4.1 General trends
To better understand the current status of enteric glia research, this study examined 514 relevant original articles in the WoSCC database over the past 20 years (2003–2022) based on bibliometrics. The rapid growth in publications and citations indicate that enteric glia as an emerging focus is receiving sustained attention from scholars around the world. We analyzed the most influential countries, institutions, and authors in the field of enteric glia. The total number of publications from the United States, Italy, and China accounted for more than half of the total, among which the United States, as the country with the highest number of publications, influence, and level of cooperation in this field, had cooperative relations with 21 countries/regions, and the closest cooperation with Italy. Although China ranked third in the world in terms of the total number of publications, its level of international cooperation was relatively inadequate. In the institutional collaboration network, University of Nantes and INSERM from France and Michigan State University from the United States were the top 3 institutions with the number of publications. Notably, most collaborations of the publications were from domestic organizations, with a noticeable absence of institutional collaboration across different countries. Therefore, strengthening exchanges and cooperation between countries and institutions is particularly important to promote further development. Michel Neunlist from INSERM and Brian D. Gulbransen from Michigan State University were the most active authors in this field. As the most influential author, Michel Neunlist was mainly engaged in the intrinsic connection between EGCs and the pathogenesis of PD (Devos et al., 2013; Clairembault et al., 2014) and CD (Le Loupp et al., 2015; Pochard et al., 2016), while Brian D. Gulbransen focused on exploring how EGCs participate in neuronal regulation of intestinal motility (Gulbransen and Sharkey, 2009; McClain et al., 2014; Delvalle et al., 2018b) and how they regulate immune cells to affect intestinal inflammation (Grubišić et al., 2020; Chow et al., 2021).
In addition, we identified the top 10 articles of enteric glia, most of which focus on the mechanism of EGCs in neuro-immune interactions or inflammation. The most cited paper was published in Neurobiology of Disease by David Devos in 2013, which innovatively found increased expression levels of enteric glial markers glial fibrillary acidic protein (GFAP) and SOX10 in the colon of PD patients and proposed a new theory that EGCs may be involved in the occurrence and development of PD and related intestinal dysfunction (Devos et al., 2013). The paper with the highest average annual citations was published in Nature Medicine in 2017 Workman et al. (2017). The study was the first to link human pluripotent stem cell-derived neural crest cells with functional ENS to human intestinal organoids and successfully differentiate into new neurons and glial cells in the intestinal mesenchyme (Workman et al., 2017). It may provide a new treatment model for diseases with enteric glia as their pathologic basis.
4.2 Knowledge base
The analysis of co-cited references was used to investigate the research foundation in the field of enteric glia. Based on the number of co-citations, we identified high-impact references in enteric glia research, which represent the main focus of researchers in the knowledge base. The top 10 most-cited references included 5 reviews and 5 original articles. These 5 reviews summarized and supplemented the new findings of enteric glia in intestinal motility, neuron-glia communication and intestinal epithelial barrier (IEB) in different years (2012–2017), and emphasized that its functional performance in enteric neuro-immune interactions and some enteral and parenteral diseases. Laranjeira et al. (2011) found that enteric glia and neurons in the mouse intestine were derived from neural crest precursor cells expressing the transcription factor Sox10. The neurogenic potential of these cells was highest in the second trimester and gradually disappeared between 1 and 3 months after birth. Interestingly, they also found that most mature EGCs continued to maintain Sox10 expression in adult mice and acquired the ability to generate functional enteric neurons in vitro culture and in vivo after enteric ganglion injury (Laranjeira et al., 2011). These findings provided a novel theoretical framework for the treatment of diseases related to intestinal neuron degeneration (such as IBD) and diseases related to intestinal neuron loss (such as Hirschsprung’s disease). McClain et al. (2014) were the first to demonstrate that connexin-43 hemichannels mediated purinergic signaling-induced Ca2+ responses in mouse colon EGCs, connexin-43 ablation resulted in Ca2+ wave propagation disorder in the glial network, consequently leading to delayed colonic transit and reduced isometric force generation in the intestine. This study offered significant molecular insights for targeting enteric glia to regulate gastrointestinal motility. By using Mosaic Analysis with Double Markers, fate mapping, lineage tracing, and Ca2+ imaging, Boesmans et al. (2015) showed that EGCs in mammals exhibited different molecular and functional characteristics based on differences in morphology and location and had a certain degree of phenotypic plasticity, which suggests that the unique microenvironment of the intestinal wall may be a determinant of the phenotype of EGCs and its typical biomarker expression. The data of Brown et al. (2016) confirmed that enteric glia promoted neuroinflammation and subsequent neuronal death. They found that intestinal neuroinflammation activated neuronal P2X7 receptors and led to the release of neuronal adenosine triphosphate (ATP) in pannexin 1 channels, which was then rapidly hydrolyzed to adenosine diphosphate (ADP) by eNTPDase2 to activate glial P2Y1 receptors and glial cell activity in the gut. They also found that glial P2Y1 receptors enhanced connexin-43 hemichannel-dependent ATP release by driving inducible nitric oxide synthase (iNOS) to produce nitric oxide (NO), thereby promoting neuronal death and neuroinflammatory processes (Brown et al., 2016). Rao et al. (2017) used the proteolipid protein 1 promoter to deplete mouse enteric glia. They found that, in addition to increasing the frequency of colonic migrating motor complexes (CMMCs) in female mice, it did not lead to changes in intestinal epithelial proliferation, intestinal barrier, and intestinal neuron survival. These results argued against the necessity of EGCs to maintain IEB integrity and survival of enteric neurons under homeostatic conditions and were consistent with Grubišić and Gulbransen (2017b).
