. typhimurium, partly because of reduced induction of 5HT production in response to butyrate and decreased secretion of antimicrobial peptides.