Role of Notch signalling in intestinal epithelium

Résumé: Chez les mammifères, les intestins sont les organes ayant le plus haut taux de renouvellement cellulaire dans l'organisme. L'épithélium intestinal se renouvelle complètement en moins d'une semaine. Il se compose de projections (villosités) et d'invaginations (cryptes) qui ont toutes deux des fonctions bien distinctes. Les cellules de l'intestin sont constamment produites à partir de cellules souches, situées dans la crypte, qui se différencient en cellules proliférantes transitoires, puis en cellules caliciformes, de Paneth, entéroendocrine ou en entérocytes. Ces cellules migrent dans leurs lieux spécifiques pour accomplir leur fonction physiologique pour finalement mourir. A cours de mon travail de thèse, j'ai étudié le rôle de la voie de signalisation de Notch dans le renouvellement cellulaire et dans le processus de l'homéostase des cellules de l'intestin marin en utilisant le système Cre-loxP pour induire la délétion des gènes Notch1, Notch2, Jaggedl et RBP-Jk. Bien que l'inactivation de Notch1 avec ou sans Jagged1, ou celle de Notch2, n'aboutissent à aucun phénotype, une déficience pour RBP-Jk, ou pour Notch1 et Notch2 simultanément, conduit au développement d'un impressionnant phénotype. Au niveau de la crypte, une rapide et importante modification des cellules apparaît: les cellules proliférantes sont devenues des cellules caliciformes qui ont perdu la capacité de se renouveler. Ces résultats impliquent la voie Notch en tant que nouvelle clé de voûte dans le maintien des cellules qui s'auto-renouvellent dans l'épithélium intestinal. Un rôle similaire a été proposé pour la voie Wnt, laquelle n'est cependant, pas affectée dans nos souris. C'est pourquoi ces deux voies sont essentielles dans le maintien de la prolifération dans les cryptes intestinales. Ce travail a aussi proposé un mécanisme par lequel la voie Notch contrôlerait l'intégrité du cycle cellulaire dans les cellules de la crypte intestinale, ceci en inhibant la transcription d'un inhibiteur du cycle cellulaire, la protéine p27KIP1. De plus, l'inactivation de RBP-Jk dans les adénomes développés par les souris APCmin induisent la différenciation de cellules tumorales en cellules caliciformes. Comme autre effet, la localisation histologique des cellules de Paneth est également affectée par la délétion de RBP-Jk ou de Notch1/Notch2, suggérant un rôle pour la voie Notch dans le compartiment des cellules de Paneth. Finalement, ce travail démontre que les cellules progénitrices de l'intestin ont besoin d'une convergence fonctionnelle des voie Wnt et Notch. Ces résultats préliminaires peuvent être considérés comme un concept pour l'utilisation d'inhibiteurs de secrétase-γ (inhibiteurs de Notch) à des fins thérapeutiques pour les cancers colorectaux. Summary The mammalian intestine has one of the highest cellular turnover rates in the body. The complete intestinal epithelium is renewed in less than a week. It is divided into spatially distinct compartments in the form of finger-like projections (villi) and flask-shaped invaginations (crypts) that are dedicated to specific functions. Intestinal cells are constantly produced from a stem cell reservoir that gives rise to proliferating transient amplifying cells, which subsequently differentiate and home to their specific compartments before dying after having fulfilled their physiological function. In this thesis project, the physiological role of the Notch signalling cascade in the marine intestine was studied. Inducible tissue specific inactivation of Notch1, Notch2, Jagged1 and RBP-Jk genes was applied to assess their role in the maintenance of intestinal homeostasis and cell fate determination. The analysis unequivocally revealed that Notch1, Notch1 and Jagged1 combined as well as Notch2 are dispensable for intestinal homeostasis and lineage differentiation. However, deficiency of RBP-Jk as well as the simultaneous inactivation of both Notch1 and Notch2 receptors unveiled a striking phenotype. In these mice, a rapid and massive conversion of proliferative crypt cells into post-mitotic goblet cells was observed. These results identify the Notch pathway as a key player for the maintenance of the proliferative crypt compartment. A similar role was implicated for the Wnt cascade, which, however, was not affected in the different tissue specific Notch signalling deficient mice. Thus, the Wnt and Notch signalling pathways are essential for the self-renewal capacity of the intestinal epithelium. Furthermore, our results suggest a molecular mechanism for Notch signalling mediated control of cell cycle regulation within the crypt. The Notch cascade inhibits expression of the cyclin-dependent kinase inhibitor p27KIP1 and thereby maintains proliferation of the intestinal progenitor cells. In addition, the inactivation of RBP-Jk in adenomas developed by APCmin mice resulted in the differentiation of tumour cells into goblet cells. Finally, Notch deficiency affected differentiated Paneth cells, suggesting that Notch may play a role in the Paneth cell compartment. In summary, this work clearly demonstrates that undifferentiated, proliferative cells in intestinal crypts require the concerted activation of the RBP-Jk-mediated Notch signalling and the Wnt cascade. In addition, our preliminary results can be considered as a "proof-of-principle" for the use of γ-secretase inhibitors for therapeutic modalities for colorectal cancer.