In order to explore the hot papers in the knowledge base of enteric glia, we also screened out the top 15 references with the strongest citation bursts, including the top 10 references with the most citations. “Isola A. M. Brown (2016)” (Brown et al., 2016), “Fernando Ochoa-Cortes (2016)” (Ochoa-Cortes et al., 2016), “Vladimir Grubisic (2017)” (Grubišić and Gulbransen, 2017a), and “Meenakshi Rao (2017)” (Rao et al., 2017), as high-impact references, remained in citation bursts until 2022, suggesting that clinical and basic research workers in enteric glia need to pay more attention to the research directions involved in these articles. In summary, the above references provide essential theoretical support for the current paradigm of understanding the role of enteric glia in digestive physiology and pathophysiology.
4.3 Research hotspots and frontiers
We further analyzed the research hotspots and frontiers in enteric glia through keyword co-occurrence analysis. The keyword co-occurrence map suggests that the understanding of enteric glia function in the past 20 years was mainly focused on neuron-glia communication, intestinal motility, inflammation, and IEB, which were associated with intestinal diseases (Hirschsprung’s disease, IBD, IBS), infectious diseases (Clostridioides difficile infection, Chagas disease) and parenteral diseases. It is worth pointing out that CD in IBD, as the keyword with the strongest burst, was one of the hot topics in early enteric glia research. Based on tissue biopsy, researchers found that the gliosis in the inflammatory area of intestinal mucosa in patients with CD was far less evident than that in patients with UC, and the EGC density in uninvolved intestinal mucosal areas was lower than that in the control group (Cornet et al., 2001; von Boyen et al., 2011). The above evidence suggests that CD may be a form of enteric gliopathy associated with disruption of the glial cell network. The studies describing the involvement of EGCs in the pathogenesis of CD highlight their cellular properties in the IEB, glia-immune interactions, and neuroplasticity. These processes are expected to drive the development of further therapeutic strategies for CD. In addition, the results of the burst detection of keywords revealed that “inflammation,” “intestinal motility,” “receptor,” “gut microbiota,” and “activation” are among the prominent areas in enteric glia research, reflecting recent advancements in this field. Here, discussion is focused on the potential of enteric glia in inflammation, intestinal motility, and gut microbiota.
4.3.1 Inflammation
The resolution of inflammation is necessary for the maintenance of mucosal integrity. Based on glial ablation animal models, earlier studies evaluating the functional significance of the glial network in intestinal homeostasis found a functional link between enteric glia and inflammation. For example, the depletion of enteric glia in transgenic mice expressing thymidine kinase gene of the herpes simplex virus (HSV-Tk) from the mouse GFAP promoter (Bush et al., 1998) and in autoimmune mice targeting CD8+ T cells (Cornet et al., 2001) led to the rapid development of fulminant jejuno-ileitis and even death. Although the absence of glia in both models is associated with severe colonic inflammation, unknown methodological effects on intestinal epithelium, neurons, and immune cells diminish the importance of the data obtained from these glial ablation animal models. Ablation of enteric glia also obscures a clear understanding of the cellular properties of specific subtypes, and more selective methods are needed to elucidate the role of glial signaling in the ENS homeostasis.
