Enteric neuronal culture system as a platform to study neuroimmune interactions in the gut

Abstract

The enteric nervous system is composed of a heterogeneous population of putative sensory neurons, interneurons, secretomotor neurons, and excitatory and inhibitory motor neurons that coordinate intestinal function and immunity. Under inflammatory conditions, specific cytokine receptor signaling in enteric neurons and subsequent neuropeptide action on the immune cells via specific neuropeptide receptors contribute to neuro-immune interactions in the gut. However, the set of all functional cytokine receptors expressed by enteric neurons and the downstream effects of their activation are not fully understood. Owing to this, it is crucial to investigate which specific receptors and signaling cascades are active in different subsets of enteric neurons. In this study, we adopted an in vitro model to differentiate enteric neurons from neurospheres formed from murine neural crest progenitors. We show that enteric neurons exhibit stimulus-specific transcriptional responses to inflammatory cues, including cytokines and bacterial lipopolysaccharide. Targeted gene expression analysis studies on in vitro differentiated enteric neurons by qPCR revealed changes in the gene expression levels of certain neuropeptides, cytokines, and cytokine receptors in response to different stimuli. In addition to this, we were able to identify distinct drivers of neuronal activation in response to inflammation. We also employed immunocytochemistry and fluorescent in situ hybridization-based characterization to study neuronal activation. We further optimized a cFos-GFP system to quantify activated neurons in culture. Together, these findings help us provide a base for building an atlas of enteric neuronal responses to inflammation and use it as a platform to study neuroimmune signaling in ENS.