Toll-like Receptor 9 Pathway Mediates Schlafen+-MDSC Polarization During Helicobacter-induced Gastric Metaplasias.

BACKGROUND & AIMS: A subset of myeloid-derived suppressor cells (MDSCs) that express murine Schlafen4 (SLFN4) or its human ortholog SLFN12L polarize in the Helicobacter-inflamed stomach coincident with intestinal or spasmolytic polypeptide-expressing metaplasia. We propose that individuals with a more robust response to damage-activated molecular patterns and increased Toll-like receptor 9 (TLR9) expression are predisposed to the neoplastic complications of Helicobacter infection. METHODS: A mouse or human Transwell co-culture system composed of dendritic cells (DCs), 2-dimensional gastric epithelial monolayers, and Helicobacter were used to dissect the cellular source of interferon-α (IFNα) in the stomach by flow cytometry. Conditioned media from the co-cultures polarized primary myeloid cells. MDSC activity was determined by T-cell suppression assays. In human subjects with intestinal metaplasia or gastric cancer, the rs5743836 TLR9T>C variant was genotyped and linked to TLR9, IFNα, and SLFN12L expression by immunohistochemistry. Nuclear factor-κB binding to the TLR9 C allele was determined by electrophoretic mobility shift assays. RESULTS: Helicobacter infection induced gastric epithelial and plasmacytoid DC expression of TLR9 and IFNα. Co-culturing primary mouse or human cells with DCs and Helicobacter induced TLR9, IFNα secretion, and SLFN+-MDSC polarization. Neutralizing IFNα in vivo mitigated Helicobacter-induced spasmolytic polypeptide-expressing metaplasia. The TLR9 minor C allele creates a nuclear factor-κB binding site associated with higher levels of TLR9, IFNα, and SLFN12L in Helicobacter-infected stomachs that correlated with a greater incidence of metaplasias and cancer. CONCLUSIONS: TLR9 plays an essential role in the production of IFNα and polarization of SLFN+ MDSCs on Helicobacter infection. Subjects carrying the rs5743836 TLR9 minor C allele are predisposed to neoplastic complications if chronically infected.

STAT3-mediated upregulation of the AIM2 DNA sensor links innate immunity with cell migration to promote epithelial tumourigenesis.

OBJECTIVE: The absent in melanoma 2 (AIM2) cytosolic pattern recognition receptor and DNA sensor promotes the pathogenesis of autoimmune and chronic inflammatory diseases via caspase-1-containing inflammasome complexes. However, the role of AIM2 in cancer is ill-defined. DESIGN: The expression of AIM2 and its clinical significance was assessed in human gastric cancer (GC) patient cohorts. Genetic or therapeutic manipulation of AIM2 expression and activity was performed in the genetically engineered gp130 F/F spontaneous GC mouse model, as well as human GC cell line xenografts. The biological role and mechanism of action of AIM2 in gastric tumourigenesis, including its involvement in inflammasome activity and functional interaction with microtubule-associated end-binding protein 1 (EB1), was determined in vitro and in vivo. RESULTS: AIM2 expression is upregulated by interleukin-11 cytokine-mediated activation of the oncogenic latent transcription factor STAT3 in the tumour epithelium of GC mouse models and patients with GC. Genetic and therapeutic targeting of AIM2 in gp130 F/F mice suppressed tumourigenesis. Conversely, AIM2 overexpression augmented the tumour load of human GC cell line xenografts. The protumourigenic function of AIM2 was independent of inflammasome activity and inflammation. Rather, in vivo and in vitro AIM2 physically interacted with EB1 to promote epithelial cell migration and tumourigenesis. Furthermore, upregulated expression of AIM2 and EB1 in the tumour epithelium of patients with GC was independently associated with poor patient survival. CONCLUSION: AIM2 can play a driver role in epithelial carcinogenesis by linking cytokine-STAT3 signalling, innate immunity and epithelial cell migration, independent of inflammasome activation.

Human Gastrointestinal Organoid Models for Studying Microbial Disease and Cancer.

One of the major discoveries in stem cell research in the past decade embraces the development of "organs in a dish," also known as "organoids." Organoids are three-dimensional cellular structures derived from primary stem cells of different organ-specific cell types which are capable of self-renewal and maintenance of the parental lineages. Researchers have developed in vitro organoid models to mimic in vivo host-microbial interactions and disease. In this review, we focus on the use of gastrointestinal organoids as models of microbial disease and cancer.

