Project Summary Helicobacter pylori is the strongest known risk factor for gastric cancer and interactions between this chronic pathogen and immune cells dysregulate gastric epithelial signaling pathways that influence carcinogenesis. One H. pylori oncogenic determinant is the cag type IV secretion system (TFSS) which translocates pro- inflammatory effectors, such as CagA, into epithelial cells. In studies supported by R01 CA 77955, we demonstrated that the cag TFSS can also translocate peptidoglycan, which is sensed by the pattern recognition receptor (PRR), NOD1. However, negative regulation of certain PRRs by chronic pathogens can increase pro-inflammatory responses and disease, and we have also demonstrated that prolonged H. pylori infection activates a NOD1-dependent negative feedback loop in gastric epithelial cells, leading to increased NF-kB activation. These data have driven our recent efforts to more broadly define the consequences of NOD1 suppression using human samples and rodent models; these findings demonstrate that 1) NOD1 expression is reduced in H. pylori-infected gastric cancer tissue compared with uninvolved tissue, 2) genetic deficiency of Nod1 significantly increases H. pylori-induced carcinogenesis and expression of the Th9 cytokine IL-9, a key effector of epithelial damage and inflammation, 3) H. pylori up-regulates expression of the IL-9 receptor in ex vivo epithelial gastroids, and 4) treatment of H. pylori-infected gastroids with IL-9 leads to enhanced cell survival within the context of Nod1 deficiency. We have established innovative models (primary gastroid:macrophage:T cell co-culture systems) that more closely recapitulate the infected gastric niche to demonstrate that H. pylori upregulates IL-9 production in a cag TFSS-dependent manner. Finally, we have gastric tissue and sera from a unique longitudinal human cohort in Colombia that includes persons who either progressed to irreversible premalignant gastric lesions or remained stable, which will provide critical clinical validation of our mechanistic studies. Our hypothesis is that suppression of NOD1 signaling contributes to the augmentation in cancer risk conferred by H. pylori by deploying immune responses (Th9) with carcinogenic potential. We will test this hypothesis via these Specific Aims: 1. Utilize novel rodent models and ex vivo systems to define IL-9-dependent host responses linked to H. pylori- induced disease progression 2. Validate and extend mechanistic studies focused on H. pylori, IL-9, and NOD1 using samples and H. pylori strains isolated from persons who did or did not progress towards gastric cancer 3. Use targeted approaches to inhibit IL-9 immune responses within the context of H. pylori infection