Dendritic cell-derived TGF-ß mediates the induction of mucosal regulatory T-cell response to Helicobacter infection essential for maintenance of immune tolerance in mice.

BACKGROUND: Helicobacter pylori infection leads to regulatory T-cell (Treg) induction in infected mice, which contributes to H. pylori immune escape. However, the mechanisms responsible for H. pylori induction of Treg and immune tolerance remain unclear. We hypothesized DC-produced TGF-ß may be responsible for Treg induction and immune tolerance. MATERIALS AND METHODS: To test this hypothesis, we generated TGF-ß∆DC mice (CD11c+ DC-specific TGF-ß deletion) and assessed the impact of DC-specific TGF-ß deletion on DC function during Helicobacter infection in vitro and in vivo. To examine the T cell-independent DC function, we crossed TGF-ß∆DC mice onto Rag1KO background to generate TGF-ß∆DC xRag1KO mice. RESULTS: When stimulated with H. pylori, TGF-ß∆DC BMDC/splenocyte cocultures showed increased levels of proinflammatory cytokines and decreased levels of anti-inflammatory cytokines compared to control, indicating a proinflammatory DC phenotype. Following 6 months of H. felis infection, TGF-ß∆DC mice developed more severe gastritis and a trend toward more metaplasia compared to TGF-ßfl/fl with increased levels of inflammatory Th1 cytokine mRNA and lower gastric H. felis colonization compared to infected TGF-ßfl/fl mice. In a T cell-deficient background using TGF-ß∆DC xRag1KO mice, H. felis colonization was significantly lower when DC-derived TGF-ß was absent, revealing a direct, innate function of DC in controlling H. felis infection independent of Treg induction. CONCLUSIONS: Our findings indicate that DC-derived TGF-ß mediates Helicobacter-induced Treg response and attenuates the inflammatory Th1 response. We also demonstrated a previously unrecognized innate role of DC controlling Helicobacter colonization via a Treg-independent mechanism. DC TGF-ß signaling may represent an important target in the management of H. pylori.

Effects of Anti-Helicobacter pylori Therapy on Incidence of Autoimmune Diseases, Including Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Helicobacter pylori induces immune tolerance and is associated with a lower risk for immune-mediated disorders, such as autoimmune and inflammatory bowel diseases (IBD). We aimed to determine the effects of treatment for H pylori infection on the incidence of autoimmune disease and IBD. METHODS: We collected data from the National Health Insurance Research Database in Taiwan on patients younger than 18 years old without a prior diagnosis of autoimmune disease or IBD. Patients with peptic ulcer disease (PUD) with treatment of H pylori infection (PUD+HPRx), PUD without H pylori treatment (PUD-HPRx), a urinary tract infection (UTI) treated with cephalosporin, or without PUD (controls) were matched for age, sex, insurance, and Charlson's comorbidity index score. RESULTS: Of the 1 million patients we collected data from in 2005, we included 79,181 patients in the study. We compared the effects of treatment for H pylori infection on the risk of autoimmunity or IBD and found that PUD+HPRx has the highest adjusted hazard risk (aHR) for autoimmunity or IBD (aHR, 2.36), compared to PUD-HPRx (aHR, 1.91) or UTI (aHRs, 1.71) (P < .001). The increased risk of autoimmune disease was not completely accounted for by antibiotic therapy alone, because PUD+HPRx had a higher aHR than UTI (P < .001). A small but significant increase in mortality was observed in the PUD+HPRx cohort (aHR, 1.11; P = .001). CONCLUSION: In an analysis of data from the National Health Insurance Research Database in Taiwan, we found that treatment for H pylori infection is associated with a significant increase in the risk for autoimmune disease, including IBD.

Indoleamine 2,3-Dioxygenase 1, Increased in Human Gastric Pre-Neoplasia, Promotes Inflammation and Metaplasia in Mice and Is Associated With Type II Hypersensitivity/Autoimmunity.