Targeted mobilization of Lrig1+ gastric epithelial stem cell populations by a carcinogenic Helicobacter pylori type IV secretion system.

Helicobacter pylori-induced gastritis is the strongest risk factor for gastric adenocarcinoma, a malignancy preceded by a series of well-defined histological stages, including metaplasia. One microbial constituent that augments cancer risk is the cag type 4 secretion system (T4SS), which translocates the oncoprotein CagA into host cells. Aberrant stem cell activation is linked to carcinogenesis, and Lrig1 (leucine-rich repeats and Ig-like domains 1) marks a distinct population of progenitor cells. We investigated whether microbial effectors with carcinogenic potential influence Lrig1 progenitor cells ex vivo and via lineage expansion within H. pylori-infected gastric mucosa. Lineage tracing was induced in Lrig1-CreERT2/+;R26R-YFP/+ (Lrig1/YFP) mice that were uninfected or subsequently infected with cag+H. pylori or an isogenic cagE- mutant (nonfunctional T4SS). In contrast to infection with wild-type (WT) H. pylori for 2 wk, infection for 8 wk resulted in significantly increased inflammation and proliferation in the corpus and antrum compared with uninfected or mice infected with the cagE- mutant. WT H. pylori-infected mice harbored significantly higher numbers of Lrig1/YFP epithelial cells that coexpressed UEA1 (surface cell marker). The number of cells coexpressing intrinsic factor (chief cell marker), YFP (lineage marker), and GSII lectin (spasmolytic polypeptide-expressing metaplasia marker) were increased only by WT H. pylori In human samples, Lrig1 expression was significantly increased in lesions with premalignant potential compared with normal mucosa or nonatrophic gastritis. In conclusion, chronic H. pylori infection stimulates Lrig1-expressing progenitor cells in a cag-dependent manner, and these reprogrammed cells give rise to a full spectrum of differentiated cells.

Gastric Metabolomics Detects Helicobacter pylori Correlated Loss of Numerous Metabolites in Both the Corpus and Antrum.

Helicobacter pylori is a chronic bacterial pathogen that thrives in several regions of the stomach, causing inflammation that can vary by site and result in distinct disease outcomes. Whether the regions differ in terms of host-derived metabolites is not known. We thus characterized the regional variation of the metabolomes of mouse gastric corpus and antrum organoids and tissue. The uninfected secreted organoid metabolites differed between the corpus and antrum in only seven metabolites as follows: lactic acid, malic acid, phosphoethanolamine, alanine, uridine, glycerol, and isoleucine. Several of the secreted chemicals were depleted upon H. pylori infection in both regions, including urea, cholesterol, glutamine, fumaric acid, lactic acid, citric acid, malic acid, and multiple nonessential amino acids. These results suggest a model in which H. pylori preferentially uses carboxylic acids and amino acids in complex environments, and these are found in both the corpus and antrum. When organoid metabolites were compared to mouse tissue, there was little overlap. The tissue corpus and antrum metabolomes were distinct, including antrum-elevated 5-methoxytryptamine, lactic acid, and caprylic acid, and corpus-elevated phospholipid products. The corpus and antrum remained distinct over an 8-month infection time course. The antrum displayed no significant changes between the time points in contrast to the corpus, which exhibited metabolite changes that were consistent with stress, tissue damage, and depletion of key nutrients, such as glutamine and fructose-6-phosphate. Overall, our results suggest that the corpus and antrum have largely but not completely overlapping metabolomes that change moderately upon H. pylori infection.

Modeling pancreatic pathophysiology using genome editing of adult stem cell-derived and induced pluripotent stem cell (iPSC)-derived organoids.

In recent years, organoids have become a novel in vitro method to study gastrointestinal organ development, physiology, and disease. An organoid, in short, may be defined as a miniaturized organ that can be grown from adult stem cells in vitro and studied at the microscopic level. Organoids have been used in multitudes of different ways to study the physiology of different human diseases including gastrointestinal cancers such as pancreatic cancer. The development of genome editing based on the bacterial defense mechanism clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a laboratory tool that provides the opportunity to study the effects of specific genetic changes on organ development, physiology, and disease. The CRISPR/Cas9 approach can be combined with organoid technology including the use of induced pluripotent stem cell (iPSC)-derived and tissue-derived organoids. The goal of this review is to provide highlights on the development of organoid technology, and the use of this culture system to study the pathophysiology of specific mutations in the development of pancreatic and gastric cancers.NEW & NOTEWORTHY The goal of this review is not only to provide highlights on the development of organoid technology but also to subsequently use this information to study the pathophysiology of those specific mutations in the formation of malignant pancreatic and gastric cancer.

