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Background & Aims: The presence of myenteric plexitis in the proximal resection margins is a predictive factor of early postoperative recurrence in Crohn's disease. To decipher the mechanisms leading to their formation, T-cell interactions with enteric neural cells were studied in vitro and in vivo., Methods: T cells close to myenteric neural cells were retrospectively quantified in ileocolonic resections from 9 control subjects with cancer and 20 patients with Crohn's disease. The mechanisms involved in T-cell adhesion were then investigated in co-cultures of T lymphocytes with enteric glial cells (glia). Finally, the implication of adhesion molecules in the development of plexitis and colitis was studied in vitro but also in vivo in Winnie mice., Results: The mean number of T cells close to glia, but not neurons, was significantly higher in the myenteric ganglia of relapsing patients with Crohn's disease (2.42 ± 0.5) as compared with controls (0.36 ± 0.08, P = .0007). Co-culture experiments showed that exposure to proinflammatory cytokines enhanced T-cell adhesion to glia and increased intercellular adhesion molecule-1 (ICAM-1) expression in glia. We next demonstrated that T-cell adhesion to glia was inhibited by an anti-ICAM-1 antibody. Finally, using the Winnie mouse model of colitis, we showed that the blockage of ICAM-1/lymphocyte function-associated antigen-1 (LFA-1) with lifitegrast reduced colitis severity and decreased T-cell infiltration in the myenteric plexus., Conclusions: Our present work argues for a role of glia-T-cell interaction in the development of myenteric plexitis through the adhesion molecules ICAM-1/LFA-1 and suggests that deciphering the functional consequences of glia-T-cell interaction is important to understand the mechanisms implicated in the development and recurrence of Crohn's disease., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Colon cancer stem cells (CSCs), considered responsible for tumor initiation and cancer relapse, are constantly exposed to regulatory cues emanating from neighboring cells present in the tumor microenvironment. Among these cells are enteric glial cells (EGCs) that are potent regulators of the epithelium functions in a healthy intestine. However, whether EGCs impact CSC-driven tumorigenesis remains unknown., Methods: Impact of human EGC primary cultures or a non-transformed EGC line on CSCs isolated from human primary colon adenocarcinomas or colon cancer cell lines with different p53, MMR system and stemness status was determined using murine xenograft models and 3D co-culture systems. Supernatants of patient-matched human primary colon adenocarcinomas and non-adjacent healthy mucosa were used to mimic tumor versus healthy mucosa secretomes and compare their effects on EGCs., Findings: Our data show that EGCs stimulate CSC expansion and ability to give rise to tumors via paracrine signaling. Importantly, only EGCs that were pre-activated by tumor epithelial cell-derived soluble factors increased CSC tumorigenicity. Pharmacological inhibition of PGE2 biosynthesis in EGCs or IL-1 knockdown in tumor epithelial cells prevented EGC acquisition of a pro-tumorigenic phenotype. Inhibition of PGE2 receptor EP4 and EGFR in CSCs inhibited the effects of tumor-activated EGCs., Interpretation: Altogether, our results show that EGCs, once activated by the tumor, acquire a pro-tumorigenic phenotype and stimulate CSC-driven tumorigenesis via a PGE2/EP4/EGFR-dependent pathway., Funding: This work was supported by grants from the French National Cancer Institute, La Ligue contre le Cancer, the 'Région des Pays de la Loire' and the UNC Lineberger Comprehensive Cancer Center., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Neuroimmune interactions are essential to maintain gut homeostasis and prevent intestinal disorders but so far, the impact of enteric glial cells (EGC) on immune cells remains a relatively unexplored area of research. As a dysregulation of critical cytokines such as interleukine-7 (IL-7) was suggested to exacerbate gut chronic inflammation, we investigated whether EGC could be a source of IL-7 in the gastrointestinal tract., Methods: Expression of IL-7 in the rat enteric nervous system was analyzed by immunochemistry and Q-PCR. IL-7 variants were cloned and specific antibodies against rat IL-7 isoforms were raised to characterize their expression in the submucosal plexus. IL-7 isoforms were produced in vitro to analyze their impact on T-cell survival., Key Results: Neurons and glial cells of the rat enteric nervous system expressed IL-7 at both mRNA and protein levels. Novel rat IL-7 isoforms with distinct C-terminal parts were detected. Three of these isoforms were found in EGC or in both enteric neurons and EGC. Exposure of EGC to pro-inflammatory cytokines (IL-1β and/or TNFα) induced an upregulation of all IL-7 isoforms. Interestingly, time-course and intensity of the upregulation varied according to the presence or absence of exon 5a in IL-7 variants. Functional analysis on T lymphocytes revealed that only canonical IL-7 protects T cells from cell death., Conclusions and Inferences: IL-7 and its variants are expressed by neurons and glial cells in the enteric nervous system. Their distinct expression and upregulation in inflammatory conditions suggest a role in gut homeostasis which could be critical in case of chronic inflammatory diseases., (© 2018 John Wiley & Sons Ltd.)

