NF-kappaB activation in infections with Helicobacter pylori or Legionella pneumophila

Bakterielle Infektionen aktivieren den Transkriptionsfaktor NF-κB. Dies trägt sowohl zur Immunantwort, als auch zum Überleben der Wirtszellen bei. In Infektionen mit dem Bakterium Helicobacter pylori stellt diese doppelte Funktion einen mechanistischen Link zwischen der chronischen Entzündung und der Krebsentstehung dar. Für das intrazelluläre Bakterium Legionella pneumophila ist das Überleben der Wirtszelle notwendig für die Vermehrung. Bisher sind jedoch bei beiden Infektionen die Signalwege, die zur NF-κB-Aktivierung führen, nicht ausreichend untersucht. Um NF-κB analysieren zu können, wurden hier monoklonale Zelllinien hergestellt, die ein Fusionsprotein der NF-κB-Untereinheit p65 mit GFP stabil exprimieren. Die Kerntranslokation von p65-GFP kann mit Fluoreszenz-Mikroskopie visualisiert und mit automatischer Bildanalyse quantifiziert werden. Mit dieser Methode konnte zum ersten Mal eine durch ein Bakterium induzierte Oszillation von p65-GFP gezeigt werden. In einem RNAi-basierten Hochdurchsatzscreen wurde diese Methode eingesetzt um neue Faktoren im NF-κB-Signalweg zu identifizieren. Dabei wurde die Infektion mit H. pylori mit den Induktoren TNFα und IL-1β verglichen. Insgesamt wurden 24 Regulatoren identifiziert. Die Identifikation dieser Faktoren vertieft nicht nur unser Verständnis des NF-κB-Signalweges, sondern bietet auch neue molekulare Ziele für mögliche zukünftige Therapien. Bei der detaillierten Analyse der L. pneumophila-induzierten NF-κB-Aktivierung konnte ein einzigartiger, zwei-phasiger Ablauf gezeigt werden. Zunächst verursachte bakterielles Flagellin eine starke, aber kurze Aktivierung. Später war p65 dauerhaft über Stunden im Kern lokalisiert, was eng an die Replikation der Bakterien gekoppelt war. Da die kontinuierliche Kernlokalisation für Transkriptionsfaktoren der NF-κB Familie sehr ungewöhnlich ist, könnte dies ein Hinweis für eine Manipulation durch das Bakterium sein, um das Überleben der Wirtszelle zu sichern.Infection with pathogens leads to activation of the transcription factor NF-κB. This contributes both to the immune response as well as survival of host cells. In infection with the bacterium Helicobacter pylori, this dual function provides a mechanistic link between the chronic inflammation and cancer development. In infection with the intracellular pathogen Legionella pneumophila, the survival oft he host cell is necessary for replication. However, in both infections, the signaling pathways leading to NF-κB activation are not well understood. Here, in order to investigate NF-κB activation, monoclonal cell lines were generated that stably express the NF-κB subunit p65 fused to GFP. Nuclear translocation of p65-GFP can be visualized by fluorescence microscopy and quantified by software-based picture analysis. Using this technology, oscillations of p65-GFP nuclear translocation could be shown after H. pylori infection. To identify new factors important for NF-κB activation, the new assay was used to conduct an RNAi-based screen. In the screen, infection with H. pylori was compared to induction with the cytokines TNFα and IL-1β. In total, 24 key regulators for NF-κB were identified. The identification of these factors broadens our understanding of NF-κB signaling and could provide targets for future therapies. Finally, detailed observation of NF-κB activation induced by L. pneumophila in single cells revealed a unique, biphasic NF-κB activation. During the first hours, bacterial flagellin induced strong but transient activation. Then, p65 translocated continuously to the nucleus over hours without oscillation. Testing an array of bacterial mutants, a tight link between bacterial replication and continuous NF-κB activation could be shown. Because this continuous nuclear localization is very unusual for a transcription factor of the NF-κB family, this indicates that L. pneumophila could manipulate NF-κB to ensure host cell survival

Adult gastric stem cells and their niches.

Adult gastric stem cells replenish the gastric epithelium throughout life. Recent studies have identified diverse populations of stem cells, progenitor cells, and even differentiated cells that can regain stem cell capacity, so highlighting an unexpected plasticity within the gastric epithelium, both in the corpus and antrum. Two niches seem to co-exist in the gastric unit: one in the isthmus region and the other at the base of the gland, although the precise features of the cell populations and the two niches are currently under debate. A variety of gastric organoid models have been established, providing new insights into niche factors required by the gastric stem cell populations. Here we review our current knowledge of gastric stem cell populations, their markers and interactions, important niche factors, and different gastric organoid systems. For further resources related to this article, please visit the WIREs website.Wellcome-Trus

