A Fluorescence-based Assay of Membrane Potential for High-throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines.

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.

High-Throughput Functional Analysis of CFTR and Other Apically Localized Proteins in iPSC-Derived Human Intestinal Organoids.

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.

Helicobacter pylori Infection.

Helicobacter pylori infection remains one of the most prevalent infections worldwide, causing significant morbidity and mortality from gastric malignancies and peptic ulcers. This article provides a summary of the microbiology and pathogenesis of this bacterium, emphasizing the complex and protean effects of H pylori on gastric epithelial cells, including stem and progenitor populations, and evasion of host immune defenses. Increasing antibiotic resistance has made management more challenging. This article discusses the appropriate diagnostic modality for different clinical scenarios, and the evolving treatment of H pylori infections, including the use of antibiotic susceptibility testing to aid regimen selection.

VacA generates a protective intracellular reservoir for Helicobacter pylori that is eliminated by activation of the lysosomal calcium channel TRPML1.

Helicobacter pylori infection is a proven carcinogen for gastric cancer. Its virulence factor vacuolating cytotoxin A (VacA) promotes more severe disease and gastric colonization. VacA, by an unknown mechanism, usurps lysosomal and autophagy pathways to generate a protected reservoir for H. pylori that confers bacterial survival in vitro. Here, we show the existence of a VacA-generated intracellular niche in vivo that protects the bacteria from antibiotic treatment and leads to infection recrudescence after therapy. Furthermore, we report that VacA targets the lysosomal calcium channel TRPML1 to disrupt endolysosomal trafficking and mediate these effects. Remarkably, H. pylori that lack toxigenic VacA colonize enlarged dysfunctional lysosomes in the gastric epithelium of trpml1-null mice, where they are protected from eradication therapy. Furthermore, a small molecule agonist directed against TRPML1 reversed the toxic effects of VacA on endolysosomal trafficking, culminating in the clearance of intracellular bacteria. These results suggest that TRPML1 may represent a therapeutic target for chronic H. pylori infection.

VacA promotes CagA accumulation in gastric epithelial cells during Helicobacter pylori infection.

Helicobacter pylori (H. pylori) is the causative agent of gastric cancer, making it the only bacterium to be recognized as a Class I carcinogen by the World Health Organization. The virulence factor cytotoxin associated gene A (CagA) is a known oncoprotein that contributes to the development of gastric cancer. The other major virulence factor vacuolating cytotoxin A (VacA), disrupts endolysosomal vesicular trafficking and impairs the autophagy pathway. Studies indicate that there is a functional interplay between these virulence factors by unknown mechanisms. We show that in the absence of VacA, both host-cell autophagy and the proteasome degrade CagA during infection with H. pylori. In the presence of VacA, CagA accumulates in gastric epithelial cells. However, VacA does not affect proteasome function during infection with H. pylori suggesting that VacA-disrupted autophagy is the predominant means by which CagA accumulates. Our studies support a model where in the presence of VacA, CagA accumulates in dysfunctional autophagosomes providing a possible explanation for the functional interplay of VacA and CagA.

Microbial Disruption of Autophagy Alters Expression of the RISC Component AGO2, a Critical Regulator of the miRNA Silencing Pathway.

BACKGROUND: Autophagy is implicated in Crohn's disease (CD) pathogenesis. Recent evidence suggests autophagy regulates the microRNA (miRNA)-induced silencing complex (miRISC). Therefore, autophagy may play a novel role in CD by regulating expression of miRISC, thereby altering miRNA silencing. As microbes associated with CD can alter autophagy, we hypothesized that microbial disruption of autophagy affects the critical miRISC component AGO2. METHODS: AGO2 expression was assessed in epithelial and immune cells, and intestinal organoids with disrupted autophagy. Microarray technology was used to determine the expression of downstream miRNAs in cells with defective autophagy. RESULTS: Increased AGO2 was detected in autophagy-deficient ATG5-/- and ATG16-/- mouse embryonic fibroblast cells (MEFs) in comparison with wild-type MEFs. Chemical agents and VacA toxin, which disrupt autophagy, increased AGO2 expression in MEFs, epithelial cells lines, and human monocytes, respectively. Increased AGO2 was also detected in ATG7-/- intestinal organoids, in comparison with wild-type organoids. Five miRNAs were differentially expressed in autophagy-deficient MEFs. Pathway enrichment analysis of the differentially expressed miRNAs implicated signaling pathways previously associated with CD. CONCLUSIONS: Taken together, our results suggest that autophagy is involved in the regulation of the critical miRISC component AGO2 in epithelial and immune cells and primary intestinal epithelial cells. We propose a mechanism by which autophagy alters miRNA expression, which likely impacts the regulation of CD-associated pathways. Furthermore, as enteric microbial products can manipulate autophagy and AGO2, our findings suggest a novel mechanism by which enteric microbes could influence miRNA to promote disease.