BACKGROUND & AIMS: Chronic gastrointestinal inflammation increases the risk of cancer by mechanisms that are not well understood. Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-binding enzyme that regulates the immune response via catabolization and regulation of tryptophan availability for immune cell uptake. IDO1 expression is increased during the transition from chronic inflammation to gastric metaplasia. We investigated whether IDO1 contributes to the inflammatory response that mediates loss of parietal cells leading to metaplasia. METHODS: Chronic gastric inflammation was induced in Ido1-/- and CB57BL/6 (control) mice by gavage with Helicobacter felis or overexpression of interferon gamma in gastric parietal cells. We also performed studies in Jh-/- mice, which are devoid of B cells. Gastric tissues were collected and analyzed by flow cytometry, immunostaining, and real-time quantitative polymerase chain reaction. Plasma samples were analyzed by enzyme-linked immunosorbent assay. Gastric tissues were obtained from 20 patients with gastric metaplasia and 20 patients without gastric metaplasia (controls) and analyzed by real-time quantitative polymerase chain reaction; gastric tissue arrays were analyzed by immunohistochemistry. We collected genetic information on gastric cancers from The Cancer Genome Atlas database. RESULTS: H felis gavage induced significantly lower levels of pseudopyloric metaplasia in Ido1-/- mice, which had lower frequencies of gastric B cells, than in control mice. Blood plasma from H felis-infected control mice had increased levels of autoantibodies against parietal cells, compared to uninfected control mice, but this increase was lower in Ido1-/- mice. Chronically inflamed stomachs of Ido1-/- mice had significantly lower frequencies of natural killer cells in contact with parietal cells, compared with stomachs of control mice. Jh-/- mice had lower levels of pseudopyloric metaplasia than control mice in response to H felis infection. Human gastric pre-neoplasia and carcinoma specimens had increased levels of IDO1 messenger RNA compared with control gastric tissues, and IDO1 protein colocalized with B cells. Co-clustering of IDO1 messenger RNA with B-cell markers was corroborated by The Cancer Genome Atlas database. CONCLUSIONS: IDO1 mediates gastric metaplasia by regulating the B-cell compartment. This process appears to be associated with type II hypersensitivity/autoimmunity. The role of autoimmunity in the progression of pseudopyloric metaplasia warrants further investigation.

Helicobacter pylori Antimicrobial Susceptibility Testing-Guided Salvage Therapy in the USA: A Real Life Experience.

BACKGROUND: The current practice guidelines recommend that Helicobacter pylori (H. pylori) culture and antimicrobial susceptibility testing (AST) be considered after patients failed the second course of H. pylori eradication therapy. AIMS: Here we report the real life experience of following this recommendation in the USA. METHODS: We established an in-house H. pylori culture protocol for AST and identified retrospectively patients who previously failed ≥ 2 courses of anti-H. pylori therapy and underwent esophagogastroduodenoscopy with AST at University of Michigan from 2010 to 2017. We determined the rate of H. pylori antibiotic resistance, the success rates of AST-guided tailored therapy, and the risk factors associated with treatment failure. RESULTS: Forty-seven patients were identified and 34 (72.3%) had successful cultures and AST. The most common antibiotic resistance was to metronidazole (79.4%), followed by clarithromycin (70.6%) and ciprofloxacin (42.9%). None of the patients were resistant to amoxicillin or tetracycline. The overall success rate of AST-guided tailored therapy was low (44.4%, 12/27). In patients infected with metronidazole-resistant H. pylori, bismuth quadruple therapy appears to be superior compared to non-bismuth quadruple therapy (6/8 or 75.0% vs. 3/14 or 21.4%, P = 0.03). High body mass index was significantly associated with tailored therapy failure (OR 1.24, 95% CI 1.00-1.54, P = 0.049). CONCLUSIONS: The success rate of AST-guided salvage therapy in the USA is low particularly in those with high BMI. Bismuth-based therapy appears to be better than non-bismuth-based regimens.

CCR2 mediates Helicobacter pylori-induced immune tolerance and contributes to mucosal homeostasis.

