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.

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.

DUOX2 variants associate with preclinical disturbances in microbiota-immune homeostasis and increased inflammatory bowel disease risk.

A primordial gut-epithelial innate defense response is the release of hydrogen peroxide by dual NADPH oxidase (DUOX). In inflammatory bowel disease (IBD), a condition characterized by an imbalanced gut microbiota-immune homeostasis, DUOX2 isoenzyme is the highest induced gene. Performing multiomic analyses using 2872 human participants of a wellness program, we detected a substantial burden of rare protein-altering DUOX2 gene variants of unknown physiologic significance. We identified a significant association between these rare loss-of-function variants and increased plasma levels of interleukin-17C, which is induced also in mucosal biopsies of patients with IBD. DUOX2-deficient mice replicated increased IL-17C induction in the intestine, with outlier high Il17c expression linked to the mucosal expansion of specific Proteobacteria pathobionts. Integrated microbiota/host gene expression analyses in patients with IBD corroborated IL-17C as a marker for epithelial activation by gram-negative bacteria. Finally, the impact of DUOX2 variants on IL-17C induction provided a rationale for variant stratification in case control studies that substantiated DUOX2 as an IBD risk gene. Thus, our study identifies an association of deleterious DUOX2 variants with a preclinical hallmark of disturbed microbiota-immune homeostasis that appears to precede the manifestation of IBD.

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.

Aim2-mediated/IFN-ß-independent regulation of gastric metaplastic lesions via CD8+ T cells.

Development of gastric cancer is often preceded by chronic inflammation, but the immune cellular mechanisms underlying this process are unclear. Here we demonstrated that an inflammasome molecule, absent in melanoma 2 (Aim2), was upregulated in patients with gastric cancer and in spasmolytic polypeptide-expressing metaplasia of chronically Helicobacter felis-infected stomachs in mice. However, we found that Aim2 was not necessary for inflammasome function during gastritis. In contrast, Aim2 deficiency led to an increase in gastric CD8+ T cell frequency, which exacerbated metaplasia. These gastric CD8+ T cells from Aim2-/- mice were found to have lost their homing receptor expression (sphingosine-1-phosphate receptor 1 [S1PR1] and CD62L), a feature of tissue-resident memory T cells. The process was not mediated by Aim2-dependent regulation of IFN-ß or by dendritic cell-intrinsic Aim2. Rather, Aim2 deficiency contributed to an increased production of CXCL16 by B cells, which could suppress S1PR1 and CD62L in CD8+ T cells. This study describes a potentially novel function of Aim2 that regulates CD8+ T cell infiltration and retention within chronically inflamed solid organ tissue. This function operates independent of the inflammasome, IFN-ß, or dendritic cells. We provide evidence that B cells can contribute to this mechanism via CXCL16.

Dietary L-serine confers a competitive fitness advantage to Enterobacteriaceae in the inflamed gut.

Metabolic reprogramming is associated with the adaptation of host cells to the disease environment, such as inflammation and cancer. However, little is known about microbial metabolic reprogramming or the role it plays in regulating the fitness of commensal and pathogenic bacteria in the gut. Here, we report that intestinal inflammation reprograms the metabolic pathways of Enterobacteriaceae, such as Escherichia coli LF82, in the gut to adapt to the inflammatory environment. We found that E. coli LF82 shifts its metabolism to catabolize L-serine in the inflamed gut in order to maximize its growth potential. However, L-serine catabolism has a minimal effect on its fitness in the healthy gut. In fact, the absence of genes involved in L-serine utilization reduces the competitive fitness of E. coli LF82 and Citrobacter rodentium only during inflammation. The concentration of luminal L-serine is largely dependent on dietary intake. Accordingly, withholding amino acids from the diet markedly reduces their availability in the gut lumen. Hence, inflammation-induced blooms of E. coli LF82 are significantly blunted when amino acids-particularly L-serine-are removed from the diet. Thus, the ability to catabolize L-serine increases bacterial fitness and provides Enterobacteriaceae with a growth advantage against competitors in the inflamed gut.

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.

Increased risk for inflammatory bowel disease in congenital hypothyroidism supports the existence of a shared susceptibility factor.

Loss-of-function mutations in dual oxidase (DUOX) 2 are the most common genetic variants found in congenital hypothyroidism (CH), and similar mutations have been recently reported in few very-early-onset inflammatory bowel disease (IBD) patients without CH. If DUOX2 variants indeed increase susceptibility for IBD, the enrichment of DUOX2 mutation carriers among CH patients should be reflected in higher risk for developing IBD. Using a database containing health insurance claims data for over 230 million patients in the United States, 42,922 subjects with CH were identified based on strict inclusion criteria using diagnostic codes. For subgroup analysis, CH patients with pharmacy records were stratified as transient or permanent CH based on the absence or presence of levothyroxine treatment, respectively. Patients were matched to an equal-sized, age- and gender-matched non-CH group. Compared to controls, CH patients had a 73% higher overall IBD prevalence (0.52% vs 0.30%; P < 0.0001). The CH-associated relative risk was higher for indeterminate or ulcerative colitis than Crohn's disease. Patients with transient CH had higher odds for IBD (OR 2.39 (95% CI 1.77-3.23) than those with permanent CH (1.69 (95% CI 1.31-2.18). We conclude that patients with CH are at an increased risk of developing IBD. The risk was highest for patients with transient CH, for which partial defects in the DUOX2 system are a particularly common finding.

