Julien Chevalier

Short-Chain Fatty Acids Regulate the Enteric Neurons and Control Gastrointestinal Motility in Rats

BACKGROUND & AIMS Little is known about the environmental and nutritional regulation of the enteric nervous system (ENS), which controls gastrointestinal motility. Short-chain fatty acids (SCFAs) such as butyrate regulate colonic mucosa homeostasis and can modulate neuronal excitability. We investigated their effects on the ENS and colonic motility. METHODS Effects of butyrate on the ENS were studied in colons of rats given a resistant starch diet (RSD) or intracecal perfusion of SCFAs. Effects of butyrate were also studied in primary cultures of ENS. The neurochemical phenotype of the ENS was analyzed with antibodies against Hu, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) and by quantitative polymerase chain reaction. Signaling pathways involved were analyzed by pharmacologic and molecular biology methods. Colonic motility was assessed in vivo and ex vivo. RESULTS In vivo and in vitro, RSD and butyrate significantly increased the proportion of ChAT- but not nNOS-immunoreactive myenteric neurons. Acetate and propionate did not reproduce the effects of butyrate. Enteric neurons expressed monocarboxylate transporter 2 (MCT2). Small interfering RNAs silenced MCT2 and prevented the increase in the proportion of ChAT- immunoreactive neurons induced by butyrate. Butyrate and trichostatin A increased histone H3 acetylation in enteric neurons. Effects of butyrate were prevented by inhibitors of the Src signaling pathway. RSD increased colonic transit, and butyrate increased the cholinergic-mediated colonic circular muscle contractile response ex vivo. CONCLUSION Butyrate or histone deacetylase inhibitors might be used, along with nutritional approaches, to treat various gastrointestinal motility disorders associated with inhibition of colonic transit.

Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF

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.

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

Activity-dependent regulation of tyrosine hydroxylase expression in the enteric nervous system.

The regulation of neuromediator expression by neuronal activity in the enteric nervous system (ENS) is currently unknown. Using primary cultures of ENS derived from rat embryonic intestine, we have characterized the regulation of tyrosine hydroxylase (TH), a key enzyme involved in the synthesis of dopamine. Depolarization induced either by 40 mm KCl, veratridine or by electrical field stimulation produced a robust and significant increase in the proportion of TH immunoreactive (TH‐IR) neurons (total neuronal population was identified with PGP9.5 or Hu) compared to control. This increase in the proportion of TH‐IR neurons was significantly reduced by the sodium channel blocker tetrodotoxin (0.5 μm), demonstrating that neuronal activity was critically involved in the effects of these depolarizing stimuli. KCl also increased the proportion of VIP‐IR but not nNOS‐IR enteric neurons. The KCl‐induced increase in TH expression was partly reduced in the presence of the nicotinic receptor antagonist hexamethonium (100 μm), of noradrenaline (1 μm) and of the α2‐adrenoreceptor agonist clonidine (1 μm). Combining pharmacological and calcium imaging studies, we have further shown that L‐type calcium channels were involved in the increase of TH expression induced by KCl. Finally, using specific inhibitors, we have shown that both protein kinases A and C as well as the extracellular signal‐regulated kinases were required for the increase in the proportion of TH‐IR neurons induced by KCl. These results are the first demonstration that TH phenotype of enteric neurons can be regulated by neuronal activity. They could also set the basis for the study of the pathways and mechanisms involved in the neurochemical plasticity observed both during ENS development and in inflammatory enteric neuropathies.

ATP‐dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice

Abstract  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 Ca2+‐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 Ca2+‐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 ADPβs. 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 ADPβs. 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.

n-3 polyunsaturated fatty acids in the maternal diet modify the postnatal development of nervous regulation of intestinal permeability in piglets

Non‐technical summary  In this study, we demonstrated that supplementation of the maternal diet with a particular fatty acid, 18:3 n‐3, the precursor of the n‐3 fatty acid family, modified intestinal permeability, probably via diet‐induced neuroplastic changes of the enteric nervous system of newborn piglets. These findings suggest that feeding fatty acids of the n‐3 family during pregnancy and lactation impact newborn intestinal barrier function. However, the beneficial versus harmful consequences of this increased intestinal permeability remain to be elucidated.

