Enteric glial cells (EGC) modulate motility, maintain gut homeostasis, and contribute to neuroinflammation in intestinal diseases and motility disorders. Damage induces a reactive glial phenotype known as "gliosis", but the molecular identity of the inducing mechanism and triggers of "enteric gliosis" are poorly understood. We tested the hypothesis that surgical trauma during intestinal surgery triggers ATP release that drives enteric gliosis and inflammation leading to impaired motility in postoperative ileus (POI). ATP activation of a p38-dependent MAPK pathway triggers cytokine release and a gliosis phenotype in murine (and human) EGCs. Receptor antagonism and genetic depletion studies revealed P2X2 as the relevant ATP receptor and pharmacological screenings identified ambroxol as a novel P2X2 antagonist. Ambroxol prevented ATPinduced enteric gliosis, inflammation, and protected against dysmotility, while abrogating enteric gliosis in human intestine exposed to surgical trauma. We identified a novel pathogenic P2X2dependent pathway of ATP-induced enteric gliosis, inflammation and dysmotility in humans and mice. Interventions that block enteric glial P2X2 receptors during trauma may represent a novel therapy in treating POI and immune-driven intestinal motility disorders.

Muscularis Externa Macrophages (ME-Macs) and enteric glial cells (EGCs) are closely associated cell types in the bowel wall, and important interactions are thought to occur between them during intestinal inflammation. They are involved in developing postoperative ileus (POI), an acute, surgery-induced inflammatory disorder triggered by IL-1 receptor type I (IL1R1)-signaling. In this study, we demonstrate that IL1R1-signaling in murine and human EGCs induces a reactive state, named enteric gliosis, characterized by a strong induction of distinct chemokines, cytokines, and the colony-stimulating factors 1 and 3. Ribosomal tagging revealed enteric gliosis as an early part of POI pathogenesis, and mice with an EGC-restricted IL1R1-deficiency failed to develop postoperative enteric gliosis, showed diminished immune cell infiltration, and were protected from POI. Furthermore, the IL1R1-deficiency in EGCs altered the surgery-induced glial activation state and reduced phagocytosis in macrophages, as well as their migration and accumulation around enteric ganglia. In patients, bowel surgery also induced IL-1-signaling, key molecules of enteric gliosis, and macrophage activation. Together, our data show that IL1R1-signaling triggers enteric gliosis, which results in ME-Mac activation and the development of POI. Intervention in this pathway might be a useful prophylactic strategy in preventing such motility disorders and gut inflammation.

Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAPcre x Ai14floxed mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor.

Background: Transcriptional profiling of specific intestinal cell populations under health and disease is generally based on traditional sorting approaches followed by gene expression analysis. Therein, specific cell populations are identified either by expressing reporter genes under a cell type-specific promotor or by specific surface antigens. This method provides adequate results for blood-derived and tissue-resident immune cells. However, in stromal cell analysis, cellular stress due to digestion often results in degraded RNA. Particularly, ramified cells integrated into the tissue, such as enteric neurons and glial cells, suffer from these procedures.These cell types are involved in various intestinal processes, including a prominent immune-regulatory role, which requires suitable approaches to generate cell-specific transcriptional profiles.Methods: Sox10 iCreERT2 and choline acetyltransferase (ChAT Cre ) mice were crossed with mice labeling the ribosomal Rpl22 protein upon Cre activity with a hemagglutinin tag (Rpl22-HA, termed RiboTag). This approach enabled cellular targeting of enteric glia and neurons and the immediate isolation of cell-specific mRNA from tissue lysates without the need for cell sorting. Key results:We verified the specific expression of Rpl22-HA in enteric glia and neurons and provided gene expression data demonstrating a successful enrichment of either Sox-10 + glial or ChAT + neuronal mRNAs by the RiboTag-mRNA procedure using qPCR and RNA-Seq analysis. Conclusions and inferences:We present a robust and selective protocol that allows the generation of cell type-specific transcriptional in vivo snapshots of distinct enteric cell populations that will be especially useful for various intestinal disease models involving peripheral neural cells.

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Background and PurposeET‐1 signalling modulates intestinal motility and inflammation, but the role of ET‐1/ETB receptor signalling is poorly understood. Enteric glia modulate normal motility and inflammation. We investigated whether glial ETB signalling regulates neural‐motor pathways of intestinal motility and inflammation.Experimental ApproachWe studied ETB signalling using: ETB drugs (ET‐1, SaTX, BQ788), activity‐dependent stimulation of neurons (high K+‐depolarization, EFS), gliotoxins, Tg (Ednrb‐EGFP)EP59Gsat/Mmucd mice, cell‐specific mRNA in Sox10CreERT2;Rpl22‐HAflx or ChATCre;Rpl22‐HAflx mice, Sox10CreERT2::GCaMP5g‐tdT, Wnt1Cre2::GCaMP5g‐tdT mice, muscle tension recordings, fluid‐induced peristalsis, ET‐1 expression, qPCR, western blots, 3‐D LSM‐immunofluorescence co‐labelling studies in LMMP‐CM and a postoperative ileus (POI) model of intestinal inflammation.Key ResultsIn the muscularis externa ETB receptor is expressed exclusively in glia. ET‐1 is expressed in RiboTag (ChAT)‐neurons, isolated ganglia and intra‐ganglionic varicose‐nerve fibres co‐labelled with peripherin or SP. ET‐1 release provides activity‐dependent glial ETB receptor modulation of Ca2+ waves in neural evoked glial responses. BQ788 reveals amplification of glial and neuronal Ca2+ responses and excitatory cholinergic contractions, sensitive to L‐NAME. Gliotoxins disrupt SaTX‐induced glial‐Ca2+ waves and prevent BQ788 amplification of contractions. The ETB receptor is linked to inhibition of contractions and peristalsis. Inflammation causes glial ETB up‐regulation, SaTX‐hypersensitivity and glial amplification of ETB signalling. In vivo BQ788 (i.p., 1 mg·kg−1) attenuates intestinal inflammation in POI.Conclusion and ImplicationsEnteric glial ET‐1/ETB signalling provides dual modulation of neural‐motor circuits to inhibit motility. It inhibits excitatory cholinergic and stimulates inhibitory nitrergic motor pathways. Amplification of glial ETB receptors is linked to muscularis externa inflammation and possibly pathogenic mechanisms of POI.

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