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

Abstract The enteric nervous system (ENS) senses microbiota-derived signals and orchestrates mucosal immunity and epithelial barrier functions, in health and disease. However, mechanistic dissections of intestinal neuro-immune-microbiota communications remain challenging and existing research methods limit experimental controllability and throughput. Here, we present a novel optogenetics-integrated gut organ culture system that enables real-time, whole-tissue stimulation of specific ENS lineages, allowing for 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 promotes gene-sets associated with type-2 immunity, tachykininergic enteric neurons differentially control mucosal defense programs. Remarkably, luminal introduction of the immunomodulatory bacterium C. ramosum significantly remodeled cholinergic-induced neuro-immunological transcription. These findings suggest that complex combinatorial signals delivered by gut microbes and enteric neurons are locally integrated to fine-tune intestinal 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 drugs, microbes, or metabolites. Short abstract The enteric nervous system senses microbiota-derived signals and orchestrates mucosal immunity and epithelial barrier functions. Mechanistic dissections of intestinal neuro-immune-microbiota communications remain challenging. We developed an optogenetics-integrated gut organ culture system for real-time neuronal stimulation and analysis. We revealed neuronal-specific activity patterns, which differentially regulate intestinal transcription and epithelial barrier integrity. Collectively, we provide a powerful platform to test neuroimmune connections and their potential modulation by drugs, microbes, or metabolites.