Gastroenterology 2014;147:1230–1237
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BRIEF REVIEW Robert F. Schwabe and John W. Wiley, Section Editors
Enteric Glial Cells: Recent Developments and Future Directions Michel Neunlist,1,2,3 Malvyne Rolli-Derkinderen,1,2,3 Rocco Latorre,4 Laurianne Van Landeghem,1,2,3 Emmanuel Coron,1,2,3 Pascal Derkinderen,1,2,3,5 and Roberto De Giorgio4 1 INSERM Unité 913, Nantes, France; 2Université Nantes, Nantes, France; 3CHU Nantes, Hôtel Dieu, Institut des Maladies de l’Appareil Digestif, Nantes, France; 4Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy; and 5 Department of Neurology, CHU Nantes, Nantes, France
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.
Keywords: Enteric Nervous System; Intestinal Epithelial Barrier; Enteric Neuron.
H
omeostasis (ὅmoio2 [similar] and ssάsi2 [status]), a concept introduced by Claude Bernard and further refined by Walter Canon, is a basic principle of life and refers to the ability of an organ to maintain its function (ie, physiology) in response to environmental challenges. The homeostasis of the gut is the result of a complex interplay between luminal (eg, nutrients, microbiota) and cellular (epithelial, immune, muscular, endothelial) factors as well as a number of regulatory neuroendocrine mechanisms. To control gut homeostasis, the gut harbors the second largest nervous system of the human body, which is also referred to as the enteric nervous system (ENS). The ENS is an integrative nervous system organized all along the gut. It is composed of 2 major plexuses: the myenteric plexus, located between the longitudinal and the circular muscle, and the submucosal plexus, located between the circular muscle and the mucosa. The ENS contributes, alone or in concert with extrinsic (parasympathetic/sympathetic)
neurons, to the regulation of virtually all gut functions, including motility, nutrient absorption, immune responses, and blood flow. Thus, it is not surprising that alterations in ENS functions, and thereby gut homeostasis, may result in intestinal and extraintestinal diseases. Major progress has been achieved over the past 40 years in understanding the role of enteric neurons in the control of gut functions in health and disease states. In contrast, it is only recently that enteric glial cells (EGCs), the major component of the ENS, have started to be investigated. Based on these studies, EGCs are increasingly recognized as the central actor of the ENS and gut homeostasis, revealing morphological and functional similarities to astrocytes of the central nervous system (CNS). This review will summarize the current knowledge on different aspects of EGC functions in health and disease.
Developmental Origin of EGCs As compared with enteric neurons, much less is known about the early development of EGCs. Some excellent reviews1,2 cover developmental aspects of EGCs beyond those discussed in this review. The ENS originates from neural crest cells that migrate in a rostrocaudal direction along the gut during embryonic stages. EGC precursors identified by brain fatty acid–binding protein3 can be detected within 24 hours after gut segments have been colonized by neural crest cells. Glial fibrillary acidic protein (GFAP), a classic marker of EGCs, appears later at the end of the mouse embryonic stage.1 This delayed differentiation of EGCs as compared with enteric neurons is reminiscent of what occurs to astrocytes during development of the CNS.4 Little is known about mechanisms controlling the development and differentiation of EGCs. The transcription factor Sox-10, which regulates multipotency during
Abbreviations used in this paper: CD, Crohn’s disease; CNS, central nervous system; EGC, enteric glial cell; ENS, enteric nervous system; GDNF, glial cell–derived neurotrophic factor; GFAP, glial fibrillary acidic protein; IEB, intestinal epithelial barrier; IEC, intestinal epithelial cell; IL, interleukin; LPS, lipopolysaccharide; STC, slow transit constipation; UC, ulcerative colitis. © 2014 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2014.09.040
development, is a central factor required for peripheral glial fate acquisition and probably also for EGC differentiation.5 Lgl4 protein or its receptor ADAM226 as well as bone morphogenetic proteins7 are involved in differentiation of EGCs as well. Notch signaling could also participate in gliogenesis, although results are contradictory.1,8,9 Besides these genetic controls, environmental factors such as nutrients could play a pivotal role in gliogenesis. Short-chain fatty acids have indeed been shown to enhance enteric neuronal maturation.10 Early life changes in nutrient composition have also been shown to enhance GFAP expression in a model of neonatal pig,11 and administration of a high-fat diet has been shown to increase the number of EGCs in juvenile mice.12
Figure 1. EGCs are distributed in different gut layers and respond to environmental challenges. (A) Immunohistochemical staining of EGCs within a myenteric ganglia (EGC displaying GFAP immunoreactivity [green]) and enteric neuronal cell bodies positive for HuC/D (red) in the rat small intestine. (B) Mucosal EGC expressing S100-b immunoreactivity are densely surrounding colonic crypts in the human colon. (C) S100-b immunoreactive EGCs ensheath capillaries in the human colon. Immunocytochemical staining of primary culture of human myenteric EGCs with GFAP shows that inflammatory mediators can modify their morphology (E) as compared with control (D). Scale bars ¼ 50 mm.
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Distribution and Organization of EGCs EGCs are closely organized with enteric neuronal structures (ganglia, interganglionic fiber strands, nerve fibers) and can be detected in all layers of the gut wall (ie, within muscle, vasculature, and epithelium) (Figure 1A–C). Increasing evidence suggests at least 4 morphologically distinct subclasses of EGCs based on their localization.13,14 Within ganglia, EGCs have star-shaped morphology (type I), whereas interganglionic EGCs are more elongated (type II). Mucosal and intramuscular EGCs have type III and IV morphology, respectively. EGCs express different markers such as Sox-10, GFAP, and S100b. Whereas Sox-10 and probably Ran-2 are general glial markers, only distinct subpopulations express GFAP and/or S100b.14 Whether these different
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subpopulations of EGC reflect developmentally programmed distinct cell types remains to be determined. Finally, the EGC-to-neuron ratio differs between the myenteric and submucosal plexus as well as between species.15
EGC Functions in the ENS and Gut Homeostasis The characterization of the functional role of EGCs has been hampered over the years by the lack of methods to directly and specifically modulate EGC functions, in contrast to neurons that are excitable cells. However, combined use of in vivo deletion models of EGCs (genetically or chemically induced) and isolation and culture of EGCs have unraveled major EGC functions. Further progress is expected in the near future with the development of in vivo imaging techniques, optogenetic16 and cell lineage tracing methods17,18 to better characterize their role, and, in particular, whether functionally distinct subclasses of EGCs exist.
Effect of EGCs on Enteric Neuronal Functions An in vivo deletion model of EGCs has revealed the presence of neurodegenerative processes, suggesting a neuroprotective role of EGCs for enteric neurons. Using in vitro primary culture models, Abdo et al have shown that EGCs can increase neuronal survival and also reduce oxidative stress–induced cell death.19 Interestingly, glial mediators, such as glutathione, 15dPGJ2, and glial cell–derived neurotrophic factor (GDNF), have been identified to exert neuroprotective effects.19,20 In addition, EGCs have been suggested to regulate expression of neuromediators because differential changes in neuronal phenotype were observed after EGC alterations.21 EGCs can also be a source of substrate for neuronal enzymes involved in neuromediator synthesis.22 The ability of EGCs to respond to different neuromediators by changes in intracellular [Ca2þ] further support a contributory role of EGCs in enteric neuronal function.13,23 However, whether Ca2þ changes affect EGCs and/or neuronal functional phenotype remains largely unknown. Finally, EGCs have the ability to regenerate the ENS in the adult and in particular to give rise not only to GFAP-positive EGCs but also to enteric neurons.17,18 EGCs participate in postnatal development of the ENS via expression of Toll-like receptor 2.24 Nevertheless, whether EGCs are, similarly to astrocytes, involved in the development and maturation of enteric neuronal circuitry remains to be identified.
