Sina Bartfeld

Tissue-Resident Adult Stem Cell Populations of Rapidly Self-Renewing Organs

The epithelial lining of the intestine, stomach, and skin is continuously exposed to environmental assault, imposing a requirement for regular self-renewal. Resident adult stem cell populations drive this renewal, and much effort has been invested in revealing their identity. Reliable adult stem cell biomarkers would accelerate our understanding of stem cell roles in tissue homeostasis and cancer. Membrane-expressed markers would also facilitate isolation of these adult stem cell populations for exploitation of their regenerative potential. Here, we review recent advances in adult stem cell biology, highlighting the promise and pitfalls of the candidate biomarkers of the various stem cell populations.

In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection

BACKGROUND & AIMS We previously established long-term, 3-dimensional culture of organoids from mouse tissues (intestine, stomach, pancreas, and liver) and human intestine and pancreas. Here we describe conditions required for long-term 3-dimensional culture of human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. METHODS We generated organoids from surgical samples of human gastric corpus. Culture conditions were developed based on those for the mouse gastric and human intestinal systems. We used microinjection to infect the organoids with H pylori. Epithelial responses were measured using microarray and quantitative polymerase chain reaction analyses. RESULTS Human gastric cells were expanded indefinitely in 3-dimensional cultures. We cultured cells from healthy gastric tissues, single-sorted stem cells, or tumor tissues. Organoids maintained many characteristics of their respective tissues based on their histology, expression of markers, and euploidy. Organoids from healthy tissue expressed markers of 4 lineages of the stomach and self-organized into gland and pit domains. They could be directed to specifically express either lineages of the gastric gland, or the gastric pit, by addition of nicotinamide and withdrawal of WNT. Although gastric pit lineages had only marginal reactions to bacterial infection, gastric gland lineages mounted a strong inflammatory response. CONCLUSIONS We developed a system to culture human gastric organoids. This system can be used to study H pylori infection and other gastric pathologies.

The Type III Secretion Effector NleE Inhibits NF-κB Activation

The complex host-pathogen interplay involves the recognition of the pathogen by the hosts innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-kappaB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-kappaB activation. We show that inhibition of NF-kappaB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro

Background and aims Helicobacter pylori is the causative agent of gastric diseases and the main risk factor in the development of gastric adenocarcinoma. In vitro studies with this bacterial pathogen largely rely on the use of transformed cell lines as infection model. However, this approach is intrinsically artificial and especially inappropriate when it comes to investigating the mechanisms of cancerogenesis. Moreover, common cell lines are often defective in crucial signalling pathways relevant to infection and cancer. A long-lived primary cell system would be preferable in order to better approximate the human in vivo situation. Methods Gastric glands were isolated from healthy human stomach tissue and grown in Matrigel containing media supplemented with various growth factors, developmental regulators and apoptosis inhibitors to generate long-lasting normal epithelial cell cultures. Results Culture conditions were developed which support the formation and quasi-indefinite growth of three dimensional (3D) spheroids derived from various sites of the human stomach. Spheroids could be differentiated to gastric organoids after withdrawal of Wnt3A and R-spondin1 from the medium. The 3D cultures exhibit typical morphological features of human stomach tissue. Transfer of sheared spheroids into 2D culture led to the formation of dense planar cultures of polarised epithelial cells serving as a suitable in vitro model of H. pylori infection. Conclusions A robust and quasi-immortal 3D organoid model has been established, which is considered instrumental for future research aimed to understand the underlying mechanisms of infection, mucosal immunity and cancer of the human stomach.

Generation of L cells in mouse and human small intestine organoids.

Upon a nutrient challenge, L cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate functional L cells from three-dimensional cultures of mouse and human intestinal crypts. We show that short-chain fatty acids selectively increase the number of L cells, resulting in an elevation of GLP-1 release. This is accompanied by the upregulation of transcription factors associated with the endocrine lineage of intestinal stem cell development. Thus, our platform allows us to study and modulate the development of L cells in mouse and human crypts as a potential basis for novel therapeutic strategies in patients with type 2 diabetes.

Temporal resolution of two-tracked NF-kappa B activation by Legionella pneumophila

The intracellular pathogen Legionella pneumophila activates the transcription factor NF‐κB in macrophages and human epithelial cells, contributing to cytokine production and anti‐apoptosis. The former is important for the innate immune response to infection, the latter for intracellular replication by securing host cell survival. Here, we demonstrate biphasic activation of NF‐κB by L. pneumophila in human epithelial cells, using a p65‐GFP expressing variant of A549 cells. Early in infection, a strong but transient nuclear translocation of p65 was observed. Only flagellin‐deficient (ΔfliA and ΔflaA) mutants could not induce this first, TLR5 and MyD88‐dependent activation. The second p65 translocation event, however, is a long‐term activation, independent of flagellin, TLR5 and MyD88, and marked by permanent nuclear localization of p65‐GFP without oscillation for 30 h. Persistent p65 translocation also involved degradation of IκBα and upregulation of anti‐apoptotic genes. L. pneumophila mutants lacking a functional Dot/Icm secretion system (ΔdotA; ΔicmB/dotO), Dot/Icm effectors (ΔsdbA; ΔlubX) and two bacterial effector mutants (ΔenhC; ΔptsP) could not induce persistent p65 translocation. Strikingly, all these mutants were deficient in intracellular replication in A549 cells. Our data underline the strong connection between NF‐κB activation and intracellular replication and hints at an active interference of NF‐κB signalling by L. pneumophila.

