Rabbit appendix consists mainly of lymphoid follicles (LF) covered by M cells, the specialized antigen-sampling cells of the mucosal immune system, and surrounded by glandular epithelium. Until now, these M cells have been characterized morphologically and histologically by using cellular markers. Here, the adhesion and transport of pathogenic bacteria were investigated to assess the function of M cells of the appendix. We used the enteroinvasive motile Salmonella typhimurium and the rabbit enteropathogenic non-motile Escherichia coli RDEC-1, which are known to target specifically rabbit M cells of Peyer's patches (PPs). We found that S. typhimurium efficiently attached and was transported through appendix M cells in vivo. In contrast to S. typhimurium, RDEC-1 targeted M cells only ex vivo, when bacteria were allowed to have direct contact with the surface of the follicle. The difference in interaction of the two bacteria with appendix M cells led us to investigate whether this could be correlated with the lack of motility of RDEC-1. We used an aflagellate mutant of S. typhimurium and found that it had the same infection phenotype as RDEC-1. Gene complementation restored the efficiency of infection to that of S. typhimurium wild-type strain. In conclusion, we show that M cells of the appendix display features of the canonical M cells of PP, since they efficiently sample luminal pathogenic bacteria. However, due to the morphology of the appendix, motile bacteria appear to be more potent in their interactions with appendix M cells.

Beta1 integrins are anchored on the basal membrane of enterocytes, but little is known about their localization in M cells, which are the main entry route into the intestinal mucosa for many bacterial pathogens. In particular, it has been suggested that adhesion of enteropathogenic Yersinia to M cells is mediated by interaction of the bacterial protein invasin and apical beta1 integrins. Using a novel in vitro model of M cells, we demonstrate an augmented apical and basolateral targeting of beta1 integrins in M cells associated with increased total alpha chain synthesis. The alpha3 and alpha6 subunits were targeted to the basal pole, but alpha2 subunit was targeted at both poles. No other alpha subunit was found associated with apical beta1 integrins on M cells. Interestingly, Y. enterocolitica still adhered to the apical surface of M cells, despite the fact that alpha2beta1 is not a receptor for invasin. We therefore studied the adhesive properties of invasin-mutant Y. enterocolitica and invasin-expressing Escherichia coli on the apical surface of M cells. We show that it is not invasin, but the product of an as yet unidentified bacterial chromosomal gene, that is involved in the adhesion of Y. enterocolitica to the apical membrane of M cells.

To explore mechanisms whereby Malpighian keratinocytes can transdifferentiate into an intestinal-like epithelium, as observed in the early steps of Barrett's esophagus (BE) development, long-standing cultures of esophageal keratinocytes derived from normal mouse esophageal explants were developed. These cells were able to form multilayers and to differentiate on filter support by the formation of differentiated layers of basal cells(cytokeratine 14 positive) on which secondary suprabasal cell layers(cytokeratine 4 positive) spontaneously developed. Thus, these cultured cells,referred to as P3E6, reproduced, at least in part, the proliferation and stratification pattern existing in the normal esophagus. Because chronic exposure to acid pH is known to be a critical factor for BE development,culture medium at pH 3.5 was added into the apical chamber of cell cultures. This led to a decrease in the overall number of cells but it did not affect cell proliferation. Furthermore, external acid environment triggered expression of the GFP reporter gene fused downstream of the cdx2 intestinal homeogene regulatory sequences in P3E6 transfected cells. Expression of the endogenous CDX2 protein, detected by western blot and immunocytochemical analysis, correlated with promoter activation. These findings demonstrate that chronic exposure of esophageal keratinocytes to acid pH induces transcription of cdx2, an intestinal specific homeobox gene known to play a critical role in the differentiation and maintenance of intestinal epithelial functions. The results suggest that chronic acid exposure can modify the fate of P3E6 esophageal keratinocytes towards an intestinal program. This can be a key step in the development of intestinal metaplasia often observed in esophagus-cardia junction.