Helicobacter pylori inhibits dendritic cell maturation via interleukin-10-mediated activation of the signal transducer and activator of transcription 3 pathway.

Helicobacter pylori infects the human gastric mucosa causing a chronic infection that is the primary risk factor for gastric cancer development. Recent studies demonstrate that H. pylori promotes tolerogenic dendritic cell (DC) development indicating that this bacterium evades the host immune response. However, the signaling pathways involved in modulating DC activation during infection remain unclear. Here, we report that H. pylori infection activated the signal transducer and activator of transcription 3 (STAT3) pathway in murine bone marrow-derived DCs (BMDCs) and splenic DCs isolated ex vivo. Isogenic cagA-, cagE-, vacA- and urease-mutants exhibited levels of phosphoSTAT3 that were comparable to in the wild-type (WT) parent strain. H. pylori-infected BMDCs produced increased immunosuppressive IL-10, which activated STAT3 in an autocrine/paracrine fashion. Neutralization of IL-10 prevented H. pylori-mediated STAT3 activation in both BMDCs and splenic DCs. In addition, anti-IL-10 treatment of infected H. pylori-BMDCs was associated with increased CD86 and MHC II expression and enhanced proinflammatory IL-1ß cytokine secretion. Finally, increased CD86 and MHC II expression was detected in H. pylori-infected STAT3 knockout DCs when compared to WT controls. Together, these results demonstrate that H. pylori infection induces IL-10 secretion in DCs, which activates STAT3, thereby modulating DC maturation and reducing IL-1ß secretion. These findings identify a host molecular mechanism by which H. pylori can manipulate the innate immune response to potentially favor chronic infection and promote carcinogenesis.

The protein ATG16L1 suppresses inflammatory cytokines induced by the intracellular sensors Nod1 and Nod2 in an autophagy-independent manner.

The peptidoglycan sensor Nod2 and the autophagy protein ATG16L1 have been linked to Crohn's disease (CD). Although Nod2 and the related sensor, Nod1, direct ATG16L1 to initiate anti-bacterial autophagy, whether ATG16L1 affects Nod-driven inflammation has not been examined. Here, we uncover an unanticipated autophagy-independent role for ATG16L1 in negatively regulating Nod-driven inflammatory responses. Knockdown of ATG16L1 expression, but not that of ATG5 or ATG9a, specifically enhanced Nod-driven cytokine production. In addition, autophagy-incompetent truncated forms of ATG16L1 regulated Nod-driven cytokine responses. Mechanistically, we demonstrated that ATG16L1 interfered with poly-ubiquitination of the Rip2 adaptor and recruitment of Rip2 into large signaling complexes. The CD-associated allele of ATG16L1 was impaired in its ability to regulate Nod-driven inflammatory responses. Overall, these results suggest that ATG16L1 is critical for Nod-dependent regulation of cytokine responses and that disruption of this Nod1- or Nod2-ATG16L1 signaling axis could contribute to the chronic inflammation associated with CD.

Modulation of autophagy by Helicobacter pylori and its role in gastric carcinogenesis.

Helicobacter pylori infection represents the strongest known risk factor for the development of gastric cancer. The vacuolating cytotoxin (VacA) plays a key role in disease pathogenesis by exerting pleiotrophic effects on the host. One effect of acute VacA exposure is the induction of autophagy. However, prolonged exposure to the toxin disrupts autophagy by preventing maturation of the autolysosome. Novel insights into the mechanism and consequences of this phenomenon have emerged, but many aspects remain largely unknown. Current evidence supports a scenario in which H. pylori-suppressed autophagy facilitates intracellular survival and persistence of the pathogen, while also generating an environment favoring carcinogenesis.

Pathogenesis of Helicobacter pylori infection.

