Optogenetics-integrated gut organ culture system connects enteric neurons dynamics and gut homeostasis

The enteric nervous system (ENS) senses microbiota-derived signals and orchestrates mucosal immunity and epithelial barrier functions. However, mechanistic dissections of intestinal neuro-immune-microbiota communications remain challenging. Here, we present an optogenetics-integrated gut organ culture system that enables real-time, whole-tissue stimulation of defined ENS lineages, and detailed analysis of their functional impact. We demonstrate that optogenetic activation of enteric cholinergic neurons rapidly modulates intestinal physiology. Interestingly, distinct neuronal firing patterns differentially modulate neuro-immunological gene expression and epithelial barrier integrity. Furthermore, diverse enteric neuronal lineages exert distinct regulatory roles. While cholinergic activation enhances gene-sets associated with type-2 immunity, tachykininergic neurons modulate distinct mucosal defense programs. Intriguingly, luminal introduction of the immunomodulatory bacterium Thomasclavelia ramosa remodeled cholinergic-induced neuro-immunological transcription. These findings suggest that microbial and neuronal signals are locally integrated to fine-tune gut immunity and barrier defense. Collectively, we provide a powerful platform for systematic discovery and mechanistic exploration of functional neuroimmune connections, and their potential modulation by microbes, drugs or metabolites.