What is clear is that EGCs are actively involved in intestinal inflammation in a variety of diseases, such as IBD, postoperative ileus (POI), and bacterial and viral infections, from which they derive a “reactive EGC phenotype”. Depending on the severity and type of inflammation, its location in the intestine, and the glial signals transmitted, the context-dependent and disease-specific enteric glial network responds to pathological perturbations by changing its molecular expression, structure, and/or function, and in severe cases reactive enteric gliosis occurs. The current assessment of reactive EGCs relies on the expression of biomarkers such as GFAP and S100β, as well as morphological changes. Notably, nuclear factor kappa-B (NF-κB) signaling associated with increased S100β, as the primary pathway of pro-inflammatory stimuli in EGCs, has been proven to be associated with dextran sodium sulphate induced colitis (Esposito et al., 2014), enteroinvasive Escherichia coli infection (Turco et al., 2014), UC(Cirillo et al., 2009; Esposito et al., 2014), human immunodeficiency virus-1-induced diarrhea (Sarnelli et al., 2018), and colon carcinoma (Seguella et al., 2019; Seguella et al., 2020). Additionally, the potential of enteric glia S100β in promoting NF-κB transcription and sequestrating pro-apoptotic wild-type p53 is considered an ideal bridge between mucosal inflammation and colorectal cancer, necessitating further investigation (Seguella et al., 2019; Seguella et al., 2020).
By releasing damaging and/or protective factors, reactive EGCs interact with the nervous and immune systems to exert a dual modulatory action in intestinal inflammation (Ochoa-Cortes et al., 2016). For example, EGCs-derived NO is a key mediator of neurodegeneration (Brown et al., 2016) and epithelial barrier dysfunction (MacEachern et al., 2015) during intestinal inflammation, with its production being tightly regulated by toll-like receptor (TLR) (Turco et al., 2014) and S100β/receptor for advanced glycation end products (RAGE) signaling pathways (Cirillo et al., 2009). Specifically, the overproduced S100β in reactive EGCs enters the cell through binding with RAGE on the surface of EGCs. Subsequently, it interacts with MyD88 downstream of TLR, leading to NF-κB activation and translocation into the nucleus. This process drives the upregulation of iNOS activity and triggers downstream release of NO. Furthermore, conclusive data confirm that the improvement of peroxisome proliferator-activated receptor-alpha (PPAR-α) agonist palmitoylethanolamide in UC and colitis mouse models is attributed to its inhibitory effect on NO production in EGCs. This finding suggests that targeting NO production in the enteric glia may be a potential new therapeutic strategy for colitis (Esposito et al., 2014). In addition, extracellular ATP, functioning as a purinergic component actively involved in neurogenic inflammation, serves as a bidirectional messenger facilitating communication between intestinal neurons and EGCs. Through pannexin 1 channels, neurons release high concentrations of the neurotransmitter ATP to drive neurogenic inflammation and stimulate glial cell activity encoded by intracellular Ca2+ responses. In turn, the Ca2+ response of enteric glia triggers the release of the gliotransmitter ATP through NO-controlled connexin-43 hemichannels, thereby inducing the continuation and amplification of neurogenic inflammation (Gulbransen et al., 2012). Some evidence suggests that P2X7 receptor (P2X7R) in the ENS are involved in the pathophysiology of intestinal inflammation. However, the location of P2X7R is still controversial. Earlier studies assumed that P2X7R in enteric neurons is involved in neuronal cell death and colitis-related motor dysfunction (Gulbransen et al., 2012; Brown et al., 2016). In contrast, a recent study pointed out that P2X7R is mainly expressed in macrophages and S100β-positive EGCs and denied the idea that P2X7R exists in enteric neurons (Jooss et al., 2023). In a short phase IIa clinical study (Eser et al., 2015), the oral P2X7R antagonist AZD9056 reduced abdominal pain in patients with moderate to severe CD, indicating that the blocking of P2X7R contributed to the reduction of visceral pain. However, P2X7R blocking resulted in an increased incidence of colitis-related cancers in mice (Hofman et al., 2015). Therefore, the efficacy and adverse effects of long-term use of P2X7R antagonists need to be further evaluated in subsequent studies.
