Philippe Aubert

Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome: involvement of soluble mediators

Background: Growing evidence suggests that patients with irritable bowel syndrome (IBS) have increased intestinal permeability. In addition, mucosal soluble mediators are involved in the pathophysiology of pain in IBS. We aimed to investigate (1) paracellular permeability in colonic biopsies of patients with IBS; and (2) the ability of soluble factors from colonic biopsies to reproduce these alterations in vitro. Methods: Paracellular permeability in colonic biopsies of healthy subjects and patients with IBS was measured by mounting the biopsies in Ussing chambers. Cleared supernatant (SUP) of the culture from colonic biopsies was collected and applied to Caco-2 cells for 48 h. Paracellular permeability and transepithelial resistance (TER) were evaluated. mRNA expression of the tight junction proteins, zonula occludens (ZO)-1 and occludin, was assessed in colonic biopsies. Abdominal pain was assessed using a validated questionnaire. Results: Permeability of colonic biopsies was significantly higher in patients with IBS compared to healthy subjects. These changes were associated with significantly lower expression of ZO-1 mRNA in biopsies of IBS as compared to healthy subjects. Compared to healthy subjects, SUP of IBS markedly reduced TER and significantly increased permeability in Caco-2 cells. SUP of IBS patients induced a significant decrease of ZO-1 mRNA in Caco-2 as compared to healthy subjects. SUP-induced increased paracellular permeability correlated with the severity of abdominal pain. Conclusions: Our study shows that colonic soluble mediators are able to reproduce functional (permeability) and molecular (ZO-1 mRNA expression) alterations observed in IBS patients. These findings might pave the way both to identify novel biomarkers as well as new therapeutic targets in IBS.

Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprungs disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione

Enteric glial cells (EGCs) are essential in the control of gastrointestinal functions. Although lesions of EGCs are associated with neuronal degeneration in animal models, their direct neuroprotective role remains unknown. Therefore, the aims of this study were to demonstrate the direct neuroprotective effects of EGCs and to identify putative glial mediators involved. First, viral targeted ablation of EGCs in primary cultures of enteric nervous system increased neuronal death both under basal conditions and in the presence of oxidative stress (dopamine, hydrogen peroxide). Second, direct or indirect coculture experiments of EGC lines with primary cultures of enteric nervous system or neuroblastoma cell lines (SH‐SY5Y) prevented neurotoxic effects induced by oxidative stress (increased membrane permeability, release of neuronal specific enolase, caspase‐3 immunoreactivity, changes in [Ca2+]i response). Finally, combining pharmacological inhibition and mRNA silencing methods, we demonstrated that neuroprotective effects of EGCs were mediated in part by reduced glutathione but not by oxidized glutathione or by S‐nitrosoglutathione. Our study identified the neuroprotective effects of EGCs via their release of reduced glutathione, extending their critical role in physiological contexts and in enteric neuropathies.—Abdo, H., Derkinderen, P., Gomes, P., Chevalier, J., Aubert, P., Masson, D., Galmiche, J.‐P., Vanden Berghe, P., Neunlist, M., Lardeux, B. Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione. FASEB J. 24, 1082‐1094 (2010). www.fasebj.org

Neurochemical plasticity in the enteric nervous system of a primate animal model of experimental Parkinsonism

Abstract  Emerging evidences suggest that the enteric nervous system (ENS) is affected by the degenerative process in Parkinson’s disease (PD). In addition lesions in the ENS could be associated with gastrointestinal (GI) dysfunctions, in particular constipation, observed in PD. However, the precise alterations of the ENS and especially the changes in the neurochemical phenotype remain largely unknown both in PD and experimental Parkinsonism. The aim of our study was thus to characterize the neurochemical coding of the ENS in the colon of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated monkeys, a well‐characterized model of PD. In the myenteric plexus, there was a significant increase in the number of neurons per ganglia (identified with Hu), especially nitric oxide synthase immunoreactives (IR) neurons in MPTP‐treated monkeys compared to controls. A concomitant 72% decrease in the number of tyrosine hydroxylase‐IR neurons was observed in MPTP‐treated monkeys compared to controls. In contrast no change in the cholinergic or vasoactive intestinal peptide‐IR population was observed. In addition, the density of enteric glial cells was not modified in MPTP‐treated monkeys. Our results demonstrate that MPTP induces major changes in the myenteric plexus and to a lesser extent in the submucosal plexus of monkeys. They further reinforce the observation that lesions of the ENS occur in the course of PD that might be related to the GI dysfunction observed in this pathology.