Increased Programmed Death-Ligand 1 is an Early Epithelial Cell Response to Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) is the major risk factor for the development of gastric cancer. Our laboratory has reported that the Sonic Hedgehog (Shh) signaling pathway is an early response to infection that is fundamental to the initiation of H. pylori-induced gastritis. H. pylori also induces programmed death ligand 1 (PD-L1) expression on gastric epithelial cells, yet the mechanism is unknown. We hypothesize that H. pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Shh signaling pathway during infection. To identify the role of Shh signaling as a mediator of H. pylori-induced PD-L1 expression, human gastric organoids generated from either induced pluripotent stem cells (HGOs) or tissue (huFGOs) were microinjected with bacteria and treated with Hedgehog/Gli inhibitor GANT61. Gastric epithelial monolayers generated from the huFGOs were also infected with H. pylori and treated with GANT61 to study the role of Hedgehog signaling as a mediator of induced PD-1 expression. A patient-derived organoid/autologous immune cell co-culture system infected with H. pylori and treated with PD-1 inhibitor (PD-1Inh) was developed to study the protective mechanism of PD-L1 in response to bacterial infection. H. pylori significantly increased PD-L1 expression in organoid cultures 48 hours post-infection when compared to uninfected controls. The mechanism was cytotoxic associated gene A (CagA) dependent. This response was blocked by pretreatment with GANT61. Anti-PD-L1 treatment of H. pylori infected huFGOs, co-cultured with autologous patient cytotoxic T lymphocytes and dendritic cells, induced organoid death. H. pylori-induced PD-L1 expression is mediated by the Shh signaling pathway within the gastric epithelium. Cells infected with H. pylori that express PD-L1 may be protected from the immune response, creating premalignant lesions progressing to gastric cancer.

Carcinogenic Helicobacter pylori Strains Selectively Dysregulate the In Vivo Gastric Proteome, Which May Be Associated with Stomach Cancer Progression.

Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly up-regulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared with gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans.

MiR130b from Schlafen4+ MDSCs stimulates epithelial proliferation and correlates with preneoplastic changes prior to gastric cancer.

The myeloid differentiation factor Schlafen4 (Slfn4) marks a subset of myeloid-derived suppressor cells (MDSCs) in the stomach during Helicobacter-induced spasmolytic polypeptide-expressing metaplasia (SPEM). OBJECTIVE: To identify the gene products expressed by Slfn4+-MDSCs and to determine how they promote SPEM. DESIGN: We performed transcriptome analyses for both coding genes (mRNA by RNA-Seq) and non-coding genes (microRNAs using NanoString nCounter) using flow-sorted SLFN4+ and SLFN4- cells from Helicobacter-infected mice exhibiting metaplasia at 6 months postinfection. Thioglycollate-elicited myeloid cells from the peritoneum were cultured and treated with IFNα to induce the T cell suppressor phenotype, expression of MIR130b and SLFN4. MIR130b expression in human gastric tissue including gastric cancer and patient sera was determined by qPCR and in situ hybridisation. Knockdown of MiR130b in vivo in Helicobacter-infected mice was performed using Invivofectamine. Organoids from primary gastric cancers were used to generate xenografts. ChIP assay and Western blots were performed to demonstrate NFκb p65 activation by MIR130b. RESULTS: MicroRNA analysis identified an increase in MiR130b in gastric SLFN4+ cells. Moreover, MIR130b colocalised with SLFN12L, a human homologue of SLFN4, in gastric cancers. MiR130b was required for the T-cell suppressor phenotype exhibited by the SLFN4+ cells and promoted Helicobacter-induced metaplasia. Treating gastric organoids with the MIR130b mimic induced epithelial cell proliferation and promoted xenograft tumour growth. CONCLUSION: Taken together, MiR130b plays an essential role in MDSC function and supports metaplastic transformation.

Role of metaplasia during gastric regeneration.