Interestingly, despite exhibiting enhanced pro-inflammatory function during intestinal inflammation, reactive EGCs release factors such as glial-derived neurotrophic factor (GDNF)(Zhang et al., 2010), glial-derived s-nitrosoglutathione (GSNO) (Savidge et al., 2007), 15-hydroxyeicosatetraenoic acid (Pochard et al., 2016), and 11β-prostaglandinF2α (Coquenlorge et al., 2016) that contribute to the preservation of intestinal barrier integrity. This suggests that reactive EGCs may represent an endeavor to preserve intestinal homeostasis and could be associated with the functional status of specific subtypes of EGC. Additionally, the activation of adenosine A2B receptors on EGCs contributes to the release of GDNF, substance P, and IL-1β, thereby reducing intestinal inflammation associated with obesity induced by high fat diet (Antonioli et al., 2020; D'Antongiovanni et al., 2020). The specific mechanism of enteric glial-immune interactions in intestinal inflammation remains poorly understood. Some data show that reactive EGCs actively participate in intestinal immune response by expressing immune response-related genes and secreting inflammatory mediators. They also play a regulatory role in both innate and adaptive immune cells, including macrophages (Grubišić et al., 2020), type 3 innate lymphoid cells (ILC3) (Ibiza et al., 2016), NK cells (Progatzky et al., 2021), and T cells (Cornet et al., 2001; Kermarrec et al., 2016). Therefore, restoring normal EGC function may be an effective strategy to suppress inflammation and may be a clinical target for intestinal inflammatory diseases such as IBS.
4.3.2 Intestinal motility
Abnormalities in intestinal motility are broadly defined as failure to maintain local homeostasis in the ENS, including EGCs. A study from 1985 initially proposed that EGCs are involved in the regulation of intestinal motility based on the phenomenon of diarrhea caused by the degeneration of EGCs in suckling mice (Aikawa and Suzuki, 1985). The pivotal role of neurons in the ENS has long been established, while the beneficial impact of EGCs on intestinal motility has only recently started to be unraveled. With the visualization of Ca2+ imaging (Gulbransen and Sharkey, 2009; Boesmans et al., 2019), researchers have discovered that neuronal excitation during intestinal movement facilitates the diffusion of EGCs activity. In contrast to neurons that generate action potentials, the excitability of EGCs responsible for sensing neuronal activity is determined by intracellular Ca2+ responses and transmitted between cells through Ca2+ signals, Ca2+ oscillations or Ca2+ waves in enteric neural circuits. In GFAPhM3Dq transgenic “DREADD” (a designer receptor activated only by the designer drug) mice, selective activation of the Gq-coupled receptors dependent Ca2+ response in GFAP-positive EGCs induced neurogenic smooth muscle contractions, CMMCs, and colonic motility, suggesting that Ca2+ signaling to enteric glia enhances motor neurocircuits within the gastrointestinal tract (McClain et al., 2015). The neural regulation of intestinal motility relies on the coordination of neuron-to-glia communication, in which EGCs activity encoded by the intracellular Ca2+ response is stimulated by the release of neurotransmitters involved in fast excitatory neurotransmission, such as ATP (McClain et al., 2014), acetylcholine (Delvalle et al., 2018b), and serotonin (Okamoto et al., 2014). The Ca2+ response of EGCs subsequently triggers the release of gliotransmitters, including ATP (Brown et al., 2016) and GABA (Fried et al., 2017), which modulate intestinal nerve reflexes. Furthermore, the involvement of EGCs in intestinal motility, particularly their interactions with non-neuronal cell types such as interstitial cells of Cajal within the intestinal wall, poses a significant question for future research regarding its impact on intestinal reflexes.
The reduction of EGCs not only contributes to the impairment of neuron-glia communication, but also compromises their ability to support neuronal survival and promote neurogenesis, all of which are crucial factors in the development of intestinal dysmotility. Current evidence suggests that a decrease in EGCs is associated with reduced intestinal motility (Aubé et al., 2006; Nasser et al., 2006; Rao et al., 2017) and linked to disturbances in movement patterns observed in individuals with slow-transit constipation, diverticular disease, Chagas disease, and idiopathic megacolon (Bassotti et al., 2005; Bassotti et al., 2006; Iantorno et al., 2007). However, the factors that exacerbate EGCs loss are unclear and appear to be influenced by dietary factors (Baudry et al., 2012) and the process of aging (McClain et al., 2014). The comprehension of the mechanisms by which EGCs facilitate neuronal survival and neurogenesis while maintaining their differentiation status holds significant implications for advancing the treatment of diseases. Additionally, reactive gliosis occurs in constipation associated with PD (Clairembault et al., 2015), POI(Stoffels et al., 2014), and intestinal dysmotility in inflammatory conditions (Ochoa-Cortes et al., 2016), in which manipulation of specific glial signaling mechanisms has a significant effect on intestinal motility. For example, the selective ablation of connexin-43 restricts purinergic signaling in the glial network and weakens colonic transit under normal intestinal reflexes (Brown et al., 2016). The antagonism of interleukin-1 (IL-1) receptor reduces the expression of interleukin-6 (IL-6) and monocyte chemotactic protein 1 in EGCs, thereby preventing local immune cell infiltration and postoperative intestinal obstruction (Stoffels et al., 2014). These EGCs signaling molecules represent potential new therapeutic targets for gastrointestinal motility disorders.
