Enteric Glia Cells Research Articles

Neuron Enteric Death Is Mediated by S100B During Intestinal Mucositis Induced by 5‐Fluorouracil

The goal of this study was to investigate whether S100B is involved in 5‐FU‐induced enteric neuron death during intestinal mucositis. This study was approved by ethical committee of Federal university of Ceará (protocol n° 80/2015). Intestinal mucositis was induced by injecting a single dose of 5‐FU (450 mg/kg, IP) and the mice were euthanized after 3 days. The mice were pretreated with Pentamidine, an S100B inhibitor, (4 mg/kg daily) for three days. The animals were euthanized and small intestine tissues (duodenum, jejunum and ileum) were collected to evaluate S100B expression by Western Blot, enteric neuron alterations by immunohistochemistry for HuC/D (neuron marker), neuronal death by TUNEL Assay and double staining of HuC/D with RAGE or NFκB NLS. A primary culture of enteric neuron from the small intestine was performed to evaluate the direct effect of 5‐FU and S100B in neuron death using a TUNEL assay. 5‐FU reduced (p<0.01) HuC/D protein expression and increased (p<0.01) S100B protein expression in the small intestine, as well as increased (p<0.01) TUNEL positive cells on jejunum of mice compared with control group. 5‐FU enhanced RAGE and NFκB NLS immunostaining in enteric neurons in the jejunum compared to control group. Pentamidine prevented (p<0.01) these alterations induced by 5‐FU in the intestine. Higher S100B concentration, but not 5‐FU, directly caused (p<0.0001) enteric neuron death. This study showed an important role for enteric glia cells in 5‐FU‐induced enteric neuron death via S100B, during intestinal mucositis. Our results suggest that enteric glial cells release S100B, which in turn activates NFκB signaling through RAGE activation in neurons, leading to neuronal cell death during intestinal mucositis induced by 5‐FU. Therefore, the S100B signaling pathway appears to be a promising pharmacological target to prevent 5‐FU‐induced enteric neuron death.Support or Funding InformationPROCAD/CAPES, CNPq, FUNCAPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute Toxoplasma gondii infection alters the number of neurons and the proportion of enteric glial cells in the duodenum in Wistar rats.

Toxoplasma gondii infection can occur through the ingestion of raw meat that contains tissue cysts or food that contains oocysts. Through the ingestion of oocysts, the parasite crosses the intestinal barrier, where the enteric nervous system is located. The objective was to investigate the kinetics of neuronal and glial responses during acute T. gondii infection. We used 45 Wistar rats that were divided into a control group and infected groups that were evaluated at 6, 12, 24, 48, 72 hours, 7 days, 10 days, and 15 days after infection. The rats received 5000 sporulated oocysts of the parasite orally. To detect neurons and enteric glia cells, the myenteric and submucosal plexuses of the duodenum underwent double-labeling immunohistochemical techniques to evaluate HuC/HuD and S100, HuC/HuD and ChAT, and HuC/HuD and nNOS. We observed a reduction of the total neuron population in the submucosal plexus 72 hours after infection. Cholinergic neurons decreased in the submucosal plexus 15 days after infection, and nitrergic neurons decreased in the myenteric plexus 72 hours after infection. A decrease in the number of glial cells was observed 7 days after infection in the submucosal plexus, and an increase in the enteric glial cell (EGC)/neuron ratio was found in both plexuses 48 hours after infection. We found decrease of neurons and increase in the EGC/neuron ratio in both plexuses caused by acute T. gondii infection, with major alterations 72 hours after oral infection. The number of cholinergic neurons decreased in the submucosal plexus, and the number of nitrergic neurons decreased in the myenteric plexus. A decrease in the number of enteric glial cells was observed in the submucosal plexus, and an increase in the enteric glial cell/neuron ratio was observed in both ganglionate plexuses of the duodenum.