Helicobacter pylori infection and disease outcome are mediated by a complex interplay between bacterial, host, and environmental factors. Over the past year, our understanding of this complex interplay has been improved by a variety of studies focusing on both host and bacterial factors. These include studies assessing novel virulence factors as well as those most frequently associated with severity of disease outcome including cagA and the cag pathogenicity island, and the vacuolating cytotoxin. Several studies have focused on regulation of virulence factors by environmental factors. In addition, mechanisms by which bacterial virulence factors influence the host response and disease, by inducing epigenetic changes, autophagy and altered oxidative stress have also been elucidated. This review highlights key findings in the pathogenesis of H. pylori infection reported over the past year.

Successful liver transplantation in an infant with stage 4S(M) neuroblastoma.

We report a 2.5-month-old infant with bilateral adrenal neuroblastoma, stage 4S(M), with liver metastases and chemotherapy-induced veno-occlusive disease leading to cirrhosis requiring liver transplantation. Despite unknown tumour histology and MYCN-amplification status, we proceeded with liver transplant. This decision was based on clinical suspicion that our patient was MYCN-negative due to significant tumour regression, and was supported by evidence indicating that MYCN-amplification is rare in infants with favourable-stage neuroblastoma. This is the second case report of neuroblastoma requiring liver transplantation; however, in the previously reported case, the diagnosis of neuroblastoma was not established until after transplantation. We discuss this unique case to justify the potential use of life-saving liver transplants in infants with neuroblastoma.

Cell culture-based assays to test for bacterial adherence and internalization.

Adherence and internalization of Helicobacter pylori into epithelial cells is a recently recognized event in the pathogen's life cycle.

Cell culture assays to evaluate bacterial toxicity and virulence.

Helicobacter pylori CagA and VacA are two critical virulence factors that modulate disease severity in the infected host. The following chapter outlines methods employed to study the effects of these virulence factors on several key signaling pathways in epithelial cells.

Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans.

Autophagy plays key roles both in host defense against bacterial infection and in tumor biology. Helicobacter pylori (H. pylori) infection causes chronic gastritis and is the single most important risk factor for the development of gastric cancer in humans. Its vacuolating cytotoxin (VacA) promotes gastric colonization and is associated with more severe disease. Acute exposure to VacA initially triggers host autophagy to mitigate the effects of the toxin in epithelial cells. Recently, we demonstrated that chronic exposure to VacA leads to the formation of defective autophagosomes that lack CTSD/cathepsin D and have reduced catalytic activity. Disrupted autophagy results in accumulation of reactive oxygen species and SQSTM1/p62 both in vitro and in vivo in biopsy samples from patients infected with VacA(+) but not VacA(-) strains. We also determined that the Crohn disease susceptibility polymorphism in the essential autophagy gene ATG16L1 increases susceptibility to H. pylori infection. Furthermore, peripheral blood monocytes from individuals with the ATG16L1 risk variant show impaired autophagic responses to VacA exposure. This is the first study to identify both a host autophagy susceptibility gene for H. pylori infection and to define the mechanism by which the autophagy pathway is affected following H. pylori infection. Collectively, these findings highlight the synergistic effects of host and bacterial autophagy factors on H. pylori pathogenesis and the potential for subsequent cancer susceptibility.

Vacuolating cytotoxin and variants in Atg16L1 that disrupt autophagy promote Helicobacter pylori infection in humans.

BACKGROUND & AIMS: The Helicobacter pylori toxin vacuolating cytotoxin (VacA) promotes gastric colonization, and its presence (VacA(+)) is associated with more-severe disease. The exact mechanisms by which VacA contributes to infection are unclear. We previously found that limited exposure to VacA induces autophagy of gastric cells, which eliminates the toxin; we investigated whether autophagy serves as a defense mechanism against H pylori infection. METHODS: We investigated the effect of VacA on autophagy in human gastric epithelial cells and primary gastric cells from mice. Expression of p62, a marker of autophagy, was also assessed in gastric tissues from patients infected with toxigenic (VacA(+)) or nontoxigenic strains. We analyzed the effect of VacA on autophagy in peripheral blood monocytes obtained from subjects with different genotypes of ATG16L1, which regulates autophagy. We performed genotyping for ATG16L1 in 2 cohorts of infected and uninfected subjects. RESULTS: Prolonged exposure of human gastric epithelial cells and mouse gastric cells to VacA disrupted induction of autophagy in response to the toxin, because the cells lacked cathepsin D in autophagosomes. Loss of autophagy resulted in the accumulation of p62 and reactive oxygen species. Gastric biopsy samples from patients infected with VacA(+), but not nontoxigenic strains of H pylori, had increased levels of p62. Peripheral blood monocytes isolated from individuals with polymorphisms in ATG16L1 that increase susceptibility to Crohn's disease had reduced induction of autophagy in response to VacA(+) compared to cells from individuals that did not have these polymorphisms. The presence of the ATG16L1 Crohn's disease risk variant increased susceptibility to H pylori infection in 2 separate cohorts. CONCLUSIONS: Autophagy protects against infection with H pylori; the toxin VacA disrupts autophagy to promote infection, which could contribute to inflammation and eventual carcinogenesis.