Role of EGCs in the Control of Non-neuronal Functions Regulation of gastrointestinal motility. Various studies have shown that EGC dysfunction in vivo can affect gastrointestinal motility, leading to reduced gastric emptying, intestinal transit, and, more recently, colonic transit.21,25,26 These changes in motility were associated, in one study but not in another one, with reduced nitrergic phenotype in the ENS and reduced nitrergic neuromuscular inhibitor response ex vivo.21 Interestingly, Ca2þ transients in EGCs, shown to be neurally mediated, occur after mucosal
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stimulation and lead to migrating myoelectric complex.27 However, a recent report suggests that glial Ca2þ transients could in turn directly modulate gut motor functions because specific deletion of connexin 43 in EGCs slowed intestinal transit, probably by altering glia-glia and/or neuro-glia adenosine triphosphate–dependent communication.25
Regulation of intestinal epithelial barrier functions. A large body of data indicated that EGCs could be a central regulator of intestinal epithelial barrier (IEB) homeostasis. In vivo, severe ablation of EGCs induced a fulminant jejunoileitis, characterized by disruption of IEB integrity.28,29 Less severe ablation of EGCs increased paracellular permeability in the absence of gut inflammation.21 In a coculture model of EGCs with a monolayer of intestinal epithelial cells (IECs), EGCs increased IEB resistance and reduced paracellular permeability, in part by modulating expression of IEC tight junction proteins, such as occludin and zona occludens 1.30 The effects of EGCs on IEB resistance were shown to be mediated in part by S-nitrosoglutathione.30 Consistently, EGCs were shown to increase IEB resistance evoked by stressors such as bacteria, inflammatory mediators, or skin burn injuries.31–33 Recent evidence suggests that EGCs could also mediate neuronal effects in a conceptually similar fashion to the cross talk between neurons, interstitial cells of Cajal, and smooth muscle cells. Indeed, vagal stimulation was reported to reinforce skin burn–induced disruption of the IEB.34 These effects were associated in vivo with an early increase in GFAP expression in EGCs, a possible marker of glial activation.34 Intriguingly, in vitro, nicotinic stimulation (used as a mediator to model vagal-ENS communication) was shown to reinforce IEB resistance only in the presence of EGCs.32 However, evidence for such a role remains indirect and needs to be confirmed. Further to the modulation of IEB resistance, EGCs can also regulate processes involved in barrier repair; both in vivo and in vitro experiments showed that EGCs enhance IEB repair after mechanical or inflammatory injury.35 These effects were mediated by EGC-derived pro–epidermal growth factor and involved spreading of IECs via activation of focal adhesion kinase–dependent pathways.35 Besides enhancing cell repair, EGCs also inhibit IEC proliferation via the release of transforming growth factor b136 and enhance IEC differentiation via the production of 15dPGJ2.37 Finally, EGCs could also be involved in secretory and/or absorptive processes of the intestinal epithelium. Indeed, EGC-derived nitric oxide was suggested to participate in electrolyte secretion,38 while glia-specific disruption of connexin-43 increased fluid content in stools and decreased motility.25 Interestingly, a recent study has shown that EGC processes make contact with enteroendocrine cells called “neuropods.” These neuropods are axon-like basal process of the enteroendocrine cells that contain the peptidesecreting vesicles, and their formation is enhanced by glial-derived mediators.39 Altogether, these data identify EGCs as a central regulator of gut homeostatic processes necessary for gut functions (Figure 2A). Nonetheless, many aspects deserve further clarification, including the mechanisms and
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Figure 2. EGCs are central regulators of gut homeostatic processes and might be actors of gut diseases. (A) Under physiological conditions, EGCs regulate various neuronal functions such as neuroprotection, neuromediator expression, or neuronal renewal via liberation of different mediators. In addition, EGCs are central regulators of intestinal epithelial barrier homeostasis via the liberation of functions specific gliomediators. Altogether, EGCs exert protective and reparative functions on the gut. (B) Under environmental stressors such as inflammatory mediators or bacterial stimulation, reactive enteric gliosis (similar to astrogliosis in the brain) can occur, which could participate in the development of intestinal inflammation but also concomitantly participate in protection/repair of IEB/neuronal lesions induced by these processes. (C) EGC death (induced by specific virus or pathogens) or altered enteric gliosis could contribute to neuronal degeneration or barrier dysfunctions observed in some chronic intestinal or extraintestinal diseases.
mediators responsible for EGC functions as well as novel functions of EGCs during life, such as their putative role in the epithelial stem cell niche, gut endothelial barrier, and development of enteric neuronal networks.
Environmentally Induced Changes in EGC Phenotype: Example of Inflammation-Induced Gliosis Similarly to the astrocytes of the CNS, EGCs undergo profound changes in response to a variety of mediators/ factors originating from the gut environment. These phenotypic changes were mainly described during intestinal inflammation and are reminiscent of astrogliosis occurring in response to injuries in the CNS (Figure 1D and E). In the adult CNS, astrogliosis is a response of astrocytes to inflammation and physical trauma characterized by cellular proliferation, morphological changes and differential expression of proteins, and in particular an up-regulation of GFAP, which is a prototypical response of gliosis.40 EGCs are equipped with various receptors that enable them to detect and respond to environmental inflammatory stimuli, such as receptors to bacterial membrane compounds (Toll-like receptors41) or cytokines (interleukin [IL]1 receptor42). Besides modifying EGC intracellular [Ca2þ],43 these mediators can also regulate expression of EGC proteins such as GFAP or S100b. In particular, inflammatory cytokines (IL-1b, tumor necrosis factor a, interferon
gamma) or bacterial compounds (lipopolysaccharide [LPS]) can increase GFAP expression or S100b expression and secretion.41,44,45 Inflammation can also alter the expression of various receptors present on EGCs such as glutamate receptors (mGluR5)46 or endothelin receptors (ET-1B),47 thereby suggesting modified EGC functions during inflammation. In addition, inflammatory mediators can also modulate EGC proliferation. In particular, IL-1b was shown to dose-dependently reduce EGC proliferation48; however, another study did not report changes in EGC proliferation.45 IL-10 had concentration-dependent biphasic effects on EGC proliferation, with low concentrations increasing and high concentrations decreasing EGC proliferation.48 LPS in combination with interferon gamma increased EGC proliferation.44 Consistently, in vivo, intestinal inflammation has been shown to stimulate myenteric EGC proliferation.49 Increasing evidence suggests that EGCs could directly contribute to the intestinal inflammatory response by producing inflammatory mediators. Indeed, after stimulation with LPS or cytokines, EGCs can release mediators such as nitric oxide,41,44 IL-6,50 IL-1b, or prostaglandin E2.43 Interestingly, in vivo, activation of IL-1 receptor in EGCs was suggested to be responsible for the inflammatory response in a mouse model of postoperative ileus.42 Further support for an active role of EGCs in inflammation includes observations that knockout mice for Toll-like receptor 2/4 that are expressed on EGCs have reduced sensitivity to dextran sulfate sodium–induced colitis.24,51
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Conversely, some data indicate that EGCs can produce “beneficial” mediators such as GDNF and nerve growth factor45,52 in response to IL-1b, tumor necrosis factor a, or LPS. These mediators can increase neuronal as well as epithelial survival.20,53–55 Altogether, these studies suggest that a similar shift in paradigm could occur for EGCs as for astrocytes in the brain. Indeed, reactive astrocytes of the CNS have long been considered to be deleterious before being increasingly recognized as essential actors of tissue repair after injury.40
Considering Digestive Diseases as Enteric Gliopathies? Based on the central role of EGCs in gut homeostasis, increasing evidence points toward a role of EGC dysfunction (or gliopathy) in many gastrointestinal diseases and also probably in extraintestinal disorders. Thus, although attractive, the concept of enteric gliopathy is still new, and further studies are warranted before it will be considered a key pathological feature.
