Are We Close to Targeting Enteric Glia in Gastrointestinal Diseases and Motility Disorders?

Understanding the mechanisms that regulate intestinal homeostasis is essential for the development of targeted therapies for intestinal diseases. New evidence shows that enteric glia, a unique type of peripheral neuroglia found in the intestine, contribute to the regulation of gastrointestinal (GI) motility, gut inflammation, and potentially other intestinal processes, such as epithelial barrier function, endocrine functions, and immunity (Figure 1).1Sharkey K.A. Emerging roles for enteric glia in gastrointestinal disorders.J Clin Invest. 2015; 125: 918-925Crossref PubMed Scopus (127) Google Scholar, 2Ochoa-Cortes F. Turco F. Liñán-Rico A. et al.Enteric glial cells: a new frontier in neurogastroenterology and clinical target for inflammatory bowel diseases.Inflamm. Bowel Dis. 2016; 22: 433-449Crossref PubMed Scopus (103) Google Scholar, 3Grubišić V. Gulbransen B.D. Enteric glia: the most alimentary of all glia.J Physiol. 2017; 15: 557-570Crossref Scopus (95) Google Scholar, 4Sundaresan S. Meininger C.A. Kang A.J. et al.Gastrin induces nuclear export and proteasome degradation of menin in enteric glial cells.Gastroenterology. 2017; 153: 1555-1567.e15Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar These attributes make gliomodulation an attractive therapeutic avenue, but how close we are to harnessing the power of glia to aid human digestive health remains debatable. Herein, we focus on several points that could provide clarity around this issue and help to guide future research in the field. Mechanistic studies show that interactions between enteric glial cells and neurons, epithelial cells, and immune cells have the potential to influence the physiologic regulation of many gut functions such as motility, secretions, and barrier function (Figure 1). For example, glia associated with neurons in the myenteric and submucosal plexuses sense neuronal activity and, in turn, modulate the activity of enteric neurons. New evidence showing that the direct activation of glial cells is sufficient to drive neurogenic reflexes controlling motility5McClain J.L. Fried D.E. Gulbransen B.D. Agonist-evoked Ca2+ signaling in enteric glia drives neural programs that regulate intestinal motility in mice.Cell Mol Gastroenterol Hepatol. 2015; 1: 631-645Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar and secretions6Grubišić V. Gulbransen B.D. Enteric glial activity regulates secretomotor function in the mouse colon but does not acutely affect gut permeability.J Physiol. 2017; 595: 3409-3424Crossref PubMed Scopus (44) Google Scholar suggests that glia have a major role in the integrated functions of the enteric nervous system. However, the role of glia in the maintenance of gut barrier function is less clear. Current data suggest that glia are recruited to the mucosa by cues from bacteria and aid in enhancing barrier function by secreting factors that influence epithelial cells,7Kabouridis P.S. Lasrado R. McCallum S. et al.Microbiota controls the homeostasis of glial cells in the gut lamina propria.Neuron. 2015; 85: 289-295Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 8Savidge T.C. Newman P. Pothoulakis C. et al.Enteric glia regulate intestinal barrier function and inflammation via release of S-nitrosoglutathione.Gastroenterology. 2007; 132: 1344-1358Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 9Neunlist M. Aubert P. Bonnaud S. et al.Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-β1-dependent pathway.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G231-G241Crossref PubMed Scopus (133) Google Scholar but a loss of this role does not create a failure of barrier function under normal conditions.6Grubišić V. Gulbransen B.D. Enteric glial activity regulates secretomotor function in the mouse colon but does not acutely affect gut permeability.J Physiol. 2017; 595: 3409-3424Crossref PubMed Scopus (44) Google Scholar, 10Rao M. Rastelli D. Dong L. et al.Enteric glia regulate gastrointestinal motility but are not required for maintenance of the epithelium in mice.Gastroenterology. 2017; 153: 1068-1081.e7Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar The supportive role of glia in barrier function may have more prominence during disease, when the barrier is compromised and glial factors such as 15-HETE are required to prevent gut permeability.11Pochard C. Coquenlorge S. Jaulin J. et al.Defects in 15-HETE production and control of epithelial permeability by human enteric glial cells from patients with Crohn’s disease.Gastroenterology. 