Innate immune receptor NOD2 mediates LGR5+ intestinal stem cell protection against ROS cytotoxicity via mitophagy stimulation

International audienceThe nucleotide-binding oligomerization domain-containing protein 2 (NOD2) agonist muramyl dipeptide (MDP), a peptidoglycan motif common to all bacteria, supports leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)+ intestinal stem cell (ISC) survival through NOD2 activation upon an otherwise lethal oxidative stress-mediated signal. However, the underlying protective mechanisms remain unknown. Here, using irradiation as stressor and primarily murine-derived intestinal organoids as a model system, we show that MDP induced a significant reduction of total and mitochondrial reactive oxygen species (ROS) within ISCs, which was associated with mitophagy induction. ATG16L1 knockout (KO) and NOD2 KO organoids did not benefit from the MDP-induced cytoprotection. We confirmed the MDP-dependent induction of ISC mitophagy upon stress in vivo. These findings elucidate the NOD2-mediated mechanism of cytoprotection involving the clearance of the lethal excess of ROS molecules through mitophagy, triggered by the coordinated activation of NOD2 and ATG16L1 by a nuclear factor κB (NF-κB)-independent pathway

Pancreatic ductal deletion of Hnf1b disrupts exocrine homeostasis, leads to pancreatitis and facilitates tumorigenesis

BACKGROUND AND AIMS: The exocrine pancreas consists of acinar cells that produce digestive enzymes transported to the intestine through a branched ductal epithelium. Chronic pancreatitis is characterized by progressive inflammation, fibrosis and loss of acinar tissue. These changes of the exocrine tissue are risk factors for pancreatic cancer. The cause of chronic pancreatitis cannot be identified in one-quarter of patients. Here, we investigated how duct dysfunction could contribute to pancreatitis development. METHODS: The transcription factor Hnf1b, first expressed in pancreatic progenitors, is strictly restricted to ductal cells from late embryogenesis. We have previously shown that Hnf1b is crucial for pancreas morphogenesis but its postnatal role still remains unelucidated. To investigate the role of pancreatic ducts in exocrine homeostasis, we inactivated Hnf1b gene in vivo in mouse ductal cells. RESULTS: We uncovered that postnatal Hnf1b inactivation in pancreatic ducts leads to chronic pancreatitis in adults. Hnf1bΔduct mutants display dilatation of ducts, loss of acinar cells, acinar-to-ductal metaplasia (ADM) and lipomatosis. We deciphered the early events involved, with downregulation of cystic disease-associated genes, loss of primary cilia, upregulation of signaling pathways, especially Yap pathway involved in ADM. Remarkably, Hnf1bΔduct mutants developed pancreatic intraepithelial neoplasia and promote PanIN progression in concert with KRAS. We further showed that adult Hnf1b inactivation in pancreatic ducts is associated with impaired regeneration after injury, with persistent metaplasia and initiation of neoplasia. CONCLUSION: Loss of Hnf1b in ductal cells leads to chronic pancreatitis and neoplasia. This reveals that Hnf1b deficiency may contribute to diseases of the exocrine pancreas and could gain further insight into the etiology of pancreatitis and tumorigenesis.Support to CH was received from theCentre National de la Recherche Scientifique (CNRS), the Universite Pierre et Marie Curie (UPMC)- Sorbonne Université , the GEFLUC - Les entreprises contre le Cancer, the Societe Francophone du Diabete (SFD)-Ypsomed, the programme Emergence UPMC. EQ was supported by a PhD fellowship from the French Ministère de la Recherche et de la Technologie. MF is an assistant engineer of the CNRS. TD and AS were supported by Sorbonne Université. MDV was supported by a PhD student fellowship from the European Marie Curie Initial Training Network (ITN)-Biology of Liver and Pancreatic Development and Disease (BOLD). O. O. was supported by a Master1 fellowship. RCP was supported by a postdoctoral fellowship from the American Heart Association (14POST20380262). MG was supported by the National Institutes of Health (U01 DK089540) and the Juvenile Diabetes Research Foundation (1-2011-592). CH is a permanent senior researcher of the Institut National de la Sante et de la Recherche Medicale (INSERM).S