Enteric glia modulate epithelial cell proliferation and differentiation through 15-deoxy-Δ12,14-prostaglandin J2

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 γ(PPARγ)‐dependent functional effects. In this context, the present study aimed at identifying whether EGC could synthesize and release the main PPARγ ligand, 15‐deoxy‐Δ12,14‐prostaglandin J2 (15dPGJ2), and regulate IEC functions such as proliferation and differentiation via a PPARγ 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 PPARγ in IEC, and both EGC and 15dPGJ2 effects upon IEC proliferation were prevented by the PPARγ antagonist GW9662. Finally, EGC induced differentiation‐related gene expression in IEC through a PPARγ‐dependent pathway. Our results identified 15dPGJ2 as a novel glial‐derived mediator involved in the control of IEC proliferation/differentiation through activation of PPARγ. 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.

Intestinal Epithelial Cell Dysfunction is Mediated by an Endothelial-Specific Radiation-Induced Bystander Effect

Abstract Gaugler, M. H., Neunlist, M., Bonnaud, S., Aubert, P., Benderitter, M. and Paris, F. Intestinal Epithelial Cell Dysfunction is Mediated by an Endothelial-Specific Radiation-Induced Bystander Effect. Radiat. Res. 167, 185–193 (2007). The response of endothelial cells (EC) to high radiation doses leads to damage of normal tissue or tumor. The precise mechanisms of the endothelial-tissue linkage are still largely unknown. We investigated the possible involvement of a bystander effect, secondary to endothelial damage, in tissue response to radiation. Proliferating human intestinal epithelial T84 cells were grown in a non-contact co-culture with confluent primary human microvascular EC (HMVEC-L). The bystander response in unirradiated T84 cells co-cultured with irradiated EC was studied by evaluating cell growth, cell death and epithelial morphology. Twenty-four hours after exposure of EC to 15 Gy, unirradiated T84 cells showed a decreased cell number (29%) and percentage in mitosis (66%) as well as increased apoptosis (1.5-fold) and cell surface area (1.5-fold), highlighting the involvement of bystander effects on T84 cells after irradiation of EC. Furthermore, the responses of T84 cells were amplified when EC and T84 cells were irradiated together, indicating that the bystander response in T84 cells adds further to direct radiation damage. As opposed to direct irradiation, the T84 cell bystander response did not involve the cell cycle-related protein p21Waf1 (CDKN1A) and pro-apoptosis protein BAX. The bystander effect was specific to EC since the irradiation of human colon fibroblasts did not induce bystander responses in unirradiated T84 cells. These results strengthen previous in vivo evidence of the role of EC in tissue damage by radiation. In addition, this study provides a suitable and useful model to identify soluble factors involved in bystander effects secondary to endothelial damage. Modulating such factors may have important clinical implications.

Effects of oral administration of rotenone on gastrointestinal functions in mice.

Background  The systemic rotenone model of Parkinson’s disease (PD) accurately replicates many aspects of the pathology of human PD, especially neurodegeneration of the substantia nigra and lesions in the enteric nervous system (ENS). Nevertheless, the precise effects of oral rotenone on the ENS have not been addressed yet. This study was therefore designed to assess the effects of a chronic oral treatment by rotenone on enteric neurochemical phenotype, gastrointestinal (GI) motility, and intestinal epithelial barrier permeability.