Gone are the days when enteric glial cells (EGC) were considered merely satellites of enteric neurons. Like their brain counterpart astrocytes, EGC express an impressive number of receptors for neurotransmitters and intercellular messengers, thereby contributing to neuroprotection and to the regulation of neuronal activity. EGC also produce different soluble factors that regulate neighboring cells, among which are intestinal epithelial cells. A better understanding of EGC response to an inflammatory environment, often referred to as enteric glial reactivity, could help define the physiological role of EGC and the importance of this reactivity in maintaining gut functions. In chronic inflammatory disorders of the gut such as Crohn's disease (CD) and ulcerative colitis, EGC exhibit abnormal phenotypes, and their neighboring cells are dysfunctional; however, it remains unclear whether EGC are only passive bystanders or active players in the pathophysiology of both disorders. The aim of the present study is to review the physiological roles and properties of EGC, their response to inflammation, and their role in the regulation of the intestinal epithelial barrier and to discuss the emerging concept of CD as an enteric gliopathy.

Background: Growing evidence indicates a wide array of cellular remodeling in the mucosal microenvironment during irritable bowel syndrome (IBS), which possibly contributes to pathophysiology and symptom generation. Here, we investigated whether enteric glial cells (EGC) may be altered, and which factors/mechanisms lead to these changes., Methods: Colonic mucosal biopsies of IBS patients (13 IBS-Constipation [IBS-C]; 10 IBS-Diarrhea [IBS-D]; 11 IBS-Mixed [IBS-M]) and 24 healthy controls (HC) were analyzed. Expression of S100β and GFAP was measured. Cultured rat EGC were incubated with supernatants from mucosal biopsies, then proliferation and Ca 2+ response to ATP were analyzed using flow cytometry and Ca 2+ imaging. Histamine and histamine 1-receptor (H1R) involvement in the effects of supernatant upon EGC was analyzed., Key Results: Compared to HC, the mucosal area immunoreactive for S100β was significantly reduced in biopsies of IBS patients, independently of the IBS subtype. IBS-C supernatants reduced EGC proliferation and IBS-D and IBS-M supernatants reduced Ca 2+ response to ATP in EGC. EGC expressed H1R and the effects of supernatant upon Ca 2+ response to ATP in EGC were blocked by pyrilamine and reproduced by histamine via H1R. IBS supernatants reduced mRNA expression of connexin-43. The S100β-stained area was negatively correlated with the frequency and intensity of pain and bloating., Conclusion and Inferences: Changes in EGC occur in IBS, involving mucosal soluble factors. Histamine, via activation of H1R-dependent pathways, partly mediates altered Ca 2+ response to ATP in EGC. These changes may contribute to the pathophysiology and the perception of pain and bloating in patients with IBS., (© 2017 John Wiley & Sons Ltd.)