During the digestive-tract phase of infection, poliovirus (PV) is found in the oropharynx and the intestine. It has been proposed that PV enters the organism by crossing M cells, which are scattered in the epithelial sheet covering lymphoid follicles of Peyer’s patches. However, PV translocation through M cells has never been demonstrated. A model of M-like cells has been previously developed using monolayers of polarized Caco-2 enterocytes cocultured with lymphocytes isolated from Peyer’s patches. In this model, lymphoepithelial interactions trigger the appearance of epithelial cells having morphological and functional characteristics of M cells. We have demonstrated efficient, temperature-dependent PV transcytosis in Caco-2 cell monolayers containing M-like cells. This experimental evidence is consistent with M cells serving as gateways allowing PV access to the basal face of enterocytes, the underlying immune follicle cells, and PV transport toward mesenteric lymph nodes.

In the intestine, the follicle-associated epithelium (FAE) of Peyer's patches (PP) performs Ag sampling as the first step in developing immune responses. Depending on the species, this epithelium contains 10-50% of M cells, which act as regulated gates in epithelial barriers that can be used opportunistically by pathogens to invade their host. However, the mechanisms involved in the differentiation and uptake processes of M cells are not known, in part because their limited number in the intestinal mucosa has hampered molecular and biochemical studies. In this work we provide evidence that PP lymphocytes can themselves modulate gene expression in PP in vivo and in an in vitro model of FAE. Transgenic mice carrying a reporter gene under the control of a modified L-pyruvate kinase promoter (SVPK) exhibit strong transgene expression in PP and FAE, but not in the adjacent villous cells. We used the mouse intestinal epithelial cell line m-IC(cl2) transfected with the SVPK promoter fused to beta-galactosidase to investigate the direct effect of PP lymphocytes on SVPK promoter activity. beta-Galactosidase expression was 4.4-fold higher in transfected m-IC(cl2) cells when they were cultured with PP lymphocytes. Conversely, green fluorescent protein expression was 1.8-fold lower in stably transfected differentiated intestinal Caco-2(cl1) cells with the sucrase isomaltase promoter fused to green fluorescent protein cDNA when they were cultured with PP lymphocytes, indicating that the in vivo FAE down-regulation of sucrase isomaltase promoter is transcriptionally regulated.

Peyer′s patch lymphocytes cocultured with Caco-2 cells trigger the phenotypic conversion of enterocytes into cells that express morphological and functional M-cell properties. We report a technical update for setting up this model, which will enable the study of M-cell biology, the identification by biochemical approaches of molecules involved in the interaction of microorganisms with M cells, and the development of vectors that would efficiently target the mucosal immune system.

BACKGROUND & AIMS:The follicle-associated epithelium (FAE) over mucosa-associated lymphoid tissues consists of distinct enterocytes and M cells concentrated at its periphery. The basement membrane composition was analyzed to test whether differences account for the distinct differentiation programs along the crypt-villus and crypt-FAE axes. To determine whether the decreased number of M cells in the FAE apex is caused by premature extrusion, we mapped the site where they undergo apoptosis.METHODS:The FAE basal lamina of Peyer's patches from BALB/c mice was analyzed by immunochemistry. M cells were identified using the Ulex europaeus agglutinin lectin. The cell proliferation and apoptotic compartments were characterized using bromodeoxyuridine incorporation and the TUNEL assay.RESULTS:The perlecan and laminin 2 stainings were different in FAE and villi. Myofibroblasts were absent beneath the FAE. The migration kinetics of cells along the FAE was similar to that along the villi. Apoptotic cells were detected exclusively at the apex of the FAE.CONCLUSIONS:FAE and M-cell differentiation is associated with a distinct basal lamina composition. FAE enterocytes express transient M-cell features as they move from the crypts toward the apoptotic compartment. M cells have a highly plastic phenotype that raises interesting questions about the control of intestinal epithelial cell differentiation.

