Canine granulomatous colitis (GC) is a severe and often fatal inflammatory bowel disease (IBD) affecting young Boxer Dogs and French Bulldogs. It is characterized by chronic diarrhea, wasting, mucosal thickening and ulceration and infiltrates of foamy PAS+ macrophages that harbor intracellular E. coli with concurrent inflammation in the ileum. Although previous seminal work from the Simpson lab helped affected dogs to achieve clinical remission, the majority of dogs now harbor E. coli strains that are multidrug resistant and therefore suffer chronic illness, tragically ending in euthanasia. There is a critical need to deepen understanding of the molecular landscape and pathway alterations in canine GC in order to develop novel strategies for effective treatment and prevention. Research in the Sethupathy lab has revealed that microRNAs (miRNAs) are important prognostic indicators of disease severity and also pathogenic drivers and candidate therapeutic targets in both pediatric and adult human IBD. However, miRNAs are completely unexplored in the context of canine GC. Also, single-cell analysis of gene expression in human IBD has revealed novel insights about pathologic mechanisms and potential cell-to-cell interactions, but this technology has not been applied to the study of canine GC. Over the past couple years, the Simpson and Sethupathy teams have teamed up to generate preliminary data which motivates the hypothesis that miRNAs are likely a critical piece to the puzzle of GC pathogenesis and treatment. In this proposal, the collaborative team will apply state-of-the-art genome-scale techniques to determine the miRNA signature of GC in a large archive of canine tissue samples and also define the single-cell gene expression landscape of GC for the first time. The findings from these studies will represent a significant advance in the field and will lay the groundwork for future investigations focused on the development of novel prognostic, preventative and therapeutic measures.