BACKGROUND: We previously demonstrated that H. pylori infection leads to increased induction of regulatory T cells in local and systemic immune compartments. Here, we investigate the role of CCR2 in the tolerogenic programing of dendritic cells in a mouse model of H. pylori infection. MATERIALS AND METHODS: CCR2 deficient (CCR2KO) mice and wild-type (Wt) mice infected with H. pylori SS1 strain were analyzed by qPCR and FACS analysis. In vitro, bone marrow-derived DC on day 6 from CCR2KO and Wt mice cocultured with or without H. pylori were examined to determine the impact of CCR2 signaling on dendritic cells function by qPCR, ELISA, and FACS analyses. RESULTS: Acute H. pylori infection was associated with a threefold increase in CCR2 mRNA expression in the gastric mucosa. H. pylori-infected CCR2KO mice exhibited a higher degree of mucosal inflammation, that is, increased gastritis scores and pro-inflammatory cytokine mRNA levels, but lower degree of H. pylori gastric colonization compared to infected Wt mice. Peripheral H. pylori-specific immune response measured in the CCR2KO spleen was characterized by a higher Th17 response and a lower Treg response. In vitro, CCR2KO bone marrow-derived DC was less mature and shown a lower Treg/Th17 ratio. Moreover, blockade of CCR2 signaling by MCP-1 neutralizing antibody inhibited H. pylori-stimulated bone marrow-derived DC maturation. CONCLUSIONS: Our results indicate that CCR2 plays an essential role in H. pylori-induced immune tolerance and shed light on a novel mechanism of CCR2-dependent DC Treg induction, which appears to be important in maintaining mucosal homeostasis during H. pylori infection.

Increased Expression of DUOX2 Is an Epithelial Response to Mucosal Dysbiosis Required for Immune Homeostasis in Mouse Intestine.

BACKGROUND & AIMS: Dual oxidase 2 (DUOX2), a hydrogen-peroxide generator at the apical membrane of gastrointestinal epithelia, is up-regulated 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 IL22 signaling. We performed 16S ribosomal RNA gene quantitative polymerase chain reaction of intestinal mucosa and mesenteric lymph nodes of Duoxa(-/-) mice that lack functional DUOX enzymes. Genes differentially expressed in Duoxa(-/-) mice compared with 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 up-regulated 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.

Tryptophan catabolism restricts IFN-γ-expressing neutrophils and Clostridium difficile immunopathology.

The interplay between Clostridium difficile and the host's metabolome is believed to influence the severity of infection. However, the mechanism for this phenomenon remains unclear. In this study, we model one of these metabolic pathways by focusing on tryptophan metabolism in the host. We found that inhibition of tryptophan catabolism in IDO1-knockout mice led to increased mucosal destruction, cecal hemorrhage, and increased production of IFN-γ in response to C. difficile infection, but no significant change in mucosal effector or regulatory T cell numbers or IL-10 mRNA expression. The increased immunopathology in infected IDO1-knockout mice was associated with a lower C. difficile burden and an increased percentage of IFN-γ-expressing neutrophils. We further demonstrated the ability of kynurenine to induce apoptosis in bone marrow-derived neutrophils, whereas the presence of tryptophan reversed this effect, providing a possible mechanism for the increased neutrophil accumulation in IDO1(-/-) mice. We conclude that C. difficile induces tryptophan catabolism in cecal lamina propria cells, which restricts C. difficile-associated immunopathology and the accumulation of IFN-γ-expressing neutrophils. This might represent a self-regulatory mechanism for neutrophils, via the IFN-γ-IDO1 pathway, to restrict their own accumulation during infection. These findings have important clinical implications because IDO inhibitors are used to treat cancer in clinical trials (in patients particularly susceptible to getting C. difficile infection), and treatment with IDO1 inhibitors may exacerbate the severity of C. difficile colitis.

Recapitulating Human Gastric Cancer Pathogenesis: Experimental Models of Gastric Cancer.

This review focuses on the various experimental models to study gastric cancer pathogenesis, with the role of genetically engineered mouse models (GEMMs) used as the major examples. We review differences in human stomach anatomy compared to the stomachs of the experimental models, including the mouse and invertebrate models such as Drosophila and C. elegans. The contribution of major signaling pathways, e.g., Notch, Hedgehog, AKT/PI3K is discussed in the context of their potential contribution to foregut tumorigenesis. We critically examine the rationale behind specific GEMMs, chemical carcinogens, dietary promoters, Helicobacter infection, and direct mutagenesis of relevant oncogenes and tumor suppressor that have been developed to study gastric cancer pathogenesis. Despite species differences, more efficient and effective models to test specific genes and pathways disrupted in human gastric carcinogenesis have yet to emerge. As we better understand these species differences, "humanized" versions of mouse models will more closely approximate human gastric cancer pathogenesis. Towards that end, epigenetic marks on chromatin, the gut microbiota, and ways of manipulating the immune system will likely move center stage, permitting greater overlap between rodent and human cancer phenotypes thus providing a unified progression model.