2016; 150: 168-180Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar In disease, glial mechanisms contribute to enteric neuropathy and to the regulation of inflammatory responses. Glia directly regulate neurodegeneration by sensing cellular danger cues such as adenosine triphosphate (ATP)12Gulbransen B.D. Bashashati M. Hirota S.A. et al.Activation of neuronal P2X7 receptor-pannexin-1 mediates death of enteric neurons during colitis.Nat Med. 2012; 18: 600-604Crossref PubMed Scopus (305) Google Scholar and subsequently enacting mechanisms that drive neuron death.13Brown I.A.M. McClain J.L. Watson R.E. et al.Enteric glia mediate neuron death in colitis through purinergic pathways that require connexin-43 and nitric oxide.Cell Mol Gastroenterol Hepatol. 2016; 2: 77-91Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar In addition, glia indirectly influence the inflammatory milieu through interactions with immune cells.14Ibiza S. García-Cassani B. Ribeiro H. et al.Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence.Nature. 2016; 535: 440-443Crossref PubMed Scopus (211) Google Scholar Interestingly, glia transform into a reactive phenotype in response to pathogenic bacteria15Turco 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: 105-115Crossref PubMed Scopus (110) Google Scholar and experience a purinergic switch from ATP to adenosine diphosphate/adenosine/uridine 5'-triphosphate (UTP) signaling,16Liñán-Rico A. Turco F. Ochoa-Cortes F. et al.Molecular signaling and dysfunction of the human reactive enteric glial cell phenotype: implications for GI infection, IBD, POI, Neurological, Motility, and GI disorders.Inflamm Bowel Dis. 2016; 22: 1812-1834Crossref PubMed Scopus (52) Google Scholar which may disrupt enteric neurotransmission by altering the function and/or survival of enteric neurons. Defective glia may, in fact, contribute to the influx of pathogenic bacteria in disease by decreasing their secretion of beneficial factors that enhance gut barrier function.11Pochard C. Coquenlorge S. Jaulin J. et al.Defects in 15-HETE production and control of epithelial permeability by human enteric glial cells from patients with Crohn’s disease.Gastroenterology. 2016; 150: 168-180Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar However, whether defective glial release of these factors is a primary cause of disease or a consequence that perpetuates disease processes is unknown. If glial cells are responsible for carrying out such widespread roles in the intestine as eluded to, why then did a recent study10Rao M. Rastelli D. Dong L. et al.Enteric glia regulate gastrointestinal motility but are not required for maintenance of the epithelium in mice.Gastroenterology. 2017; 153: 1068-1081.e7Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar observe only relatively minor effects in a transgenic mouse model of glial depletion? There are several potential explanations for this observation, but deciphering this outcome requires a clearer understanding of how other cells react and adapt to the death of their neighbors and how many glial cells are actually required to maintain functions. The lack of major consequences suggests that plasticity occurring during the death of enteric glia and the subsequent immune response required to clear the cellular debris is capable of maintaining most essential gut functions, but this is only one of many possible explanations for these observations. Integrating these observations with the body of evidence provided supports the concept that glia play a major role in motility circuits, where they are both necessary and sufficient to drive normal enteric reflexes, but only provide a supportive role at the epithelium where their influence may become more important during disease processes. Glia are extraordinarily plastic cells. This property presents an interesting challenge when studying the various types of enteric glia and their roles in disease processes. Glial cells respond to a wide range of extracellular cues and this is the main driving force influencing glial phenotype. This, of course, means that the context under which the glial cell is studied is a critical aspect to consider when interpreting results because it will play a major role in dictating unique glial phenotypes. This aspect of gliobiology is clear from work in the central nervous system showing extraordinary complexity in glial phenotypic diversity during health and disease.17Anderson M.A. Burda J.E. Ren Y. et al.Astrocyte scar formation aids central nervous system axon regeneration.Nature. 2016; 532: 195-200Crossref PubMed Scopus (1075) Google Scholar, 18Liddelow S.A. Guttenplan K.A. Clarke L.E. et al.Neurotoxic reactive astrocytes are induced by activated microglia.Nature. 2017; 541: 481-487Crossref PubMed Scopus (3356) Google Scholar, 19Gosselin D. Skola D. Coufal N.G. et al.An environment-dependent transcriptional network specifies human microglia identity.Science. 