Key Points: Unlike astrocytes in the brain, the potential role of enteric glial cells (EGCs) in the formation of the enteric neuronal circuit is currently unknown. To examine the role of EGCs in the formation of the neuronal network, we developed a novel neuron-enriched culture model from embryonic rat intestine grown in indirect coculture with EGCs. We found that EGCs shape axonal complexity and synapse density in enteric neurons, through purinergic- and glial cell line-derived neurotrophic factor-dependent pathways. Using a novel and valuable culture model to study enteric neuron-glia interactions, our study identified EGCs as a key cellular actor regulating neuronal network maturation., Abstract: In the nervous system, the formation of neuronal circuitry results from a complex and coordinated action of intrinsic and extrinsic factors. In the CNS, extrinsic mediators derived from astrocytes have been shown to play a key role in neuronal maturation, including dendritic shaping, axon guidance and synaptogenesis. In the enteric nervous system (ENS), the potential role of enteric glial cells (EGCs) in the maturation of developing enteric neuronal circuit is currently unknown. A major obstacle in addressing this question is the difficulty in obtaining a valuable experimental model in which enteric neurons could be isolated and maintained without EGCs. We adapted a cell culture method previously developed for CNS neurons to establish a neuron-enriched primary culture from embryonic rat intestine which was cultured in indirect coculture with EGCs. We demonstrated that enteric neurons grown in such conditions showed several structural, phenotypic and functional hallmarks of proper development and maturation. However, when neurons were grown without EGCs, the complexity of the axonal arbour and the density of synapses were markedly reduced, suggesting that glial-derived factors contribute strongly to the formation of the neuronal circuitry. We found that these effects played by EGCs were mediated in part through purinergic P2Y 1 receptor- and glial cell line-derived neurotrophic factor-dependent pathways. Using a novel and valuable culture model to study enteric neuron-glia interactions, our study identified EGCs as a key cellular actor required for neuronal network maturation., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Enteric glial cells (EGC) have trophic and neuroregulatory functions in the enteric nervous system, but whether they exert a direct effect on immune cells is unknown. Here, we used co-cultures to show that human EGC can inhibit the proliferation of activated T lymphocytes. Interestingly, EGC from Crohn's patients were effective at one EGC for two T cells whereas EGC from control patients required a ratio of 1:1. These data suggest that EGC contribute to local immune homeostasis in the gastrointestinal wall. They also raise the possibility that EGC have particular immunosuppressive properties in inflammatory bowel diseases such as Crohn's disease., (Copyright © 2016. Published by Elsevier B.V.)

The postnatal period is crucial for the development of gastrointestinal (GI) functions. The enteric nervous system is a key regulator of GI functions, and increasing evidences indicate that 1) postnatal maturation of enteric neurons affect the development of GI functions, and 2) microbiota-derived short-chain fatty acids can be involved in this maturation. Although enteric glial cells (EGC) are central regulators of GI functions, the postnatal evolution of their phenotype remains poorly defined. We thus characterized the postnatal evolution of EGC phenotype in the colon of rat pups and studied the effect of short-chain fatty acids on their maturation. We showed an increased expression of the glial markers GFAP and S100β during the first postnatal week. As demonstrated by immunohistochemistry, a structured myenteric glial network was observed at 36 days in the rat colons. Butyrate inhibited EGC proliferation in vivo and in vitro but had no effect on glial marker expression. These results indicate that the EGC myenteric network continues to develop after birth, and luminal factors such as butyrate endogenously produced in the colon may affect this development., (Copyright © 2016 the American Physiological Society.)

