PROJECT SUMMARY Intestinal epithelial cells protect the host from pathogens and prevent exaggerated inflammatory responses that cause collateral injury. Defining the cellular mechanisms that regulate intestinal inflammation is an essential first step to uncover therapeutic targets that can manipulate these physiological processes to the host's advantage. Nuclear hormone receptors are ligand-activated transcription factors that regulate metabolism and many other aspects of cellular physiology. However, little is known about how nuclear hormone receptors influence intestinal inflammation or the innate recognition of bacterial pathogens. In C. elegans, the family of nuclear hormone receptors has dramatically expanded compared to other metazoans, particularly those that are homologous to the human intestinal nuclear hormone receptor hepatocyte nuclear factor 4 (HNF4). These proteins may, therefore, have critical roles in nematode physiology. In this competitive renewal application, we aim to determine how nuclear hormone receptors regulate immune activation and pathogen resistance. We will test the conceptually innovative hypothesis that nuclear hormone receptors function as pathogen sensors that directly activate innate immune defenses in intestinal epithelial cells. C. elegans does not seem to rely on canonical pattern recognition receptors (e.g., Toll-like receptors) to sense pathogen infection and activate immune defense in intestinal epithelial cells. Thus, the key implication of our findings is that the nuclear hormone receptor family in C. elegans expanded, at least in part, because of their function in pathogen detection and immune regulation. In the first funding period, we identified a homolog of mammalian HNF4 that surveys the chemical environment and activates anti-pathogen immune defenses. In this competitive renewal application, we will test the conceptually innovative hypothesis that sensing of a pathogen-derived bacterial metabolite by this nuclear hormone receptor is an ancient mechanism utilized by C. elegans to detect toxigenic pathogens in its environment that have grown to dangerous levels and are poised to cause disease (Aim 1). In Aim 2, we will determine if this pathogen-derived metabolite is a natural ligand for NHR-86 that directly binds to and activates this nuclear hormone receptor, an effort that will characterize the first bona fide innate immune pattern recognition receptor and its natural ligand in C. elegans and de-orphan a nuclear hormone receptor. Finally, in Aim 3, we will characterize the role of another HNF4 homolog in innate immunity, which will provide important support for the innovative idea that members of this protein family function as pathogen sensors in an organism that lacks canonical mechanisms of pattern recognition. The proposed study will reveal fundamental insights into previously unrecognized, evolutionary ancient strategies of immune activation and regulation that will have broad implications for...