2017; (eaal3222)Crossref PubMed Scopus (588) Google Scholar These studies show that glial phenotypes are regionally specific, that glial reactivity is insult specific, and that glial phenotypes represent a spectrum rather than a binary switch. Overlapping, but distinct, molecular pathways are responsible for producing these unique phenotypes and understanding core regulators of these pathways could uncover ways to control glia phenotypes on demand. Enteric glia share this plastic potential and the enteric glial phenotype is heavily influenced by the gut microenvironment. Recent transcriptional profiling of enteric glia suggests that the default genetic program of enteric glia is more similar to oligodendrocytes and Schwann cells than astrocytes,20Rao M. Nelms B.D. Dong L. et al.Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system.Glia. 2015; : 1-18PubMed Google Scholar and these data are supported by cell culture experiments showing that enteric glia in culture adopt some markers of Schwann cells.21Jessen K.R. Mirsky R. Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia.J Neurosci. 1983; 3: 2206-2218Crossref PubMed Google Scholar However, the gut microenvironment shifts this default program toward an astrocyte-like translational profile, morphology, and function in vivo.3Grubišić V. Gulbransen B.D. Enteric glia: the most alimentary of all glia.J Physiol. 2017; 15: 557-570Crossref Scopus (95) Google Scholar For example, enteric glia express astrocytic cell surface markers, an astrocyte-associated determinant of GFAP that is not expressed by Schwann cells, do not form myelin, and use similar mechanisms to detect and modulate neuronal activity, such as neurotransmitter receptors and the subsequent release of gliotransmitters.3Grubišić V. Gulbransen B.D. Enteric glia: the most alimentary of all glia.J Physiol. 2017; 15: 557-570Crossref Scopus (95) Google Scholar, 21Jessen K.R. Mirsky R. Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia.J Neurosci. 1983; 3: 2206-2218Crossref PubMed Google Scholar, 22Jessen K.R. Mirsky R. Glial fibrillary acidic polypeptides in peripheral glia. Molecular weight, heterogeneity and distribution.J Neuroimmunol. 1985; 8: 377-393Abstract Full Text PDF PubMed Scopus (64) Google Scholar The enteric nervous system is the most autonomous branch of the peripheral nervous system because of the integrative capacity of enteric neurocircuits. It is likely that enteric glia adopt a more astrocyte-like role in this environment than other types of peripheral glia because these circuits are more “brain-like” than other areas of the peripheral nervous system. However, it is also clear that enteric glia are a unique class of neuroglia and that properties of other glia cannot be blindly generalized to enteric glia without supportive evidence. What then does this mean for in vitro studies where glia are plucked from their native environment and placed in an artificial context? The dependence of glial phenotype on extracellular cues creates a major challenge for cell culture models. Some of these issues may be circumvented by using co-culture models to maintain a more natural environment for glia. It is also likely that some populations of enteric glia may be more amenable to modeling in cell culture than others. For example, cultures of human enteric glial cells (hEGCs) have been used successfully to model key mechanisms of intercellular communication used by myenteric and submucosal glia that are conserved between mice and men such as the expression of connexin-43 and the propagation of Ca2+ waves by purines.16Liñán-Rico A. Turco F. Ochoa-Cortes F. et al.Molecular signaling and dysfunction of the human reactive enteric glial cell phenotype: implications for GI infection, IBD, POI, Neurological, Motility, and GI disorders.Inflamm Bowel Dis. 2016; 22: 1812-1834Crossref PubMed Scopus (52) Google Scholar, 23Ochoa-Cortes F. Rico A.L. Zuleta-Alarcon A. et al.Sa1689 Hemichannels, GJICs and ATP signaling in human enteric glial cells (hEGC).Gastroenterology. 2016; 150: S346-S347Abstract Full Text PDF Google Scholar, 24McClain J.L. Grubišić V. Fried D. et al.Ca2+ responses in enteric glia are mediated by connexin-43 hemichannels and modulate colonic transit in mice.Gastroenterology. 2014; 146: 497-507.e1Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 25Zhang W. Segura B.J. Lin T.R. et al.Intercellular calcium waves in cultured enteric glia from neonatal guinea pig.Glia. 2003; 42: 252-262Crossref PubMed Scopus (56) Google Scholar, 26Rico A.L. Zuleta-Alarcon A. Ochoa-Cortes F. et al.UTP is a potential physiological regulator of human enteric glial cell communication by activating P2Y4 and P2Y6 receptors.Gastroenterology. 