Changes in EGCs in Gastrointestinal Diseases EGC abnormalities have been identified in inflammatory56,57 and functional58 gastrointestinal disorders. Several lines of evidence indicate that changes in EGCs occur in inflammatory bowel diseases. First, in ulcerative colitis (UC), GFAP and GDNF expression were significantly higher in the mucosa of patients with UC (and infectious colitis) as compared with those of controls and of noninflamed UC segments,57 expanding previous data from Cornet et al.29 Furthermore, in UC, increased expression and secretion of S100b leading to enhanced NO production was observed in inflamed areas as compared with noninflamed areas.56 Taken together, the findings support the idea that EGCs may contribute to mucosal inflammation in UC. In contrast to UC, a reduced number of GFAP-positive EGCs has been shown in noninflamed gut specimens of patients with Crohn’s disease (CD) by Cornet et al, thus lending further support that EGC loss is associated with increased susceptibility of the IEB to aggression by pathogens.29 von Boyen et al consistently reported a less prominent increase of GFAP (and GDNF) in patients with CD compared with UC.57 It is tentative to speculate that an altered glial reactivity (as possibly observed in CD) or excessive gliosis (as possibly observed in UC) could contribute to different pathological conditions. Another setting in which EGCs show changes is that of infectious enteritis/colitis. Specifically, ex vivo human models of Shigella flexneri infection have shown that EGC loss is associated with neurodegeneration.59 Moreover, S flexneri evoked neuronal injury and loss via N-methyl-Daspartate receptors, suggesting that reduced glutamate reuptake as a result of EGC damage could be responsible for neuronal cell death. Conversely, other pathogens (eg, Entamoeba histolytica) can elicit neurodegeneration without altering EGCs.60 Concerning functional bowel disorders, EGC abnormalities have been detailed in 2 severe gut dysmotility
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disorders: slow transit constipation (STC)/colonic inertia and megacolon. STC refers to a subset of patients characterized by a marked delay of gut transit that usually does not or responds poorly to medical treatment. The histopathological analysis of colonic full-thickness specimens obtained from patients with intractable STC revealed significant qualitative and quantitative changes characterized by enteric neuron (ether in myenteric and submucosal plexus), interstitial cells of Cajal, and EGC abnormalities compared with colonic specimens of patients who underwent surgery for uncomplicated colorectal cancer.61 The coexistent damage to EGCs and enteric neurons led the investigators to hypothesize that a gliopathy first occurred in the evolution of the disease and that this event affected neuronal (and perhaps also ICC) survival and maintenance in severe STC. Although exciting, whether impairment of EGCs precedes neuronal dysfunction deserves further confirmatory evidence in STC and in other functional gastrointestinal disorders. EGC abnormalities (with or without ICC depletion) have been also reported in patients with megacolon (either idiopathic or secondary to Chagas disease)62 and diverticular disease.63 Further reinforcing the role of EGCs in gut diseases is recent evidence that EGCs can be a target of disease. Selgrad et al64 aimed to investigate neurotropic viruses, in particular John Cunningham virus, in tissues from patients with severe gut dysmotility (ie, chronic intestinal pseudoobstruction).65 This study showed that 8 of 10 patients with an idiopathic neurogenic chronic intestinal pseudoobstruction had John Cunningham virus–related T antigen DNA sequences, whereas 7 of 10 patients had expression of T antigen protein in the microdissected myenteric plexuses of ileal or colonic specimens. Only 3 of 31 control subjects had T antigen protein DNA (but no T antigen protein) expression. Notably, other neurotropic viruses were absent in chronic intestinal pseudo-obstruction and control specimens. The John Cunningham virus capsid protein VP1 immunolabeling was identified in GFAP-labeled cells, providing evidence that the virus specifically infected myenteric EGCs in patients with chronic intestinal pseudoobstruction. These findings are in line with other data presented herein and showing that other viruses (ie, the adenovirus) infect EGCs rather than neurons, thus providing evidence for the existence of a viral gliotropism.19 Whether neurotropic viruses may actually lead to enteric neuron abnormalities remains an open issue. However, the data of Selgrad et al64 and Abdo et al19 pave the way for additional studies aimed at establishing the pathogenetic role of neurotropic viruses in gastrointestinal motility disorders. Glioplastic changes have also recently been reported to occur in extraintestinal diseases in which gut dysfunctions probably play a central role. For instance, although reported in animal models, a Western diet was shown to increase the density of EGCs in gastric myenteric ganglia.12 In contrast, high-fat diet–induced obesity evoked a decline of GFAP and S100b expression in EGCs.66 Whether these changes depend on diet composition or difference in age remains to be determined. Another disease in which changes in EGCs were recently observed is Parkinson disease. Indeed, similarly to
the brain, GFAP expression is increased in colonic biopsy specimens from patients with Parkinson disease as compared with patients with atypical parkinsonism or healthy controls.67 The functional consequences of these changes on gut functions or their meaning as potential biomarkers of disease evolution remain to be determined.
Conclusions and Future Perspectives EGCs have been considered mere supportive and passive cells for decades. Conversely, current data clearly indicate that EGCs play a central role in gut homeostasis, that is, highly integrated processes such as motility, secretion, absorption, and intestinal barrier function. However, much remains to be discovered in terms of EGC phenotype and physiological properties. The increasing use of genetic tools (eg, lineage tracing, glial-specific gene targeting) and/or novel in vivo imaging methods (optogenetics) are setting the basis for a rapid increase in our understating of the role of EGCs in the control of gut functions and cell biology. From a clinical standpoint, the pathophysiological role of EGCs in gut dysfunction observed with both intestinal and even extraintestinal diseases deserves further elucidation. Progresses in this research area will allow us to establish whether EGC abnormalities can be biomarkers of disease evolution, severity, or response to treatment. Ultimately, EGCs could represent a novel therapeutic target for the treatment of gastrointestinal diseases.
References 1. Hao MM, Young HM. Development of enteric neuron diversity. J Cell Mol Med 2009;13:1193–1210. 2. Lake JI, Heuckeroth RO. Enteric nervous system development: migration, differentiation, and disease. Am J Physiol Gastrointest Liver Physiol 2013;305:G1–G24. 3. Britsch S, Goerich DE, Riethmacher D, et al. The transcription factor Sox10 is a key regulator of peripheral glial development. Genes Dev 2001;15:66–78. 4. Sloan SA, Barres BA. Mechanisms of astrocyte development and their contributions to neurodevelopmental disorders. Curr Opin Neurobiol 2014;27C:75–81. 5. Bondurand N, Sham MH. The role of SOX10 during enteric nervous system development. Dev Biol 2013; 382:330–343. 6. Nishino J, Saunders TL, Sagane K, et al. Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system. J Neurosci 2010;30:15228–15240. 7. Chalazonitis A, D’Autreaux F, Pham TD, et al. Bone morphogenetic proteins regulate enteric gliogenesis by modulating ErbB3 signaling. Dev Biol 2011;350:64–79. 8. Okamura Y, Saga Y. Notch signaling is required for the maintenance of enteric neural crest progenitors. Development 2008;135:3555–3565. 9. Taylor MK, Yeager K, Morrison SJ. Physiological Notch signaling promotes gliogenesis in the developing peripheral and central nervous systems. Development 2007;134:2435–2447.
