Project Summary / Abstract Cardiovascular disease and heart failure are highly prevalent among the veteran population. Cardiac transplantation remains a preferred therapy for patients who suffer from end-stage heart failure. However, outcomes after heart transplantation are adversely impacted by primary graft dysfunction, a consequence of ischemia reperfusion injury. Primary graft dysfunction causes immediate tissue damage and can also augment adaptive immune responses that trigger graft rejection. Currently, there are no effective therapies for primary graft dysfunction after heart transplantation and the management of these patients is mostly supportive. Our laboratory has developed intravital imaging platforms that has allowed us to visualize the infiltration of leukocytes into murine heart grafts in real time. Through these approaches we have uncovered cellular and molecular cues that regulate the trafficking of neutrophils and monocytes, innate immune cells that are known to mediate tissue damage, into transplanted hearts. Our findings raise the intriguing prospect that targeting immune pathways and cell populations within the donor graft can control the initial immune response following heart transplantation. During the previous funding period we have discovered that ferroptosis, a non- apoptotic form of cell death mediates the early inflammatory response after reperfusion of heart grafts. We have reported that graft endothelial cells and tissue-resident CCR2+ macrophages play critical and complementary roles in driving the recruitment of neutrophils to the transplanted heart. Now we have generated preliminary data showing that additional donor immune cell populations and signaling pathways regulate leukocyte recruitment to cardiac grafts. In this proposal, we will use state-of-the-art techniques including intravital two-photon microscopy, new positron emission tomography probes, single cell RNA sequencing and novel murine strains to perform studies that will define the role of donor non-classical monocytes (Aim 1) and TREM-1/3 signaling (Aim 2) in promoting inflammatory responses after reperfusion of ischemic heart grafts. Our studies will lay the foundation for novel therapies that will improve outcomes for heart transplant recipients and patients who suffer from myocardial ischemia reperfusion injury due to other conditions.