Summary/Abstract of the diversity supplement to the parent project. Despite recent advances in the understanding host-microbiome interactions in the pathogenesis of Inflammatory Bowel Diseases (IBD), the complexity of the host’s response to changing gut microbiota is daunting and still incompletely understood. In this proposal, we show that poly-ADP-ribosylation (PARylation), a post-translational modification that involves the enzymatic transfer of ADP-ribose (ADPr) from NAD+ to specific amino acids of target proteins, plays key roles as a mediator of inflammatory response in the gut. In the parent proposal, we provided evidence that PARP1 is the primary PAR writer in the colon, where it serves as a powerful transcriptional modulator. Commensal bacteria are necessary for mucosal PARP1 activity and PARylation and, reciprocally, PARP1 controls the microbial composition and metabolic activity, modulates colonic epithelial barrier function, and restricts the mucosal Treg compartment. Importantly, human and murine colitis is associated with mucosal hyperPARylation, which can be transferred to germ-free mice with complex microbial community from IBD patients. Total or epithelial-specific knockout of PARP1 (or pharmacological inhibition) protect from and promote recovery from mucosal injury. In this administrative supplement, the work conducted by Ms. Catherine Ellis, PhD candidate in the Kiela lab, further expands these findings and describes the screening process using untargeted metabolomic and lipidomic analysis of stool samples from healthy and IBD patients, and the identification of protein and lipid fraction as responsible for hyperPARylation in the colonic epithelium. Among lipid luminal metabolites, fecal lactosylceramide (highly elevated in IBD samples and described by others as a reliable marker of IBD) appears to be the key driver of hyperPARylation in the inflamed gut. Based on these novel preliminary observations, we expand our hypothesis and plan to test the following: 1. Define the role of lactosylceramide and related sphingolipids in promoting epithelial hyperPARylation. 2. Mechanistically define the PARylation-dependent effects of lactosylceramide on epithelial cell dysfunction under inflammatory stress.