Secretory IgA: an actor of the interplay between the commensal flora and the intestinal immune system?

In the gastro-intestinal tract,Peyers patches have been describedas a major inductive site for mucosalsecretory IgA (SIgA) responses directedagainst pathogens. The classicalview is that SIgAserves as the firstline of defense against microorganismsby agglutining potential invadersand faciliting their clearance byperistaltic and mucociliary movements,a mechanism called immuneexclusion. Our laboratory has shownthat SIgA is not only able to be"retrotransported" into Peyers patchesvia the associated M cells, but also todeliver sizeable cargos in the form ofSIgA-based immune complexes, resultingin the onset of non-inflammatorytype of responses. Such a novelfunction raises the question of thepossible role of mucosal SIgA in theinterplay with commensal bacteriaand the contribution of the antibody inbacterial homeostasis. To address thisquestion, Lactobacillus rhamnosus(LPR) was administered into a mouseligated loop comprising a Peyerspatch, in association or not with SIgA.The fate of fluorescently labelled bacteriawas followed by laser scanningconfocal microscopy at different incubationtimes. After 2 hours of incubationin the loop, LPR bacteria arefound more abundantly in thesubepithelial dome (SED) regionwhen they are coated with SIgA thanLPR administered alone despite theyare absent from neighboring villi.Herein, it is shown that this mechanismof entry involves M cells inPeyers pathes. After their sampling byM cells, bacteria are engulfed by thedendritic cells of the subjacent SEDregion. Interestingly, LPR bacteriaare found coated by the endogenousnatural SIgA present in mice intestinalsecretions, confirming the requirementof SIgA for this type of entry.The subsequent effect on the maturationof dendritic cells after interactionwith LPR was investigated in vitroin presence or not of SIgA by measuringthe expression of CD40, CD80and CD86 surface markers with flowcytometry analyses. Results show thatDCs respond in the same way in presenceof SIgA than with LPR bacteriaalone, indicating that SIgA does notmodulate the interaction betweenDCs and bacteria in this context. Thiswork gives new evidences about theinvolvement of SIgA in the mechanismby which the intestinal immunesystem permanently checks the contentof the intestine.

Targeting of secretory IgA to Peyer's patch dendritic and T cells after transport by intestinal M cells.

In addition to being instrumental to the protection of mucosal epithelia, secretory IgA (SIgA) adheres to and is transported by intestinal Peyer's patch (PP) M cells. The possible functional reason for this transport is unknown. We have thus examined in mice the outcome of SIgA delivered from the intestinal lumen to the cells present in the underlying organized mucosa-associated lymphoreticular tissue. We show selective association of SIgA with dendritic cells and CD4(+) T and B lymphocytes recovered from PP in vitro. In vivo, exogenously delivered SIgA is able to enter into multiple PP lining the intestine. In PP, SIgA associates with and is internalized by dendritic cells in the subepithelial dome region, whereas the interaction with CD4(+) T cells is limited to surface binding. Interaction between cells and SIgA is mediated by the IgA moiety and occurs for polymeric and monomeric molecular forms. Thus, although immune exclusion represents the main function of SIgA, transport of the Ab by M cells might promote Ag sampling under neutralizing conditions essential to the homeostasis of mucosal surfaces.

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Age, smoking and overweight contribute to the development of intestinal metaplasia of the cardia.

AIM: To assess the role of Helicobacter pylori (H. pylori), gastroesophageal reflux disease (GERD), age, smoking and body weight on the development of intestinal metaplasia of the gastric cardia (IMC).¦METHODS: Two hundred and seventeen patients scheduled for esophagogastroduodenoscopy were enrolled in this study. Endoscopic biopsies from the esophagus, gastroesophageal junction and stomach were evaluated for inflammation, the presence of H. pylori and intestinal metaplasia. The correlation of these factors with the presence of IMC was assessed using logistic regression.¦RESULTS: IMC was observed in 42% of the patients. Patient age, smoking habit and body mass index (BMI) were found as potential contributors to IMC. The risk of developing IMC can be predicted in theory by combining these factors according to the following formula: Risk of IMC = a + s - 2B where a = 2,...6 decade of age, s = 0 for non-smokers or ex-smokers, 1 for < 10 cigarettes/d, 2 for > 10 cigarettes/d and B = 0 for BMI < 25 kg/m² (BMI < 27 kg/m² in females), 1 for BMI > 25 kg/m² (BMI > 27 kg/m² in females). Among potential factors associated with IMC, H. pylori had borderline significance (P = 0.07), while GERD showed no significance.¦CONCLUSION: Age, smoking and BMI are potential factors associated with IMC, while H. pylori and GERD show no significant association. IMC can be predicted in theory by logistic regression analysis.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND:: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18. METHODS:: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS:: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS:: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations. (Inflamm Bowel Dis 2010).

