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IntroductionEnteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed.MethodsWe examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3’ bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice.ResultsWe identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1β treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation.DiscussionTogether, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.

Enteric glia are a heterogeneous population of peripheral glia within the enteric nervous system and play pivotal roles in gut homeostasis, tissue integrity, coordination of motility, and intestinal immune responses. Under physiological conditions, they communicate with enteric neurons to control intestinal motility. In contrast, enteric glia undergo reactive changes in response to inflammatory signals during enteric neuroinflammation and participate in immune control. In this state, these glia are called reactive enteric glia, which promote cytokine and chemokine secretion and perpetuate immune cell recruitment, thereby affecting disease progression. Interestingly, reactive glia exhibit a huge plasticity and adapt to or shape the immune environment towards a resolving phenotype during inflammation and neuropathies. Recent studies revealed a bidirectional communication between enteric glia and resident and infiltrating immune cells under healthy conditions and in the context of inflammation‐based intestinal disorders and neuropathies. While recent reviews give a superb general overview of enteric glial reactivity, we herein discuss the latest evidence on enteric glial reactivity in two prominent inflammatory conditions: acute postoperative inflammation, resulting in postoperative ileus, and chronic inflammation in inflammatory bowel diseases. We define their plasticity during inflammation and the interplay between reactive enteric glia and intestinal macrophages. Finally, we sketch important questions that should be addressed to clarify further the impact of enteric glial reactivity on intestinal inflammation. [ABSTRACT FROM AUTHOR]