Project summary The immune response to Toxoplasma gondii, a prevalent foodborne pathogen, relies heavily on the cytokine IFN-γ. Previous research has primarily focused on the role of dendritic cells and macrophages in triggering IFN- γ responses. However, the contribution of non-myeloid cells, particularly intestinal epithelial cells (IECs), in IFN- γ-mediated host defense remains poorly understood. Our preliminary data suggest that IECs play a central role in inducing intestinal immunopathology during T. gondii infection, characterized by tissue damage without a significant increase in parasite burden. We have also identified a novel mTOR-dependent mechanism of Paneth cell death, a subset of IECs, in IFN-γ-induced immunopathological responses. However, the responses of other IECs to IFN-γ are largely unknown. In this competitive renewal, our goal is to gain a mechanistic understanding of IEC-specific IFN-γ-mediated immunity to T. gondii by investigating both protective and immunopathological responses. We have two specific aims: Aim 1: Determine intestinal epithelial cell-specific responses to IFN-γ during T. gondii infection. In this aim, we aim to identify the specific IECs that mediate IFN-γ-dependent immunopathology triggered by T. gondii infection and define the lineage-specific responses of IECs to IFN-γ during mucosal infection. Aim 2: Identify the IFN-γ-induced metabolic adaptations of epithelial cells contributing to the pathological response during T. gondii infection. Building upon our previous findings of mTOR inactivation and Paneth cell death, we will investigate the metabolic changes in IECs triggered by IFN-γ. By the completion of this aim, we will have a comprehensive understanding of the cellular and molecular mechanisms underlying IFN-γ-mediated intestinal pathology during T. gondii infection. The proposed project is significant and innovative due to its novel concept of IFN-mediated immunity and immunopathology at the site of infection, and the experimental tools developed to accomplish the goal of this proposal during the previous funding period.