BACKGROUND AND AIMS: Enteric glial cells (EGCs) regulate gastrointestinal homeostasis and inflammation. While activated EGCs have been shown to support epithelial and immune balance in preclinical models, their functional status and turnover in inflammatory bowel diseases (IBD) remain poorly defined. This study aimed to identify EGC activation markers and assess their susceptibility to cytokine-driven death in IBD.

METHODS: We analysed 390 intestinal samples from IBD patients using bulk and single-nucleus RNA sequencing and validated findings across public datasets comprising over 1,160 patients and 19,000 EGC transcriptomes. We used multiple mouse models of gut inflammation, reporter-based glial sorting, transcriptomics, and glia-specific Casp8 deletion to dissect mechanisms of EGC activation and death. Ex vivo stimulation of sorted EGCs was used to assess cytokine-specific effects.

RESULTS: We identified novel IBD subtype- and location-specific EGC activation markers, including osteopontin (SPP1), enriched in ulcerative colitis. Single-nucleus and single-cell data revealed that activated EGC clusters selectively upregulate cell death signatures with IBD EGC displaying necroptosis via phosphorylation of MLKL. In mice, acute Th1/Th17-driven inflammation rapidly induced EGC activation and necroptosis, impairing intestinal motility. Ex vivo, IFN-γ and TNF co-stimulation, but not individual cytokines, induced MLKL-dependent necroptosis in EGCs. Casp8-deficient EGCs were hypersensitive to TNF-induced death, confirming a Caspase-8 -dependent survival checkpoint.

CONCLUSION: A proportion of activated EGC are selectively eliminated in IBD via cytokine-mediated necroptosis, driven by a coordinated IFN-γ/TNF axis. This process compromises enteric glial support functions and may contribute to IBD-associated dysmotility. Targeting glial survival may represent a novel therapeutic avenue.

The coordinated transit of intestinal contents is crucial for digestion and host defense, and is regulated by cross-talk between neural circuits, the muscular gut wall and luminal factors. Here we show that androgen signaling to Nos1+ enteric neurons and Scn10a+ spinal afferent neurons is required for normal intestinal transit in mice and is microbiome dependent. Microbial depletion with antibiotics abolished androgen receptor expression in enteric neurons, diminished serum testosterone and caused dysmotility. Androgens were necessary for antibiotics to affect transit and partly sufficient to rescue dysmotility. Nos1 neurons upregulate androgen receptor upon puberty in parallel with shifts in fecal bacterial beta-glucuronidase (GUS) enzymes that can deconjugate steroid glucuronides in mice and humans. Intracolonic administration of a GUS enzyme found to metabolize androgen glucuronides was sufficient to restore neuronal androgen signaling in microbe-depleted mice. Thus, gut microbial reactivation of host-excreted androgens via GUS enzymes represents a dynamic microbe-host interaction that is essential for peripheral nervous system function in homeostasis.

One of the largest glial populations outside the brain is in the gut. These enteric glia are involved in many functions, from intestinal peristalsis to immunity, yet it is unclear whether subtypes exist with distinct roles in homeostasis. Comparing glia from divergent microenvironments in the mouse intestine, we found that mucosal glia most resembled microglia, while muscularis glia resembled satellite glia. Tacr3, encoding the receptor for neuropeptide neurokinin B (NKB), was enriched within muscularis glia associated with neuronal soma and was undetectable in extraintestinal glia. Genetic or pharmacological manipulation of NKB-TACR3 signaling disrupted the establishment of enteric glial populations during postnatal development and dynamically modulated intestinal motor behaviors in adult mice. Collectively, we delineate spatially, transcriptionally, and functionally distinct populations of enteric glia; identify one as an unanticipated target of TACR3 antagonists in clinical use; and establish this pathway as necessary for enteric glial diversification and function.

Enteric glia, integral to the enteric nervous system (ENS), are crucial for intestinal motility, secretomotor function, and immunity. These glia occupy diverse niches from the serosa to the lumen, yet their transcriptional diversity across these compartments remains incompletely understood. Traditionally, studies on enteric glia have mostly focused on the myenteric plexus, omitting the large populations of glia located in the circular muscle and mucosa. Here, we present a method to rapidly isolate enteric glia from the muscularis externa and mucosal compartments of the mouse intestine in tandem, allowing direct comparative and compartment-specific analyses of each population. This protocol circumvents the need for traditional and labor-intensive longitudinal muscle myenteric plexus (LMMP) peeled tissue preparations by enabling rapid cell isolation suitable for many downstream applications, ranging from transcriptional profiling to cell culture. We detail a protocol for mechanically separating adult small intestinal layers, dissociating cells from the mucosal and muscularis compartments, and sorting these cells by flow cytometry. This method is applicable to both small and large intestines and has been validated in mice from weaning to adulthood. The ability to isolate distinct populations of enteric glia will enable functional interrogation of their niche-specific roles, advancing our understanding of their diverse contributions to ENS biology and pathology.

Intestinal stem cells (ISCs) reside in regionally variable niches that provide diverse microenvironmental cues such as tissue oxygen status, and morphogen signaling. Integration of these cues with ISC metabolism and fate remains poorly understood. Here, we show that cellular redox balance orchestrates niche factors with metabolic state to govern cell fate decisions. We demonstrate that hypoxia and Wnt signaling synergistically restrict the reactive oxygen species generating enzyme NADPH oxidase 1 (NOX1) regionally to the crypt base in the distal colon. NOX1 enables maintenance of an oxidative cell state that licenses cell cycle entry, altering the balance of asymmetric ISC self-renewal and lineage commitment. Mechanistically, cell redox state directs a self-reinforcing circuit that connects hypoxia inducible factor 1α-dependent signaling with post-translational regulation of the metabolic enzyme isocitrate dehydrogenase 1. Our studies show redox balance acts as a cellular rheostat that is central and causative for metabolic control of the ISC cell-cycle.