DRG-Derived Neural Progenitors Differentiate into Functional Enteric Neurons Following Transplantation in the Postnatal Colon

Cell therapy has great promise for treating gastrointestinal motility disorders caused by intestinal nervous system (ENS) diseases. However, appropriate sources, other than enteric neural stem cells and human embryonic stem cells, are seldom reported. Here, we show that neural progenitors derived from the dorsal root ganglion (DRG) of EGFP mice survived, differentiated into enteric neurons and glia cells, migrated widely from the site of injection, and established neuron-muscle connections following transplantation into the distal colon of postnatal mice. The exogenous EGFP+ neurons were physiologically functional as shown by the activity of calcium imaging. This study shows that that other tissues besides the postnatal bowel harbor neural crest stem cells or neural progenitors that have the potential to differentiate into functional enteric neurons in vivo and can potentially be used for intestinal nerve regeneration. These DRG-derived neural progenitor cells may be a choice for cell therapy of ENS disease as an allograft. The new knowledge provided by our study is important for the development of neural crest stem cell and cell therapy for the treatment of intestinal neuropathy.

Advances in Enteric Neurobiology: The "Brain" in the Gut in Health and Disease.

The enteric nervous system (ENS) is a large, complex division of the peripheral nervous system that regulates many digestive, immune, hormonal, and metabolic functions. Recent advances have elucidated the dynamic nature of the mature ENS, as well as the complex, bidirectional interactions among enteric neurons, glia, and the many other cell types that are important for mediating gut behaviors. Here, we provide an overview of ENS development and maintenance, and focus on the latest insights gained from the use of novel model systems and live-imaging techniques. We discuss major advances in the understanding of enteric glia, and the functional interactions among enteric neurons, glia, and enteroendocrine cells, a large class of sensory epithelial cells. We conclude by highlighting recent work on muscularis macrophages, a group of immune cells that closely interact with the ENS in the gut wall, and the importance of neurological-immune system communication in digestive health and disease.

Expression profile of some neuronal and glial cell markers in the ovine ileal enteric nervous system during prenatal development

The enteric nervous system (ENS) is a network of neurons and glia found in the gut wall and governs this gastrointestinal function independently from the central nervous system (CNS). ENS comprises the myenteric plexus (MP) and the submucous plexus (SP). In this study, we examined the expression profile of neurofilament heavy chain (NF-H), neuron-specific enolase (NSE), calcyclin (S100A6), vimentin and glial fibril acidic protein (GFAP) in ovine ileal enteric neurons and enteric glia cells (EGCs) during prenatal development using an immunohistochemical method. The material of the study consisted of 15 different fetal ileum tissues obtained between days 60 and 150 of pregnancy. NF-H was observed in the majority of ganglion cells in SP and MP throughout the fetal period. It was determined that there was no NF-H reaction in some ganglion cells in Peyer’s patches of internal submucosal plexus (ISPF). In the early stage of pregnancy (60–90 days), there was no expression of NSE and S1006 in ileum. After this period, NSE and S1006 were expressed in the ganglion cells of the plexus, indicating an increase in the amount of expression towards the end of pregnancy. In the early period, vimentin expression was only detected in intramuscular interstitial cells (ICs) (60–90 days), but later (90–150 days) it was also seen in the cells around the ganglion cells in the plexus. On days 60–90 of gestation, GFAP expression only occurred in MP, but in later stages, staining was also detected in SP. In the plexus, an immunoreactivity was present in EGCs forming a network around the ganglion cell. During the last period of gestation (120–150 days), the number of GFAP-positive plexus increased, with the majority of these stained cells being observed in MP. Interestingly, weak staining or reaction did not occur in ISPF, unlike other plexuses. In conclusion, this is the first study that demonstrated the expression of NF-H, vimentin, S100A6, NSE and glial fibril acidic protein (GFAP) in ovine ileal ENS in the prenatal period. In the last period of gestation (120–150 days), the expression profile of ENS was similar to that of adult animals. The expression of the used markers increased toward the end of pregnancy. Our results suggest that neurons and EGCs show heterogeneity, and GFAP and NF-H cannot be used as panenteric glial or panneuronal markers, respectively. We also demonstrated, for the first time, the prenatal expression of S100A6 in enteric neurons and the possibility of using this protein for the identification of enteric neurons.