2017; 152: S179Abstract Full Text PDF Google Scholar These mechanisms play key roles in the control of gut motility and inflammation in mice13Brown I.A.M. McClain J.L. Watson R.E. et al.Enteric glia mediate neuron death in colitis through purinergic pathways that require connexin-43 and nitric oxide.Cell Mol Gastroenterol Hepatol. 2016; 2: 77-91Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 24McClain J.L. Grubišić V. Fried D. et al.Ca2+ responses in enteric glia are mediated by connexin-43 hemichannels and modulate colonic transit in mice.Gastroenterology. 2014; 146: 497-507.e1Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 27Bhave S. Gade A. Kang M. et al.Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.FASEB J. 2017; 31: 2649-2660Crossref PubMed Scopus (32) Google Scholar and using hEGCs to study these mechanism in more detail could provide novel insight into therapeutic targets and strategies. In addition, hEGCs may provide opportunities to test how proinflammatory signals such as s100β, IL-1β, and bacterial products from Escherichia coli or Clostridium difficile disrupt Ca2+ waves, purinergic signaling, mechanosensitivity, and alter the molecular phenotype of glia. Such mechanistic studies would provide important insight into how glia are affected by, and contribute to, gut inflammation. However, modeling subpopulations of mucosal glia in cell culture may prove more problematic because this population requires a more diverse array of extracellular cues from both host and microbiome to maintain their phenotype.7Kabouridis P.S. Lasrado R. McCallum S. et al.Microbiota controls the homeostasis of glial cells in the gut lamina propria.Neuron. 2015; 85: 289-295Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar More complex organoid model systems are currently being developed that may circumvent some of these issues and afford the opportunity to study the responses of mucosal glial in vitro to important pathogens such as E coli or C difficile toxins.28Workman M.J. Mahé M.M. Trisno S. et al.Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.Nat Med. 2017; 23: 49-59Crossref PubMed Scopus (345) Google Scholar However, the high potential for glial transformations in any in vitro glial model requires that mechanisms addressed in culture must be confirmed in situ by histologic and functional analyses. We, and other investigators, are currently developing in vitro strategies to facilitate translation from animals to humans. In vitro models ranging from single cells to intact gut surgical tissue provide complimentary information on how glia function individually or within networks and data from these models are providing new insights into human gliophysiology. Future work would benefit from using clinical samples in these models to establish clear links between reactive glia and motility disorders, slow transit constipation, opioid- or Parkinson disease-induced constipation, and postoperative ileus. Recent work has identified important glial mechanisms that contribute to gut physiology and modulate GI disease that can now be pursued to advance treatment outcomes. Here, we introduce several glial therapeutic targets and the available drugs and strategies to modulate these targets (Figure 2). The therapeutic tools are at various stages of development and while some could be enacted immediately (with relevant drugs already in clinical trials), others would require additional targeting strategies (and development of more selective agents). Cellular activity encoded by intracellular Ca2+ signaling is a fundamental feature of enteric glia that is maintained across experimental animals, humans, and culture models. The processes downstream of glial Ca2+ responses involve intercellular signaling mediated by glial connexin-43 channels and contribute to enhancing key intestinal reflexes that regulate motility and secretions.5McClain J.L. Fried D.E. Gulbransen B.D. Agonist-evoked Ca2+ signaling in enteric glia drives neural programs that regulate intestinal motility in mice.Cell Mol Gastroenterol Hepatol. 2015; 1: 631-645Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 6Grubišić V. Gulbransen B.D. Enteric glial activity regulates secretomotor function in the mouse colon but does not acutely affect gut permeability.J Physiol. 2017; 595: 3409-3424Crossref PubMed Scopus (44) Google Scholar, 24McClain J.L. Grubišić V. Fried D. et al.Ca2+ responses in enteric glia are mediated by connexin-43 hemichannels and modulate colonic transit in mice.Gastroenterology. 