Spasmolytic polypeptide/trefoil factor 2 (TFF2)-expressing metaplasia (SPEM) is a mucous-secreting reparative lineage that emerges at the ulcer margin in response to gastric injury. Under conditions of chronic inflammation with parietal cell loss, SPEM has been found to emerge and evolve into neoplasia. Cluster-of-differentiation gene 44 (CD44) is known to coordinate normal and metaplastic epithelial cell proliferation. In particular, CD44 variant isoform 9 (CD44v9) associates with the cystine-glutamate transporter xCT, stabilizes the protein, and provides defense against reactive oxygen species (ROS). xCT stabilization by CD44v9 leads to defense against ROS by cystine uptake, glutathione (GSH) synthesis, and maintenance of the redox balance within the intracellular environment. Furthermore, p38 signaling is a known downstream ROS target, leading to diminished cell proliferation and migration, two vital processes of gastric epithelial repair. CD44v9 emerges during repair of the gastric epithelium after injury, where it is coexpressed with other markers of SPEM. The regulatory mechanisms for the emergence of CD44v9 and the role of CD44v9 during the process of gastric epithelial regeneration are largely unknown. Inflammation and M2 macrophage infiltration have recently been demonstrated to play key roles in the induction of SPEM after injury. The following review proposes new insights into the functional role of metaplasia in the process of gastric regeneration in response to ulceration. Our insights are extrapolated from documented studies reporting oxyntic atrophy and SPEM development and our current unpublished findings using the acetic acid-induced gastric injury model.

Modelling human development and disease in pluripotent stem-cell-derived gastric organoids.

Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world's population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signalling and induction of epithelial proliferation. Together, these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.

CD44 variant isoform 9 emerges in response to injury and contributes to the regeneration of the gastric epithelium.

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a funcitonal role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-deficient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14-20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile anlayzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastic epithelium CD44v9 emerges within a regenerative cell lineage that coincides with macrophage inflitration within the injured mucosa. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hedgehog signalling in myeloid cells impacts on body weight, adipose tissue inflammation and glucose metabolism.

AIMS/HYPOTHESIS: Recently, hedgehog (Hh) was identified as a crucial player in adipose tissue development and energy expenditure. Therefore, we tested whether Hh ligands are regulated in obesity. Further, we aimed at identifying potential target cells of Hh signalling and studied the functional impact of Hh signalling on adipose tissue inflammation and glucose metabolism. METHODS: Hh ligands and receptors were analysed in adipose tissue or serum from lean and obese mice as well as in humans. To study the impact on adipose tissue inflammation and glucose metabolism, Hh signalling was specifically blocked in myeloid cells using a conditional knockout approach (Lys-Smo -/-). RESULTS: Desert Hh (DHH) and Indian Hh (IHH) are local Hh ligands, whereas Sonic Hh is not expressed in adipose tissue from mice or humans. In mice, obesity leads to a preferential upregulation of Hh ligands (Dhh) and signalling components (Ptch1, Smo and Gli1) in subcutaneous adipose tissue. Further, adipose tissue macrophages are Hh target cells owing to the expression of Hh receptors, such as Patched1 and 2. Conditional knockout of Smo (which encodes Smoothened, a mandatory Hh signalling component) in myeloid cells increases body weight and adipose tissue inflammation and attenuates glucose tolerance, suggesting an anti-inflammatory effect of Hh signalling. In humans, adipose tissue expression of DHH and serum IHH decrease with obesity and type 2 diabetes, which might be explained by the intake of metformin. Interestingly, metformin reduced Dhh and Ihh expression in mouse adipose tissue explants. CONCLUSIONS/INTERPRETATION: Hh signalling in myeloid cells affects adipose tissue inflammation and glucose metabolism and may be a potential target to treat type 2 diabetes.

Organoid Models of Human Gastrointestinal Development and Disease.

We have greatly advanced our ability to grow a diverse range of tissue-derived and pluripotent stem cell-derived gastrointestinal (GI) tissues in vitro. These systems, broadly referred to as organoids, have allowed the field to move away from the often nonphysiological, transformed cell lines that have been used for decades in GI research. Organoids are derived from primary tissues and have the capacity for long-term growth. They contain varying levels of cellular complexity and physiological similarity to native organ systems. We review the latest discoveries from studies of tissue-derived and pluripotent stem cell-derived intestinal, gastric, esophageal, liver, and pancreatic organoids. These studies have provided important insights into GI development, tissue homeostasis, and disease and might be used to develop personalized medicines.

CD44 plays a functional role in Helicobacter pylori-induced epithelial cell proliferation.