Functions of Enteric Glia in Health and Disease 1235 10. Suply E, de Vries P, Soret R, et al. Butyrate enemas enhance both cholinergic and nitrergic phenotype of myenteric neurons and neuromuscular transmission in newborn rat colon. Am J Physiol Gastrointest Liver Physiol 2012;302:G1373–G1380. 11. van Haver ER, de Vooght L, Oste M, et al. Postnatal and diet-dependent increases in enteric glial cells and VIPcontaining neurones in preterm pigs. Neurogastroenterol Motil 2008;20:1070–1079. 12. Baudry C, Reichardt F, Marchix J, et al. Diet-induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line-derived neurotrophic factor. J Physiol 2012; 590:533–544. 13. Gulbransen BD, Sharkey KA. Novel functional roles for enteric glia in the gastrointestinal tract. Nat Rev Gastroenterol Hepatol 2012;9:625–632. 14. Boesmans W, Lasrado R, Vanden Berghe P, et al. Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system. Glia 2014 Aug 26 [Epub ahead of print]. 15. Hoff S, Zeller F, von Weyhern CW, et al. Quantitative assessment of glial cells in the human and guinea pig enteric nervous system with an anti-Sox8/9/10 antibody. J Comp Neurol 2008;509:356–371. 16. Boesmans W, Martens MA, Weltens N, et al. Imaging neuron-glia interactions in the enteric nervous system. Front Cell Neurosci 2013;7:183. 17. Joseph NM, He S, Quintana E, et al. Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut. J Clin Invest 2011;121:3398–3411. 18. Laranjeira C, Sandgren K, Kessaris N, et al. Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury. J Clin Invest 2011;121: 3412–3424. 19. Abdo H, Derkinderen P, Gomes P, et al. Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione. FASEB J 2010;24:1082–1094. 20. Anitha M, Gondha C, Sutliff R, et al. GDNF rescues hyperglycemia-induced diabetic enteric neuropathy through activation of the PI3K/Akt pathway. J Clin Invest 2006;116:344–356. 21. Aube AC, Cabarrocas J, Bauer J, et al. Changes in enteric neurone phenotype and intestinal functions in a transgenic mouse model of enteric glia disruption. Gut 2006;55:630–637. 22. Nagahama M, Semba R, Tsuzuki M, et al. L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum. Biol Signals Recept 2001;10: 336–340. 23. Boesmans W, Cirillo C, Van den Abbeel V, et al. Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells. Neurogastroenterol Motil 2013;25:e151–e160. 24. Brun P, Giron MC, Qesari M, et al. Toll-like receptor 2 regulates intestinal inflammation by controlling integrity of the enteric nervous system. Gastroenterology 2013; 145:1323–1333. 25. McClain JL, Grubisic V, Fried D, et al. Ca2þ responses in enteric glia are mediated by connexin-43 hemichannels
REVIEWS AND PERSPECTIVES
December 2014
1236 Neunlist et al
REVIEWS AND PERSPECTIVES
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
and modulate colonic transit in mice. Gastroenterology 2014;146:497–507.e1. Nasser Y, Fernandez E, Keenan CM, et al. Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function. Am J Physiol Gastrointest Liver Physiol 2006;291:G912–G927. Broadhead MJ, Bayguinov PO, Okamoto T, et al. Ca2þ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine. J Physiol 2012;590:335–350. Bush TG, Savidge TC, Freeman TC, et al. Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice. Cell 1998;93:189–201. Cornet A, Savidge TC, Cabarrocas J, et al. Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn’s disease? Proc Natl Acad Sci U S A 2001;98:13306–13311. Savidge TC, Newman P, Pothoulakis C, et al. Enteric glia regulate intestinal barrier function and inflammation via release of S-nitrosoglutathione. Gastroenterology 2007; 132:1344–1358. Cheadle GA, Costantini TW, Lopez N, et al. Enteric glia cells attenuate cytomix-induced intestinal epithelial barrier breakdown. PLoS One 2013;8:e69042. Costantini TW, Krzyzaniak M, Cheadle GA, et al. Targeting alpha-7 nicotinic acetylcholine receptor in the enteric nervous system: a cholinergic agonist prevents gut barrier failure after severe burn injury. Am J Pathol 2012;181:478–486. Flamant M, Aubert P, Rolli-Derkinderen M, et al. Enteric glia protect against Shigella flexneri invasion in intestinal epithelial cells: a role for S-nitrosoglutathione. Gut 2011; 60:473–484. Costantini TW, Bansal V, Krzyzaniak M, et al. Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells. Am J Physiol Gastrointest Liver Physiol 2010;299: G1308–G1318. Van Landeghem L, Chevalier J, Mahe MM, et al. Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF. Am J Physiol Gastrointest Liver Physiol 2011;300:G976–G987. Neunlist M, Aubert P, Bonnaud S, et al. Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGFbeta1-dependent pathway. Am J Physiol Gastrointest Liver Physiol 2007;292:G231–G241. Bach-Ngohou K, Mahe MM, Aubert P, et al. Enteric glia modulate epithelial cell proliferation and differentiation through 15-deoxy-12,14-prostaglandin J2. J Physiol 2010;588:2533–2544. MacEachern SJ, Patel BA, McKay DM, et al. Nitric oxide regulation of colonic epithelial ion transport: a novel role for enteric glia in the myenteric plexus. J Physiol 2011; 589:3333–3348. Bohorquez DV, Samsa LA, Roholt A, et al. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy. PLoS One 2014;9:e89881. Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci 2009; 32:638–647.
Gastroenterology Vol. 147, No. 6 41. Turco F, Sarnelli G, Cirillo C, et al. Enteroglial-derived S100B protein integrates bacteria-induced Toll-like receptor signalling in human enteric glial cells. Gut 2014; 63:105–115. 42. Stoffels B, Hupa KJ, Snoek SA, et al. Postoperative ileus involves interleukin-1 receptor signaling in enteric glia. Gastroenterology 2014;146:176–187.e1. 43. Murakami M, Ohta T, Ito S. Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin-1beta from enteric glial cells. J Neurosci Res 2009;87:2095–2104. 44. Cirillo C, Sarnelli G, Turco F, et al. Proinflammatory stimuli activates human-derived enteroglial cells and induces autocrine nitric oxide production. Neurogastroenterol Motil 2011;23:e372–e382. 45. von Boyen GB, Steinkamp M, Reinshagen M, et al. Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia. Gut 2004;53:222–228. 46. Nasser Y, Keenan CM, Ma AC, et al. Expression of a functional metabotropic glutamate receptor 5 on enteric glia is altered in states of inflammation. Glia 2007; 55:859–872. 47. von Boyen GB, Degenkolb N, Hartmann C, et al. The endothelin axis influences enteric glia cell functions. Med Sci Monit 2010;16:BR161–BR167. 48. Ruhl A, Franzke S, Stremmel W. IL-1beta and IL-10 have dual effects on enteric glial cell proliferation. Neurogastroenterol Motil 2001;13:89–94. 49. Bradley JS Jr, Parr EJ, Sharkey KA. Effects of inflammation on cell proliferation in the myenteric plexus of the guinea-pig ileum. Cell Tissue Res 1997;289:455–461. 50. Ruhl A, Franzke S, Collins SM, et al. Interleukin-6 expression and regulation in rat enteric glial cells. Am J Physiol Gastrointest Liver Physiol 2001;280:G1163–G1171. 51. Esposito G, Capoccia E, Turco F, et al. Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-alpha activation. Gut 2014;63:1300–1312. 52. von Boyen GB, Steinkamp M, Reinshagen M, et al. Nerve growth factor secretion in cultured enteric glia cells is modulated by proinflammatory cytokines. J Neuroendocrinol 2006;18:820–825. 53. Steinkamp M, Geerling I, Seufferlein T, et al. Glial-derived neurotrophic factor regulates apoptosis in colonic epithelial cells. Gastroenterology 2003;124:1748–1757. 54. Steinkamp M, Gundel H, Schulte N, et al. GDNF protects enteric glia from apoptosis: evidence for an autocrine loop. BMC Gastroenterol 2012;12:6. 55. von Boyen GB, Steinkamp M, Geerling I, et al. Proinflammatory cytokines induce neurotrophic factor expression in enteric glia: a key to the regulation of epithelial apoptosis in Crohn’s disease. Inflamm Bowel Dis 2006;12:346–354. 56. Cirillo C, Sarnelli G, Esposito G, et al. Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein. Neurogastroenterol Motil 2009;21. 1209–e112. 57. von Boyen GB, Schulte N, Pfluger C, et al. Distribution of enteric glia and GDNF during gut inflammation. BMC Gastroenterol 2011;11:3.
58. Bassotti G, Villanacci V, Antonelli E, et al. Enteric glial cells: new players in gastrointestinal motility? Lab Invest 2007;87:628–632. 59. Coron E, Flamant M, Aubert P, et al. Characterisation of early mucosal and neuronal lesions following Shigella flexneri infection in human colon. PLoS One 2009;4:e4713. 60. Lourenssen S, Houpt ER, Chadee K, et al. Entamoeba histolytica infection and secreted proteins proteolytically damage enteric neurons. Infect Immun 2010;78: 5332–5340. 61. Bassotti G, Villanacci V, Maurer CA, et al. The role of glial cells and apoptosis of enteric neurones in the neuropathology of intractable slow transit constipation. Gut 2006; 55:41–46. 62. Iantorno G, Bassotti G, Kogan Z, et al. The enteric nervous system in chagasic and idiopathic megacolon. Am J Surg Pathol 2007;31:460–468. 63. Bassotti G, Battaglia E, Bellone G, et al. Interstitial cells of Cajal, enteric nerves, and glial cells in colonic diverticular disease. J Clin Pathol 2005;58:973–977. 64. Selgrad M, De Giorgio R, Fini L, et al. JC virus infects the enteric glia of patients with chronic idiopathic intestinal pseudo-obstruction. Gut 2009;58:25–32. 65. De Giorgio R, Cogliandro RF, Barbara G, et al. Chronic intestinal pseudo-obstruction: clinical features, diagnosis,
Functions of Enteric Glia in Health and Disease 1237 and therapy. Gastroenterol Clin North Am 2011;40: 787–807. 66. Stenkamp-Strahm C, Patterson S, Boren J, et al. High-fat diet and age-dependent effects on enteric glial cell populations of mouse small intestine. Auton Neurosci 2013;177:199–210. 67. Clairembault T, Kamphuis W, Leclair-Visonneau L, et al. Enteric GFAP expression and phosphorylation in Parkinson’s disease. J Neurochem 2014;130:805–815.
Received July 2, 2014. Accepted September 12, 2014. Reprint requests Address requests for reprints to: Michel Neunlist, PhD, UMR INSERM Unité 913, 1, rue Gaston Veil, 44000 Nantes, France. e-mail: [email protected]. Acknowledgments The authors thank past and present lab members for their contribution to the research presented in this review. Conflicts of interest The authors disclose no conflicts. Funding Work in our laboratory (INSERM Unité 913) is supported by grants from INSERM, Région Pays de la Loire, Agence National pour la Recherche, Fondation SantéDige, Michael J. Fox Foundation for Parkinson’s Research, La Ligue contre le Cancer, Fondation de France, and France Parkinson. M.R.-D. is supported by the Centre National de la Recherche Scientifique.
