ß-adrenergic signaling triggers enteric glial reactivity and acute enteric gliosis during surgery.

BACKGROUND: Enteric glia contribute to the pathophysiology of various intestinal immune-driven diseases, such as postoperative ileus (POI), a motility disorder and common complication after abdominal surgery. Enteric gliosis of the intestinal muscularis externa (ME) has been identified as part of POI development. However, the glia-restricted responses and activation mechanisms are poorly understood. The sympathetic nervous system becomes rapidly activated by abdominal surgery. It modulates intestinal immunity, innervates all intestinal layers, and directly interfaces with enteric glia. We hypothesized that sympathetic innervation controls enteric glia reactivity in response to surgical trauma. METHODS: Sox10iCreERT2/Rpl22HA/+ mice were subjected to a mouse model of laparotomy or intestinal manipulation to induce POI. Histological, protein, and transcriptomic analyses were performed to analyze glia-specific responses. Interactions between the sympathetic nervous system and enteric glia were studied in mice chemically depleted of TH+ sympathetic neurons and glial-restricted Sox10iCreERT2/JellyOPfl/+/Rpl22HA/+ mice, allowing optogenetic stimulation of ß-adrenergic downstream signaling and glial-specific transcriptome analyses. A laparotomy model was used to study the effect of sympathetic signaling on enteric glia in the absence of intestinal manipulation. Mechanistic studies included adrenergic receptor expression profiling in vivo and in vitro and adrenergic agonism treatments of primary enteric glial cell cultures to elucidate the role of sympathetic signaling in acute enteric gliosis and POI. RESULTS: With ~ 4000 differentially expressed genes, the most substantial enteric glia response occurs early after intestinal manipulation. During POI, enteric glia switch into a reactive state and continuously shape their microenvironment by releasing inflammatory and migratory factors. Sympathetic denervation reduced the inflammatory response of enteric glia in the early postoperative phase. Optogenetic and pharmacological stimulation of ß-adrenergic downstream signaling triggered enteric glial reactivity. Finally, distinct adrenergic agonists revealed ß-1/2 adrenoceptors as the molecular targets of sympathetic-driven enteric glial reactivity. CONCLUSIONS: Enteric glia act as early responders during post-traumatic intestinal injury and inflammation. Intact sympathetic innervation and active ß-adrenergic receptor signaling in enteric glia is a trigger of the immediate glial postoperative inflammatory response. With immune-activating cues originating from the sympathetic nervous system as early as the initial surgical incision, adrenergic signaling in enteric glia presents a promising target for preventing POI development.

BQ788 reveals glial ETB receptor modulation of neuronal cholinergic and nitrergic pathways to inhibit intestinal motility: Linked to postoperative ileus.

BACKGROUND AND PURPOSE: ET-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 APPROACH: We 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 RESULTS: In 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 IMPLICATIONS: Enteric 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.

IL-1-dependent enteric gliosis guides intestinal inflammation and dysmotility and modulates macrophage function.

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.

Application of a RiboTag-based approach to generate and analyze mRNA from enteric neural cells.

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: Sox10iCreERT2 and choline acetyltransferase (ChATCre ) 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.

Intestinal Epithelial Barrier Maturation by Enteric Glial Cells Is GDNF-Dependent.

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.

A novel P2X2-dependent purinergic mechanism of enteric gliosis in intestinal inflammation.

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 ATP-induced enteric gliosis, inflammation, and protected against dysmotility, while abrogating enteric gliosis in human intestine exposed to surgical trauma. We identified a novel pathogenic P2X2-dependent 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.