2014; 146: 497-507.e1Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar These properties make glial Ca2+ responses and connexin-43 channels prime targets for therapeutics aimed at enhancing motility in individuals with functional GI disorders, such as constipation-predominant irritable bowel syndrome and chronic constipation. Work in animal models shows that genetic approaches could be used to selectively activate glial mechanisms to relieve constipation. For example, the expression of designer receptors exclusively activated by designer drugs allows for the selective activation of glial Ca2+ responses with the result of increasing gut motility and secretions.5McClain J.L. Fried D.E. Gulbransen B.D. Agonist-evoked Ca2+ signaling in enteric glia drives neural programs that regulate intestinal motility in mice.Cell Mol Gastroenterol Hepatol. 2015; 1: 631-645Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 6Grubišić V. Gulbransen B.D. Enteric glial activity regulates secretomotor function in the mouse colon but does not acutely affect gut permeability.J Physiol. 2017; 595: 3409-3424Crossref PubMed Scopus (44) Google Scholar However, similar genetic targeting and activation strategies are not yet available for use in humans. Glial connexin-43 channels are also involved in neuroinflammation in the intestine and contribute to neurodegeneration by releasing ATP that subsequently activates neuronal P2X7 receptors and pannexin-1 channels.12Gulbransen B.D. Bashashati M. Hirota S.A. et al.Activation of neuronal P2X7 receptor-pannexin-1 mediates death of enteric neurons during colitis.Nat Med. 2012; 18: 600-604Crossref PubMed Scopus (305) Google Scholar, 13Brown I.A.M. McClain J.L. Watson R.E. et al.Enteric glia mediate neuron death in colitis through purinergic pathways that require connexin-43 and nitric oxide.Cell Mol Gastroenterol Hepatol. 2016; 2: 77-91Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Targeting glial connexin-43 and/or neuronal P2X7 receptors and pannexin-1 channels could be beneficial in a number of inflammatory conditions in the intestine. Drugs that modulate connexin-43 such as carbenoxolone are currently in use for peptic, esophageal, and oral ulceration and inflammation. However, carbenoxolone displays poor selectivity for connexin-43 and lacks the ability to selectively target glial connexin-43 rather than connexin-43 gap junctions expressed by intestinal smooth muscle cells or interstitial cells of Cajal where the inhibition of connexin-43 could compromise gut motility. Drugs are available to modulate neuronal mechanisms downstream of glial ATP release such as the P2X7 receptor antagonist AZD9056 and the pannexin-1 antagonist spironolactone. AZD9056 did show promise in a phase IIa clinical trial in 34 patients for moderate to severe Crohn’s disease. A decrease in disease activity was observed for pain and general well-being, but there was no improvement in remission rates.29Eser A. Colombel J.-F. Rutgeerts P. et al.Safety and efficacy of an oral inhibitor of the purinergic receptor P2X7 in adult patients with moderately to severely active crohn's disease: a randomized placebo-controlled, double-blind, phase IIa study.Inflamm Bowel Dis. 2015; 21: 2247-2253PubMed Google Scholar Further clinical studies are essential to determine if AZD9056 or other P2X7R antagonist drugs such as CE-224,535 or GSK1482160 are an effective therapy for inflammatory bowel disease (IBD) or other GI disorders. The inhibition of neuronal pannexin-1 channels with spironolactone could provide a novel therapeutic avenue for glial-driven neuroinflammatory mechanisms. Spironolactone is currently used for the treatment of low-renin hypertension, hypokalemia, and Conn’s syndrome, but its effects on gut inflammation are not known. The potential impact of other glial mediators is less well defined, but some such as IL-1β clearly contribute to the pathophysiology of conditions like postoperative ileus.30Stoffels B. Hupa K.J. Snoek S.A. et al.Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.Gastroenterology. 2014; 146: 176-187.e1Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Drugs that target IL-1β signaling via IL-1R such as anakinra are already in clinical use for rheumatoid arthritis and their therapeutic benefit in postoperative ileus and other inflammatory conditions such as IBD could be tested immediately. Likewise, the glial s100β protein is involved in inflammation caused by pathogenic bacteria in the human gut, Clostridium difficile infections and ulcerative colitis and celiac disease.31Esposito G. Capoccia E. Turco F. et al.Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-α activation.Gut. 2013; 63: 1300-1312Crossref PubMed Scopus (187) Google Scholar Anti-s100β antibodies are available and could be used to reduce the inflammatory actions of this glial protein. Bacterial products activate the Toll-like receptor 4/s100β–RAGE/nitric oxide signaling pathway via nuclear factor-κB32Esposito G. Cirillo C. Sarnelli G. et al.Enteric glial-derived S100B protein stimulates nitric oxide production in celiac disease.Gastroenterology. 