Background & Aims: Enteric glial cells (EGCs) produce soluble mediators that regulate homeostasis and permeability of the intestinal epithelial barrier (IEB). We investigated the profile of polyunsaturated fatty acid (PUFA) metabolites produced by EGCs from rats and from patients with Crohn's disease (CD), compared with controls, along with the ability of one of these metabolites, 15-hydroxyeicosatetraenoic acid (15-HETE), to regulate the permeability of the IEB., Methods: We isolated EGCs from male Sprague-Dawley rats, intestinal resections of 6 patients with CD, and uninflamed healthy areas of intestinal tissue from 6 patients who underwent surgery for colorectal cancer (controls). EGC-conditioned media was analyzed by high-sensitivity liquid-chromatography tandem mass spectrometry to determine PUFA signatures. We used immunostaining to identify 15-HETE-producing enzymes in EGCs and tissues. The effects of human EGCs and 15-HETE on permeability and transepithelial electrical resistance of the IEB were measured using Caco-2 cells; effects on signal transduction proteins were measured with immunoblots. Levels of proteins were reduced in Caco-2 cells using short-hairpin RNAs or proteins were inhibited pharmacologically. Rats were given intraperitoneal injections of 15-HETE or an inhibitor of 15-lipoxygenase (the enzyme that produces 15-HETE); colons were collected and permeability was measured., Results: EGCs expressed 15-lipoxygenase-2 and produced high levels of 15-HETE, which increased IEB resistance and reduced IEB permeability. 15-HETE production was reduced in EGCs from patients with CD compared with controls. EGCs from patients with CD were unable to reduce the permeability of the IEB; the addition of 15-HETE restored permeability to levels of control tissues. Inhibiting 15-HETE production in rats increased the permeability of the IEB in colon tissues. We found that 15-HETE regulates IEB permeability by inhibiting an adenosine monophosphate-activated protein kinase and increasing expression of zonula occludens-1., Conclusions: Enteric glial cells from patients with CD have reduced production of 15-HETE, which controls IEB permeability by inhibiting adenosine monophosphate-activated protein kinase and increasing expression of zonula occludens-1., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Recent works provide evidence of the importance of the prostaglandin D2 (PGD2) metabolic pathway in inflammatory bowel diseases. We investigated the expression of PGD2 metabolic pathway actors in Crohn's disease (CD) and the ability of the enteric nervous system (ENS) to produce PGD2 in inflammatory conditions., Methods: Expression of key actors involved in the PGD2 metabolic pathway and its receptors was analyzed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in colonic mucosal biopsies of patients from three groups: controls, quiescent and active CD patients. To determine the ability of the ENS to secrete PGD2 in proinflammatory conditions, Lipocalin-type prostaglandin D synthase (L-PGDS) expression by neurons and glial cells was analyzed by immunostaining. PGD2 levels were determined in a medium of primary culture of ENS and neuro-glial coculture model treated by lipopolysaccharide (LPS)., Results: In patients with active CD, inflamed colonic mucosa showed significantly higher COX2 and L-PGDS mRNA expression, and significantly higher PGD2 levels than healthy colonic mucosa. On the contrary, peroxysome proliferator-activated receptor Gamma (PPARG) expression was reduced in inflamed colonic mucosa of CD patients with active disease. Immunostaining showed that L-PGDS was expressed in the neurons of human myenteric and submucosal plexi. A rat ENS primary culture model confirmed this expression. PGD2 levels were significantly increased on primary culture of ENS treated with LPS. This production was abolished by AT-56, a specific competitive L-PGDS inhibitor. The neuro-glial coculture model revealed that each component of the ENS, ECG and neurons, could contribute to PGD2 production., Conclusions: Our results highlight the activation of the PGD2 metabolic pathway in Crohn's disease. This study supports the hypothesis that in Crohn's disease, enteric neurons and glial cells form a functional unit reacting to inflammation by producing PGD2.

Lewy pathology has been described in neurons of the enteric nervous system in nearly all Parkinson's disease (PD) patients at autopsy. The enteric nervous system not only contains a variety of functionally distinct enteric neurons but also harbors a prominent component of glial cells, the so-called enteric glial cells, which, like astrocytes of the central nervous system, contribute to support, protect, and maintain the neural network. A growing body of evidence supports a role for enteric glial cells in the pathophysiology of gastrointestinal disorders such as inflammatory bowel disease and chronic constipation. We have recently shown that enteric glial cell dysfunction occurs in PD. In the present review, we discuss the possible implications of enteric glia in PD-related gut dysfunction as well as in disease initiation and development., (© 2014 International Parkinson and Movement Disorder Society.)