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BRIEF REVIEW Robert F. Schwabe and John W. Wiley, Section Editors
Enteric Glial Cells: Recent Developments and Future Directions Michel Neunlist,1,2,3 Malvyne Rolli-Derkinderen,1,2,3 Rocco Latorre,4 Laurianne Van Landeghem,1,2,3 Emmanuel Coron,1,2,3 Pascal Derkinderen,1,2,3,5 and Roberto De Giorgio4 1 INSERM Unité 913, Nantes, France; 2Université Nantes, Nantes, France; 3CHU Nantes, Hôtel Dieu, Institut des Maladies de l’Appareil Digestif, Nantes, France; 4Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy; and 5 Department of Neurology, CHU Nantes, Nantes, France
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.
Keywords: Enteric Nervous System; Intestinal Epithelial Barrier; Enteric Neuron.
H
omeostasis (ὅmoio2 [similar] and ssάsi2 [status]), a concept introduced by Claude Bernard and further refined by Walter Canon, is a basic principle of life and refers to the ability of an organ to maintain its function (ie, physiology) in response to environmental challenges. The homeostasis of the gut is the result of a complex interplay between luminal (eg, nutrients, microbiota) and cellular (epithelial, immune, muscular, endothelial) factors as well as a number of regulatory neuroendocrine mechanisms. To control gut homeostasis, the gut harbors the second largest nervous system of the human body, which is also referred to as the enteric nervous system (ENS). The ENS is an integrative nervous system organized all along the gut. It is composed of 2 major plexuses: the myenteric plexus, located between the longitudinal and the circular muscle, and the submucosal plexus, located between the circular muscle and the mucosa. The ENS contributes, alone or in concert with extrinsic (parasympathetic/sympathetic)
neurons, to the regulation of virtually all gut functions, including motility, nutrient absorption, immune responses, and blood flow. Thus, it is not surprising that alterations in ENS functions, and thereby gut homeostasis, may result in intestinal and extraintestinal diseases. Major progress has been achieved over the past 40 years in understanding the role of enteric neurons in the control of gut functions in health and disease states. In contrast, it is only recently that enteric glial cells (EGCs), the major component of the ENS, have started to be investigated. Based on these studies, EGCs are increasingly recognized as the central actor of the ENS and gut homeostasis, revealing morphological and functional similarities to astrocytes of the central nervous system (CNS). This review will summarize the current knowledge on different aspects of EGC functions in health and disease.
Developmental Origin of EGCs As compared with enteric neurons, much less is known about the early development of EGCs. Some excellent reviews1,2 cover developmental aspects of EGCs beyond those discussed in this review. The ENS originates from neural crest cells that migrate in a rostrocaudal direction along the gut during embryonic stages. EGC precursors identified by brain fatty acid–binding protein3 can be detected within 24 hours after gut segments have been colonized by neural crest cells. Glial fibrillary acidic protein (GFAP), a classic marker of EGCs, appears later at the end of the mouse embryonic stage.1 This delayed differentiation of EGCs as compared with enteric neurons is reminiscent of what occurs to astrocytes during development of the CNS.4 Little is known about mechanisms controlling the development and differentiation of EGCs. The transcription factor Sox-10, which regulates multipotency during
Abbreviations used in this paper: CD, Crohn’s disease; CNS, central nervous system; EGC, enteric glial cell; ENS, enteric nervous system; GDNF, glial cell–derived neurotrophic factor; GFAP, glial fibrillary acidic protein; IEB, intestinal epithelial barrier; IEC, intestinal epithelial cell; IL, interleukin; LPS, lipopolysaccharide; STC, slow transit constipation; UC, ulcerative colitis. © 2014 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2014.09.040
development, is a central factor required for peripheral glial fate acquisition and probably also for EGC differentiation.5 Lgl4 protein or its receptor ADAM226 as well as bone morphogenetic proteins7 are involved in differentiation of EGCs as well. Notch signaling could also participate in gliogenesis, although results are contradictory.1,8,9 Besides these genetic controls, environmental factors such as nutrients could play a pivotal role in gliogenesis. Short-chain fatty acids have indeed been shown to enhance enteric neuronal maturation.10 Early life changes in nutrient composition have also been shown to enhance GFAP expression in a model of neonatal pig,11 and administration of a high-fat diet has been shown to increase the number of EGCs in juvenile mice.12
Figure 1. EGCs are distributed in different gut layers and respond to environmental challenges. (A) Immunohistochemical staining of EGCs within a myenteric ganglia (EGC displaying GFAP immunoreactivity [green]) and enteric neuronal cell bodies positive for HuC/D (red) in the rat small intestine. (B) Mucosal EGC expressing S100-b immunoreactivity are densely surrounding colonic crypts in the human colon. (C) S100-b immunoreactive EGCs ensheath capillaries in the human colon. Immunocytochemical staining of primary culture of human myenteric EGCs with GFAP shows that inflammatory mediators can modify their morphology (E) as compared with control (D). Scale bars ¼ 50 mm.
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Distribution and Organization of EGCs EGCs are closely organized with enteric neuronal structures (ganglia, interganglionic fiber strands, nerve fibers) and can be detected in all layers of the gut wall (ie, within muscle, vasculature, and epithelium) (Figure 1A–C). Increasing evidence suggests at least 4 morphologically distinct subclasses of EGCs based on their localization.13,14 Within ganglia, EGCs have star-shaped morphology (type I), whereas interganglionic EGCs are more elongated (type II). Mucosal and intramuscular EGCs have type III and IV morphology, respectively. EGCs express different markers such as Sox-10, GFAP, and S100b. Whereas Sox-10 and probably Ran-2 are general glial markers, only distinct subpopulations express GFAP and/or S100b.14 Whether these different
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subpopulations of EGC reflect developmentally programmed distinct cell types remains to be determined. Finally, the EGC-to-neuron ratio differs between the myenteric and submucosal plexus as well as between species.15
EGC Functions in the ENS and Gut Homeostasis The characterization of the functional role of EGCs has been hampered over the years by the lack of methods to directly and specifically modulate EGC functions, in contrast to neurons that are excitable cells. However, combined use of in vivo deletion models of EGCs (genetically or chemically induced) and isolation and culture of EGCs have unraveled major EGC functions. Further progress is expected in the near future with the development of in vivo imaging techniques, optogenetic16 and cell lineage tracing methods17,18 to better characterize their role, and, in particular, whether functionally distinct subclasses of EGCs exist.
Effect of EGCs on Enteric Neuronal Functions An in vivo deletion model of EGCs has revealed the presence of neurodegenerative processes, suggesting a neuroprotective role of EGCs for enteric neurons. Using in vitro primary culture models, Abdo et al have shown that EGCs can increase neuronal survival and also reduce oxidative stress–induced cell death.19 Interestingly, glial mediators, such as glutathione, 15dPGJ2, and glial cell–derived neurotrophic factor (GDNF), have been identified to exert neuroprotective effects.19,20 In addition, EGCs have been suggested to regulate expression of neuromediators because differential changes in neuronal phenotype were observed after EGC alterations.21 EGCs can also be a source of substrate for neuronal enzymes involved in neuromediator synthesis.22 The ability of EGCs to respond to different neuromediators by changes in intracellular [Ca2þ] further support a contributory role of EGCs in enteric neuronal function.13,23 However, whether Ca2þ changes affect EGCs and/or neuronal functional phenotype remains largely unknown. Finally, EGCs have the ability to regenerate the ENS in the adult and in particular to give rise not only to GFAP-positive EGCs but also to enteric neurons.17,18 EGCs participate in postnatal development of the ENS via expression of Toll-like receptor 2.24 Nevertheless, whether EGCs are, similarly to astrocytes, involved in the development and maturation of enteric neuronal circuitry remains to be identified.