4.3.3 Gut microbiota
Under physiological conditions, the commensal microbiota residing in the intestine assists the absorption of nutrients and prevents the damage of IEB by pathogenic bacteria through competitive inhibition [74]. As an important component of intestinal homeostasis, gut microbiota also contributes to the maintenance of the ENS development and maturation (Kabouridis and Pachnis, 2015; Kayama et al., 2020). Recent data support for the role of gut microbiota in promoting migration of EGCs from the nerve plexus within the intestinal wall to the intestinal mucosa (Kabouridis et al., 2015). Furthermore, it has been observed that depletion of gut microbiota due to antibiotic exposure results in a non-selective loss of intestinal neurons and region-specific reduction in EGCs(Vicentini et al., 2021), thereby raising additional concerns regarding the intricate relationship between gut microbiota and EGCs. The immune tolerance induced by regulatory T cells and anti-inflammatory cytokines enables the commensal microbiota in the gut to evade immune activation, while being closely monitored by host immunity (Kayama et al., 2020). The increased intestinal permeability and barrier dysfunction associated with microbial dysbiosis result in a diminished host tolerance to gut microbiota, in which EGCs are thought to be related to the intestinal immune response against invasive microorganisms. Studies in humans have reported the abnormal colonization of adherent-invasive E. coli (AIEC) in individuals with CD (Palmela et al., 2018). Furthermore, the activation of AIEC in EGCs induces TLR/S100B-RAGE dependent NO signaling, which may be an important pathway for CD infection (Turco et al., 2014). Interestingly, EGCs also exert a protective effect in the invasion of intestinal pathogens. For example, data obtained from human colonic explants and intestinal epithelial cells indicate that GSNO reduces damage to the intestinal epithelial barrier and inflammatory response caused by Shigella flexneri invasion (Flamant et al., 2011). GDNF also controls neurotrophic factors and innate IL-22 by activating RET in ILC3, thereby regulating mucosal homeostasis and anti-microbial defense of the intestine (Artis and Spits, 2015; Ibiza et al., 2016).
The rapid development of microbiome technology has revolutionized the traditional understanding of the gut-brain (GB) axis into a systematic biological perspective of MGB axis interactions (Mayer et al., 2022). Through the bidirectional action of the GB axis, gut microbiota disorder repeatedly impacts psychological states via central nervous system (CNS) feedback, while emotional stress in turn affects intestinal motility, permeability, and visceral sensitivity (Mayer et al., 2022). The interaction of EGCs with gut microbiota in neuroinflammation and psychiatric disorders has led to a reevaluation of the pathophysiology of several diseases, with a focus on PD (Claudino Dos Santos et al., 2023) and IBS(Jeffery et al., 2012; Meira de-Faria et al., 2021; Han et al., 2022). For example, gastrointestinal manifestations associated with gut microbiota precede motor symptoms and disease diagnosis in PD, accompanied by an upregulation in GFAP expression and phosphorylation levels associated with EGCs (Clairembault et al., 2014). According to the Braak hypothesis, reactive gliosis driven by α-synuclein or other neurotropic agents may contribute to the progression of the progression of PD to the CNS by exacerbating local inflammatory responses and synaptic dysfunction in the gut (Claudino Dos Santos et al., 2023). The prevalence of mood disorders in IBS patients and their general neglect significantly contribute to the poor actual benefit of clinical treatments (Staudacher et al., 2021). Altered gut microbiota may be both a product of IBS pathology and a potential factor in the onset and progression of the disease. microbe-associated molecular patterns of gut microbiota, such as lipopolysaccharides, lipoproteins and/or flagellin, initiate a sustained inflammatory state in the ENS and CNS by contacting pattern recognition receptors in intestinal immune cells or EGCs to trigger the release of local cytokines, ultimately leading to increased visceral pain perception and sensitivity (Aguilera-Lizarraga et al., 2022). Future targets may involve the effects of gut microbiota on specific EGCs, which may be involved in the pathogenesis of GB-related diseases. In conclusion, exploring the role of EGCs interactions with gut microbiota in gut physiology and pathophysiology may unveil novel and intriguing avenues for research.