Etude du rôle des gènes Iroquois au cours de la régionalisation du cerveau chez le poisson-zèbre

Au cours de sa formation, le cerveau des vertébrés est progressivement régionalisé selon l axe antéroposterieur en prosencéphale, mésencéphale et rhombencéphale. Le rhombencéphale est transitoirement subdivisé en 7 rhombomères (r1-r7). Certains facteurs de trancription sont requis pour la formation des différents rhombomères. Krox20 est ainsi nécessaire à la mise en place de r3 et r5. Au cours de ma thèse, je me suis intéressée au rôle de deux gènes de la famille Iroquois, irx7 et irx1b, dans la régionalisation du cerveau chez le poisson-zèbre. J ai montré qu ils étaient nécessaires à la formation du rhombencéphale antérieur, en activant notamment l expression du gène krox20 dans r3. j ai également participé à la recherche des cibles directes d Irx7 réalisée par immunoprécipitation de chromatine couplée à un séquençage massif (ChIP-seq) et démontré que le gène cyp26a1 était une cible positive d Irx7. cyp26a1 code pour une enzyme de dégradation de l acide rétinoïque qui joue une fonction primordiale dans la régionalisation du cerveau. Ces résultats démontrent donc le rôle essentiel joué par les Iroquois au cours de ce processus.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Decrypting the communication between microbes and the intestinal mucosa—A brief review on Pathogénie Microbienne Moléculaire's latest research

International audienceOver the past 10 years, the "Pathogénie Microbienne Moléculaire" unit of Professor Philippe Sansonetti has studied the molecular cross talk between the intestinal micro-biota and the gut epithelium, aiming to better understand how this mutualistic symbi-osis delineates homoeostasis and, when perturbed, prompts pathology. To do so, the unit has manipulated both bacterial and epithelial cells, and used cutting-edge technology. More recently, the lab has turned its focus also on studying the intestinal crypt and more specifically the intestinal stem cell for their role in epithelial regener-ation and long-term epithelium renewal. Here, we provide a brief review summarising recent results obtained from the lab, with particular focus on the intestinal crypt

Le dialogue microbiote-cellules souches

Le microbiote intestinal est un des plus denses et complexes du corps humain. Son impact sur la physiologie de l’hôte s’exerce à la fois localement, où il joue d’importantes fonctions métaboliques et de consolidation de la barrière intestinale, et à l’échelle systémique de l’organisme, puisqu’il influence des processus aussi fondamentaux que la croissance ou la maturation du système immunitaire. Une caractéristique de l’épithélium intestinal est son extraordinaire capacité de régénération, qui repose sur la présence de cellules souches résidant dans le fond des cryptes de Lieberkühn. Plusieurs études ont montré que le microbiote intestinal participait à ce processus de régénération, suggérant qu’il peut influencer la capacité proliférative des cellules souches intestinales (CSI). Les cellules immunitaires résidant dans l’intestin sont des médiateurs importants du dialogue microbiote-épithélium. Le microbiote peut également exercer des fonctions régulatrices sur les CSI au moyen de modes de communication plus directs, passant par exemple par la liaison de ligands bactériens aux récepteurs de l’immunité innée exprimés par les CSI. Dans cette revue, nous explorons les différents modes de communication qui peuvent s’établir entre les cellules souches et le microbiote, et leur impact sur l’homéostasie intestinale

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

International audienceThe present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS. ABSTRACT: Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural byproducts of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cell

Initiation of cyp26a1 Expression in the Zebrafish Anterior Neural Plate by a Novel Cis-Acting Element