Ribosomal preparations of pathogenic micro-organisms of the upper respiratory tract can be delivered orally for the prevention of recurrent infectious episodes, because they induce mucosal and protective immune responses. The mechanism of mucosal barrier translocation is difficult to study in animal models, little is therefore known about this process. In order to circumvent these problems, we have examined the uptake of ribosomal preparations in three experimental systems that model human intestinal cells. We found that M-like cells displayed a 8.7-fold increase in the uptake of a ribosomal immunostimulant when compared to absorptive or crypt enterocyte-like cells. The product was taken up, translocated, and delivered in the basolateral compartment by cultured M-like cells. No translocation was observed across monolayers of T84 cells (model of crypt cells). Only minimal translocation occured through monolayers of Caco-2 cells (model of absorptive enterocytes). This suggests that, in vivo, colyophilisat is delivered mainly through the M cells overlying lymphoid follicles (Peyer's patches) or nodules of the gut-associated mucosal lymphoid tissue, which are the major inductor sites of mucosal responses. Use of the M-like cell cultured model could be a key step for the development of even more efficient immunostimulators in animals and human.

Yersinia enterocolitica cross the intestinal epithelium via translocation through M cells, which are located in the follicle-associated epithelium (FAE) of Peyer's patches (PP). To investigate the molecular basis of this process, studies were performed using a recently developed in vitro model, in which the enterocyte-like cell line Caco-2 and PP lymphocytes are co-cultured in order to establish FAE-like structures including M cells. Here, we demonstrate that Y. enterocolitica does not adhere significantly to the apical membrane of differentiated enterocyte-like Caco-2 cells that express binding sites for Ulex europaeus agglutinin (UEA)-1. In contrast, Y. enterocolitica adhered to, and was internalized by, cells that lacked UEA-1 binding sites and displayed a disorganized brush border. These cells were considered to be converted to M-like cells. Further analysis revealed that part of these cells expressed beta 1 integrins at their apical surface and, as revealed by comparison of wild-type and mutant strains, interacted with invasin of Y. enterocolitica. Consistently, anti-beta 1 integrin antibodies significantly inhibited internalization of inv-expressing yersiniae. Experiments with Yersinia mutant strains deficient in YadA or Yop secretion revealed that these virulence factors play a minor role in this process. After internalization, yersiniae were transported within LAMP-1-negative vacuoles to, and released at, the basal surface. Internalization and transport of yersiniae was inhibited by cytochalasin D, suggesting that F-actin assembly is required for this process. These results provide direct evidence that expression of beta 1 integrins at the apical surface of M cells enables interaction with the invasin of Y. enterocolitica, and thereby initiates internalization and translocation of bacteria.

The maintenance during adult life of a large spectrum of pluripotency by stem cells originating from the endoderm seems to be the grounds for the striking plasticity of the digestive epithelium, which is able to drastically modify its differentiation pattern depending on the microenvironment. As a paradigm, Peyer's patch M cell development appears to be induced by crosstalk between lymphoid cells and/or microorganisms. Examples of pathological transdifferentiation of epithelia, also described as 'metaplasia' and affecting various organs, support the concept of intestinal plasticity. Though, the molecular processes involved in epithelial transdifferentiation have not been identified, histological analyses of these metaplastic tissues and experimental induction of transdifferentiation of normal epithelia provide lines of evidence suggesting that a modification of the local environment, such as occurs during contact of the epithelium with lymphoid cells or microorganisms, plays a key role in this process.

ABSTRACT: Differentiation of specific epithelial cell lineages during development, as well as epithelial plasticity in response to heterologous cell‐to‐cell cross talk during adult life, accounts for the large variety of functions which are performed by the mucosal surfaces found in the human body. Among its functions, the digestive mucosa is able to sample antigens and microorganisms through M cells of Peyer's patches follicle‐associated epithelium, in order to trigger the development of either tolerance or immune responses. At least in the gut, M‐cell formation is immunoregulated. Close contact between immune cells and intestinal epithelium modifies the permeability of the epithelial barrier by inducing the conversion of enterocytes into M cells, offering at the same time an opportunistic way of invasion for pathogens. These lympho‐epithelial interactions triggering M‐cell formation have now been modeled in culture.