Mo1590 - Purinergic Signaling in Enteric Glia Cells During Inflammation

Mo1590 - Purinergic Signaling in Enteric Glia Cells During Inflammation

Gene Editing with CRISPR/Cas9 Technology before Cell Transplantation Using a Mouse Model of Hirschsprung's Disease

Hirschsprung's disease (HSCR) is a common congenital gastrointestinal motility disorder presented with the regional absence or reduction of enteric neurons in the colon. The Dominant megacolon (Sox10Dom) mouse is an animal model of HSCR, where a frameshift mutation in the Sox10 gene has been located. Recently, the generation of induced pluripotent stem cells (iPSCs) from patients has raised the possibility of using autologous cell transplantation to treat HSCR patients, if the gene mutation in patient‐derived iPSCs can be corrected before transplantation. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) gene editing technology was used to correct the Sox10 mutation in iPSCs derived from Sox10Dom/Dom embryos. 4 sgRNAs targeting the mutation site were designed, and a common donor plasmid carrying the correct sequence for the homology directed repair (HDR) was constructed. A SmaI digestion site was introduced to the correct sequence of the donor plasmid for detecting the HDR efficiency. The T7E1 assay demonstrated that the sgRNAs 1, 2 and 3 had high indel efficiency, and SmaI digestion showed 23.9% HDR efficiency. Using SmaI digestion, 8 homozygous and 4 heterozygous isogenic lines with the corrected Sox10 sequence were obtained, among which 3 homozygous corrected lines did not show meaningful off‐targets. Immunofluorescence staining showed that stem cell markers Oct4, Sox2 and SSEA‐1 were highly expressed in corrected iPSCs (Sox10Cor/Cor iPSCs). Neural crest cells (NCCs) are the progenitors of enteric neurons and glia cells. To determine the ability of iPSCs to differentiate into NCCs before and after the gene correction, a three‐step protocol was used to induce mouse iPSCs into NCCs, which were found to exhibit properties similar to enteric neural crest cells within the developing gut. Immunofluorescence staining showed that NCC markers Sox10, Nestin, AP2α and Ednrb were expressed in NCCs derived from Sox10+/+ iPSCs. Real time RT‐PCR also demonstrated that Sox10, p75 and Nestin were highly expressed in Sox10+/+ iNCCs. Flow cytometric cell analyses showed 25% p75‐positive cells in Sox10+/+ iPSCs‐induced NCCs. Immunofluorescence staining also showed that NCC markers Sox10, Nestin and Ednrb were expressed in NCCs derived from Sox10Dom/Dom and Sox10Cor/Cor iPSCs. Sox10+/+, Sox10Dom/Dom and Sox10Cor/Cor iPSCs‐derived NCCs were successfully directed to differentiate into neurons, glia, myocytes, osteoblasts and chondrocytes in vitro. Real‐time RT‐PCR showed rescued expression of nestin, p75 and Ednrb in Sox10Cor/Cor iPSCs‐derived NCCs when compared those derived from Sox10Dom/Dom. Our results demonstrated the successful use of CRISPR/Cas9‐mediated gene editing to correct the Sox10 mutation in Sox10Dom/Dom mouse iPSCs. After gene editing, the iPSCs could be induced to form neural crest cells for the potential use in cell transplantation to generate enteric neurons in the gut.Support or Funding InformationThe work was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No.: CUHK 14102214)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction of Human Enterochromaffin Cells with Human Enteric Adenovirus 41 Leads to Serotonin Release and Subsequent Activation of Enteric Glia Cells.

Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells.IMPORTANCE The nonenveloped human adenovirus 41 causes diarrhea, vomiting, dehydration, and low-grade fever mainly in children under 2 years of age. Even though acute gastroenteritis is well described, how human adenovirus 41 causes diarrhea is unknown. In our study, we analyzed the effect of human adenovirus 41 infection on human enterochromaffin cells and found it stimulates serotonin secretion in the cells, which is involved in regulation of intestinal secretion and gut motility and can also activate enteric glia cells, which are found in close proximity to enterochromaffin cells in vivo This disruption of gut barrier homeostasis as maintained by these cells following human adenovirus 41 infection might be a mechanism in enteric adenovirus pathogenesis in humans and could indicate a possible serotonin-dependent cross talk between human adenovirus 41, enterochromaffin cells, and enteric glia cells.

The Role of Enteric Glia Cells in Intestinal Regeneration After Mesenteric Ischemia and Reperfusion

Background: Acute occlusive mesenteric ischemia is a life-threatening disease and remains to be incompletely understood in modern gastrointestinal surgery. Recently it has been shown, that specifically enteric glia cells (EGCs) are important to maintain epithelial homeostasis in the intestine. After EGC ablation, lethal intestinal inflammation occurs. EGCs of various subtypes and locations interact with the gut microbiome, synthesize trophic factors (i.e. TGF-ß, GDNF) and potentially protect enteric neurons from I/R and oxidative stress. We hypothesize that EGC function may therefore be pivotal for regeneration of epithelial integrity, cell protection and recovery of intestinal function after I/R. EGC activation after intestinal ischemia and reperfusion (I/R) has been suggested, but the relevance of such a reactive “gliosis” in the intestine in analogy to an “astrocytosis” in the brain (i.e. after ischemic stroke) is not fully understood. We therefore aim to investigate the role of EGCs after I/R. Methods: We studied a superior mesenteric artery (SMA)-occlusion model in C57Bl/6 mice as well as in GFAP-GFP and GFAP-Td-Tomato reporter-mice, using the glial fibrillary acidic protein GFAP as an EGC marker. Anesthetized mice underwent median laparotomy and the SMA was identified and occluded with a microvascular clamp for 45 minutes. Intestinal samples were procured after 3 hours and 24 hours of reperfusion. To assess I/R injury, Parks’ score was performed in H/E stained paraffin slides and a standardized segment of ileum was tested for FITC dextran diffusion in an in-vitro assay confirming epithelial barrier breakdown. Immunofluorescence staining in intestinal whole mounts was performed for EGC markers. Intestinal samples were also analyzed for EGC related gene expression in the tunica muscularis. For further mechanistic studies, primary EGC cultures were established and subjected to in-vitro ischemic conditions. Results: Parks’ score showed a significant I/R injury in the intestines of SMA-clamped animals at both timepoints, concomitant to an epithelial barrier breakdown as tested in an in-vitro FITC dextran diffusion assay, compared to sham animals. Immunfluorescence staining of the EGC marker GFAP in intestinal tunica muscularis whole mounts showed a morphologic upregulation of GFAP filaments after 45 minutes of intestinal ischemia at 3h and 24h of reperfusion (mean fluorescence intensity MFI: clamp3h vs. sham3h and clamp24h vs. sham24; One Way Anova p<0.001). GFAP gene expression in the tunica muscularis of animals with SMA-clamping was significantly upregulated after 3h of reperfusion. Furthermore, in GFAP reporter mice (GFAP-GFP) gene expression of the well-established transcription factor and hypoxia marker HIF1-α was significantly upregulated after 3h and 24h of reperfusion (clamp3h vs. sham3h, unpaired t-test p<0.05; clamp24h vs. sham24h, unpaired t-test p<0.01), concomitantly to HIF1-α dependent genes such as TGFß1 and VEGF-α. It was also found, that several proinflammatory cytokines (IL-6, Il-1ß) and chemokines (MCP-1) were significantly upregulated after limited mesenteric ischemia both in the animal and in-vitro models.FigureFigureConclusion: Mesenteric ischemia and reperfusion activates GFAP-positive EGC potentially via HIF-1α dependent pathways and produces an inflammatory micro-environment. Further study of the interactions of EGC and the intestinal epithelium may provide new insights into the regenerative mechanisms of the bowel after limited mesenteric ischemia.

Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.

Morphine is one of the most widely used drugs for the treatment of pain. However, side effects, including persistent constipation and antinociceptive tolerance, limit its clinical efficacy. Prolonged morphine treatment results in a "leaky" gut, predisposing to colonic inflammation that is facilitated by microbial dysbiosis and associated bacterial translocation. In this study, we examined the role of enteric glia in mediating this secondary inflammatory response to prolonged treatment with morphine. We found that purinergic P2X receptor activity was significantly enhanced in enteric glia that were isolated from mice with long-term morphine treatment (in vivo) but not upon direct exposure of glia to morphine (in vitro). LPS, a major bacterial product, also increased ATP-induced currents, as well as expression of P2X4, P2X7, IL6, IL-1β mRNA in enteric glia. LPS increased connexin43 (Cx43) expression and enhanced ATP release from enteric glia cells. LPS-induced P2X currents and proinflammatory cytokine mRNA expression were blocked by the Cx43 blockers Gap26 and carbenoxolone. Likewise, colonic inflammation related to prolonged exposure to morphine was significantly attenuated by carbenoxolone (25 mg/kg). Carbenoxolone also prevented gut wall disruption and significantly reduced morphine-induced constipation. These findings imply that enteric glia activation is a significant modulator of morphine-related inflammation and constipation.-Bhave, S., Gade, A., Kang, M., Hauser, K. F., Dewey, W. L., Akbarali, H. I. Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.

Enteric glia cells are critical to limiting the intestinal inflammatory response after injury.

Vagal nerve stimulation (VNS) has been shown to limit intestinal inflammation following injury; however, a direct connection between vagal terminals and resident intestinal immune cells has yet to be identified. We have previously shown that enteric glia cell (EGC) expression is increased after injury through a vagal-mediated pathway to help restore gut barrier function. We hypothesize that EGCs modulate immune cell recruitment following injury and relay vagal anti-inflammatory signals to resident immune cells in the gut. EGCs were selectively ablated from an isolated segment of distal bowel with topical application of benzalkonium chloride (BAC) in male mice. Three days following BAC application, mice were subjected to an ischemia-reperfusion injury (I/R) by superior mesenteric artery occlusion for 30 min. VNS was performed in a separate cohort of animals. EGC+ and EGC- segments were compared utilizing histology, flow cytometry, immunohistochemistry, and intestinal permeability. VNS significantly reduced immune cell recruitment after I/R injury in EGC+ segments with cell percentages similar to sham. VNS failed to limit immune cell recruitment in EGC- segments. Histologic evidence of gut injury was diminished with VNS application in EGC+ segments, whereas EGC- segments showed features of more severe injury. Intestinal permeability increased following I/R injury in both EGC+ and EGC- segments. Permeability was significantly lower after VNS application compared with injury alone in EGC+ segments only (95.1 ± 30.0 vs. 217.6 ± 21.7 μg/ml, P < 0.05). Therefore, EGC ablation uncouples the protective effects of VNS, suggesting that vagal-mediated signals are translated to effector cells through EGCs.NEW & NOTEWORTHY Intestinal inflammation is initiated by local immune cell activation and epithelial barrier breakdown, resulting in the production of proinflammatory mediators with subsequent leukocyte recruitment. Vagal nerve stimulation (VNS) has been shown to limit intestinal inflammation following injury; however, direct connection between vagal terminals and resident intestinal immune cells has yet to be identified. Here, we demonstrate that intact enteric glia cells are required to transmit the gut anti-inflammatory effects of VNS.