Since their discovery at the end of the 19th century, enteric glial cells (EGCs), the major cellular component of the enteric nervous system, have long been considered mere supportive cells for neurons. However, recent evidence has challenged this view and highlighted their central role in the regulation of gut homeostasis as well as their implication in digestive and extradigestive diseases. In this review, we summarize emerging concepts as to how EGCs regulate neuromediator expression, exert neuroprotective roles, and even act as neuronal as well as glial progenitors in the enteric nervous system. A particularly crucial property of EGCs is their ability to maintain the integrity of the intestinal epithelial barrier, a role that may have important clinical implications not only for digestive diseases, such as postoperative ileus and inflammatory bowel diseases, but also for extradigestive diseases, such as Parkinson disease or obesity. EGCs could also contribute directly to disease processes (eg, inflammation) by their ability to secrete chemokines/cytokines in response to bacterial or inflammatory challenges. Defining the pleiotropic roles exerted by EGCs may reveal better knowledge and help develop new targeted therapeutic options for a variety of gastrointestinal diseases., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Enteric glial cells (EGC) are major regulators of neuronal and intestinal epithelial cell (IEC) functions. Simple isolation methods of EGC, especially human tissues, remain scarce and limit their study. We present herein a method to isolate EGC and we characterize EGC phenotype and their functional impact on IEC., Methods: Longitudinal muscle and myenteric plexus preparations of rat, mouse, or human intestine were obtained by microdissection. After mechanical and enzymatic dissociation, individual ganglionic or interganglionic structures were seeded into plates, maintained in culture several weeks and passaged up to 4 times. Purity of cultures was assessed by immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), S100β and Sox10 or smooth muscle actin. Effects of adenosine triphosphate (ATP) on intracellular Ca²⁺ signaling in EGC were studied. Co-cultures of EGC with IEC line, Caco-2, were performed for 2-6 days to analyze their impact on monolayer resistance, cell proliferation, and cell spreading., Key Results: More than 80% of DAPI-positive cells were GFAP, S100β, and Sox10-immunoreactive. EGC expressed these glial markers over 4 consecutive passages, and the majority of them responded to ATP by an increase in intracellular Ca²⁺ concentration. In addition, rat, mouse, and human EGC increased intestinal barrier resistance, IEC size, and reduced IEC number., Conclusions & Inferences: We have developed a simple method to isolate and culture human, rat, or mouse EGC. EGC exhibit similar functional properties on the intestinal barrier independently of the species. This study sets the basis for exploring glial biology and functions in human health and diseases., (© 2013 John Wiley & Sons Ltd.)

The monolayer of columnar epithelial cells lining the gastrointestinal tract--the intestinal epithelial barrier (IEB)--is the largest exchange surface between the body and the external environment. The permeability of the IEB has a central role in the regulation of fluid and nutrient intake as well as in the control of the passage of pathogens. The functions of the IEB are highly regulated by luminal as well as internal components, such as bacteria or immune cells, respectively. Evidence indicates that two cell types of the enteric nervous system (ENS), namely enteric neurons and enteric glial cells, are potent modulators of IEB functions, giving rise to the novel concept of a digestive 'neuronal-glial-epithelial unit' akin to the neuronal-glial-endothelial unit in the brain. In this Review, we summarize findings demonstrating that the ENS is a key regulator of IEB function and is actively involved in pathologies associated with altered barrier function.

The enteric nervous system (ENS), a major regulatory system for gastrointestinal function, is composed of neurons and enteric glial cells (EGCs). Enteric glia have long been thought to provide only structural support to neurons. However, recent evidence indicates enteric glia-neuron cross talk significantly contributes to neuronal maintenance, survival, and function. Thus damage to EGCs may trigger neurodegenerative processes thought to play a role in gastrointestinal dysfunctions and symptoms. The purpose of this review is to provide an update on EGCs, particularly focusing on their possible neuroprotective features and the resultant enteric neuron abnormalities subsequent to EGC damage. These neuroprotective mechanisms may have pathogenetic relevance in a variety of functional and inflammatory gut diseases. Basic and clinical (translational) studies support a neuroprotective role mediated by EGCs. Different models have been developed to test whether selective EGC damage/ablation has an impact on gut functions and the ENS. Preclinical data indicated that selective EGC alterations were associated with changes in gut physiology related to enteric neuron abnormalities. In humans, a substantial loss of EGCs was described in patients with various functional and/or inflammatory gastrointestinal diseases. However, whether EGC changes precede or follow neuronal degeneration and loss and how this damage occurs is not defined. Additional studies on EGC neuroprotective capacity are expected to improve knowledge of gut diseases and pave the way for targeted therapeutic strategies of underlying neuropathies.

