PROJECT SUMMARY Our overarching goal is to understand how the epithelial-specific ER stress sensor IRE1b (ERN2) and its ubiquitously expressed paralogue IRE1a (ERN1) contribute to intestinal homeostasis and host defense. A notable adaptation of the ER unique to epithelial cells lining mucosal surfaces, especially in the gut, is expression of the ER stress sensor IRE1b, a close paralogue of the major and most evolutionarily conserved ER stress sensor IRE1a. We recently discovered, however, that IRE1b functions distinctly from IRE1a in enzymatic activity, enabling it to act as a non-competitive inhibitor of IRE1a signaling in the ER stress response. Our new and ongoing studies demonstrate in vivo that IRE1b operates critically in goblet cell differentiation and assembly of the mucus barrier - with both activities dependent on the normal gut microbiota: gut microbes and their products stimulate IRE1b expression, and IRE1b in turn affects the assembly, taxonomy, and functional output of colonizing gut microbes. These data frame our central hypothesis that IRE1b acts as an environmentally induced mediator of innate epithelial host defense by affecting the ER stress response via IRE1a, and then goblet cell development, mucus assembly, and host epithelial-microbe interactions that shape the structure and function of the colon. In Aim 1, we will use recombinant proteins and biochemical and biophysical approaches to test how IRE1a and IRE1b may be activated by gut lumenal factors, explain how IRE1b oligomerizes with IRE1a, and elucidate what structural features and post-translational modifications modify this assembly, IRE1b signaling, and mechanisms for IRE1b activation and suppression of IRE1a function. In Aim 2, we will use wt and IRE1b-/- mice and colonoids prepared from these animals to determine how IRE1b links the gut microbiota with epithelial stress and development responses and identify core microbial pathways and metabolites that regulate IRE1b expression and perhaps function. In Aim 3, we will explain how the lack of IRE1b attenuates recovery from acute infection with C. rodentium. We found that IRE1b-/- mice have impaired assembly of the colonic mucus layer and dysbiosis - mimicking defects in the colonic mucosa seen in humans with ulcerative colitis (UC). As modeled by Citrobacter rodentium, the altered mucus layer in IRE1b-/- mice was associated with impaired recovery following normal clearance of the pathogen. We will (i) test if the dysbiotic gut microbiota, or unresolved epithelial ER stress, or both, drive the sustained inflammation following C. rodentium infection in IRE1b-/- mice; and (ii) determine if microbiota obtained from humans with UC phenocopy these effects and fail to induce IRE1b, goblet cell development, or an effective mucus barrier when reconstituted in germ-free wt IRE1b+/+ mice. ER stress is implicated in the chronic inflammatory and other intestinal diseases. The outcomes of these studies will be informative of the hos...