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Mononuclear phagocytes (MNPs) such as dendritic cells and macrophages perform key sentinel functions in mucosal tissues and are responsible for inducing and maintaining adaptive immune responses to mucosal pathogens. Positioning of MNPs at the epithelial interface facilitates their access to luminally-derived antigens and regulates MNP function through soluble mediators or surface receptor interactions. Therefore, accurately quantifying the distribution of MNPs within mucosal tissues as well as their spatial relationship with other cells is important to infer functional cellular interactions in health and disease. In this study, we developed and validated a MATLAB-based tissue cytometry platform, termed "MNP mapping application" (MNPmApp), that performs high throughput analyses of MNP density and distribution in the gastrointestinal mucosa based on digital multicolor fluorescence microscopy images and that integrates a Monte Carlo modeling feature to assess randomness of MNP distribution. MNPmApp identified MNPs in tissue sections of the human gastric mucosa with 98 ± 2% specificity and 76 ± 15% sensitivity for HLA-DR

Helicobacter pylori infection is the main risk factor for the development of gastric cancer, the third leading cause of cancer death worldwide. H. pylori colonizes the human gastric mucosa and persists for decades. The inflammatory response is ineffective in clearing the infection, leading to disease progression that may result in gastric adenocarcinoma. We have shown that polyamines are regulators of the host response to H. pylori, and that spermine oxidase (SMOX), which metabolizes the polyamine spermine into spermidine plus H2O2, is associated with increased human gastric cancer risk. We now used a molecular approach to directly address the role of SMOX, and demonstrate that Smox-deficient mice exhibit significant reductions of gastric spermidine levels and H. pylori-induced inflammation. Proteomic analysis revealed that cancer was the most significantly altered functional pathway in Smox-/- gastric organoids. Moreover, there was also less DNA damage and β-catenin activation in H. pylori-infected Smox-/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and β-catenin activation was confirmed in human gastric organoids that were treated with a novel SMOX inhibitor. These findings indicate that SMOX promotes H. pylori-induced carcinogenesis by causing inflammation, DNA damage, and activation of β-catenin signaling.

Helicobacter pylori infection is commonly treated with a combination of antibiotics and proton pump inhibitors. However, since H. pylori is becoming increasingly resistant to standard antibiotic regimens, novel treatment strategies are needed. Previous studies have demonstrated that black and red berries may have antibacterial properties. Therefore, we analyzed the antibacterial effects of black and red raspberries and blackberries on H. pylori. Freeze-dried powders and organic extracts from black and red raspberries and blackberries were prepared, and high-performance liquid chromatography was used to measure the concentrations of anthocyanins, which are considered the major active ingredients. To monitor antibiotic effects of the berry preparations on H. pylori, a high-throughput metabolic growth assay based on the Biolog system was developed and validated with the antibiotic metronidazole. Biocompatibility was analyzed using human gastric organoids. All berry preparations tested had significant bactericidal effects in vitro, with MIC90 values ranging from 0.49 to 4.17%. Antimicrobial activity was higher for extracts than powders and appeared to be independent of the anthocyanin concentration. Importantly, human gastric epithelial cell viability was not negatively impacted by black raspberry extract applied at the concentration required for complete bacterial growth inhibition. Our data suggest that black and red raspberry and blackberry extracts may have potential applications in the treatment and prevention of H. pylori infection but differ widely in their MICs. Moreover, we demonstrate that the Biolog metabolic assay is suitable for high-throughput antimicrobial susceptibility screening of H. pylori.

Helicobacter pylori is an important bacterial pathogen that causes chronic infection of the human stomach, leading to gastritis, peptic ulcer disease and gastric cancer. Treatment with appropriate antibiotics can eliminate H. pylori infection and reduce the risk for severe disease outcomes. However, since H. pylori is becoming increasingly resistant to standard antibiotic regimens, novel treatment strategies are needed. Previous studies have demonstrated that black and red berries may have antibacterial properties. Therefore, we analyzed organic extracts and powders from black and red raspberries and blackberries and determined their antibacterial effects on multiple H. pylori strains. We used high-performance liquid chromatography to measure berry anthocyanins, which are considered the major active ingredients. To monitor antibiotic effects of the berry preparations on H. pylori, we developed a high-throughput metabolic growth assay based on the OmniLog™ system. All berry preparations tested had significant bactericidal effects in vitro, with MIC90 values ranging from 0.49 to 4.17%. We next used human gastric epithelial organoids to evaluate biocompatibility of the berry preparations and showed that black raspberry extract, which had the strongest antimicrobial activity, was non-toxic at the concentration required for complete bacterial growth inhibition. To determine whether dietary black raspberry application could eliminate H. pylori infection in vivo, mice were infected with H. pylori and then were placed on a diet containing 10% black raspberry powder. However, this treatment did not significantly impact bacterial infection rates or gastric pathology. In summary, our data indicate that black and red raspberry and blackberry products have potential applications in the treatment and prevention of H. pylori infection, because of their antibacterial effects and good biocompatibility. However, delivery and formulation of berry compounds needs to be optimized to achieve significant antibacterial effects in vivo.