Alteration of colonic excitatory tachykininergic motility and enteric inflammation following dopaminergic nigrostriatal neurodegeneration.

BackgroundParkinson’s disease (PD) is frequently associated with gastrointestinal (GI) symptoms, including constipation and defecatory dysfunctions. The mechanisms underlying such disorders are still largely unknown, although the occurrence of a bowel inflammatory condition has been hypothesized. This study examined the impact of central dopaminergic degeneration, induced by intranigral injection of 6-hydroxydopamine (6-OHDA), on distal colonic excitatory tachykininergic motility in rats.MethodsAnimals were euthanized 4 and 8 weeks after 6-OHDA injection. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. SP, tachykininergic NK1 receptor, and glial fibrillary acidic protein (GFAP) expression, as well as the density of eosinophils and mast cells in the colonic wall, were examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay), TNF, and IL-1β (ELISA assay) levels were also examined. The polarization of peritoneal macrophages was evaluated by real-time PCR.ResultsIn colonic preparations, electrically and SP-evoked tachykininergic contractions were increased in 6-OHDA rats. Immunohistochemistry displayed an increase in SP and GFAP levels in the myenteric plexus, as well as NK1 receptor expression in the colonic muscle layer of 6-OHDA rats. MDA, TNF, and IL-1β levels were increased also in colonic tissues from 6-OHDA rats. In 6-OHDA rats, the number of eosinophils and mast cells was increased as compared with control animals, and peritoneal macrophages polarized towards a pro-inflammatory phenotype.ConclusionsThe results indicate that the induction of central nigrostriatal dopaminergic degeneration is followed by bowel inflammation associated with increased oxidative stress, increase in pro-inflammatory cytokine levels, activation of enteric glia and inflammatory cells, and enhancement of colonic excitatory tachykininergic motility.

The enteric nervous system in patients with calculous and acalculous gallbladder

BackgroundIt is generally thought that gallbladder motility plays a more or less important role in the pathogenesis of gallstones. Some studies have shown that some abnormalities of its intrinsic innervations, but these studies were usually limited to one cell component. AimsWe investigated the main cell components of gallbladder intrinsic innervation in patients with and without gallstones. MethodsArchival gallbladder specimens from 39 patients, 27 with gallstones (age range 45–69 yrs) and 12 patients without gallstones (age range 39–71 yrs) were obtained. Full thickness sections were obtained from the gallbladder neck and immunohistochemistry was carried out for enteric neurons (neuron-specific enolase and calretinin), enteric glia (S100) and interstitial cells of Cajal (CD117 and CD34); tryptase staining was also done to distinguish the latter from mast cells. ResultsApart from calretinin-positive neurons, patients with gallstones featured a significant decrease of neurons, enteric glial cells (EGC) and mast cells compared to those without gallstones; interstitial cells of Cajal were extremely few and only found in two patients, one for each group. ConclusionsThe intrinsic innervations of the gallbladder is abnormal in gallstone patients, and this may contribute to gallstone formation in these subjects.

P015. Interaction between enteric glia and myeloid cells as critical players in intestinal immune homeostasis.

P015. Interaction between enteric glia and myeloid cells as critical players in intestinal immune homeostasis.

Glial Fibrillary Acidic Protein-Expressing Glia in the Mouse Lung.