Increasing evidence suggests that enteric glial cells (EGCs) are critical for enteric neuron survival and functions. In particular, EGCs exert direct neuroprotective effects mediated in part by the release of glutathione. However, other glial factors such as those identified as regulating the intestinal epithelial barrier and in particular the omega-6 fatty acid derivative 15-deoxy-Δ¹²,¹⁴-prostaglandin J2 (15d-PGJ2) could also be involved in EGC-mediated neuroprotection. Therefore, our study aimed to assess the putative role of EGC-derived 15d-PGJ2 in their neuroprotective effects. We first showed that pretreatment of primary cultures of enteric nervous system(ENS)or humann euroblastoma cells (SH-SY5Y)with 15d-PGJ2 dose dependently prevented hydrogen peroxide neurotoxicity. Furthermore, neuroprotective effects of EGCs were significantly inhibited following genetic invalidation in EGCs of the key enzyme involved in 15d-PGJ2 synthesis, i.e. L-PGDS. We next showed that 15d-PGJ2 effects were mediated by an Nrf2 dependent pathway but were not blocked by PPARγ inhibitor (GW9662) in SH-SY5Y cells and enteric neurons. Finally, 15d-PGJ2 induced a significant increase in glutamate cysteine ligase expression and intracellular glutathione in SH cells and enteric neurons. In conclusion, we identified 15d-PGJ2 as a novel glial-derived molecule with neuroprotective effects in the ENS. This study further supports the concept that omega-6 derivatives such as 15d-PGJ2 might be used in preventive and/or therapeutic strategies for the treatment of enteric neuropathies.

Wound healing of the gastrointestinal mucosa is essential for the maintenance of gut homeostasis and integrity. Enteric glial cells play a major role in regulating intestinal barrier function, but their role in mucosal barrier repair remains unknown. The impact of conditional ablation of enteric glia on dextran sodium sulfate (DSS)-induced mucosal damage and on healing of diclofenac-induced mucosal ulcerations was evaluated in vivo in GFAP-HSVtk transgenic mice. A mechanically induced model of intestinal wound healing was developed to study glial-induced epithelial restitution. Glial-epithelial signaling mechanisms were analyzed by using pharmacological inhibitors, neutralizing antibodies, and genetically engineered intestinal epithelial cells. Enteric glial cells were shown to be abundant in the gut mucosa, where they associate closely with intestinal epithelial cells as a distinct cell population from myofibroblasts. Conditional ablation of enteric glia worsened mucosal damage after DSS treatment and significantly delayed mucosal wound healing following diclofenac-induced small intestinal enteropathy in transgenic mice. Enteric glial cells enhanced epithelial restitution and cell spreading in vitro. These enhanced repair processes were reproduced by use of glial-conditioned media, and soluble proEGF was identified as a secreted glial mediator leading to consecutive activation of epidermal growth factor receptor and focal adhesion kinase signaling pathways in intestinal epithelial cells. Our study shows that enteric glia represent a functionally important cellular component of the intestinal epithelial barrier microenvironment and that the disruption of this cellular network attenuates the mucosal healing process.

Background: Enteric glial cells (EGCs) are important regulators of intestinal epithelial barrier (IEB) functions. EGC-derived S-nitrosoglutathione (GSNO) has been shown to regulate IEB permeability. Whether EGCs and GSNO protect the IEB during infectious insult by pathogens such as Shigella flexneri is not known., Methods: S flexneri effects were characterised using in vitro coculture models of Caco-2 cells and EGCs (or GSNO), ex vivo human colonic mucosa, and in vivo ligated rabbit intestinal loops. The effect of EGCs on S flexneri-induced changes in the invasion area and the inflammatory response were analysed by combining immunohistochemical, ELISA and PCR methods. Expression of small G-proteins was analysed by western blot. Expression of ZO-1 and localisation of bacteria were analysed by fluorescence microscopy., Results: EGCs significantly reduced barrier lesions and inflammatory response induced by S flexneri in Caco-2 monolayers. The EGC-mediated effects were reproduced by GSNO, but not by reduced glutathione, and pharmacological inhibition of pathways involved in GSNO synthesis reduced EGC protecting effects. Furthermore, expression of Cdc42 and phospho-PAK in Caco-2 monolayers was significantly reduced in the presence of EGCs or GSNO. In addition, changes in ZO-1 expression and distribution induced by S flexneri were prevented by EGCs and GSNO. Finally, GSNO reduced S flexneri-induced lesions of the IEB in human mucosal colonic explants and in a rabbit model of shigellosis., Conclusion: These results highlight a major protective function of EGCs and GSNO in the IEB against S flexneri attack. Consequently, this study lays the scientific basis for using GSNO to reduce barrier susceptibility to infectious or inflammatory challenge.