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

Normal kinetics of intestinal glucose absorption in the absence of GLUT2: evidence for a transport pathway requiring glucose phosphorylation and transfer into the endoplasmic reticulum.

Glucose is absorbed through the intestine by a transepithelial transport system initiated at the apical membrane by the cotransporter SGLT-1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Here, we evaluated the impact of GLUT2 gene inactivation on this transepithelial transport process. We report that the kinetics of transepithelial glucose transport, as assessed in oral glucose tolerance tests, was identical in the presence or absence of GLUT2; that the transport was transcellular because it could be inhibited by the SGLT-1 inhibitor phlorizin, and that it could not be explained by overexpression of another known glucose transporter. By using an isolated intestine perfusion system, we demonstrated that the rate of transepithelial transport was similar in control and GLUT2(-/-) intestine and that it was increased to the same extent by cAMP in both situations. However, in the absence, but not in the presence, of GLUT2, the transport was inhibited dose-dependently by the glucose-6-phosphate translocase inhibitor S4048. Furthermore, whereas transport of [(14)C]glucose proceeded with the same kinetics in control and GLUT2(-/-) intestine, [(14)C]3-O-methylglucose was transported in intestine of control but not of mutant mice. Together our data demonstrate the existence of a transepithelial glucose transport system in GLUT2(-/-) intestine that requires glucose phosphorylation and transfer of glucose-6-phosphate into the endoplasmic reticulum. Glucose may then be released out of the cells by a membrane traffic-based pathway similar to the one we previously described in GLUT2-null hepatocytes.

Epithelium-Intrinsic NAIP/NLRC4 Inflammasome Drives Infected Enterocyte Expulsion to Restrict Salmonella Replication in the Intestinal Mucosa.

The gut mucosal epithelium separates the host from the microbiota, but enteropathogens such as Salmonella Typhimurium (S.Tm) can invade and breach this barrier. Defenses against such acute insults remain incompletely understood. Using a murine model of Salmonella enterocolitis, we analyzed mechanisms limiting pathogen loads in the epithelium during early infection. Although the epithelium-invading S.Tm replicate initially, this intraepithelial replicative niche is restricted by expulsion of infected enterocytes into the lumen. This mechanism is compromised if inflammasome components (NAIP1-6, NLRC4, caspase-1/-11) are deleted, or ablated specifically in the epithelium, resulting in ∼100-fold higher intraepithelial loads and accelerated lymph node colonization. Interestingly, the cytokines downstream of inflammasome activation, interleukin (IL)-1α/β and IL-18, appear dispensable for epithelial restriction of early infection. These data establish the role of an epithelium-intrinsic inflammasome, which drives expulsion of infected cells to restrict the pathogen's intraepithelial proliferation. This may represent a general defense mechanism against mucosal infections.

Vincristine and intestinal pseudo-obstruction in children: report of 5 cases, literature review, and suggested management.

Summary: Intestinal pseudo-obstruction is a rare complication resulting from a variety of disorders. Symptoms include abdominal pain, nausea, vomiting, diarrhea, constipation, and malnutrition. Vincristine-related pseudo-obstruction has been reported in the literature, but its description in children and recommendations for management are lacking. A review of the literature revealed 21 reported pediatric cases of vincristine-related pseudo-obstruction. Most have, however, been attributed to a drug interaction with itraconazole, accidental vincristine overdose, or liver failure. Potential genetic causes are rarely addressed. We present here 5 cases of pseudo-obstruction related to vincristine without any identifiable predisposing factors, and a suggested algorithm for management

Agglutinating Secretory IgA Preserves Intestinal Epithelial Cell Integrity during Apical Infection by Shigella flexneri.

Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-κB nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion.

Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) that overlies Peyer's patches (PPs) exhibits distinct features compared with the adjacent villus epithelium. Besides the presence of antigen-sampling membranous M cells and the down-regulation of digestive functions, it constitutively expresses the chemokine CCL20. The mechanisms that induce FAE differentiation and CCL20 expression are poorly understood. The aim of this work was to test whether lymphotoxin beta receptor signaling (LTbetaR), which plays a central role in PPs' organogenesis, mediates CCL20 gene expression in intestinal epithelial cells. METHODS: CCL20, lymphotoxin beta (LTbeta) and LTbetaR expression were monitored during embryonic development by in situ hybridization of mouse intestine. The human intestinal epithelial cell line T84 was used to study CCL20 expression following LTalpha(1)/beta(2) stimulation. In vivo CCL20 expression following agonistic anti-LTbetaR antibody treatment was studied by laser microdissection and quantitative RT-PCR. RESULTS: CCL20 was expressed in the FAE before birth at the time when the first hematopoietic CD4(+)CD3(-) appeared in the PP anlage. LTbetaR was expressed in the epithelium during PP organogenesis, making it a putative target for LTalpha(1)beta(2)signals. In vitro, CCL20 was induced in T84 cells upon LTbetaR signaling, either using an agonistic ligand or anti-LTbeta receptor agonistic antibody. LTalpha(1)beta(2)-induced CCL20 expression was found to be NF-kappaB dependent. LTbetaR signaling up-regulated CCL20 expression in the small intestinal epithelium in vivo. CONCLUSIONS: Our results show that LTbetaR signaling induces CCL20 expression in intestinal epithelial cells, suggesting that this pathway triggers constitutive production of CCL20 in the FAE.

Epidemiology of small intestinal atresia in Europe: a register-based study.

BACKGROUND: The epidemiology of congenital small intestinal atresia (SIA) has not been well studied. This study describes the presence of additional anomalies, pregnancy outcomes, total prevalence and association with maternal age in SIA cases in Europe. METHODS: Cases of SIA delivered during January 1990 to December 2006 notified to 20 EUROCAT registers formed the population-based case series. Prevalence over time was estimated using multilevel Poisson regression, and heterogeneity between registers was evaluated from the random component of the intercept. RESULTS: In total 1133 SIA cases were reported among 5126, 164 registered births. Of 1044 singleton cases, 215 (20.6%) cases were associated with a chromosomal anomaly. Of 829 singleton SIA cases with normal karyotype, 221 (26.7%) were associated with other structural anomalies. Considering cases with normal karyotype, the total prevalence per 10 000 births was 1.6 (95% CI 1.5 to 1.7) for SIA, 0.9 (95% CI 0.8 to 1.0) for duodenal atresia and 0.7 (95% CI 0.7 to 0.8) for jejunoileal atresia (JIA). There was no significant trend in SIA, duodenal atresia or JIA prevalence over time (RR=1.0, 95% credible interval (CrI): 1.0 to 1.0 for each), but SIA and duodenal atresia prevalence varied by geographical location (p=0.03 and p=0.04, respectively). There was weak evidence of an increased risk of SIA in mothers aged less than 20 years compared with mothers aged 20 to 29 years (RR=1.3, 95% CrI: 1.0 to 1.8). CONCLUSION: This study found no evidence of a temporal trend in the prevalence of SIA, duodenal atresia or JIA, although SIA and duodenal atresia prevalence varied significantly between registers.

Intestinal bacteria condition dendritic cells to promote IgA production.

Immunoglobulin (Ig) A represents the predominant antibody isotype produced at the intestinal mucosa, where it plays an important role in limiting the penetration of commensal intestinal bacteria and opportunistic pathogens. We show in mice that Peyer's Patch-derived dendritic cells (PP-DC) exhibit a specialized phenotype allowing the promotion of IgA production by B2 cells. This phenotype included increased expression of the retinaldehyde dehydrogenase 1 (RALDH1), inducible nitric oxide synthase (iNOS), B cell activating factor of the tumor necrosis family (BAFF), a proliferation-inducing ligand (APRIL), and receptors for the neuropeptide vasoactive intestinal peptide (VIP). The ability of PP-DC to promote anti-CD40 dependent IgA was partially dependent on retinoic acid (RA) and transforming growth factor (TGF)-beta, whilst BAFF and APRIL signaling were not required. Signals delivered by BAFF and APRIL were crucial for CD40 independent IgA production, although the contribution of B2 cells to this pathway was minimal. The unique ability of PP-DC to instruct naïve B cells to differentiate into IgA producing plasma cells was mainly imparted by the presence of intestinal commensal bacteria, and could be mimicked by the addition of LPS to the culture. These data indicate that exposure to pathogen-associated molecular patterns present on intestinal commensal bacteria condition DC to express a unique molecular footprint that in turn allows them to promote IgA production.