Intestinal epithelium has the property to renew very quickly. The crypts present at the base of the villi contain stem cells that give rise to the different cell types present in the intestinal epithelium. Stem cells have a high proliferative activity with a small mitotic potential. As the cells migrate along the villi they differentiate and acquire intestinal properties. It was previously thought that intestinal differentiation resulted from autonomous regulation: here we summarize studies showing that intestinal differentiation is the result of many complex cross talks between the different partners forming the intestinal environment. In this review we concentrate on the activity of two of these partners; one is the lymphoid system and the other comprises the microbiota. We have also analyzed the latest results on the identification and the implications of transcription factors on the induction of intestinal properties with a special focus on the role of the extracellular matrix on the expression of specific intestinal transcription factors.

The epithelium that lines the gut is impermeable to macromolecules and microorganisms, except in Peyer's patches (PPs), where the lymphoid follicle-associated epithelium (FAE) contains M cells that transport antigens and microorganisms. A cultured system that reproduces the main characteristics of FAE and M cells was established by cultivation of PP lymphocytes with the differentiated human intestinal cell line Caco-2. Lymphocytes settled into the epithelial monolayer, inducing reorganization of the brush border and a temperature-dependent transport of particles and Vibrio cholerae. This model system could prove useful for intestinal physiology, vaccine research, and drug delivery studies.

The mucosal epithelia of the digestive tract acts as a selective barrier, permeable to ions, small molecules and macromolecules. These epithelial cells aid the digestion of food and absorption of nutrients. They contribute to the protection against pathogens and undergo continuous cell renewal which facilitates the elimination of damaged cells. Both innate and adaptive defence mechanisms protect the gastrointestinal-mucosal surfaces against pathogens. Interaction of microorganisms with epithelial cells triggers a host response by activating specific transcription factors which control the expression of chemokines and cytokines. This host response is characterized by the recruitment of macrophages and neutrophils at the site of infection. Disruption of epithelial signalling pathways that recruit migratory immune cells results in a chronic inflammatory response. The adaptive defence mechanism relies on the collaboration of epithelial cells (resident sampling system) with antigen-presenting and lymphoid cells (migratory sampling system); in order to obtain samples of foreign antigen, these samples must be transported across the barriers without affecting the integrity of the barrier. These sampling systems are regulated by both environmental and host factors. Fates of the antigen may differ depending on the way in which they cross the epithelial barrier, i.e. via interaction with motile dendritic cells or epithelial M cells in the follicle-associated epithelium.

Reactive oxygen species modulate the cell growth of a wide variety of mammalian cells. To determine whether oxidative metabolism is altered during the differentiation process, we studied the expression of pro- and antioxidant proteins in proliferating and differentiated CaCo-2 cells, a human colon adenocarcinoma cell line. Nitric oxide synthase type 2 (iNOS) produces nitric oxide (NO). Depending on its rate of synthesis, NO may either promote cellular and DNA damage or reduce the ability of other free radicals to induce cell injury. Using Western and Northern blot analysis and arginine conversion assay, we demonstrate that the expression of iNOS decreases when cells undergo differentiation. This biological event entails a diminished production of NO metabolites and correlates with the loss of activation of soluble guanylate cyclase activity. In differentiated cells, a 2-fold down-regulation of the nuclear factor kappa B activity was observed, suggesting that nuclear factor kappa B could be one of the iNOS gene regulatory factors in the CaCo-2 model. In parallel, we studied the expression of other antioxidant proteins including glutathione S-transferase alpha (GST alpha), bcl-2, and the metallothioneins (MTs). We show that the protein levels of GST alpha and MT increase during the differentiation of CaCo-2 cells, whereas bcl-2 levels decrease. Our investigation indicates that the expression of iNOS, GST alpha, bcl-2, and MT is associated with the enterocytic differentiation. The shift in the expression of specific antioxidant genes during CaCo-2 cell differentiation may occur to avoid alterations in the cell redox potential.