Déterminer le rôle de C1orf106, un gène associé aux maladies inflammatoires de l’intestin

Les maladies inflammatoires de l’intestin (MIIs, [MIM 266600]) sont caractérisées par une inflammation chronique au niveau du tube gastro-intestinal. Les deux principales formes sont la maladie de Crohn (MC) et la colite ulcéreuse (CU). Les MIIs résulteraient d’un défaut du système immunitaire et de l’épithélium intestinal. Ce dernier forme une barrière physique et biochimique qui sépare notre système immunitaire des microorganismes commensaux et pathogènes de la microflore intestinale. Un défaut dans la barrière épithéliale intestinale pourrait donc mener à une réponse immunitaire soutenue contre notre microflore intestinale. Les études d’association pangénomiques (GWAS) ont permis d’identifier 201 régions de susceptibilité aux MIIs. Parmi celles-ci, la région 1q32 associée à la MC (p<2x10-11) et à la CU (p<6x10-7) contient 4 gènes, dont C1orf106, un gène codant pour une protéine de fonction inconnue. Le re-séquençage de la région 1q32 a permis d’identifier une variante génétique rare de C1orf106 (MAF˂1%) associée aux MIIs (p=0,009), Y333F. Nous avons démontré que la substitution de la tyr333 par une phénylalanine semble avoir un effet sur la stabilité protéique de C1orf106 tel que démontré lors de l’inhibition de la synthèse protéique induite par le cycloheximide. Nous avons déterminé que C1orf106 est exprimé dans le côlon et l’intestin grêle. De plus, son expression est augmentée lors de la différenciation des cellules épithéliales Caco-2 en épithélium intestinal polarisé. Son profil d’expression correspond aux types cellulaires et tissulaires affectés dans les MIIs. De plus, C1orf106 est partiellement co-localisée avec le marqueur des jonctions serrées, ZO-1. Toutefois, son marquage reproduit parfaitement celui du marqueur des jonctions adhérentes, E-cadhérine. Les jonctions serrées et adhérentes sont localisées du côté apical de la jonction intercellulaire et sont toutes deux impliquées dans l’établissement de la barrière épithéliale. Nous avons donc testé l’impact de C1orf106 sur la perméabilité de l’épithélium intestinal. Nous avons observé une augmentation de la perméabilité épithéliale chez un épithélium intestinal formé par des cellules Caco-2 sous-exprimant C1orf106. Nos résultats suggèrent que C1orf106 pourrait être le gène causal de la région 1q32.

Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

Isolation and culture of mouse intestinal cells.

Complex cell signal transduction mechanisms regulate intestinal epithelial shape, polarity, motility, organelles, cell membrane components as well as physical and mechanical properties to influence alimentary digestion, absorption, secretion, detoxification and fluid balance. Interactions between the epithelial cells and adjacent mesenchyme are central to intestinal homeostasis although the key regulatory molecules of specific differentiation steps remain unclear. Isolation and primary culture of heterotypic murine intestinal cells provides a model system for elucidation of essential molecular cross-talk between epithelium and mesenchyme that may provide several biological and practical advantages over transformed cell lines. An in vitro primary culture system for neonatal rat or mouse intestinal cells has been established that forms monolayers, expresses intestine-specific epithelial features including intestinal brush borders and appropriate hydrolase enzymes. Our studies confirm the promise of this method which may advance our understanding of heterotypic cellular interactions implicated in intestinal function and may provide important insights into the pathobiology of disease.

Lactobacilli antagonize the growth, motility, and adherence of brachyspira pilosicoli: a potential intervention against avian intestinal spirochetosis

Avian intestinal spirochetosis (AIS) results from the colonization of the ceca and colorectum of poultry by pathogenic Brachyspira species. The number of cases of AIS has increased since the 2006 European Union ban on the use of antibiotic growth promoters, which, together with emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Probiotics have been reported as protecting livestock against infection with common enteric pathogens, and here we investigate which aspects of the biology of Brachyspira they antagonize in order to identify possible interventions against AIS. The cell-free supernatants (CFS) of two Lactobacillus strains, Lactobacillus reuteri LM1 and Lactobacillus salivarius LM2, suppressed the growth of Brachyspira pilosicoli B2904 in a pH-dependent manner. In in vitro adherence and invasion assays with HT29-16E three-dimensional (3D) cells and in a novel avian cecal in vitro organ culture (IVOC) model, the adherence and invasion of B. pilosicoli in epithelial cells were reduced significantly by the presence of lactobacilli (P < 0.001). In addition, live and heat-inactivated lactobacilli inhibited the motility of B. pilosicoli, and electron microscopic observations indicated that contact between the lactobacilli and Brachyspira was crucial in inhibiting both adherence and motility. These data suggest that motility is essential for B. pilosicoli to adhere to and invade the gut epithelium and that any interference of motility may be a useful tool for the development of control strategies.