Recent advances in Helicobacter pylori pathogenesis.

PURPOSE OF REVIEW: Helicobacter pylori is implicated in numerous gastric pathologies; however, the prevalence of infection is declining in developed countries. Therefore, it is important to understand the complex mechanism of its interaction with the host and how the changing epidemiology of infection may impact on disease. In this review, we systemically revisit the major novel discoveries of the last year relating to H. pylori disease pathogenesis. RECENT FINDINGS: Novel pathways have been implicated in H. pylori cytotoxin-associated gene (CagA) mediated carcinogenesis, highlighting the aberrant regulation of proliferation and apoptosis. Furthermore, the human microbiome was implicated as having a key role in H. pylori-related disease development. Several studies have begun to delineate the mechanisms behind the epidemiologically inverse correlation of H. pylori infection with asthma and inflammatory bowel disease. SUMMARY: The recent findings enable researchers to focus on novel and previously unsuspected mechanisms in the development of disease, and prompt further research into possible therapeutic approaches. The potential beneficial aspects of H. pylori colonization and the role bacterial flora play in promoting disease have yet to be elucidated, but promise to have a great impact on patient care.

Helicobacter pylori activates calpain via toll-like receptor 2 to disrupt adherens junctions in human gastric epithelial cells.

Helicobacter pylori is a risk factor for the development of gastritis, gastroduodenal ulcers, and gastric adenocarcinoma. H. pylori-induced disruption of epithelial adherens junctions (AJs) is thought to promote the development of severe disease; however, the mechanisms whereby H. pylori alters AJ structure remain incompletely understood. The present study demonstrates that H. pylori infection in human patients is associated with elevated serum levels of an 80-kDa E-cadherin ectodomain, whose presence is independent of the presence of serum antibodies against CagA. In vitro, a heat-labile H. pylori surface component activates the host protease calpain in human gastric MKN45 cells independently of the virulence factors CagA and VacA. H. pylori-induced calpain activation results in cleavage of E-cadherin to produce a 100-kDa truncated form and induce relocalization of E-cadherin and ß-catenin. Stimulation of MKN45 cells with the toll-like receptor 2 (TLR2) ligand P3C activated calpain and disrupted E-cadherin and ß-catenin in a pattern similar to that induced by H. pylori. Inhibition of TLR2 prevented H. pylori-induced calpain activation and AJ disassembly. Together, these findings identify a novel pathway whereby H. pylori activates calpain via TLR2 to disrupt gastric epithelial AJ structure.

Interleukin-1 receptor phosphorylation activates Rho kinase to disrupt human gastric tight junctional claudin-4 during Helicobacter pylori infection.

Helicobacter pylori infects more than half of the human population worldwide. In the absence of treatment, this persistent infection leads to asymptomatic gastritis, which in some cases can progress into gastric ulcers and adenocarcinomas. The host-microbial interactions that govern the clinical outcome of infection remain incompletely understood. H. pylori is known to disrupt gastric epithelial tight junctions, which may represent a significant component of disease pathogenesis. The present study demonstrates that H. pylori disrupt epithelial tight junctional claudin-4 in a Rho kinase (ROCK)-dependent manner in human gastric epithelial (HGE-20) cell monolayers, independently of the virulence factors CagA and VacA, and without altering claudin-4 transcription. In the same epithelial cell model, interleukin (IL)-1beta, mediated a similar ROCK-dependent pattern of tight junction disruption. Further experiments revealed that H. pylori infection induced IL-1 receptor type I (IL-1RI) phosphorylation, independently of epithelial secretion of its endogenous ligands IL-1alpha, IL-1beta or IL-18. Finally, inhibition of IL-1RI activation prevented H. pylori-induced ROCK activation and claudin-4 disruption. Taken together, these findings identify a novel pathophysiological mechanism by which H. pylori disrupts gastric epithelial barrier structure via IL-1RI-dependent activation of ROCK, which in turn mediates tight junctional claudin-4 disruption.