Role of EGCs in the Control of Non-neuronal Functions Regulation of gastrointestinal motility. Various studies have shown that EGC dysfunction in vivo can affect gastrointestinal motility, leading to reduced gastric emptying, intestinal transit, and, more recently, colonic transit.21,25,26 These changes in motility were associated, in one study but not in another one, with reduced nitrergic phenotype in the ENS and reduced nitrergic neuromuscular inhibitor response ex vivo.21 Interestingly, Ca2þ transients in EGCs, shown to be neurally mediated, occur after mucosal
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stimulation and lead to migrating myoelectric complex.27 However, a recent report suggests that glial Ca2þ transients could in turn directly modulate gut motor functions because specific deletion of connexin 43 in EGCs slowed intestinal transit, probably by altering glia-glia and/or neuro-glia adenosine triphosphate–dependent communication.25
Regulation of intestinal epithelial barrier functions. A large body of data indicated that EGCs could be a central regulator of intestinal epithelial barrier (IEB) homeostasis. In vivo, severe ablation of EGCs induced a fulminant jejunoileitis, characterized by disruption of IEB integrity.28,29 Less severe ablation of EGCs increased paracellular permeability in the absence of gut inflammation.21 In a coculture model of EGCs with a monolayer of intestinal epithelial cells (IECs), EGCs increased IEB resistance and reduced paracellular permeability, in part by modulating expression of IEC tight junction proteins, such as occludin and zona occludens 1.30 The effects of EGCs on IEB resistance were shown to be mediated in part by S-nitrosoglutathione.30 Consistently, EGCs were shown to increase IEB resistance evoked by stressors such as bacteria, inflammatory mediators, or skin burn injuries.31–33 Recent evidence suggests that EGCs could also mediate neuronal effects in a conceptually similar fashion to the cross talk between neurons, interstitial cells of Cajal, and smooth muscle cells. Indeed, vagal stimulation was reported to reinforce skin burn–induced disruption of the IEB.34 These effects were associated in vivo with an early increase in GFAP expression in EGCs, a possible marker of glial activation.34 Intriguingly, in vitro, nicotinic stimulation (used as a mediator to model vagal-ENS communication) was shown to reinforce IEB resistance only in the presence of EGCs.32 However, evidence for such a role remains indirect and needs to be confirmed. Further to the modulation of IEB resistance, EGCs can also regulate processes involved in barrier repair; both in vivo and in vitro experiments showed that EGCs enhance IEB repair after mechanical or inflammatory injury.35 These effects were mediated by EGC-derived pro–epidermal growth factor and involved spreading of IECs via activation of focal adhesion kinase–dependent pathways.35 Besides enhancing cell repair, EGCs also inhibit IEC proliferation via the release of transforming growth factor b136 and enhance IEC differentiation via the production of 15dPGJ2.37 Finally, EGCs could also be involved in secretory and/or absorptive processes of the intestinal epithelium. Indeed, EGC-derived nitric oxide was suggested to participate in electrolyte secretion,38 while glia-specific disruption of connexin-43 increased fluid content in stools and decreased motility.25 Interestingly, a recent study has shown that EGC processes make contact with enteroendocrine cells called “neuropods.” These neuropods are axon-like basal process of the enteroendocrine cells that contain the peptidesecreting vesicles, and their formation is enhanced by glial-derived mediators.39 Altogether, these data identify EGCs as a central regulator of gut homeostatic processes necessary for gut functions (Figure 2A). Nonetheless, many aspects deserve further clarification, including the mechanisms and
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Figure 2. EGCs are central regulators of gut homeostatic processes and might be actors of gut diseases. (A) Under physiological conditions, EGCs regulate various neuronal functions such as neuroprotection, neuromediator expression, or neuronal renewal via liberation of different mediators. In addition, EGCs are central regulators of intestinal epithelial barrier homeostasis via the liberation of functions specific gliomediators. Altogether, EGCs exert protective and reparative functions on the gut. (B) Under environmental stressors such as inflammatory mediators or bacterial stimulation, reactive enteric gliosis (similar to astrogliosis in the brain) can occur, which could participate in the development of intestinal inflammation but also concomitantly participate in protection/repair of IEB/neuronal lesions induced by these processes. (C) EGC death (induced by specific virus or pathogens) or altered enteric gliosis could contribute to neuronal degeneration or barrier dysfunctions observed in some chronic intestinal or extraintestinal diseases.
mediators responsible for EGC functions as well as novel functions of EGCs during life, such as their putative role in the epithelial stem cell niche, gut endothelial barrier, and development of enteric neuronal networks.
Environmentally Induced Changes in EGC Phenotype: Example of Inflammation-Induced Gliosis Similarly to the astrocytes of the CNS, EGCs undergo profound changes in response to a variety of mediators/ factors originating from the gut environment. These phenotypic changes were mainly described during intestinal inflammation and are reminiscent of astrogliosis occurring in response to injuries in the CNS (Figure 1D and E). In the adult CNS, astrogliosis is a response of astrocytes to inflammation and physical trauma characterized by cellular proliferation, morphological changes and differential expression of proteins, and in particular an up-regulation of GFAP, which is a prototypical response of gliosis.40 EGCs are equipped with various receptors that enable them to detect and respond to environmental inflammatory stimuli, such as receptors to bacterial membrane compounds (Toll-like receptors41) or cytokines (interleukin [IL]1 receptor42). Besides modifying EGC intracellular [Ca2þ],43 these mediators can also regulate expression of EGC proteins such as GFAP or S100b. In particular, inflammatory cytokines (IL-1b, tumor necrosis factor a, interferon
gamma) or bacterial compounds (lipopolysaccharide [LPS]) can increase GFAP expression or S100b expression and secretion.41,44,45 Inflammation can also alter the expression of various receptors present on EGCs such as glutamate receptors (mGluR5)46 or endothelin receptors (ET-1B),47 thereby suggesting modified EGC functions during inflammation. In addition, inflammatory mediators can also modulate EGC proliferation. In particular, IL-1b was shown to dose-dependently reduce EGC proliferation48; however, another study did not report changes in EGC proliferation.45 IL-10 had concentration-dependent biphasic effects on EGC proliferation, with low concentrations increasing and high concentrations decreasing EGC proliferation.48 LPS in combination with interferon gamma increased EGC proliferation.44 Consistently, in vivo, intestinal inflammation has been shown to stimulate myenteric EGC proliferation.49 Increasing evidence suggests that EGCs could directly contribute to the intestinal inflammatory response by producing inflammatory mediators. Indeed, after stimulation with LPS or cytokines, EGCs can release mediators such as nitric oxide,41,44 IL-6,50 IL-1b, or prostaglandin E2.43 Interestingly, in vivo, activation of IL-1 receptor in EGCs was suggested to be responsible for the inflammatory response in a mouse model of postoperative ileus.42 Further support for an active role of EGCs in inflammation includes observations that knockout mice for Toll-like receptor 2/4 that are expressed on EGCs have reduced sensitivity to dextran sulfate sodium–induced colitis.24,51
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Conversely, some data indicate that EGCs can produce “beneficial” mediators such as GDNF and nerve growth factor45,52 in response to IL-1b, tumor necrosis factor a, or LPS. These mediators can increase neuronal as well as epithelial survival.20,53–55 Altogether, these studies suggest that a similar shift in paradigm could occur for EGCs as for astrocytes in the brain. Indeed, reactive astrocytes of the CNS have long been considered to be deleterious before being increasingly recognized as essential actors of tissue repair after injury.40
Considering Digestive Diseases as Enteric Gliopathies? Based on the central role of EGCs in gut homeostasis, increasing evidence points toward a role of EGC dysfunction (or gliopathy) in many gastrointestinal diseases and also probably in extraintestinal disorders. Thus, although attractive, the concept of enteric gliopathy is still new, and further studies are warranted before it will be considered a key pathological feature.
