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. ### Competing Interest Statement The authors have declared no competing interest.

The intestinal epithelial barrier facilitates homeostatic host–microbiota interactions and immunological tolerance. However, mechanistic dissections of barrier dynamics following luminal stimulation pose a substantial challenge. Here, we describe an ex vivo intestinal permeability assay, X-IPA, for quantitative analysis of gut permeability dynamics at the whole-tissue level. We demonstrate that specific gut microbes and metabolites induce rapid, dose-dependent increases to gut permeability, thus providing a powerful approach for precise investigation of barrier functions.

T cells that express the transcription factor RORγ, regulatory (Treg), or conventional (Th17) are strongly influenced by intestinal symbionts. In a genetic approach to identify mechanisms underlying this influence, we performed a screen for microbial genes implicated, in germfree mice monocolonized with Escherichia coli Nissle. The loss of capsule-synthesis genes impaired clonal expansion and differentiation of intestinal RORγ + T cells. Mechanistic exploration revealed that the capsule-less mutants remained able to induce species-specific immunoglobulin A (IgA) and were highly IgA-coated. They could still trigger myeloid cells, and more effectively damaged epithelial cells in vitro. Unlike wild-type microbes, capsule-less mutants were mostly engulfed in intraluminal casts, large agglomerates composed of myeloid cells extravasated into the gut lumen. We speculate that sequestration in luminal casts of potentially harmful microbes, favored by IgA binding, reduces the immune system’s actual exposure, preserving host–microbe equilibrium. The variable immunostimulation by microbes that has been charted in recent years may not solely be conditioned by triggering molecules or metabolites but also by physical limits to immune system exposure.