2007; 133: 918-925Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar and glial nitric oxide is involved in neural dysfunction.13Brown I.A.M. McClain J.L. Watson R.E. et al.Enteric glia mediate neuron death in colitis through purinergic pathways that require connexin-43 and nitric oxide.Cell Mol Gastroenterol Hepatol. 2016; 2: 77-91Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Drugs targeting RAGE such as PF-04494700 are beneficial in Alzheimer’s disease and could be combined with drugs targeting upstream or downstream proteins such as nitric oxide synthase for synergistic effects.33Wong V.W.C. Lerner E. Nitric oxide inhibition strategies.Future Sci OA. 2015; 1: 1420Crossref Scopus (37) Google Scholar An alternate anti-inflammatory strategy could be to activate glial PPAR-α receptors with palmitoylethanolamide (PEA). This strategy has shown success in animal models and could be relevant in the treatment of IBD.31Esposito G. Capoccia E. Turco F. et al.Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-α activation.Gut. 2013; 63: 1300-1312Crossref PubMed Scopus (187) Google Scholar PEA is currently available as a nutritional supplement and its availability over the counter for the relief of intestinal complaints, including IBD, makes it an attractive treatment strategy. However, more selective PPAR-α agonists are still needed because PEA acts on several receptor pathways in the gut, such as cannabinoid receptors and TRP channels. Significant progress is being made in the field. However, several key technical and conceptual hurdles need attention. For example, understanding the molecular phenotypes of enteric glia and how the molecular phenotypes of glia change in response to various disease states would be a major advance toward identifying therapeutically relevant pathways and modulators. To this end, we recently used RiboTag mice to isolate glial ribosomal RNA from the colon myenteric plexus in a cell type-specific manner and analyzed the glial “translatome” in healthy animals and during acute colitis.34Delvalle N.M. Dharshika C. Morales-Soto W. et al.Communication between enteric neurons, glia, and nociceptors underlies the effects of tachykinins on neuroinflammation.Cell Mol Gastroenterol Hepatol. 2018; (Accepted in press)https://doi.org/10.1016/j.jcmgh.2018.05.009Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar However, the efficacy of this model depends on the specificity of the mouse line driving Cre-recombinase expression and more specific driver lines to selectively target enteric, and not central, glia are desperately needed. This development requires the identification of unique molecular and functional markers of enteric glia. Likewise, methods to address the various subtypes of enteric glia are needed to assess the molecular phenotypes of these populations. Overcoming the technical difficulties involved with isolating and maintaining these populations in vitro or targeting them in vivo could produce novel insights into their functions in health and disease. Translating data from cells or laboratory animals to humans is challenging for a myriad reasons and clearly “the human is not always a good model for the mouse.” These translational issues between animal models and humans often limit the success of clinical trials. One issue has to do with a species difference in glia between a human and a mouse—that is the ratio of glia to neurons in human gut is 7:1 versus 1:1 for mice, implying that glia are likely to exert a much greater influence on neuronal function in a human than a mouse. Another potential issue is that our understanding of human enteric glia, myenteric in particular, is based on human tissue derived from apparently healthy margins of diseased individuals that may or may not have normal gut physiology and glia. For instance, in jejunal surgical tissue obtained from Roux-en-Y bypass surgeries, the patient is morbidly obese, and the gut cannot be considered normal. Even tissue obtained from surgical polyp cases from healthy regions is influenced by gut surgical manipulation and trauma during the operation30Stoffels B. Hupa K.J. Snoek S.A. et al.Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.Gastroenterology. 