Enteric nervous system (ENS)-derived neuropeptides modulate immune cell function, yet our understanding of how inflammatory cues directly influence enteric neuron responses during infection is considerably lacking. Here, we characterized a primary enteric sensory neuron (PSN) subset producing the neuropeptides neuromedin U (NMU) and calcitonin gene-related peptide β (CGRPβ) and coexpressing receptors for the type 2 cytokines interleukin-4 (IL-4) and IL-13. Type 2 cytokines amplified NMU and CGRPβ expression in PSNs, in vitro and in vivo, which was abrogated by PSN-specific Il13ra1 deletion. Deletion of Il13ra1 in PSNs impaired host defense to the gastrointestinal helminth Heligmosomoides polygyrus and blunted muscularis immune responses. Co-administration of NMU23 and CGRPβ rescued helminth clearance deficits and restored anti-helminth immunity, highlighting the essential bi-directional neuro-immune crosstalk regulating intestinal type 2 inflammation.

Neurons innervating the gut are on the frontlines of host-microbe interactions and thus exposed to a myriad of inflammatory and infectious insults. In this issue of Neuron, Forster, Jakob et al.1 reveal that diverse populations of gut-innervating neurons exhibit conserved responses to inflammation, linking interferon signaling to ferroptosis.

Enteric glia are a unique type of peripheral neuroglia that accompany neurons in the enteric nervous system (ENS) of the digestive tract. The ENS displays integrative neural circuits that are capable of governing moment-to-moment gut functions independent of input from the central nervous system. Enteric glia are interspersed with neurons throughout these intrinsic gut neural circuits and are thought to fulfill complex roles directed at maintaining homeostasis in the neuronal microenvironment and at neuroeffector junctions in the gut. Changes to glial functions contribute to a wide range of gastrointestinal diseases, but the precise roles of enteric glia in gut physiology and pathophysiology are still under examination. This review summarizes current concepts regarding enteric glial development, diversity, and functions in health and disease.

Glial cells of the enteric nervous system (ENS) interact closely with the intestinal epithelium and secrete signals that influence epithelial cell proliferation and barrier formation in vitro. Whether these interactions are important in vivo, however, is unclear because previous studies reached conflicting conclusions (Prochera and Rao, 2023). To better define the roles of enteric glia in steady state regulation of the intestinal epithelium, we characterized the glia in closest proximity to epithelial cells and found that the majority express the gene Proteolipid protein 1 (PLP1) in both mice and humans. To test their functions using an unbiased approach, we genetically depleted PLP1+ cells in mice and transcriptionally profiled the small and large intestines. Surprisingly, glial loss had minimal effects on transcriptional programs and the few identified changes varied along the gastrointestinal tract. In the ileum, where enteric glia had been considered most essential for epithelial integrity, glial depletion did not drastically alter epithelial gene expression but caused a modest enrichment in signatures of Paneth cells, a secretory cell type important for innate immunity. In the absence of PLP1+ glia, Paneth cell number was intact, but a subset appeared abnormal with irregular and heterogenous cytoplasmic granules, suggesting a secretory deficit. Consistent with this possibility, ileal explants from glial-depleted mice secreted less functional lysozyme than controls with corresponding effects on fecal microbial composition. Collectively, these data suggest that enteric glia do not exert broad effects on the intestinal epithelium but have an essential role in regulating Paneth cell function and gut microbial ecology.

BACKGROUND & AIMS: RET tyrosine kinase is necessary for enteric nervous system development. Loss-of-function RET mutations cause Hirschsprung disease (HSCR), in which infants are born with aganglionic bowel. Despite surgical correction, patients with HSCR often experience chronic defecatory dysfunction and enterocolitis, suggesting that RET is important after development. To test this hypothesis, we determined the location of postnatal RET and its significance in gastrointestinal (GI) motility.

METHODS: RetCFP/+ mice and human transcriptional profiling data were studied to identify the enteric neuronal and epithelial cells that express RET. To determine whether RET regulates gut motility in vivo, genetic, and pharmacologic approaches were used to disrupt RET in all RET-expressing cells, a subset of enteric neurons, or intestinal epithelial cells.

RESULTS: Distinct subsets of enteric neurons and enteroendocrine cells expressed RET in the adult intestine. RET disruption in the epithelium, rather than in enteric neurons, slowed GI motility selectively in male mice. RET kinase inhibition phenocopied this effect. Most RET+ epithelial cells were either enterochromaffin cells that release serotonin or L-cells that release peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), both of which can alter motility. RET kinase inhibition exaggerated PYY and GLP-1 release in a nutrient-dependent manner without altering serotonin secretion in mice and human organoids. PYY receptor blockade rescued dysmotility in mice lacking epithelial RET.

CONCLUSIONS: RET signaling normally limits nutrient-dependent peptide release from L-cells and this activity is necessary for normal intestinal motility in male mice. These effects could contribute to dysmotility in HSCR, which predominantly affects males, and uncovers a mechanism that could be targeted to treat post-prandial GI dysfunction.