The role of enteric glia in acute gut inflammation

Abstract

<strong>Background: </strong> Enteric glia, a heterogeneous type of neuroglia of the enteric nervous system, are contributors to the etiology of several immune-related gut diseases, such as the acute motility disorder postoperative ileus (POI). These cells are tightly integrated into the tissue and require dedicated novel approaches to analyze their cell-specific transcriptomes. While other tissue-resident cells, such as <em>muscularis externa</em> (<em>ME</em>) macrophages, have been analyzed, enteric glia and their modes of activation, such as their switch to a reactive phenotype during postoperative trauma, are still elusive. Herein, immediate signals from the sympathetic nervous system (SNS) acting on enteric glia might be the initial trigger of their reactivity. As IL-1 receptor signaling, and the release of ATP after trauma are important drivers of POI symptoms, their actions on enteric glia might exacerbate the initial reactivity and promote a gliosis phenotype. <br /> <strong>Methods: </strong> Glial <em>RiboTag</em> mice, created by crossing <em>Sox10^iCreERT2</em> with <em>Rpl22^HA/+</em> mice, were used to generate cell-specific mRNA. Mice (<em>C57/Bl6</em>, glial <em>RiboTag</em>, <em>IL-1R1^flox/GFAP^Cre</em>) underwent an established POI mouse model involving laparotomy with or without mechanical disturbance of the small bowel. Postoperative outcome was additionally modified by pharmacological modulation of different pathways. <em>RiboTag</em> mice carrying a <em>JellyOP^fl/+</em> construct were used for optogenetic stimulation of the G_s protein cascade in enteric glia and analysis of glial-specific transcriptomes. Primary cell cultures from human and murine enteric glia were used to analyze various active compounds related to the adrenergic, IL-1, or ATP pathways. Primary immune cell cultures were used to investigate their interaction with glial-derived factors. <em>Ex vivo</em> tissue cultures and calcium imaging were used to elucidate the effects further. Histology and protein analyses were conducted to investigate glial changes regarding acute inflammation and the pathways of choice. <br /> <strong>Results: </strong> Specific expression of the glial <em>RiboTag</em> was validated, and protocols for its isolation from <em>ME</em> tissue were optimized. Longitudinal analysis of glial-specific transcriptomes revealed an early postoperative reactivity shaping the inflammatory microenvironment. Modulation of sympathetic signaling affected glial reactivity, especially during disease onset, with β-1/2 adrenoceptors as potential lynchpins of immediate postoperative gliosis. IL-1R1-signaling triggered gliosis in mice and humans accompanied by the upregulation of migratory mediators, while genetic deletion of IL-1R1 in glia abolished gliosis, changed the immune cell status, and protected from POI. Purinergic receptor activation by ATP triggered gliosis in a P2X2-dependent manner, and Ambroxol was identified as a novel P2X2 antagonist that can ameliorate gliosis and POI symptoms in murine and human specimens. <br /> <strong>Conclusions: </strong> We developed and refined a distinct protocol enabling time-point- and glia-specific <em>in vivo</em> mRNA snapshots. We revealed enteric glia as one of the earliest responding cell types after surgery through their innervation by the SNS, thereby evoking gliosis and POI initiation. This gliosis signature is further exacerbated by glial IL-1R1 signaling and ATP on glial P2X2. Modulation of either of these pathways reduced gliosis, ameliorated POI, and thus offered new avenues of POI prevention and curation.