2014; 146: 176-187.e1Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar that is known to cause an inflammatory response. This circumstance can be circumvented for the most part, by restricting studies to gut surgical polyp specimens obtained from minimal invasive laparoscopic surgery rather than open laparotomy. Yet, in every patient, we still cannot control for medications and other comorbidities that can influence glial function. Full-thickness tissues from resections are necessary to study myenteric glia and obtaining these samples in a normal state or from healthy individuals is nearly impossible. One potential solution to this issue is to study glia in translational large animal models such as pigs and primates, where myenteric glia can be studied in healthy tissue. Studies of mucosal and submucosal glia in humans are less problematic because these populations can be collected in mucosal biopsies from healthy individuals who undergo routine endoscopic screening for cancer. These samples provide a source of healthy tissue that can be used to study glia in culture or intact submucous ganglia. Although many of the main glial signaling pathways seem to be conserved between mice and humans, some of the specific molecular components involved do differ. For example, purinergic P2Y1 receptors are involved with glial signaling in both mice and humans, whereas human enteric glia and neurons also use P2Y6, P2Y4, and P2X2.16Liñán-Rico A. Turco F. Ochoa-Cortes F. et al.Molecular signaling and dysfunction of the human reactive enteric glial cell phenotype: implications for GI infection, IBD, POI, Neurological, Motility, and GI disorders.Inflamm Bowel Dis. 2016; 22: 1812-1834Crossref PubMed Scopus (52) Google Scholar Receptors themselves also display species-specific characteristics. For example, P2Y4 receptors are expressed by enteric glia in rodents35Gulbransen B.D. Sharkey K.A. Purinergic neuron-to-glia signaling in the enteric nervous system.Gastroenterology. 2009; 136: 1349-1358Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar and humans (Christofi et al, unpublished observations). In humans, UTP activates P2Y4 and ATP acts as an antagonist, whereas in rodents, both UTP and ATP are agonists. Understanding these differences is important when developing targeted therapeutics. However, recognizing conserved mechanisms shared by glia across species is paramount to understanding their key functions. Mechanisms that are conserved between cell culture, mouse, and human should be viewed as particularly important and are likely central to glial, and gut, physiology. Enteric glia fulfill diverse roles in the intestine and understanding these roles holds great promise for identifying novel ways to improve human health. To this end, basic mechanisms of glial physiology such as those regulated by glial Ca2+ waves should be thoroughly investigated to form a solid knowledge base. This information is of intrinsic value to our understanding of intestinal motility and GI transit. Similar work with glia in the brain has identified important mechanisms that contribute to diseases of the nervous system.36Tong X. Ao Y. Faas G.C. et al.Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice.Nat Neurosci. 2014; 17: 694-703Crossref PubMed Scopus (378) Google Scholar Understanding the downstream functional consequences of glial signaling in health and disease is essential to understand the significance of glia in the intestine and to identify functionally relevant mechanisms for future studies. Translational research should focus on glial targets that are shared by mice and humans and also evaluate glial molecular targets that are unique to humans. Addressing the various molecular phenotypes of enteric glia and the epigenetic landscape will be a challenge in future work, but holds great promise for the identification of important pathways in disease. Similar work in the brain recently provided a template for these studies and showed that human microglial phenotypes are dictated by an environment-dependent transcriptional network.19Gosselin D. Skola D. Coufal N.G. et al.An environment-dependent transcriptional network specifies human microglia identity.Science. 2017; (eaal3222)Crossref PubMed Scopus (588) Google Scholar Understanding similar responses of enteric glia could have great impacts on irritable bowel syndrome, motility disorders, and GI diseases. The National Institutes of Health recently reaffirmed its strong commitment to basic scientific discovery and recognized that many of the most important medical advances trace back to basic research that had no explicit disease link37Collins F.S. Anderson J.M. Austin C.P. et al.Basic science: bedrock of progress.Science. 2016; 351 (1405–1405)Crossref Scopus (19) Google Scholar (NIH, NIH-Wide Strategic Plan; available from: www.nih.gov/about-nih/nih-wide-strategic-plan). Much work is still needed to understand the basic characteristics and functions of enteric glia. Current data suggest that understanding these basic qualities holds great potential for new treatments. Future work by interdisciplinary teams of basic and clinician scientists can then use this information to design translational therapies to improve human intestinal health. The enteric glial field represents a new frontier in neurogastroenterology, with a growing number of opportunities for young investigators and trainees in laboratories around the world.