A critical role for macrophage ferroptosis in promoting fungal invasion in lung transplant recipients

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PROJECT SUMMARY/ ABSTRACT This application focuses on the contribution of transplant hemorrhage-induced iron overload in the dysregulation of pulmonary macrophages (mɸs) and the promotion of invasive aspergillosis. Aspergillus fumigatus (Af) is a ubiquitous mold that releases airborne spores (conidia) and affects nearly 20 million people worldwide. One-in- three lung transplant recipients (LTRs) suffers from Aspergillus-related pulmonary disease. While lung transplantation can be a life-saving treatment for thousands of people, survival post-transplant is often limited by Af infection. To better understand the transplant (host)-Af (pathogen) relationship, we developed a murine orthotopic tracheal transplant (OTT) model of Af infection. We have shown that transplant rejection-mediated microhemorrhage increases tissue iron levels and determines Af invasion. However, the exact interaction between immunity, iron overload and infection are still poorly understood. Mɸs are the first line of defense against Af and are also central to restoring tissue iron homeostasis. Importantly, our preliminary results indicate that microhemorrhage-mediated iron overload: (i) profoundly impacts the ability of mfs to kill Af conidia through a defect in lysosomal acidification; (ii) the innate immune response is polarized toward a pro-inflammatory mɸs state that results in high levels of tissue damaging reactive oxygen species (ROS); and (iii) iron promotes mɸ ferroptosis. Ferroptosis is a newly recognized form of regulated cell death that results from the production of iron toxic lipid ROS. Ferroptosis was first recognized in cancer but is now known to contribute to Alzheimer’s and Parkinson’s disease, ischemia reperfusion injury, atherosclerosis, acute kidney injury and the response to acute hemorrhage. However, the role of ferroptosis in lowering the host’s defense against pathogens, if any, remains unknown. The proposed studies are designed to address these questions in terms of Af invasion. The central hypothesis is that transplant microhemorrhage-mediated iron overload induces mf ferroptosis and polarization into an unrestrained pro-inflammatory phenotype that promotes Af invasion. Specific aim 1 utilizes in vitro and in vivo experiments to investigate the concept that ferroptosis is dictated by mɸs polarization state and contributes to the inability of transplant mɸs to mitigate Af infection and studies the role of iron lowering agents and anti-ferroptotic drugs to decrease fungal invasion. Specific aim 2 uses state-of-art omics techniques to define iron induced Af proteases and tests the concept that fungal protease inhibition can mitigate ferroptosis and improve outcomes in the tracheal transplant model. Specific aim 3 studies the ability of alveolar mɸs isolated from human LTRs compared to non-lung transplants to kill Af conidia and correlates the ability of mɸs to kill conidia with mɸs-polarization state and ferroptosis, using mass cytometry. This aim will provide a direct translatability of the hypothesis that iron overload induces ferroptosis and a pro-inflammatory phenotype that promotes fungal invasion. Successful completion of these studies will allow for the discovery of a fundamental new biology and provide novel targets for the treatment of fungal infections.