Dendritic cell (DC) expression of CD103, the α subunit of αEβ7 integrin, is thought to enable DC interactions with E-cadherin-expressing gastrointestinal epithelia for improved mucosal immunosurveillance. In the stomach, efficient DC surveillance of the epithelial barrier is crucial for the induction of immune responses to H. pylori, the causative agent of peptic ulcers and gastric cancer. However, gastric DCs express only low levels of surface CD103, as we previously showed. We here tested the hypothesis that intracellular pools of CD103 in human gastric DCs can be redistributed to the cell surface for engagement of epithelial cell-expressed E-cadherin to promote DC-epithelial cell adhesion. In support of our hypothesis, immunofluorescence analysis of tissue sections showed that CD103+ gastric DCs were preferentially localized within the gastric epithelial layer. Flow cytometry and imaging cytometry revealed that human gastric DCs expressed intracellular CD103, corroborating our previous findings in monocyte-derived DCs (MoDCs). Using confocal microscopy, we show that CD103 was present in endosomal compartments, where CD103 partially co-localized with clathrin, early endosome antigen-1 and Rab11, suggesting that CD103 undergoes endosomal trafficking similar to β1 integrins. Dynamic expression of CD103 on human MoDCs was confirmed by internalization assay. To analyze whether DC-expressed CD103 promotes adhesion to E-cadherin, we performed adhesion and spreading assays on E-cadherin-coated glass slides. In MoDCs generated in the presence of retinoic acid, which express increased CD103, intracellular CD103 significantly redistributed toward the E-cadherin-coated glass surface. However, DCs spreading and adhesion did not differ between E-cadherin-coated slides and slides coated with serum alone. In adhesion assays using E-cadherin-positive HT-29 cells, DC binding was significantly improved by addition of Mn2+ and decreased in the presence of EGTA, consistent with the dependence of integrin-based interactions on divalent cations. However, retinoic acid failed to increase DC adhesion, and a CD103 neutralizing antibody was unable to inhibit DC binding to the E-cadherin positive cells. In contrast, a blocking antibody to DC-expressed E-cadherin significantly reduced DC binding to the epithelium. Overall, these data indicate that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin, but is not a major driver of DC adhesion to gastrointestinal epithelia.

Background & Aims: Gastric dendritic cells (DCs) control the adaptive response to infection with Helicobacter pylori, a major risk factor for peptic ulcer disease and gastric cancer. We hypothesize that DC interactions with the gastric epithelium position gastric DCs for uptake of luminal H pylori and promote DC responses to epithelial-derived mediators. The aim of this study was to determine whether the gastric epithelium actively recruits DCs using a novel co-culture model of human gastric epithelial spheroids and monocyte-derived DCs. Methods: Spheroid cultures of primary gastric epithelial cells were infected with H pylori by microinjection. Co-cultures were established by adding human monocyte-derived DCs to the spheroid cultures and were analyzed for DC recruitment and antigen uptake by confocal microscopy. Protein array, gene expression polymerase chain reaction array, and chemotaxis assays were used to identify epithelial-derived chemotactic factors that attract DCs. Data from the co-culture model were confirmed using human gastric tissue samples. Results: Human monocyte-derived DCs co-cultured with gastric spheroids spontaneously migrated to the gastric epithelium, established tight interactions with the epithelial cells, and phagocytosed luminally applied H pylori. DC recruitment was increased upon H pylori infection of the spheroids and involved the activity of multiple chemokines including CXCL1, CXCL16, CXCL17, and CCL20. Enhanced chemokine expression and DC recruitment to the gastric epithelium also was observed in H pylori–infected human gastric tissue samples. Conclusions: Our results indicate that the gastric epithelium actively recruits DCs for immunosurveillance and pathogen sampling through chemokine-dependent mechanisms, with increased recruitment upon active H pylori infection. Keywords: Stomach, Organoid, Mononuclear Phagocyte, In Vitro Model