Autonomic nerves regulate important functions in visceral organs, including the lung. The postganglionic portion of these nerves is ensheathed by glial cells known as non-myelinating Schwann cells. In the brain, glia play important functional roles in neurotransmission, neuroinflammation, and maintenance of the blood brain barrier. Similarly, enteric glia are now known to have analogous roles in gastrointestinal neurotransmission, inflammatory response, and barrier formation. In contrast to this, very little is known about the function of glia in other visceral organs. Like the gut, the lung forms a barrier between airborne pathogens and the bloodstream, and autonomic lung innervation is known to affect pulmonary inflammation and lung function. Lung glia are described as non-myelinating Schwann cells but their function is not known, and indeed no transgenic tools have been validated to study them in vivo. The primary goal of this research was, therefore, to investigate the relationship between non-myelinating Schwann cells and pulmonary nerves in the airways and vasculature and to validate existing transgenic mouse tools that would be useful for studying their function. We focused on the glial fibrillary acidic protein promoter, which is a cognate marker of astrocytes that is expressed by enteric glia and non-myelinating Schwann cells. We describe the morphology of non-myelinating Schwann cells in the lung and verify that they express glial fibrillary acidic protein and S100, a classic glial marker. Furthermore, we characterize the relationship of non-myelinating Schwann cells to pulmonary nerves. Finally, we report tools for studying their function, including a commercially available transgenic mouse line.

The Role of Toll‐Like Receptor 4 in Enteric Glia

Toll‐like receptors (TLRs) are a class of pattern recognition receptors that play an important role in mediating inflammatory responses to pathogens. TLR4 recognizes lipopolysaccharide (LPS), a membrane component of gram negative bacteria. In the GI tract, neuronal integrity may be dependent on basal activation of TLR4 receptors suggesting a protective role for TLR4. A compromised gut epithelial barrier leads to enhanced entry of bacterial proteins into the lamina propria, leading to neuronal toxicity. Thus pro‐survival vs pro‐toxic effects of LPS may be concentration‐dependent. Enteric glia are important for the maintenance of neuronal integrity. We determined the expression of pro‐inflammatory cytokines in rat enteric glia cells in response to low and high concentrations of LPS. LPS dose‐dependently increased mRNA expression of IL‐6 and TNF‐α with 0.1 μg/ml inducing a 25‐fold and 10‐fold IL‐6 and TNF‐α mRNA expression respectively as compared to untreated glia. A 100‐fold higher concentration of LPS (1‐100 μg/ml) also induced ATP release indicative of cell damage. Whole cell voltage clamp studies showed that this high concentration of LPS increased ATP‐induced inward currents with a corresponding increase in P2X1,2,3,4,and 7 receptor mRNA expression up to 50‐fold as compared to untreated glia. Thus low concentrations of LPS can induce cytokine release whereas high concentrations of LPS induce ATP release which increases P2X receptor activity and further enhances the release of pro‐inflammatory cytokines. Understanding the role of P2X in LPS induced enteric glia activation will point to new targets to control gut bacterial inflammation while still maintaining the beneficial effects of basal TLR4 signaling. Supported by NIH DK046367, DA024009.

Cholinergic signaling in the gut: a novel mechanism of barrier protection through activation of enteric glia cells.

Enteric glia cells (EGCs) play an important role in maintaining proper intestinal barrier function. We have shown that vagal nerve stimulation (VNS) increases EGC activation, which is associated with better gut barrier integrity. Enteric neurons communicate with EGCs through nicotinic cholinergic signaling, which may represent a pathway by which VNS activates EGCs. This study sought to define further the mechanism by which VNS prevents intestinal barrier failure using an in vitro model. We hypothesized that a nicotinic cholinergic agonist would increase EGC activation, prevent intestinal nuclear factor kappa-B (NF-κB) activation, and result in better intestinal barrier function. Cultured EGCs were exposed to the nicotinic cholinergic agonist nicotine. Expression of glial fibrillary acidic protein (GFAP) was measured by immunoblot to determine changes in EGC activation. Caco-2 cells were grown to confluence and incubated alone or in co-culture with EGCs. Cells were then stimulated with Cytomix for 24 h in the presence or absence of nicotine, and barrier integrity was assessed by permeability to 4-kDa FITC-dextran. Changes in phosphorylated inhibitor of NF-κb (P-IκBα) and phosphorylated NF-κB (P-NF-κB) were assessed by immunoblot. Stimulation with nicotine resulted in EGC activation, as demonstrated by an increase in GFAP expression. Cytomix stimulation increased permeability in Caco-2 cells cultured alone or with EGCs. Treatment of stimulated Caco-2/EGC co-cultures with nicotine reduced permeability similar to control. Nicotine failed to prevent barrier permeability in Caco-2 cells alone. Co-culture of stimulated Caco-2 cells with nicotine-activated EGCs prevented Cytomix-induced increases in P-IκBα and P-NF-κB expression. A pharmacologic nicotinic cholinergic agonist increased EGC activation and improved intestinal epithelial barrier function in an in vitro model of intestinal injury. Nicotine-activated EGCs appear to modulate barrier function by preventing the activation of the NF-κB pathway. Therapies aimed at activating EGCs may have important clinical applications for improving intestinal barrier function after injury.