International audienceEarly patterning of the vertebrate neural plate involves a complex hierarchy of inductive interactions orchestrated by signalling molecules and their antagonists. The morphogen ret-inoic acid, together with the Cyp26 enzymes which degrade it, play a central role in this process. The cyp26a1 gene expressed in the anterior neural plate thus contributes to the fine modulation of the rostrocaudal retinoic acid gradient. Despite this important role of cyp26a1 in early brain formation, the mechanisms that control its expression in the anterior neural plate are totally unknown. Here, we present the isolation of a 310-base-pair DNA element adjacent to cyp26a1 promoter, displaying enhancer activity restricted to the anterior neural plate of the zebrafish gastrula. We show that unlike that of cyp26a1, expression driven by this cyp26a1 anterior neural plate element (cANE) is independent of retinoic acid. Through deletion analysis, we identify a 12-nucleotide motif essential for cANE activity. A consensus bipartite binding site for SoxB:Oct transcription factors overlaps with this motif. Mutational analysis suggests that SoxB binding is essential for its activity. We discuss the contribution of this study to the elucidation of the regulatory hierarchy involved in early neural plate patterning

Compensation between Wnt-driven tumorigenesis and cellular responses to ribosome biogenesis inhibition in the murine intestinal epithelium

International audienceRibosome biogenesis inhibition causes cell cycle arrest and apoptosis through the activation of tumor suppressor-dependent surveillance pathways. These responses are exacerbated in cancer cells, suggesting that targeting ribosome synthesis may be beneficial to patients. Here, we characterize the effect of the loss-of-function of Notchless (Nle), an essential actor of ribosome biogenesis, on the intestinal epithelium undergoing tumor initiation due to acute Apc loss-of-function. We show that ribosome biogenesis dysfunction strongly alleviates Wnt-driven tumor initiation by restoring cell cycle exit and differentiation in Apc-deficient progenitors. Conversely Wnt hyperactivation attenuates the cellular responses to surveillance pathways activation induced by ribosome biogenesis dysfunction, as proliferation was maintained at control-like levels in the stem cells and progenitors of double mutants. Thus, our data indicate that, while ribosome biogenesis inhibition efficiently reduces cancer cell proliferation in the intestinal epithelium, enhanced resistance of Apc-deficient stem and progenitor cells to ribosome biogenesis defects may be an important concern when using a therapeutic strategy targeting ribosome production for the treatment of Wnt-dependent tumorigenesis

Characterization of cANE activity as an early neural plate specific enhancer.

<p>(A-I,A’-I’) Compared expression patterns of <i>cyp26a1</i> (A-I) and <i>egfp</i> driven by cANE (A’-I’) during early embryonic development. (J) Double in situ hybridization showing <i>barhl2</i> expression domain (blue) exactly filling the gap in the <i>cyp26a1</i> expression domain (red). (A,B,C,E,G,A’,B’,C’,E’,G’) are lateral views. (d,f,h,I,D’,f’,h’,I’) are animal pole views. White arrowheads: anterior neural plate. Black arrowheads: blastoderm marginal zone. Arrows in (H-J): gap in the anterior neural plate domain of <i>cyp26a1</i> expression. All stages are indicated in the pictures. (k,k’,l,l’) The effect of 100 nM retinoic acid (RA) treatment between 2,5 hpf and 8,5 hpf on on stable transgenic cANE_endo:::<i>egfp</i> (K-K’) and cANE::<i>egfp</i> (L-L’) expression. (M) EGFP fluorescence in a 12 hpf stable transgenic cANE:::<i>egfp</i> embryo. Lateral view with dorsal to the left. (N) Schematic representation of cANE and all three reported retinoic acid responsive elements (R1, R2, R3) identified previously. cANE is located from -504 bp to -195 bp relative to <i>cyp26a1</i> ATG codon. An: animal, Vg: vegetal; V: ventral; D: dorsal, A: anterior, P: posterior, L: left, R: right, CTRL: control embryo, RA: retinoic-acid treated embryo.</p