The enteric nervous system (ENS) and its major component, enteric glial cells (EGCs), have recently been identified as a major regulator of intestinal epithelial barrier functions. Indeed, EGCs inhibit intestinal epithelial cell (IEC) proliferation and increase barrier resistance and IEC adhesion via the release of EGC-derived soluble factors. Interestingly, EGC regulation of intestinal epithelial barrier functions is reminiscent of previously reported peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent functional effects. In this context, the present study aimed at identifying whether EGC could synthesize and release the main PPARgamma ligand, 15-deoxy-(12,14)-prostaglandin J2 (15dPGJ2), and regulate IEC functions such as proliferation and differentiation via a PPARgamma dependent pathway. First, we demonstrated that the lipocalin but not the haematopoetic form for prostaglandin D synthase (PGDS), the enzyme responsible of 15dPGJ2 synthesis, was expressed in EGCs of the human submucosal plexus and of the subepithelium, as well as in rat primary culture of ENS and EGC lines. Next, 15dPGJ2 was identified in EGC supernatants of various EGC lines. 15dPGJ2 reproduced EGC inhibitory effects upon IEC proliferation, and inhibition of lipocalin PGDS expression by shRNA abrogated these effects. Furthermore, EGCs induced nuclear translocation of PPARgamma in IEC, and both EGC and 15dPGJ2 effects upon IEC proliferation were prevented by the PPARgamma antagonist GW9662. Finally, EGC induced differentiation-related gene expression in IEC through a PPARgamma-dependent pathway. Our results identified 15dPGJ2 as a novel glial-derived mediator involved in the control of IEC proliferation/differentiation through activation of PPARgamma. They also suggest that alterations of glial PGDS expression may modify intestinal epithelial barrier functions and be involved in the development of pathologies such as cancer or inflammatory bowel diseases.

Background: Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies., Results: EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC., Conclusion: This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions.

The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

Inflammatory bowel disease (IBD) is a multifactorial disease in which environmental, immune and genetic factors are involved in the pathogenesis. Although biological therapies (antibodies anti-tumour necrosis factor-alpha or anti-integrin) have considerably improved the symptoms and quality of life of IBD patients, some drawbacks have emerged limiting their long-term use. In addition, prevention of relapses and treatment of resistant ulcers remains a clinical challenge. In this context, a better understanding of the pathophysiology of IBD and the development of novel therapeutic intervention would benefit from further basic and preclinical research into the role of the cellular microenvironment and the interaction between its cellular constituents. In this context, the role of the enteric nervous system (ENS) in the regulation of the intestinal epithelial barrier (IEB) and the gut immune response has fuelled an increased interest in the last few years. Recent advances, summarized in this review, have highlighted the ENS as playing a key role in the control of IEB functions and gut immune homeostasis, and that alterations of the ENS could be directly associated in the development of IBD and its associated symptoms.

