Project Summary The overarching objective of this proposal is to establish a new and innovative strategy for Inflammatory bowel disease (IBD) treatment that leverages the important role of cholinergic innervation in mediating intestinal inflammation. Current treatments of IBD have limited efficacy and fail to promote sustained disease remission, necessitating surgical intervention in the majority of patients. The development of an effective, durable, and well-tolerated therapy is a major unmet need. Developing a neuroimmune therapy for IBD will add a valuable and complementary new approach to treating this disease and hopefully reducing its long- term consequences. It has been shown that intestinal inflammation can lead to enteric neuronal injury and subsequent intestinal dysfunction, including disorders of gut motility. Damage to the enteric nervous system (ENS) may also contribute to the development and severity of intestinal inflammation. Therefore, the ENS emerges as a critical participant in immunomodulation within the gut. Previous studies have demonstrated that cholinergic neurons directly interact with muscularis macrophages, and their numbers are reduced in inflammation. Our preliminary data show for the first time that selective optogenetic stimulation of cholinergic neurons attenuates colitis, while ablation of enteric cholinergic neurons worsens inflammation in DSS- induced acute colitis. We hypothesize that enteric cholinergic signalling can be harnessed as a novel therapeutic strategy for the treatment of IBD. In the proposed study, we will first determine the mechanism by which cholinergic signaling attenuates inflammation by investigating the impact of cholinergic neuronal activation on muscularis macrophage polarization and cytokine production. Second, we will use optogenetics to selectively stimulate enteric cholinergic neurons in Winnie mice, a well-established model of chronic colitis that closely resembles human IBD to determine the effect of cholinergic signaling on disease onset and the severity of inflammation. As parameters of anti-inflammatory activity, we will investigate changes in morphology and inflammatory indices, as well as changes in smooth muscle contractility and motility as measured by organ bath experiments and electromyography. These studies will provide important insights into the mechanisms by which cholinergic neurons can improve colitis and offer a potential novel treatment strategy for IBD.