The cytotoxin-associated gene (Cag) pathogenicity island is a strain-specific constituent of Helicobacter pylori (H. pylori) that augments cancer risk. CagA translocates into the cytoplasm where it stimulates cell signaling through the interaction with tyrosine kinase c-Met receptor, leading cellular proliferation. Identified as a potential gastric stem cell marker, cluster-of-differentiation (CD) CD44 also acts as a co-receptor for c-Met, but whether it plays a functional role in H. pylori-induced epithelial proliferation is unknown. We tested the hypothesis that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation. To assay changes in gastric epithelial cell proliferation in relation to the direct interaction with H. pylori, human- and mouse-derived gastric organoids were infected with the G27 H. pylori strain or a mutant G27 strain bearing cagA deletion (∆CagA::cat). Epithelial proliferation was quantified by EdU immunostaining. Phosphorylation of c-Met was analyzed by immunoprecipitation followed by Western blot analysis for expression of CD44 and CagA. H. pylori infection of both mouse- and human-derived gastric organoids induced epithelial proliferation that correlated with c-Met phosphorylation. CagA and CD44 co-immunoprecipitated with phosphorylated c-Met. The formation of this complex did not occur in organoids infected with ∆CagA::cat. Epithelial proliferation in response to H. pylori infection was lost in infected organoids derived from CD44-deficient mouse stomachs. Human-derived fundic gastric organoids exhibited an induction in proliferation when infected with H. pylori that was not seen in organoids pre-treated with a peptide inhibitor specific to CD44. In the well-established Mongolian gerbil model of gastric cancer, animals treated with CD44 peptide inhibitor Pep1, resulted in the inhibition of H. pylori-induced proliferation and associated atrophic gastritis. The current study reports a unique approach to study H. pylori interaction with the human gastric epithelium. Here, we show that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation.

Motility and chemotaxis mediate the preferential colonization of gastric injury sites by Helicobacter pylori.

Helicobacter pylori (H. pylori) is a pathogen contributing to peptic inflammation, ulceration, and cancer. A crucial step in the pathogenic sequence is when the bacterium first interacts with gastric tissue, an event that is poorly understood in vivo. We have shown that the luminal space adjacent to gastric epithelial damage is a microenvironment, and we hypothesized that this microenvironment might enhance H. pylori colonization. Inoculation with 106 H. pylori (wild-type Sydney Strain 1, SS1) significantly delayed healing of acetic-acid induced ulcers at Day 1, 7 and 30 post-inoculation, and wild-type SS1 preferentially colonized the ulcerated area compared to uninjured gastric tissue in the same animal at all time points. Gastric resident Lactobacillus spp. did not preferentially colonize ulcerated tissue. To determine whether bacterial motility and chemotaxis are important to ulcer healing and colonization, we analyzed isogenic H. pylori mutants defective in motility (ΔmotB) or chemotaxis (ΔcheY). ΔmotB (10(6)) failed to colonize ulcerated or healthy stomach tissue. ΔcheY (10(6)) colonized both tissues, but without preferential colonization of ulcerated tissue. However, ΔcheY did modestly delay ulcer healing, suggesting that chemotaxis is not required for this process. We used two-photon microscopy to induce microscopic epithelial lesions in vivo, and evaluated accumulation of fluorescently labeled H. pylori at gastric damage sites in the time frame of minutes instead of days. By 5 min after inducing damage, H. pylori SS1 preferentially accumulated at the site of damage and inhibited gastric epithelial restitution. H. pylori ΔcheY modestly accumulated at the gastric surface and inhibited restitution, but did not preferentially accumulate at the injury site. H. pylori ΔmotB neither accumulated at the surface nor inhibited restitution. We conclude that bacterial chemosensing and motility rapidly promote H. pylori colonization of injury sites, and thereby biases the injured tissue towards sustained gastric damage.

Helicobacter pylori targets cancer-associated apical-junctional constituents in gastroids and gastric epithelial cells.