Helicobacter pylori outer membrane protein HopQ identified as a novel T4SS‐associated virulence factor

Helicobacter pylori is a bacterial pathogen that colonizes the gastric niche of ∼ 50% of the human population worldwide and is known to cause peptic ulceration and gastric cancer. Pathology of infection strongly depends on a cag pathogenicity island (cagPAI)‐encoded type IV secretion system (T4SS). Here, we aimed to identify as yet unknown bacterial factors involved in cagPAI effector function and performed a large‐scale screen of an H. pylori transposon mutant library using activation of the pro‐inflammatory transcription factor NF‐κB in human gastric epithelial cells as a measure of T4SS function. Analysis of ∼ 3000 H. pylori mutants revealed three non‐cagPAI genes that affected NF‐κB nuclear translocation. Of these, the outer membrane protein HopQ from H. pylori strain P12 was essential for CagA translocation and for CagA‐mediated host cellresponses such as formation of the hummingbird phenotype and cell scattering. Besides that, deletion of hopQ reduced T4SS‐dependent activation of NF‐κB, induction of MAPK signalling and secretion of interleukin 8 (IL‐8) in the host cells, but did not affect motility or the quantity of bacteria attached to host cells. Hence, we identified HopQ as a non‐cagPAI‐encoded cofactor of T4SS function.

Helicobacter pylori HP0518 affects flagellin glycosylation to alter bacterial motility

Helicobacter pylori is a human gastric pathogen associated with gastric and duodenal ulcers as well as gastric cancer. Mounting evidence suggests this pathogens motility is prerequisite for successful colonization of human gastric tissues. Here, we isolated an H. pylori G27 HP0518 mutant exhibiting altered motility in comparison to its parental strain. We show that the mutants modulated motility is linked to increased levels of O‐linked glycosylation on flagellin A (FlaA) protein. Recombinant HP0518 protein decreased glycosylation levels of H. pylori flagellin in vitro, indicating that HP0518 functions in deglycosylation of FlaA protein. Furthermore, mass spectrometric analysis revealed increased glycosylation of HP0518 FlaA was due to a change in pseudaminic acid (Pse) levels on FlaA; HP0518 mutant‐derived flagellin contained approximately threefold more Pse than the parental strain. Further phenotypic and molecular characterization demonstrated that the hyper‐motile HP0518 mutant exhibits superior colonization capabilities and subsequently triggers enhanced CagA phosphorylation and NF‐κB activation in AGS cells. Our study shows that HP0518 is involved in the deglycosylation of flagellin, thereby regulating pathogen motility. These findings corroborate the prominent function of H. pylori flagella in pathogen–host cell interactions and modulation of host cell responses, likely influencing the pathogenesis process.

High-throughput and single-cell imaging of NF-κB oscillations using monoclonal cell lines

BackgroundThe nuclear factor-κB (NF-κB) family of transcription factors plays a role in a wide range of cellular processes including the immune response and cellular growth. In addition, deregulation of the NF-κB system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF-κB activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-κB-induced genes is regulated by differential dynamics of single NF-κB subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-κB activation have been observed in response to the cytokine tumor necrosis factor α (TNFα), little is known about the occurrence of oscillations in response to bacterial infections.ResultsTo quantitatively assess NF-κB dynamics we generated human and murine monoclonal cell lines that stably express the NF-κB subunit p65 fused to GFP. Furthermore, a high-throughput assay based on automated microscopy coupled to image analysis to quantify p65-nuclear translocation was established. Using this assay, we demonstrate a stimulus- and cell line-specific temporal control of p65 translocation, revealing, for the first time, oscillations of p65 translocation in response to bacterial infection. Oscillations were detected at the single-cell level using real-time microscopy as well as at the population level using high-throughput image analysis. In addition, mathematical modeling of NF-κB dynamics during bacterial infections predicted masking of oscillations on the population level in asynchronous activations, which was experimentally confirmed.ConclusionsTaken together, this simple and cost effective assay constitutes an integrated approach to infer the dynamics of NF-κB kinetics in single cells and cell populations. Using a single system, novel factors modulating NF-κB can be identified and analyzed, providing new possibilities for a wide range of applications from therapeutic discovery and understanding of disease to host-pathogen interactions.

Helicobacter pylori-induced modification of the histone H3 phosphorylation status in gastric epithelial cells reflects its impact on cell cycle regulation

Post-translational modifications of core histones are important components of the epigenetic landscape. Recent investigations of bacterial or toxin-induced effects on histone phosphorylation and acetylation in host cells have linked the changes to transcriptional alterations of key cellular response pathways. However, these changes may have other reasons and functional consequences. Here, we show that infection of gastric epithelial cell lines with the carcinogenic bacterium Helicobacter pylori leads to changes in histone H3 phosphorylation: type IV secretion system (T4SS)-dependent decreases of H3 phosphorylation levels at serine 10 (pH3Ser10) and threonine 3 (pH3Thr3) were observed. Immunofluorescence experiments with pH3Ser10 and cyclin B1 revealed that a H. pylori-induced transient pre-mitotic arrest was responsible for the observed reduction. This causal link was substantiated further by showing that H. pylori causes a strong decrease of the cell division cycle 25 (CDC25C) phosphatase. As a consequence, mitotic histone H3 kinases such as vaccinia-related kinase 1 (VRK1) and Aurora B were not fully activated in infected cells. We show that VRK1 activity, measured using a kinase activity assay, was reduced after H. pylori infection by approximately 40%. Moreover, over–expression of VRK1, but not Aurora B, compensated for the H. pylori-induced decrease of pH3Ser10. Rephosphorylation of H3Ser10 was IκB kinase α (IKKα)-dependent and occurred at later time points of infection. Taken together, our work highlights the impact of bacterial pathogens on host cell chromatin; this modulation reflects the subversion of key cellular processes such as cell cycle progression.