PROJECT SUMMARY Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn disease, is a chronic and progressive inflammatory disorder of the gastrointestinal (GI) tract most often diagnosed in adolescence and young adulthood. While significant progress has been made, current treatments for IBD are still often unsatisfactory, associated with long-term failure and side effects. Investigation into the underlying pathogenesis and mechanisms of this condition are warranted to identify new targets to develop novel treatment strategies. The autonomic nervous system regulates key functions of the GI tract, including both intrinsic (enteric nervous system, ENS) and extrinsic (sympathetic and parasympathetic) control of the intestine. Abnormalities of autonomic nerve function have been described in patients with IBD and several studies suggest that the ENS may play a role in the development and severity of intestinal inflammation. Acetylcholine (ACh) from extrinsic parasympathetic nerves is known to have an anti-inflammatory effect on the gut via the cholinergic anti-inflammatory pathway (CAIP). However, it is unknown whether ACh or other neurotransmitters derived from the neurons of the ENS have a similar role. In preliminary experiments, we find that the severity of experimentally induced colitis is worse in regions of focal ENS ablation. Therefore, we hypothesize that the intrinsic neurons of the ENS can exert an anti- inflammatory effect in the colon and that this can be leveraged for therapeutic applications. To test this, we propose the following aims: (1) to determine the mechanisms by which intrinsic enteric neurons attenuate inflammation in experimental colitis and to (2) examine whether ENS cell transplantation can reduce inflammation in a mouse model of IBD. We will utilize a novel IBD mouse with colonic aganglionosis that we recently developed by local injection of human diphtheria toxin (DT) into transgenic mice that express DT receptor in the ENS. Focal ablation of either all enteric neurons or the cholinergic enteric neurons will be followed by induction of colitis to determine the anti-inflammatory roles of the ENS (Aim 1). We will then purify enteric neuronal stem cells, enteric neurons, and enteric cholinergic neurons and transplant these into recipient mice with focal colonic aganglionosis following induction of colitis. The impact of cell therapy on the severity of inflammation will be examined (Aim 2). The results obtained will provide new insights into the anti-inflammatory role of the ENS and identify new strategies for treating IBD.