Changes in EGCs in Gastrointestinal Diseases EGC abnormalities have been identified in inflammatory56,57 and functional58 gastrointestinal disorders. Several lines of evidence indicate that changes in EGCs occur in inflammatory bowel diseases. First, in ulcerative colitis (UC), GFAP and GDNF expression were significantly higher in the mucosa of patients with UC (and infectious colitis) as compared with those of controls and of noninflamed UC segments,57 expanding previous data from Cornet et al.29 Furthermore, in UC, increased expression and secretion of S100b leading to enhanced NO production was observed in inflamed areas as compared with noninflamed areas.56 Taken together, the findings support the idea that EGCs may contribute to mucosal inflammation in UC. In contrast to UC, a reduced number of GFAP-positive EGCs has been shown in noninflamed gut specimens of patients with Crohn’s disease (CD) by Cornet et al, thus lending further support that EGC loss is associated with increased susceptibility of the IEB to aggression by pathogens.29 von Boyen et al consistently reported a less prominent increase of GFAP (and GDNF) in patients with CD compared with UC.57 It is tentative to speculate that an altered glial reactivity (as possibly observed in CD) or excessive gliosis (as possibly observed in UC) could contribute to different pathological conditions. Another setting in which EGCs show changes is that of infectious enteritis/colitis. Specifically, ex vivo human models of Shigella flexneri infection have shown that EGC loss is associated with neurodegeneration.59 Moreover, S flexneri evoked neuronal injury and loss via N-methyl-Daspartate receptors, suggesting that reduced glutamate reuptake as a result of EGC damage could be responsible for neuronal cell death. Conversely, other pathogens (eg, Entamoeba histolytica) can elicit neurodegeneration without altering EGCs.60 Concerning functional bowel disorders, EGC abnormalities have been detailed in 2 severe gut dysmotility
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disorders: slow transit constipation (STC)/colonic inertia and megacolon. STC refers to a subset of patients characterized by a marked delay of gut transit that usually does not or responds poorly to medical treatment. The histopathological analysis of colonic full-thickness specimens obtained from patients with intractable STC revealed significant qualitative and quantitative changes characterized by enteric neuron (ether in myenteric and submucosal plexus), interstitial cells of Cajal, and EGC abnormalities compared with colonic specimens of patients who underwent surgery for uncomplicated colorectal cancer.61 The coexistent damage to EGCs and enteric neurons led the investigators to hypothesize that a gliopathy first occurred in the evolution of the disease and that this event affected neuronal (and perhaps also ICC) survival and maintenance in severe STC. Although exciting, whether impairment of EGCs precedes neuronal dysfunction deserves further confirmatory evidence in STC and in other functional gastrointestinal disorders. EGC abnormalities (with or without ICC depletion) have been also reported in patients with megacolon (either idiopathic or secondary to Chagas disease)62 and diverticular disease.63 Further reinforcing the role of EGCs in gut diseases is recent evidence that EGCs can be a target of disease. Selgrad et al64 aimed to investigate neurotropic viruses, in particular John Cunningham virus, in tissues from patients with severe gut dysmotility (ie, chronic intestinal pseudoobstruction).65 This study showed that 8 of 10 patients with an idiopathic neurogenic chronic intestinal pseudoobstruction had John Cunningham virus–related T antigen DNA sequences, whereas 7 of 10 patients had expression of T antigen protein in the microdissected myenteric plexuses of ileal or colonic specimens. Only 3 of 31 control subjects had T antigen protein DNA (but no T antigen protein) expression. Notably, other neurotropic viruses were absent in chronic intestinal pseudo-obstruction and control specimens. The John Cunningham virus capsid protein VP1 immunolabeling was identified in GFAP-labeled cells, providing evidence that the virus specifically infected myenteric EGCs in patients with chronic intestinal pseudoobstruction. These findings are in line with other data presented herein and showing that other viruses (ie, the adenovirus) infect EGCs rather than neurons, thus providing evidence for the existence of a viral gliotropism.19 Whether neurotropic viruses may actually lead to enteric neuron abnormalities remains an open issue. However, the data of Selgrad et al64 and Abdo et al19 pave the way for additional studies aimed at establishing the pathogenetic role of neurotropic viruses in gastrointestinal motility disorders. Glioplastic changes have also recently been reported to occur in extraintestinal diseases in which gut dysfunctions probably play a central role. For instance, although reported in animal models, a Western diet was shown to increase the density of EGCs in gastric myenteric ganglia.12 In contrast, high-fat diet–induced obesity evoked a decline of GFAP and S100b expression in EGCs.66 Whether these changes depend on diet composition or difference in age remains to be determined. Another disease in which changes in EGCs were recently observed is Parkinson disease. Indeed, similarly to
the brain, GFAP expression is increased in colonic biopsy specimens from patients with Parkinson disease as compared with patients with atypical parkinsonism or healthy controls.67 The functional consequences of these changes on gut functions or their meaning as potential biomarkers of disease evolution remain to be determined.
Conclusions and Future Perspectives EGCs have been considered mere supportive and passive cells for decades. Conversely, current data clearly indicate that EGCs play a central role in gut homeostasis, that is, highly integrated processes such as motility, secretion, absorption, and intestinal barrier function. However, much remains to be discovered in terms of EGC phenotype and physiological properties. The increasing use of genetic tools (eg, lineage tracing, glial-specific gene targeting) and/or novel in vivo imaging methods (optogenetics) are setting the basis for a rapid increase in our understating of the role of EGCs in the control of gut functions and cell biology. From a clinical standpoint, the pathophysiological role of EGCs in gut dysfunction observed with both intestinal and even extraintestinal diseases deserves further elucidation. Progresses in this research area will allow us to establish whether EGC abnormalities can be biomarkers of disease evolution, severity, or response to treatment. Ultimately, EGCs could represent a novel therapeutic target for the treatment of gastrointestinal diseases.
References 1. Hao MM, Young HM. Development of enteric neuron diversity. J Cell Mol Med 2009;13:1193–1210. 2. Lake JI, Heuckeroth RO. Enteric nervous system development: migration, differentiation, and disease. Am J Physiol Gastrointest Liver Physiol 2013;305:G1–G24. 3. Britsch S, Goerich DE, Riethmacher D, et al. The transcription factor Sox10 is a key regulator of peripheral glial development. Genes Dev 2001;15:66–78. 4. Sloan SA, Barres BA. Mechanisms of astrocyte development and their contributions to neurodevelopmental disorders. Curr Opin Neurobiol 2014;27C:75–81. 5. Bondurand N, Sham MH. The role of SOX10 during enteric nervous system development. Dev Biol 2013; 382:330–343. 6. Nishino J, Saunders TL, Sagane K, et al. Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system. J Neurosci 2010;30:15228–15240. 7. Chalazonitis A, D’Autreaux F, Pham TD, et al. Bone morphogenetic proteins regulate enteric gliogenesis by modulating ErbB3 signaling. Dev Biol 2011;350:64–79. 8. Okamura Y, Saga Y. Notch signaling is required for the maintenance of enteric neural crest progenitors. Development 2008;135:3555–3565. 9. Taylor MK, Yeager K, Morrison SJ. Physiological Notch signaling promotes gliogenesis in the developing peripheral and central nervous systems. Development 2007;134:2435–2447.