OBJECTIVE: Helicobacter pylori strains that express the oncoprotein CagA augment risk for gastric cancer. However, the precise mechanisms through which cag(+) strains heighten cancer risk have not been fully delineated and model systems that recapitulate the gastric niche are critical for understanding pathogenesis. Gastroids are three-dimensional organ-like structures that provide unique opportunities to study host-H. pylori interactions in a preclinical model. We used gastroids to inform and direct in vitro studies to define mechanisms through which H. pylori modulates expression of the cancer-associated tight junction protein claudin-7. DESIGN: Gastroids were infected by luminal microinjection, and MKN28 gastric epithelial cells were cocultured with H. pylori wild-type cag(+) strains or isogenic mutants. ß-catenin, claudin-7 and snail localisation was determined by immunocytochemistry. Proliferation was assessed using 5-ethynyl-2'-deoxyuridine, and levels of claudin-7 and snail were determined by western blot and flow cytometry. RESULTS: Gastroids developed into a self-organising differentiation axis and H. pylori induced mislocalisation of claudin-7 and increased proliferation in a CagA- and ß-catenin-dependent manner. In MKN28 cells, H pylori-induced suppression of claudin-7 was regulated by ß-catenin and snail. Similarly, snail expression was increased and claudin-7 levels were decreased among H. pylori-infected individuals. CONCLUSIONS: H. pylori increase proliferation in a strain-specific manner in a novel gastroid system. H. pylori also alter expression and localisation of claudin-7 in gastroids and human epithelial cells, which is mediated by ß-catenin and snail activation. These data provide new insights into molecular interactions with carcinogenic potential that occur between H. pylori and epithelial cells within the gastric niche.

The use of murine-derived fundic organoids in studies of gastric physiology.

KEY POINTS: An in vitro approach to study gastric development is primary mouse-derived epithelium cultured as three-dimensional spheroids known as organoids. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Organoids maintained in co-culture with immortalized stomach mesenchymal cells express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. We report the use of these models for studies of epithelial cell biology and cell damage and repair. ABSTRACT: Studies of gastric function and disease have been limited by the lack of extended primary cultures of the epithelium. An in vitro approach to study gastric development is primary mouse-derived antral epithelium cultured as three-dimensional spheroids known as organoids. There have been no reports on the use of organoids for gastric function. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Both models were generated from single glands dissociated from whole fundic tissue and grown in basement membrane matrix (Matrigel) and organoid growth medium. Model 1 enriches for a stem cell-like niche via simple passage of the organoids. Maintained in Matrigel and growth medium, proliferating organoids expressed high levels of stem cell markers CD44 and Lgr5. Model 2 is a system of gastric organoids co-cultured with immortalized stomach mesenchymal cells (ISMCs). Organoids maintained in co-culture with ISMCs express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. Thus, we report the use of these models for studies of epithelial cell biology and cell damage and repair.

Indian Hedgehog mediates gastrin-induced proliferation in stomach of adult mice.

BACKGROUND & AIMS: Loss of expression of Sonic Hedgehog (Shh) from parietal cells results in hypergastrinemia in mice, accompanied by increased expression of Indian Hedgehog (Ihh) and hyperproliferation of surface mucous cells. We investigated whether hypergastrinemia induces gastric epithelial proliferation by activating Ihh signaling in mice. METHODS: We studied mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and hypergastrinemia, crossed with gastrin-deficient (GKO) mice (PC-Shh(KO)/GKO). When mice were 3-4 months old, gastric tissues were collected and analyzed by histology, for incorporation of bromodeoxyuridine, and for expression of the surface mucous cell marker Ulex europaeus. PC-Shh(KO)/GKO mice were given gastrin infusions for 7 days; gastric surface epithelium was collected and expression of Ihh was quantified by laser capture microdissection followed by quantitative reverse transcriptase polymerase chain reaction. Mouse stomach-derived organoids were incubated with or without inhibitors of WNT (DKK1) or Smoothened (vismodegib) and then cocultured with immortalized stomach mesenchymal cells, to assess proliferative responses to gastrin. RESULTS: Gastric tissues from PC-Shh(KO)/GKO mice with hypergastrinemia had an expanded surface pit epithelium, indicated by a significant increase in numbers of bromodeoxyuridine- and Ulex europaeus-positive cells, but there was no evidence for hyperproliferation. Gastrin infusion of PC PC-Shh(KO)/GKO mice increased expression of Ihh and proliferation within the surface epithelium compared with mice given infusions of saline. In gastric organoids cocultured with immortalized stomach mesenchymal cells, antagonists of WNT and Smoothened inhibited gastrin-induced proliferation and WNT activity. Activity of WNT in media collected from immortalized stomach mesenchymal cells correlated with increased expression of glioma-associated oncogene homolog 1, and was inhibited by DKK1 or vismodegib. CONCLUSIONS: Ihh signaling mediates gastrin-induced proliferation of epithelial cells in stomachs of adult mice.