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 ß- galactosidase gene under the control of a BMP-responsive element and BMP4(ßgal/+) 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-ß-galactosidase, or BMP-4(ßgal/+) 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 NFκß 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.

Gastrin - active participant or bystander in gastric carcinogenesis?

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.

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.

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.

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.

Transgenic expression of interferon-γ in mouse stomach leads to inflammation, metaplasia, and dysplasia.

Gastric adenocarcinoma is one of the leading causes of cancer mortality worldwide. It arises through a stepwise process that includes prominent inflammation with expression of interferon-γ (IFN-γ) and multiple other pro-inflammatory cytokines. We engineered mice expressing IFN-γ under the control of the stomach-specific H(+)/K(+) ATPase ß promoter to test the potential role of this cytokine in gastric tumorigenesis. Stomachs of H/K-IFN-γ transgenic mice exhibited inflammation, expansion of myofibroblasts, loss of parietal and chief cells, spasmolytic polypeptide expressing metaplasia, and dysplasia. Proliferation was elevated in undifferentiated and metaplastic epithelial cells in H/K-IFN-γ transgenic mice, and there was increased apoptosis. H/K-IFN-γ mice had elevated levels of mRNA for IFN-γ target genes and the pro-inflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α. Intracellular mediators of IFN-γ and IL-6 signaling, pSTAT1 and pSTAT3, respectively, were detected in multiple cell types within stomach. H/K-IFN-γ mice developed dysplasia as early as 3 months of age, and 4 of 39 mice over 1 year of age developed antral polyps or tumors, including one adenoma and one adenocarcinoma, which expressed high levels of nuclear ß-catenin. Our data identified IFN-γ as a pivotal secreted factor that orchestrates complex changes in inflammatory, epithelial, and mesenchymal cell populations to drive pre-neoplastic progression in stomach; however, additional alterations appear to be required for malignant conversion.

The Cxcr2+ subset of the S100a8+ gastric granylocytic myeloid-derived suppressor cell population (G-MDSC) regulates gastric pathology.

Introduction: Gastric myeloid-derived suppressor cells (MDSCs) are a prominent population that expands during gastric pre-neoplastic and neoplastic development in humans and mice. However, the heterogeneity of this population has circumvented the ability to study these cells or understand their functions. Aside from Schlafen-4+ (Slfn-4+) MDSCs in mouse studies, which constitute a subset of this population, limitations exist in characterizing the heterogeneity of the gastric CD11b+Ly6G+ population and targeting its different subsets. Here we identify S100a8 as a pan-specific marker for this population and utilize it to study the role of the S100a8+Cxcr2+ subset. Methods: We profiled gastric CD11b+Ly6G+ versus CD11b+Ly6G- myeloid cells by transcriptomic and single-cell RNA sequencing. We identified S100a8 as a pan-specific marker of the gastric granulocytic MDSC (G-MDSC) population, and generated S100a8CreCxcr2flox/flox to study the effects of Cxcr2 knockdown. Results: Following 6-months of Helicobacter felis infection, gastric CD11b+Ly6G+ G-MDSCs were highly enriched for the expression of S100a8, S100a9, Slfn4, Cxcr2, Irg1, Il1f9, Hcar2, Retnlg, Wfdc21, Trem1, Csf3R, Nlrp3, and Il1b. The expression of these distinct genes following 6mo H. felis infection marked heterogeneous subpopulations, but they all represented a subset of S100a8+ cells. S100a8 was identified as a pan-marker for CD11b+Ly6G+ cells arising in chronic inflammation, but not neutrophils recruited during acute gut infection. 6mo Helicobacter felis-infected S100a8CreCxcr2flox/flox mice exhibited worsened gastric metaplastic pathology than Cxcr2flox/flox mice, which was associated with dysregulated lipid metabolism and peroxidation. Conclusion: S100a8 is a pan-specific marker that can be used to target gastric G-MDSC subpopulations, of which the Cxcr2+ subset regulates gastric immunopathology and associates with the regulation of lipid peroxidation.

IFNγ contributes to the development of gastric epithelial cell metaplasia in Huntingtin interacting protein 1 related (Hip1r)-deficient mice.