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.

The effect of CT26 tumor-derived TGF-ß on the balance of tumor growth and immunity.

INTRODUCTION: TGF-ß is an important target for many cancer therapies under development. In addition to suppressing anti-tumor immunity, it has pleiotropic direct pro- and anti- tumor effects. The actions of increased endogenous TGF-ß production remain unclear, and may affect the outcomes of anti-TGF-ß cancer therapy. We hypothesize that tumor-derived TGF-ß (td-TGF-ß) plays an important role in maintaining tumor remission by controlling tumor proliferation in vivo, and that decreasing td-TGF-ß in the tumor microenvironment will result in tumor progression. The aim of this study was to examine the effect of TGF-ß in the tumor microenvironment on the balance between its anti-proliferative and immunosuppressive effects. METHODS: A murine BALB/c spontaneous colon adenocarcinoma cell line (CT26) was genetically engineered to produce increased active TGF-ß (CT26-TGF-ß), a dominant-negative soluble TGF-ß receptor (CT26-TGF-ß-R), or the empty neomycin cassette as control (CT26-neo). In vitro proliferation rates were measured. For in vivo studies, the three cell lines were injected into syngeneic BALB/c mice, and tumor growth was measured over time. Immunodeficient BALB/c nude mice were used to investigate the role of T and B cells. RESULTS: In vitro, CT26-TGF-ß-R and CT26-TGF-ß cells showed increased and suppressed proliferation, respectively, compared to control (CT26-neo), confirming TGF-ß has direct anti-tumor effects. In vivo, we found that CT26-TGF-ß-R cells displayed slower growth compared to control, likely secondary to reduced suppression of anti-tumor immunity, as this effect was ablated in immunodeficient BALB/c nude mice. However, CT26-TGF-ß cells (excess TGF-ß) exhibited rapid early growth compared to control, but later failed to progress. The same pattern was shown in immunodeficient BALB/c nude mice, suggesting the effect on tumor growth is direct, with minimal immune system involvement. There was minimal effect on systemic antitumor immunity as determined by peripheral antigen-specific splenocyte type 1 cytokine production and tumor growth rate of CT26-neo on the contralateral flank of the same mice. CONCLUSION: Although TGF-ß has opposing effects on tumor growth, this study showed that excessive td-TGF-ß in the tumor microenvironment renders the tumor non-proliferative. Depleting excess td-TGF-ß may release this endogenous tumor suppressive mechanism, thus triggering the progression of the tumor. Therefore, our findings support cautions against using anti-TGF-ß strategies in treating cancer, as this may tip the balance of anti-immunity vs. anti-tumor effects of TGF-ß, leading to tumor progression instead of remission.

Role of Dietary Metabolites in Regulating the Host Immune Response in Gastrointestinal Disease.

The host immune response to gastrointestinal (GI) infections, hypersensitivity reactions, or GI cancers comprises numerous pathways that elicit responses on different host cells. Some of these include (1) the stimulation of mast cells via their IgE receptor, (2) the production of antibodies leading to antibody-mediated cytotoxic T/natural killer cell killing, (3) the activation of the complement pathway, and (4) the activation of the adaptive immune response via antigen-presenting cell, T cell, and B cell interactions. Within the plethora of these different responses, several host immune cells represent major key players such as those of myeloid lineage (including neutrophils, macrophages, myeloid-derived suppressor cells) or lymphoid lineage (including T and B cells). In this review, we focus on newly identified metabolites and metabolite receptors that are expressed by either myeloid or lymphoid lineages. Irrespective of their source, these metabolites can in certain instances elicit responses on a wide range of cell types. The myeloid-expressed metabolic enzymes and receptors which we will discuss in this review include arginase 2 (Arg2), indoleamine-2,3-dioxygenase 1 (IDO1), hydroxycarboxylic acid receptor 2 (Hcar2; also called GPR109A), and immunoresponsive gene 1 (Irg1). We will also review the role of the lymphoid-expressed metabolite receptor that binds to the sphingosine-1-phosphate (S1P) sphingolipid. Moreover, we will describe the synthesis and metabolism of retinoic acid, and its effect on T cell activation. The review will then discuss the function of these metabolites in the context of GI disease. The review provides evidence that metabolic pathways operate in a disease- and context-dependent manner-either independently or concomitantly-in the GI tract. Therefore, an integrated approach and combinatorial analyses are necessary to devise new therapeutic strategies that can synergistically improve prognoses.

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.

Functional Characterization of Inflammatory Bowel Disease-Associated Gut Dysbiosis in Gnotobiotic Mice.

BACKGROUND & AIMS: Gut 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). METHODS: Germ-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. RESULTS: Although 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. CONCLUSIONS: Dysbiosis potentially contributes to the pathogenesis of IBD by augmenting host proinflammatory immune responses. Transcript profiling: GSE73882.

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.

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.

Does stress induce bowel dysfunction?

Psychological stress is known to induce somatic symptoms. Classically, many gut physiological responses to stress are mediated by the hypothalamus-pituitary-adrenal axis. There is, however, a growing body of evidence of stress-induced corticotrophin-releasing factor (CRF) release causing bowel dysfunction through multiple pathways, either through the HPA axis, the autonomic nervous systems, or directly on the bowel itself. In addition, recent findings of CRF influencing the composition of gut microbiota lend support for the use of probiotics, antibiotics, and other microbiota-altering agents as potential therapeutic measures in stress-induced bowel dysfunction.

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.