Interaction of enterohemorrhagic Escherichia coli O157 : H7 with mouse intestinal mucosa

In this study, we used mouse ileal loops to investigate the interaction of enterohemorrhagic Escherichia coli (EHEC) O157:H7 with the mouse intestinal mucosa. With a dose of 10(9) and 3 h incubation, EHEC O157 was detected in the lumen and to a lesser extent associated with the epithelium. Typical attaching and effacing (A/E) lesions were seen, albeit infrequently. While the effector protein Tir was essential for A/E lesion formation, the bacterial type III secretion system adaptor protein TccP was dispensable. These results suggest that A/E lesions on mouse intestinal mucosa can be formed independently of robust actin polymerization.

Structural changes in the epithelium of the small intestine and immune cell infiltration of enteric ganglia following acute mucosal damage and local inflammation

An acute enteritis is commonly followed by intestinal neuromuscular dysfunction, including prolonged hyperexcitability of enteric neurons. Such motility disorders are associated with maintained increases in immune cells adjacent to enteric ganglia and in the mucosa. However, whether the commonly used animal model, trinitrobenzene sulphonate (TNBS)-induced enteritis, causes histological and immune cell changes similar to human enteric neuropathies is not clear. We have made a detailed study of the mucosal damage and repair and immune cell invasion following intralumenal administration of TNBS. Intestines from untreated, sham-operated and TNBS-treated animals were examined at 3 h to 56 days. At 3 h, the mucosal surface was completely ablated, by 6 h an epithelial covering was substantially restored and by 1 day there was full re-epithelialisation. The lumenal epithelium developed from a squamous cell covering to a fully differentiated columnar epithelium with mature villi at about 7 days. Prominent phagocytic activity of enterocytes occurred at 1-7 days. A surge of eosinophils and T lymphocytes associated with the enteric nerve ganglia occurred at 3 h to 3 days. However, elevated immune cell numbers occurred in the lamina propria of the mucosa until 56 days, when eosinophils were still three times normal. We conclude that the disruption of the mucosal surface that causes TNBS-induced ileitis is brief, a little more than 6 h, and causes a transient immune cell surge adjacent to enteric ganglia. This is much briefer than the enteric neuropathy that ensues. Ongoing mucosal inflammatory reaction may contribute to the persistence of enteric neuropathy.

Efeitos do espaço de alojamento e da idade sobre desenvolvimento, integridade e proteção da mucosa intestinal de frangos de corte

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative Study Between Porcine Small Intestinal Submucosa and Buccal Mucosa in a Partial Urethra Substitution in Rabbits

Purpose: Several urethral conditions may require tissue substitution. One collagen-base biomaterial that recently emerged as an option is small intestinal submucosa (SIS). The aim of this study was to compare the results of SIS and buccal mucosa for urethral substitution in rabbits.Materials and Methods: Thirty-six North Folk male rabbits were randomized into three groups. In all animals, a 10 x 5 mm urethral segment was excised, and the urethral defect was repaired using a one-layer SIS patch (group I [GI]); four-layer SIS (group II [GII]); or buccal mucosa (group III [GIII]). Urethrography was performed preoperatively and after 12 weeks. After sacrifice, graft retraction was objectively measured using Scion Image (R) computer analysis and by calculation of ellipse area. The grade of fibrosis, inflammatory reaction, vascular/epithelial regeneration, and collagen III/I ratio were analyzed by hematoxylin/eosin and Picrosirius red staining.Results: Urethrography confirmed a wide urethral caliber without any signs of strictures after surgery. Urethral fistulae was diagnosed in 8.3% of cases (1 animal each group). Average graft shrinkage was 55.2% in GI; 44.2% in GII; and 57.2% in GIII (p < 0.05). The intensity of chronic inflammation, fibrosis, epithelium regeneration, and neovascularization was similar in all groups (p > 0.05). Collagen III/I ratio was higher in GII (GI: 119.6; GII: 257.2 and GIII: 115.0); p < 0.01.Conclusions: The four-layer SIS is more advantageous than the one-layer SIS and buccal mucosa for urethral substitution in rabbits.

Morphometric study of the midgut epithelium in larvae of Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae)

A larva de Diatraea saccharalis Fabricius (broca-da-cana) tem grande interesse econômico, pois afeta o cultivo e aproveitamento industrial da cana-de-açúcar. Entretanto, poucos são os estudos sobre a morfologia interna desse inseto. O objetivo deste trabalho foi estudar, morfometricamente, o seu epitélio intestinal, ao longo de seu comprimento, visando caracterizar regiões estruturalmente diferentes. O intestino médio de larvas no último instar foi subdividido em três regiões: proximal, mediana e distal e os fragmentos foram processados para observação em microscopia de luz. Os cortes histológicos foram analisados em sistema computadorizado de análise de imagens para medir comprimento, largura e área do epitélio, das diferentes células epiteliais, dos seus respectivos núcleos e do lúmen intestinal. Os dados obtidos foram submetidos ao teste estatístico de Kruskal-Wallis e à análise multivariada. Nossos resultados mostraram que o intestino médio apresentou-se constituído, morfometricamente, por duas diferentes regiões, proximal e distal; a região mediana apresentou valores coincidentes tanto com a região proximal quanto com a distal, sugerindo ser região intermediária. As células epiteliais (colunares, caliciformes e regenerativas), quando avaliadas pela análise estatística multivariada, não apresentaram diferença morfométrica nas diferentes regiões do intestino médio. Entretanto, a análise de variância, realizada para variáveis isoladas, mostrou que as células regenerativas apresentaram maior variabilidade morfométrica.