Huntingtin interacting protein 1 related (Hip1r) is an F-actin- and clathrin-binding protein involved in vesicular trafficking that is crucial for parietal cell function and epithelial cell homeostasis in the stomach. Gastric parietal cells in Hip1r-deficient mice are lost by apoptotic cell death, which leads to a progressive epithelial cell derangement, including glandular hypertrophy, zymogenic cell loss and expansion of a metaplastic mucous cell lineage known as spasmolytic polypeptide-expressing metaplasia (SPEM). The epithelial cell changes are associated with infiltration of inflammatory cells. As inflammatory mediators, such as IFNγ, have been shown to contribute to the development of the gastric epithelial cell metaplasia after Helicobacter infection, we tested whether IFNγ played a role in the spontaneous progressive epithelial metaplasia observed in Hip1r-deficient mice. Hip1r-deficient mice were crossed with IFNγ-deficient mice and single- and double-mutant mice were analyzed at 3 and 12 months of age. Histopathology scoring showed that loss of IFNγ tempered the spontaneous development of metaplastic lesions in Hip1r-deficient mice. Loss of IFNγ was observed to abrogate the glandular hypertrophy evident in Hip1r mutant stomach, although increased epithelial cell proliferation and elevated gastrin levels were not affected by the presence or absence of this pro-inflammatory cytokine. An analysis of cell lineage markers in the double-mutant mice demonstrated that IFNγ specifically affected the development of metaplastic mucous cells in the neck region, whereas the parietal cell, surface mucous cell and zymogenic cell alterations remained similar to the histopathology in the Hip1r mutant. Morphometric analysis showed that IFNγ was required for the mucous cell hypertrophy and hyperplasia observed in Hip1r-deficient mice. Together, these findings demonstrate that IFNγ is critical for the development of the gastric epithelial cell metaplasia that results from parietal cell atrophy in the Hip1r-deficient mice.

The impact of vaccination on the burden of invasive pneumococcal disease from a nationwide surveillance program in Lebanon: an unexpected increase in mortality driven by non-vaccine serotypes.

BACKGROUND: The impact of pneumococcal conjugate vaccines (PCVs) on the burden of invasive pneumococcal disease (IPD) and serotype distribution was examined across age groups from data collected by the Lebanese Inter-Hospital Pneumococcal Surveillance Program. METHODS: Between 2005 and 2020, 593 invasive Streptococcus pneumoniae isolates were collected from 79 hospitals throughout Lebanon. Serotypes and antimicrobial resistance (AMR) profiles were identified, and trends compared over 3 eras: PCV7, post-PCV7/ pre-PCV13, and PCV13 eras. RESULTS: The prevalence of PCV7 serotypes decreased significantly from 43.6% in the PCV7 era to 17.8% during the PCV13 era (p<0.001). PCV13-only serotypes remained stable in the PCV13 compared to the post-PCV7 eras, especially serotypes 1 and 3, whereas non-vaccine types (NVT) increased throughout the study period, especially 24 and 16F. The mortality rate increased substantially from 12.5% (PCV7 era) to 24.8% (PCV13 era). A significant decrease in AMR was observed across the three study eras. CONCLUSION: PCVs substantially impacted IPD and AMR in vaccinated and unvaccinated populations despite an increase in mortality driven by NVT. Broadening the recommendation of vaccination to include older age-groups, using higher valency vaccines, and implementing stringent antimicrobial stewardship are likely to further impact the burden of IPD.

Intracellular calcium release and protein kinase C activation stimulate sonic hedgehog gene expression during gastric acid secretion.

BACKGROUND & AIMS: Hypochlorhydria during Helicobacter pylori infection inhibits gastric Sonic Hedgehog (Shh) expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through intracellular calcium (Ca2(+)(i))-dependent protein kinase C (PKC) or cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation. METHODS: We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1, a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) + 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), PKC-overexpressing adenoviruses, and PKC inhibitors were used to modulate Ca(2+)(i)-release, PKC activity, and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H(+)/K(+)-ß-cholera-toxin-overexpressing mice. RESULTS: Mice that expressed secreted hedgehog-interacting protein-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression also was repressed in the hyperchlorhydric H(+)/K(+)-ß-cholera-toxin model with increased cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca(2+)(i) release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin-, and carbachol-mediated release of Ca(2+)(i) induced Shh expression. Ca(2+)-chelation with BAPTA + EGTA reduced Shh expression. Overexpression of PKC-α, -ß, and -δ (but not PKC-ϵ) induced an Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. CONCLUSIONS: Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca(2+)(i)-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin.

Interleukin-1beta 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.

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 ß-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.