Organoide

Zusammenfassung Organoide sind dreidimensionale, aus Stammzellen entwickelte Modellsysteme für unterschiedliche Organe, die großes Potenzial für Forschung und Medizin bergen. Sie werfen wissenschaftliche, aber auch philosophische, ethische und juristische Fragen auf, die bislang in Deutschland wenig diskutiert werden. Der Themenband der interdisziplinären Arbeitsgruppe (IAG) Gentechnologiebericht an der Berlin-Brandenburgischen Akademie der Wissenschaften bietet eine Übersicht über aktuelle wissenschaftliche Entwicklungen, ihre derzeitigen und potenziellen Anwendungsmöglichkeiten sowie wissenschaftstheoretische, ethische und juristische Reflexionen. Hiermit möchte die IAG einen Anstoß zu einer interdisziplinären und gesamtgesellschaftlichen Debatte liefern. Mit Beiträgen von Cantas Alev, Aileen-Diane Bamford, Sina Bartfeld, Andreia S. Batista-Rocha, Ali H. Brivanlou, Thomas Burgold, Cindrilla Chumduri, Stephan Clemens, Emrecan Dilmen, Tobias Erb, Fred Etoc, Melinda B. Fagan, Heiner Fangerau, Boris Fehse, Nina Frey, Tristan Frum, Anne Grapin-Botton, Navin Gupta, Jürgen Hampel, Ferdinand Hucho, Özge Kayisoglu, Rashmiparvathi Keshara, Yung Hae Kim, Bon-Kyoung Koo, Martin Korte, Yaroslav Koshelev, Kai Kretzschmar, Allison Lewis, Lilian Marx-Stölting, Fruzsina Molnár-Gábor, Ryuji Morizane, Stefan Mundlos, Paola Nicolas, Angela Osterheider, In-Hyun Park, Anja Pichl, Sandra Pilat-Carotta, Jens Reich, Marlen Reinschke, Hannah Schickl, Silke Schicktanz, Nicolas Schlegel, Jason R. Spence, Yoshiaki Tanaka, Jochen Taupitz, Isaree Teriyapirom, Margherita Y. Turco, Jörn Walter, Eva Winkler, Martin Zenke. Abstract Organoids are developed from stem cells and serve as three-dimensional model systems for different organs. They have great potential for research and medicine, but also raise philosophical, ethical and legal questions which have rarely been discussed in Germany so far. This thematic study by the interdisciplinary research group (IAG) Gene Technology Report at the Berlin-Brandenburg Academy of Sciences and Humanities offers an overview of current scientific developments, their present and potential application, as well as epistemological, ethical and legal reflections. Hereby, the IAG wants to provide impetus for an interdisciplinary and society-wide debate on this general subject. With contributions by Cantas Alev, Aileen-Diane Bamford, Sina Bartfeld, Andreia S. Batista-Rocha, Ali H. Brivanlou, Thomas Burgold, Cindrilla Chumduri, Stephan Clemens, Emrecan Dilmen, Tobias Erb, Fred Etoc, Melinda B. Fagan, Heiner Fangerau, Boris Fehse, Nina Frey, Tristan Frum, Anne Grapin-Botton, Navin Gupta, Jürgen Hampel, Ferdinand Hucho, Özge Kayisoglu, Rashmiparvathi Keshara, Yung Hae Kim, Bon-Kyoung Koo, Martin Korte, Yaroslav Koshelev, Kai Kretzschmar, Allison Lewis, Lilian Marx-Stölting, Fruzsina Molnár-Gábor, Ryuji Morizane, Stefan Mundlos, Paola Nicolas, Angela Osterheider, In-Hyun Park, Anja Pichl, Sandra Pilat-Carotta, Jens Reich, Marlen Reinschke, Hannah Schickl, Silke Schicktanz, Nicolas Schlegel, Jason R. Spence, Yoshiaki Tanaka, Jochen Taupitz, Isaree Teriyapirom, Margherita Y. Turco, Jörn Walter, Eva Winkler, Martin Zenke

Modeling infectious diseases and host-microbe interactions in gastrointestinal organoids

Advances in stem cell research have allowed the development of 3-dimensional (3D) primary cell cultures termed organoid cultures, as they closely mimic the in vivo organization of different cell lineages. Bridging the gap between 2-dimensional (2D) monotypic cancer cell lines and whole organisms, organoids are now widely applied to model development and disease. Organoids hold immense promise for addressing novel questions in host-microbe interactions, infectious diseases and the resulting inflammatory conditions. Researchers have started to use organoids for modeling infection with pathogens, such as Helicobacter pylori or Salmonella enteritica, gut- microbiota interactions and inflammatory bowel disease. Future studies will broaden the spectrum of microbes used and continue to establish organoids as a standard model for human host-microbial interactions. Moreover, they will increasingly exploit the unique advantages of organoids, for example to address patient-specific responses to microbes

Realizing the potential of organoids — an interview with Hans Clevers

No abstract available

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells

Organoids as Model for Infectious Diseases : Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells

Stem cell-derived organoids and their application for medical research and patient treatment

3D culture has allowed the initiation and expansion of organ-like structures, called organoids, from either tissue-resident adult stem cells or pluripotent stem cells. Today, organoids can be grown to resemble a wide variety of organs, exhibiting remarkable similarity to their in vivo counterparts. As successful organoid generation is possible from virtually every patient, organoids hold a great promise for medical research and the development of new treatments. They have already found their way into the clinic, enabling personalized medicine in small patient trials. In this review, we provide an update on current organoid technology and summarize their application in basic research, disease modelling, drug development, personalized treatment and regenerative medicine

Gastrointestinal epithelial innate immunity-regionalization and organoids as new model

The human gastrointestinal tract is in constant contact with microbial stimuli. Its barriers have to ensure co-existence with the commensal bacteria, while enabling surveillance of intruding pathogens. At the centre of the interaction lies the epithelial layer, which marks the boundaries of the body. It is equipped with a multitude of different innate immune sensors, such as Toll-like receptors, to mount inflammatory responses to microbes. Dysfunction of this intricate system results in inflammation-associated pathologies, such as inflammatory bowel disease. However, the complexity of the cellular interactions, their molecular basis and their development remains poorly understood. In recent years, stem cell-derived organoids have gained increasing attention as promising models for both development and a broad range of pathologies, including infectious diseases. In addition, organoids enable the study of epithelial innate immunity in vitro. In this review, we focus on the gastrointestinal epithelial barrier and its regional organization to discuss innate immune sensing and development