Glugacon-like peptide-2: broad receptor expression, limited therapeutic effect on intestinal inflammation and novel role in liver regeneration

The glucagon-like peptide 2 (GLP-2) is an intestinotrophic hormone with growth promoting and anti-inflammatory actions. However, the full biological functions of GLP-2 and the localization of its receptor (GLP-2R) remain controversial. Among cell lines tested, the expression of GLP-2R transcript was detected in human colonic myofibroblasts (CCD-18Co) and in primary culture of rat enteric nervous system but not in intestinal epithelial cell lines, lymphocytes, monocytes, or endothelial cells. Surprisingly, GLP-2R was expressed in murine (GLUTag), but not human (NCI-H716) enteroendocrine cells. The screening of GLP-2R mRNA in mice organs revealed an increasing gradient of GLP-2R toward the distal gut. An unexpected expression was detected in the mesenteric fat, mesenteric lymph nodes, bladder, spleen, and liver, particularly in hepatocytes. In two mice models of trinitrobenzene sulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced colitis, the colonic expression of GLP-2R mRNA was decreased by 60% compared with control mice. Also, GLP-2R mRNA was significantly downregulated in intestinal tissues of inflammatory bowel disease patients. Therapeutically, GLP-2 showed a weak restorative effect on intestinal inflammation during TNBS-induced colitis as assessed by macroscopic score and inflammatory markers. Finally, GLP-2 treatment accelerated mouse liver regeneration following partial hepatectomy as assessed by histological and molecular analyses. In conclusion, the limited therapeutic effect of GLP-2 on colonic inflammation dampens its utility in the management of severe inflammatory intestinal disorders. However, the role of GLP-2 in liver regeneration is a novelty that might introduce GLP-2 into the management of liver diseases and emphasizes on the importance of elucidating other extraintestinal functions of GLP-2.

Modulation of lipopolysaccharide-induced neuronal response by activation of the enteric nervous system

BackgroundEvidence continues to mount concerning the importance of the enteric nervous system (ENS) in controlling numerous intestinal functions in addition to motility and epithelial functions. Nevertheless, little is known concerning the direct participation of the ENS in the inflammatory response of the gut during infectious or inflammatory insults. In the present study we analyzed the ENS response to bacterial lipopolysaccharide, in particular the production of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α).MethodsTNF-α expression (measured by qPCR, quantitative Polymerase Chain Reaction) and production (measured by ELISA) were measured in human longitudinal muscle-myenteric plexus (LMMP) and rat ENS primary cultures (rENSpc). They were either treated or not treated with lipopolysaccharide (LPS) in the presence or not of electrical field stimulation (EFS). Activation of extracellular signal-regulated kinase (ERK) and 5’-adenosine monophosphate-activated protein kinase (AMPK) pathways was analyzed by immunocytochemistry and Western blot analysis. Their implications were studied using specific inhibitors (U0126, mitogen-activated protein kinase kinase, MEK, inhibitor and C compound, AMPK inhibitor). We also analyzed toll-like receptor 2 (TLR2) expression and interleukin-6 (IL-6) production after LPS treatment simultaneously with EFS or TNF-α-neutralizing antibody.ResultsTreatment of human LMMP or rENSpc with LPS induced an increase in TNF-α production. Activation of the ENS by EFS significantly inhibited TNF-α production. This regulation occurred at the transcriptional level. Signaling analyses showed that LPS induced activation of ERK but not AMPK, which was constitutively activated in rENSpc neurons. Both U0126 and C compound almost completely prevented LPS-induced TNF-α production. In the presence of LPS, EFS inhibited the ERK and AMPK pathways. In addition, we demonstrated using TNF-α-neutralizing antibody that LPS-induced TNF-α production increased TLR2 expression and reduced IL-6 production.ConclusionsOur results show that LPS induced TNF-α production by enteric neurons through activation of the canonical ERK pathway and also in an AMPK-dependent manner. ENS activation through the inhibition of these pathways decreased TNF-α production, thereby modulating the inflammatory response induced by endotoxin.

α-Synuclein expression is induced by depolarization and cyclic AMP in enteric neurons

J. Neurochem. (2010) 115, 694–706.

Postnatal development of the myenteric glial network and its modulation by butyrate

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.