Functions of Enteric Glia in Health and Disease 1235 10. Suply E, de Vries P, Soret R, et al. Butyrate enemas enhance both cholinergic and nitrergic phenotype of myenteric neurons and neuromuscular transmission in newborn rat colon. Am J Physiol Gastrointest Liver Physiol 2012;302:G1373–G1380. 11. van Haver ER, de Vooght L, Oste M, et al. Postnatal and diet-dependent increases in enteric glial cells and VIPcontaining neurones in preterm pigs. Neurogastroenterol Motil 2008;20:1070–1079. 12. Baudry C, Reichardt F, Marchix J, et al. Diet-induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line-derived neurotrophic factor. J Physiol 2012; 590:533–544. 13. Gulbransen BD, Sharkey KA. Novel functional roles for enteric glia in the gastrointestinal tract. Nat Rev Gastroenterol Hepatol 2012;9:625–632. 14. Boesmans W, Lasrado R, Vanden Berghe P, et al. Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system. Glia 2014 Aug 26 [Epub ahead of print]. 15. Hoff S, Zeller F, von Weyhern CW, et al. Quantitative assessment of glial cells in the human and guinea pig enteric nervous system with an anti-Sox8/9/10 antibody. J Comp Neurol 2008;509:356–371. 16. Boesmans W, Martens MA, Weltens N, et al. Imaging neuron-glia interactions in the enteric nervous system. Front Cell Neurosci 2013;7:183. 17. Joseph NM, He S, Quintana E, et al. Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut. J Clin Invest 2011;121:3398–3411. 18. Laranjeira C, Sandgren K, Kessaris N, et al. Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury. J Clin Invest 2011;121: 3412–3424. 19. Abdo H, Derkinderen P, Gomes P, et al. Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione. FASEB J 2010;24:1082–1094. 20. Anitha M, Gondha C, Sutliff R, et al. GDNF rescues hyperglycemia-induced diabetic enteric neuropathy through activation of the PI3K/Akt pathway. J Clin Invest 2006;116:344–356. 21. Aube AC, Cabarrocas J, Bauer J, et al. Changes in enteric neurone phenotype and intestinal functions in a transgenic mouse model of enteric glia disruption. Gut 2006;55:630–637. 22. Nagahama M, Semba R, Tsuzuki M, et al. L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum. Biol Signals Recept 2001;10: 336–340. 23. Boesmans W, Cirillo C, Van den Abbeel V, et al. Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells. Neurogastroenterol Motil 2013;25:e151–e160. 24. Brun P, Giron MC, Qesari M, et al. Toll-like receptor 2 regulates intestinal inflammation by controlling integrity of the enteric nervous system. Gastroenterology 2013; 145:1323–1333. 25. McClain JL, Grubisic V, Fried D, et al. Ca2þ responses in enteric glia are mediated by connexin-43 hemichannels
REVIEWS AND PERSPECTIVES
December 2014
1236 Neunlist et al
REVIEWS AND PERSPECTIVES
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
and modulate colonic transit in mice. Gastroenterology 2014;146:497–507.e1. Nasser Y, Fernandez E, Keenan CM, et al. Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function. Am J Physiol Gastrointest Liver Physiol 2006;291:G912–G927. Broadhead MJ, Bayguinov PO, Okamoto T, et al. Ca2þ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine. J Physiol 2012;590:335–350. Bush TG, Savidge TC, Freeman TC, et al. Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice. Cell 1998;93:189–201. Cornet A, Savidge TC, Cabarrocas J, et al. Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn’s disease? Proc Natl Acad Sci U S A 2001;98:13306–13311. Savidge TC, Newman P, Pothoulakis C, et al. Enteric glia regulate intestinal barrier function and inflammation via release of S-nitrosoglutathione. Gastroenterology 2007; 132:1344–1358. Cheadle GA, Costantini TW, Lopez N, et al. Enteric glia cells attenuate cytomix-induced intestinal epithelial barrier breakdown. PLoS One 2013;8:e69042. Costantini TW, Krzyzaniak M, Cheadle GA, et al. Targeting alpha-7 nicotinic acetylcholine receptor in the enteric nervous system: a cholinergic agonist prevents gut barrier failure after severe burn injury. Am J Pathol 2012;181:478–486. Flamant M, Aubert P, Rolli-Derkinderen M, et al. Enteric glia protect against Shigella flexneri invasion in intestinal epithelial cells: a role for S-nitrosoglutathione. Gut 2011; 60:473–484. Costantini TW, Bansal V, Krzyzaniak M, et al. Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells. Am J Physiol Gastrointest Liver Physiol 2010;299: G1308–G1318. Van Landeghem L, Chevalier J, Mahe MM, et al. Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF. Am J Physiol Gastrointest Liver Physiol 2011;300:G976–G987. Neunlist M, Aubert P, Bonnaud S, et al. Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGFbeta1-dependent pathway. Am J Physiol Gastrointest Liver Physiol 2007;292:G231–G241. Bach-Ngohou K, Mahe MM, Aubert P, et al. Enteric glia modulate epithelial cell proliferation and differentiation through 15-deoxy-12,14-prostaglandin J2. J Physiol 2010;588:2533–2544. MacEachern SJ, Patel BA, McKay DM, et al. Nitric oxide regulation of colonic epithelial ion transport: a novel role for enteric glia in the myenteric plexus. J Physiol 2011; 589:3333–3348. Bohorquez DV, Samsa LA, Roholt A, et al. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy. PLoS One 2014;9:e89881. Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci 2009; 32:638–647.
Gastroenterology Vol. 147, No. 6 41. Turco F, Sarnelli G, Cirillo C, et al. Enteroglial-derived S100B protein integrates bacteria-induced Toll-like receptor signalling in human enteric glial cells. Gut 2014; 63:105–115. 42. Stoffels B, Hupa KJ, Snoek SA, et al. Postoperative ileus involves interleukin-1 receptor signaling in enteric glia. Gastroenterology 2014;146:176–187.e1. 43. Murakami M, Ohta T, Ito S. Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin-1beta from enteric glial cells. J Neurosci Res 2009;87:2095–2104. 44. Cirillo C, Sarnelli G, Turco F, et al. Proinflammatory stimuli activates human-derived enteroglial cells and induces autocrine nitric oxide production. Neurogastroenterol Motil 2011;23:e372–e382. 45. von Boyen GB, Steinkamp M, Reinshagen M, et al. Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia. Gut 2004;53:222–228. 46. Nasser Y, Keenan CM, Ma AC, et al. Expression of a functional metabotropic glutamate receptor 5 on enteric glia is altered in states of inflammation. Glia 2007; 55:859–872. 47. von Boyen GB, Degenkolb N, Hartmann C, et al. The endothelin axis influences enteric glia cell functions. Med Sci Monit 2010;16:BR161–BR167. 48. Ruhl A, Franzke S, Stremmel W. IL-1beta and IL-10 have dual effects on enteric glial cell proliferation. Neurogastroenterol Motil 2001;13:89–94. 49. Bradley JS Jr, Parr EJ, Sharkey KA. Effects of inflammation on cell proliferation in the myenteric plexus of the guinea-pig ileum. Cell Tissue Res 1997;289:455–461. 50. Ruhl A, Franzke S, Collins SM, et al. Interleukin-6 expression and regulation in rat enteric glial cells. Am J Physiol Gastrointest Liver Physiol 2001;280:G1163–G1171. 51. Esposito G, Capoccia E, Turco F, et al. Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-alpha activation. Gut 2014;63:1300–1312. 52. von Boyen GB, Steinkamp M, Reinshagen M, et al. Nerve growth factor secretion in cultured enteric glia cells is modulated by proinflammatory cytokines. J Neuroendocrinol 2006;18:820–825. 53. Steinkamp M, Geerling I, Seufferlein T, et al. Glial-derived neurotrophic factor regulates apoptosis in colonic epithelial cells. Gastroenterology 2003;124:1748–1757. 54. Steinkamp M, Gundel H, Schulte N, et al. GDNF protects enteric glia from apoptosis: evidence for an autocrine loop. BMC Gastroenterol 2012;12:6. 55. von Boyen GB, Steinkamp M, Geerling I, et al. Proinflammatory cytokines induce neurotrophic factor expression in enteric glia: a key to the regulation of epithelial apoptosis in Crohn’s disease. Inflamm Bowel Dis 2006;12:346–354. 56. Cirillo C, Sarnelli G, Esposito G, et al. Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein. Neurogastroenterol Motil 2009;21. 1209–e112. 57. von Boyen GB, Schulte N, Pfluger C, et al. Distribution of enteric glia and GDNF during gut inflammation. BMC Gastroenterol 2011;11:3.
58. Bassotti G, Villanacci V, Antonelli E, et al. Enteric glial cells: new players in gastrointestinal motility? Lab Invest 2007;87:628–632. 59. Coron E, Flamant M, Aubert P, et al. Characterisation of early mucosal and neuronal lesions following Shigella flexneri infection in human colon. PLoS One 2009;4:e4713. 60. Lourenssen S, Houpt ER, Chadee K, et al. Entamoeba histolytica infection and secreted proteins proteolytically damage enteric neurons. Infect Immun 2010;78: 5332–5340. 61. Bassotti G, Villanacci V, Maurer CA, et al. The role of glial cells and apoptosis of enteric neurones in the neuropathology of intractable slow transit constipation. Gut 2006; 55:41–46. 62. Iantorno G, Bassotti G, Kogan Z, et al. The enteric nervous system in chagasic and idiopathic megacolon. Am J Surg Pathol 2007;31:460–468. 63. Bassotti G, Battaglia E, Bellone G, et al. Interstitial cells of Cajal, enteric nerves, and glial cells in colonic diverticular disease. J Clin Pathol 2005;58:973–977. 64. Selgrad M, De Giorgio R, Fini L, et al. JC virus infects the enteric glia of patients with chronic idiopathic intestinal pseudo-obstruction. Gut 2009;58:25–32. 65. De Giorgio R, Cogliandro RF, Barbara G, et al. Chronic intestinal pseudo-obstruction: clinical features, diagnosis,
Functions of Enteric Glia in Health and Disease 1237 and therapy. Gastroenterol Clin North Am 2011;40: 787–807. 66. Stenkamp-Strahm C, Patterson S, Boren J, et al. High-fat diet and age-dependent effects on enteric glial cell populations of mouse small intestine. Auton Neurosci 2013;177:199–210. 67. Clairembault T, Kamphuis W, Leclair-Visonneau L, et al. Enteric GFAP expression and phosphorylation in Parkinson’s disease. J Neurochem 2014;130:805–815.
Received July 2, 2014. Accepted September 12, 2014. Reprint requests Address requests for reprints to: Michel Neunlist, PhD, UMR INSERM Unité 913, 1, rue Gaston Veil, 44000 Nantes, France. e-mail: [email protected]. Acknowledgments The authors thank past and present lab members for their contribution to the research presented in this review. Conflicts of interest The authors disclose no conflicts. Funding Work in our laboratory (INSERM Unité 913) is supported by grants from INSERM, Région Pays de la Loire, Agence National pour la Recherche, Fondation SantéDige, Michael J. Fox Foundation for Parkinson’s Research, La Ligue contre le Cancer, Fondation de France, and France Parkinson. M.R.-D. is supported by the Centre National de la Recherche Scientifique.
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