Although recent studies have shown that enteric neurons control intestinal barrier function, the role of enteric glial cells (EGCs) in this control remains unknown. Therefore, our goal was to characterize the role of EGCs in the control of intestinal epithelial cell proliferation using an in vivo transgenic and an in vitro coculture model. Assessment of intestinal epithelial cell proliferation after ablation of EGCs in transgenic mice demonstrated a significant increase in crypt cell hyperplasia. Furthermore, mucosal glial network (assessed by immunohistochemical detection of S-100beta) is altered in colon adenocarcinoma compared with control tissue. In an in vitro coculture model of subconfluent Caco-2 cells seeded onto Transwell filters with EGCs, Caco-2 cell density and [3H]thymidine incorporation were significantly lower than in control (Caco-2 cultured alone). Flow cytometry analysis showed that EGCs had no effect on Caco-2 cell viability. EGCs induced a significant increase in Caco-2 cell surface area without any sign of cellular hypertrophy. These effects by EGCs were also seen in various transformed or nontransformed intestinal epithelial cell lines. Furthermore, TGF-beta1 mRNA was expressed, and TGF-beta1 was secreted by EGCs. Exogenously added TGF-beta1 reproduced partly the EGC-mediated effects on cell density and surface area. In addition, EGC effects on Caco-2 cell density were significantly reduced by a neutralizing TGF-beta antibody. In conclusion, EGCs have profound antiproliferative effects on intestinal epithelial cells. Functional alterations in EGCs may therefore modify intestinal barrier functions and be involved in pathologies such as cancer or inflammatory bowel diseases.

Aims: The influence of enteric glia on the regulation of intestinal functions is unknown. Our aim was to determine the phenotype of enteric neurones in a model of glia alterations and the putative changes in intestinal motility and permeability., Methods: Transgenic mice expressing haemagglutinin (HA) in glia were used. Glia disruption was induced by injection of activated HA specific CD8+ T cells. Control mice consisted of non-transgenic littermates injected with activated HA specific CD8+ T cells. Immunohistochemical staining for choline acetyltransferase (ChAT), substance P (SP), vasoactive intestinal peptide (VIP), and nitric oxide synthase (NOS) was performed on jejunal submucosal plexus (SMP) and myenteric plexus (MP). Neurally induced jejunal muscle activity was characterised in vitro. Gastrointestinal transit and paracellular permeability were measured using fluorescein isothiocyanate-dextran markers., Results: CD3 positive T cells infiltrates were observed in the MP of transgenic mice. In the SMP, the proportions of VIP and SP positive neurones decreased in transgenic mice compared with control mice. ChAT remained unchanged. In the MP, the proportions of ChAT and NOS positive neurones increased and decreased, respectively, in transgenic mice. In contrast, VIP and SP remained unchanged. Neurally mediated jejunal relaxation was lower in transgenic mice than in controls. This relaxation was reduced by NG-nitro-L-arginine methyl ester in control mice but not in transgenic mice. Gastrointestinal transit was delayed and intestinal permeability increased in transgenic mice compared with control mice., Conclusion: Glia disruption induces changes in the neurochemical coding of enteric neurones, which may partly be responsible for dysfunctions in intestinal motility and permeability.

Although recent studies have shown that enteric neurons control intestinal barrier function, the role of enteric glial cells (EGCs) in this control remains unknown. Therefore, our goal was to characterize the role of EGCs in the control of intestinal epithelial cell proliferation using an in vivo transgenic and an in vitro coculture model. Assessment of intestinal epithelial cell proliferation after ablation of EGCs in transgenic mice demonstrated a significant increase in crypt cell hyperplasia. Furthermore, mucosal glial network (assessed by immunohistochemical detection of S-100β) is altered in colon adenocarcinoma compared with control tissue. In an in vitro coculture model of subconfluent Caco-2 cells seeded onto Transwell filters with EGCs, Caco-2 cell density and [³H]thyrnidine incorporation were significantly lower than in control (Caco-2 cultured alone). Flow cytometry analysis showed that EGCs had no effect on Caco-2 cell viability. EGGs induced a significant increase in Caco-2 cell surface area without any sign of cellular hypertrophy. These effects by EGCs were also seen in various transformed or nontransformed intestinal epithelial cell lines. Furthermore, TGF-β1 mRNA was expressed, and TGF-β1 was secreted by EGCs. Exogenously added TGF-β1 reproduced partly the EGG-mediated effects on cell density and surface area. In addition, EGC effects on Caco-2 cell density were significantly reduced by a neutralizing TGF-β antibody. In conclusion, EGCs have profound anti- proliferative effects on intestinal epithelial cells. Functional alterations in EGCs may therefore modify intestinal barrier functions and be involved in pathologies such as cancer or inflammatory bowel diseases. [ABSTRACT FROM AUTHOR]