Characterisation of Early Mucosal and Neuronal Lesions Following Shigella flexneri Infection in Human Colon

Background Shigella, an enteroinvasive bacteria induces a major inflammatory response responsible for acute rectocolitis in humans. However, early effect of Shigella flexneri (S. flexneri) infection upon the human mucosa and its microenvironement, in particular the enteric nervous system, remains currently unknown. Therefore, in this study, we sought to characterize ex vivo the early events of shigellosis in a model of human colonic explants. In particular, we aimed at identifying factors produced by S. flexneri and responsible for the lesions of the barrier. We also aimed at determining the putative lesions of the enteric nervous system induced by S. flexneri. Methodology/Principal Findings We first showed that, following 3 h of infection, the invasive but not the non-invasive strain of S. flexneri induced significant desquamation of the intestinal epithelial barrier and a reduction of epithelial height. These changes were significantly reduced following infection with SepA deficient S. flexneri strains. Secondly, S. flexneri induced rapid neuronal morphological alterations suggestive of cell death in enteric submucosal neurones. These alterations were associated with a significant increase in the proportion of vasoactive intestinal peptide (VIP) immunoreactive (IR) neurons but not in total VIP levels. The NMDA receptor antagonist MK-801 blocked neuronal morphological changes induced by S. flexneri, but not the increase in the proportion of VIP-IR. Conclusions/Significance This human explant model can be used to gain better insight into the early pathogenic events following S. flexneri infection and the mechanisms involved.

Neurochemical coding of myenteric neurones in the human gastric fundus

Abstract  The major functions of the stomach are under the control of the enteric nervous system (ENS), but the neuronal circuits involved in this control are largely unknown in humans. Enteric neurones can be characterized by their neuromediator or marker content, i.e. by neurochemical coding. The purpose of this study was to characterize the presence and co‐localization of neurotransmitters in myenteric neurones of the human gastric fundus. Choline acetyltransferase (ChAT), neurone‐specific enolase (NSE), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), substance P (SP) were detected by immunohistochemical methods in whole mounts of gastric fundus myenteric plexus (seven patients). Antibodies against ChAT and NOS labelled the majority of myenteric neurones identified by NSE (57.2 ± 5.6% and 40.8 ± 4.5%, respectively; mean ± SD). The proportions of VIP‐ and SP‐immunoreactive neurones were significantly smaller, constituting 19.6 ± 6.9% and 16.0 ± 3.7%, respectively. Co‐localization studies revealed five major populations representing over 75% of the myenteric neurones: ChAT/‐, 30.1 ± 6.1%; NOS/‐, 24.2 ± 4.4%; ChAT/SP/‐, 8.3 ± 3.1%; NOS/VIP/‐, 7.2 ± 6.0%; ChAT/VIP/‐, 4.9 ± 2.6. Some similarities are apparent in the neurochemical coding of myenteric neurones in the stomach and intestine of humans, and between the stomach of humans and animals, but striking differences exist. The precise functional role of the neurochemically identified classes of neurones remains to be determined.

Food allergy enhances allergic asthma in mice

BackgroundAtopic march refers to the typical transition from a food allergy in early childhood to allergic asthma in older children and adults. However the precise interplay of events involving gut, skin and pulmonary inflammation in this process is not completely understood.ObjectivesTo develop a mouse model of mixed food and respiratory allergy mimicking the atopic march and better understand the impact of food allergies on asthma.MethodsFood allergy to ovalbumin (OVA) was induced through intra-peritoneal sensitization and intra-gastric challenge, and/or a respiratory allergy to house dust mite (HDM) was obtained through percutaneous sensitization and intra-nasal challenges with dermatophagoides farinae (Der f) extract. Digestive, respiratory and systemic parameters were analyzed.ResultsOVA-mediated gut allergy was associated with an increase in jejunum permeability, and a worsening of Der f-induced asthma with stronger airway hyperresponsiveness and pulmonary cell infiltration, notably eosinophils. There was overproduction of the pro-eosinophil chemokine RANTES in broncho-alveolar lavages associated with an enhanced Th2 cytokine secretion and increased total and Der f-specific IgE when the two allergies were present. Both AHR and lung inflammation increased after a second pulmonary challenge.ConclusionGut sensitization to OVA amplifies Der f-induced asthma in mice.