In vivo transplantation of neurosphere-like bodies derived from the human postnatal and adult enteric nervous system: a pilot study.

Recent advances in the in vitro characterization of human adult enteric neural progenitor cells have opened new possibilities for cell-based therapies in gastrointestinal motility disorders. However, whether these cells are able to integrate within an in vivo gut environment is still unclear. In this study, we transplanted neural progenitor-containing neurosphere-like bodies (NLBs) in a mouse model of hypoganglionosis and analyzed cellular integration of NLB-derived cell types and functional improvement. NLBs were propagated from postnatal and adult human gut tissues. Cells were characterized by immunohistochemistry, quantitative PCR and subtelomere fluorescence in situ hybridization (FISH). For in vivo evaluation, the plexus of murine colon was damaged by the application of cationic surfactant benzalkonium chloride which was followed by the transplantation of NLBs in a fibrin matrix. After 4 weeks, grafted human cells were visualized by combined in situ hybridization (Alu) and immunohistochemistry (PGP9.5, GFAP, SMA). In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature. Contractility of treated guts was assessed in organ bath after electrical field stimulation. NLBs could be reproducibly generated without any signs of chromosomal alterations using subtelomere FISH. NLB-derived cells integrated within the host tissue and showed expected differentiated phenotypes i.e. enteric neurons, glia and smooth muscle-like cells following in vivo transplantation. Our data suggest biological effects of the transplanted NLB cells on tissue contractility, although robust statistical results could not be obtained due to the small sample size. Further, it is unclear, which of the NLB cell types including neural progenitors have direct restoring effects or, alternatively may act via ‘bystander’ mechanisms in vivo. Our findings provide further evidence that NLB transplantation can be considered as feasible tool to improve ENS function in a variety of gastrointestinal disorders.

An Enteroendocrine Cell – Enteric Glia Connection Revealed by 3D Electron Microscopy

The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY – both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia – the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

Enteric glial cells and their role in the intestinal epithelial barrier.

The intestinal epithelium constitutes a physical and functional barrier between the external environment and the host organism. It is formed by a continuous monolayer of intestinal epithelial cells maintained together by intercellular junctional complex, limiting access of pathogens, toxins and xenobiotics to host tissues. Once this barrier integrity is disrupted, inflammatory disorders and tissue injury are initiated and perpetuated. Beneath the intestinal epithelial cells lies a population of astrocyte-like cells that are known as enteric glia. The morphological characteristics and expression markers of these enteric glia cells were identical to the astrocytes of the central nervous system. In the past few years, enteric glia have been demonstrated to have a trophic and supporting relationship with intestinal epithelial cells. Enteric glia lesions and/or functional defects can be involved in the barrier dysfunction. Besides, factors secreted by enteric glia are important for the regulation of gut barrier function. Moreover, enteric glia have an important impact on epithelial cell transcriptome and induce a shift in epithelial cell phenotype towards increased cell adhesion and cell differentiation. Enteric glia can also preserve epithelial barrier against intestinal bacteria insult. In this review, we will describe the current body of evidence supporting functional roles of enteric glia on intestinal barrier.