Project Summary Lung transplantation is a life-saving treatment for people with severe lung disease. However, complications like primary graft dysfunction (PGD) can occur due to ischemia reperfusion injury (IRI), which limits the success of the transplant. PGD often affects other organs, like the kidneys, and can reduce the 5-year survival rate of patients to less than 50%. Currently, treatments that deplete neutrophils, which contribute to PGD, are not feasible due to their role in fighting infections. The goal of this research project is to build on seminal findings during the prior funding cycle and better understand how donor nonclassical monocytes contribute to PGD and injury in other organs. By understanding the mechanisms behind PGD and remote organ injury, new therapeutic strategies to improve outcomes for lung transplant recipients can be developed. The first aim of the project is to investigate how classical monocytes in the recipients contribute to the retention of donor nonclassical monocytes in other organs, leading to injury. Donor nonclassical monocytes migrate to other organs, such as the kidneys and native lung, and cause damage similar to what is observed in the lung allograft. We will study the pathways involved in classical monocyte activation and how they promote donor nonclassical monocyte retention. Through these experiments, we aim to identify potential therapeutic targets to prevent bystander organ injury. The second aim is to understand the different mechanisms of activation and contrasting roles of donor and recipient nonclassical monocytes in the development of PGD. We have found that donor nonclassical monocytes, but not recipient nonclassical monocytes, contribute to lung allograft damage. We investigate the factors that activate donor nonclassical monocytes during the phases of ischemia-reperfusion and their impact on tissue injury. Additionally, we will study the involvement of mitochondrial reactive oxygen species released from donor lung epithelium in upregulating TLR transcription and cell surface translocation on donor nonclassical monocytes. We will also explore our exciting new discovery that Nod2 signaling can promote the conversion of recipient inflammatory classical monocytes into anti-inflammatory nonclassical monocytes. The goal is to develop new therapeutic approaches to mitigate PGD and promote resolution of lung injury. Overall, this study aims to provide a comprehensive understanding of the interactions between donor nonclassical monocytes, host classical monocytes, and other immune cells in the context of PGD and bystander organ injury. The insights gained will help develop new strategies to improve outcomes in lung transplantation and minimize the impact of PGD on graft and recipient survival.