Growth performance and intestinal histomorphology of Nile tilapia juveniles fed probiotics

The objective of this study was to assess the growth performance and the histomorphometric characterization of the middle intestine of GIFT strain Nile tilapia, Oreochromis niloticus juveniles fed probiotics, added either before or after the feed processing. The experimental design was completely randomized, with five treatments and five replications: pelleted feed without any probiotic; pelleted feed with inclusion of probiotic before the processing; pelleted feed with inclusion of probiotic after the processing; extruded feed without any probiotic and extruded feed with inclusion of probiotic after the processing. Two hundred and fifty juveniles were distributed in 25 aquaria (20L) and fed for 63 days. Differences in the mean values of total weight were found at the end of the experiment. After 42 days of feed intake, significant differences in feed conversion were verified for treatments with extruded diets when compared to the pelleted ones. The fishes fed diets supplemented with probiotic presented increase in the thickness of the epithelium of the intestine. The best zootechnical performance was observed in the extruded diet supplemented with probiotic added after the feed processing. The inclusion of 4.0 x 10(8) CFU g-1 of Bacillus subtilis and 4.0 x 10(8) CFU g-1 of Bacillus toyoi in both the pelleted and the extruded diets promoted an increase in the thickness of the epithelial layer of the middle intestine.

Intestinal IgG uptake by small intestine of goat kid fed goat or lyophilized bovine colostrum

The immunoglobulin G (IgG) uptake and enterocyte nucleus position in the villous were studied in newborn goat kids fed goat or lyophilized bovine colostrum. Two groups of 15 newborn goat kids, each received 5% of body weight of goat colostrum (GC) or lyophilized bovine colostrum (LBC) containing 55 mg/mL of immunoglobulin G (IgG) at 0, 7 and 14 h of life. Three animals were sampled just after birth, receiving no colostrum intake, to be used as control. Samples of duodenum, medium jejunum and ileum were collected at 0, 18, 36 and 96 h of life. IgG vacuoles were not observed in the duodenum throughout the experiment regardless of all the experimental time points. In this segment, at 0, 18 and 36 h of life, nuclei were found in the apical, medial and basal positions in the enterocytes, and localized in the upper, medial and lower parts in the villous, respectively. At 96 h, a basal nuclei position was observed in the enterocytes, throughout the villous. In jejunum, IgG vacuoles were distributed along the villous at 18 and 36 h. In this segment at Oh the nuclei were positioned predominantly apically in the enterocytes, throughout the villous. At 18 and 36 h, no consistent nuclei pattern was verified: however at 96 h, the nuclei were positioned basally in the enterocytes, throughout the jejunal villous. In the ileum at 0, 18 and 36 h, a great number of vacuoles without IgG were verified in the medial-apical part of the villous. In this segment, at Oh of life and 96 h of life, the predominance of basal nuclei was observed. Nuclei were positioned in medial-apically part of the ileal enterocytes in the upper part of the villous at 18 and 36 h. It was found that the jejunal epithelium was the most important segment related to absorption process. The IgG absorption and nucleus position in the newborn goats were dependent on the small intestine segments and experimental time points, regardless of the colostrum source. GC or LCB. Considering the IgG uptake mechanism observed in the present study, the lyophilized bovine colostrum might be used instead of goat colostrum. (C) 2011 Elsevier B.V. All rights reserved.

Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression

Background Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity. Methodology/Principal Findings Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. Conclusion We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.

Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease

Epithelial cells in the human small intestine express meprin, an astacin-like metalloprotease, which accumulates normally at the brush border membrane and in the gut lumen. Therefore, meprin is targeted towards luminal components. In coeliac disease patients, peptides from ingested cereals trigger mucosal inflammation in the small intestine, disrupting epithelial cell differentiation and function. Using in situ hybridisation on duodenal tissue sections, we observed a marked shift of meprin mRNA expression from epithelial cells, the predominant expression site in normal mucosa, to lamina propria leukocytes in coeliac disease. Meprin thereby gains access to the substrate repertoire present beneath the epithelium.

The mucosal firewalls against commensal intestinal microbes

Mammals coexist with an extremely dense microbiota in the lower intestine. Despite the constant challenge of small numbers of microbes penetrating the intestinal surface epithelium, it is very unusual for these organisms to cause disease. In this review article, we present the different mucosal firewalls that contain and allow mutualism with the intestinal microbiota.